Physiol Rev
82: 769 – 824, 2002; 10.1152/physrev.00008.2002.
Cyclic Nucleotide-Gated Ion Channels
U. BENJAMIN KAUPP AND REINHARD SEIFERT
Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany
Introduction
Basic Functional Properties: An Overview
Excursion on Cyclic Nucleotides
Cellular Function
A. CNG channels in vertebrate and invertebrate photoreceptors
B. CNG channels in chemosensory cells
C. CNG channels in the brain
D. CNG channels in spermatozoa
E. Miscellaneous nonneuronal tissues
F. Conclusions
V. Molecular Diversity of Cyclic Nucleotide-Gated Channel Subunits
A. Diversity of subunit genes
B. Functional domains
C. Posttranslational modifications
VI. Molecular Composition and Tissue Distribution
A. Molecular composition of CNG channels in photoreceptors and olfactory neurons
B. Subunit distribution in the brain and nonneuronal tissues
VII. Ligand Sensitivity and Selectivity
A. cNMP-binding site
B. C-linker region
C. NH2-terminal region
D. Pore
E. Conformational changes of protein domains during activation
F. Kinetic models
VIII. Ion Selectivity
A. Selectivity for alkali ions
B. Blockage by divalent cations
C. Ca2⫹ permeation
D. Comparison of CNG channels and voltage-gated Ca2⫹ channels
IX. Channel Modulation
A. Phosphorylation/dephosphorylation
B. Ca2⫹-binding proteins
C. Miscellaneous
X. Pharmacology
XI. Channelopathies and Knock-out Models
XII. Outlook
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I.
II.
III.
IV.
Kaupp, U. Benjamin, and Reinhard Seifert. Cyclic Nucleotide-Gated Ion Channels. Physiol Rev 82: 769 – 824,
2002; 10.1152/physrev.00008.2002.—Cyclic nucleotide-gated (CNG) channels are nonselective cation channels first
identified in retinal photoreceptors and olfactory sensory neurons (OSNs). They are opened by the direct binding of
cyclic nucleotides, cAMP and cGMP. Although their activity shows very little voltage dependence, CNG channels
belong to the superfamily of voltage-gated ion channels. Like their cousins the voltage-gated K⫹ channels, CNG
channels form heterotetrameric complexes consisting of two or three different types of subunits. Six different genes
encoding CNG channels, four A subunits (A1 to A4) and two B subunits (B1 and B3), give rise to three different
channels in rod and cone photoreceptors and in OSNs. Important functional features of these channels, i.e., ligand
sensitivity and selectivity, ion permeation, and gating, are determined by the subunit composition of the respective
channel complex. The function of CNG channels has been firmly established in retinal photoreceptors and in OSNs.
Studies on their presence in other sensory and nonsensory cells have produced mixed results, and their purported
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
roles in neuronal pathfinding or synaptic plasticity are not as well understood as their role in sensory neurons.
Similarly, the function of invertebrate homologs found in Caenorhabditis elegans, Drosophila, and Limulus is
largely unknown, except for two subunits of C. elegans that play a role in chemosensation. CNG channels are
nonselective cation channels that do not discriminate well between alkali ions and even pass divalent cations, in
particular Ca2⫹. Ca2⫹ entry through CNG channels is important for both excitation and adaptation of sensory cells.
CNG channel activity is modulated by Ca2⫹/calmodulin and by phosphorylation. Other factors may also be involved
in channel regulation. Mutations in CNG channel genes give rise to retinal degeneration and color blindness. In
particular, mutations in the A and B subunits of the CNG channel expressed in human cones cause various forms
of complete and incomplete achromatopsia.
I. INTRODUCTION
II. BASIC FUNCTIONAL PROPERTIES:
AN OVERVIEW
Physiol Rev • VOL
Cyclic nucleotides directly activate CNG channels by
binding to a site on the channel protein. The dependence
of channel activation on the ligand concentration is steep,
indicating that several, most probably four, molecules of
the ligand are required to fully open the channels. All CNG
channels respond to some extent to both cAMP and
cGMP. In rods and cones, CNG channels sharply discriminate between cAMP and cGMP, whereas channels in
chemosensitive cilia of OSNs by and large respond equally
well to both ligands. The ability to discriminate between
ligands is commonly referred to as ligand selectivity. Selectivity can be achieved either by differential control of
ligand affinity or efficacy or a combination of both. Ligand
affinity is a measure of how tightly cyclic nucleotides bind
to the channel. Efficacy refers to the ability to open the
channel once the ligand has been seated in the binding
cavity. The molecular basis of ligand affinity, efficacy, and
selectivity is discussed in section VII.
CNG channels are nonselective cation channels that
poorly discriminate between alkali ions and even allow
the passage of divalent cations, in particular Ca2⫹. Therefore, at rest (⫺60 mV) and when bathed in a physiological
ion milieu, CNG channels conduct mixed inward currents
carried by Na⫹ and Ca2⫹. To permeate, the ions bind to a
site inside the channel pore. The dwell time at this binding
site is significantly longer for Ca2⫹ than for monovalent
cations. As an important result, Ca2⫹ blocks the current of
the more permeant Na⫹. The pronounced Ca2⫹ permeability and the concomitant blockage of Na⫹ current by
divalent cations is crucially important for the channel’s
function and underlies, for example, the ability of rod
photoreceptors to detect single photons and to adapt to
steady illumination. The interaction of Ca2⫹ with the
channel pore and the consequences for the physiology of
cells is discussed in section VIII.
Unlike ligand-gated neurotransmitter receptors, CNG
channels do not desensitize in the continuous presence of
the ligand. Both the cooperative and sustained activation
predestines CNG channels to serve as a molecular switch
that faithfully tracks the cAMP or cGMP concentration in
a cell. Although the channels do not desensitize, their
activity is nonetheless modulated, notably by the Ca2⫹-
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Ion channels that are directly activated by cyclic
nucleotides [cyclic nucleotide-gated (CNG) channels] are
relatively recent arrivals in the world of ion channels.
Their discovery was intimately tied with the quest for the
intracellular messenger that mediates the photoresponse
in retinal photoreceptors. Since the late 1960s two messenger molecules, Ca2⫹ and cGMP, were the most likely
candidates to control the “light-sensitive conductance” in
the outer segement envelope from rod photoreceptors
(for review, see Ref. 431). Because the prevailing dogma
was that cyclic nucleotides control the activity of proteins
through phosphorylation mediated by cyclic nucleotidedependent kinases, it came as a surprise when in 1985
Fesenko et al. (103) reported that cGMP can directly
activate the light-dependent channel of rods.
Within a relatively short time, similar channels were
identified in cone photoreceptors (153), chemosensitive
cilia of olfactory sensory neurons (OSNs) (293), and the
pineal gland (94). Molecular cloning of CNG channels
became possible when the channel protein was purified
and unequivocally identified by functional reconstitution
into artificial liposomes and lipid bilayers (79, 151). Partial
amino acid information derived from the purified protein
allowed the successful cloning and functional expression
of the first CNG channel gene (188). The molecular identification of a CNG channel has sparked much progress
over the past several years. It is now obvious that CNG
channels are not unique to photoreceptors and OSNs, but
are expressed in other neurons and nonneuronal tissues
alike. CNG channels belong to a heterogeneous gene superfamily of ion channels that share a common transmembrane topology and pore structure and that harbor in their
COOH-terminal region a binding domain for nucleoside
3⬘,5⬘-cyclic monophosphates (cNMPs). Other members of
this superfamily are the so-called hyperpolarization-activated and cyclic nucleotide-gated (HCN) pacemaker
channels (for review, Ref. 189), the ether-a-gogo (EAG)
and human eag-related gene (HERG) family of voltageactivated K⫹ channels (for review, see Ref. 117), and
several plant K⫹ channels commonly referred to as KAT,
AKT, and KST channels (for review, see Ref. 359). The
focus of this review is on CNG channels.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
binding protein calmodulin and by phosphorylation. The
potential mechanisms of this modulation and its physiological significance are presented in section IX.
III. EXCURSION ON CYCLIC NUCLEOTIDES
Physiol Rev • VOL
cAMP- and cGMP-dependent processes including phosphorylation by PKA and PKG, activation or inhibition of
some PDE isoforms, and eventually cAMP-dependent regulation of gene expression. In addition, hydrolysis-resistant analogs may behave as competitive antagonists that
bind to the catalytic site of PDE and thereby hinder
hydrolysis of both cAMP and cGMP. As a result, endogenous cyclic nucleotides may accumulate during the
course of the experiment. Finally, the infusion of cells
with cyclic nucleotides either from a pipette in the whole
cell configuration or by bathing in a medium containing
membrane-permeable analogs is inherently slow in relation to the speed of action on kinases, PDEs, and CNG
channels. Therefore, it will be exceedingly difficult to
experimentally dissect the action of cyclic nucleotides on
CNG channels from other cellular effects.
This kind of experiment can be ameliorated using
chemical derivatives “dubbed” caged cyclic nucleotides.
Caged compounds are molecules whose biological activity has been disabled by chemical modification. Photolysis cleaves the modifying group (“uncaging”), thereby rapidly releasing the active molecule. (For a collection of
reviews on caged compounds, see Reference 261.) Four
different classes of caging groups have been used: 4,5dimethoxy-2-nitrobenzyl (DMNB), 1-(2-nitrophenyl)ethyl
(NPE), desoxybenzoinyl (Desyl), and derivatives of (7methoxy-coumarin-4-yl)methyl (MCM) (Fig. 1). Synthesis
of caged cyclic nucleotides by esterification of the phosphodiester group produces mixtures of axial and equatorial forms. The diastereomers differ significantly in solubility and solvolytic stability (150). We recommend that
the pure isomeric forms are used for experiments. To be
useful, caged cyclic nucleotides must meet specific requirements.
First, they should dissolve well in aqueous solution
(⬃100 ␮M-10 mM). The higher the concentration of the
caged compound inside the cell, the more cAMP or cGMP
is released per flash. Second, caged compounds must be
resistant toward solvolysis (the caging group is attached
to the cyclic nucleotide through an ester group that can
undergo hydrolysis in an aqueous medium). Otherwise,
the free cyclic nucleotide is produced during the course of
an experiment. Third, caged cyclic nucleotides should
display high photoefficiencies, i.e., high molar absorptivities and high quantum yields. Finally, the photochemical
reaction that releases the cyclic nucleotide should be fast
(ⱕ1 ms) compared with the physiological reaction under
study.
The NPE- and DMNB-caged compounds either photolyze relatively slowly (NPE) or display rather low photoefficiencies (DMNB) (Table 1). Desyl-caged cAMP is
very sensitive to solvolysis in aqueous buffer solution
(Table 1), and the MCM-caged cyclic nucleotides are
poorly soluble (148). However, caged derivatives of MCM
combine a set of favorable properties rendering them
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Studies on cellular cAMP and cGMP signaling frequently relied on the use of chemical derivatives of cAMP
and cGMP. Ever since the first substances have been
deployed for studies of cAMP- and cGMP-dependent protein kinases (PKA and PKG, respectively) and phosphodiesterases (PDE), the list of new derivatives has grown
long. More importantly for the subject of this review,
derivatives of cyclic nucleotides were also instrumental
for functional studies of CNG channels in various cellular
systems. cAMP and cGMP have been modified at the
cyclic phosphodiester group, the 2⬘- and 3⬘-hydroxyls of
the ribofuranose moiety, and at the adenine and guanine
ring systems (see Fig. 12). Phosphorothioate derivatives
of cAMP and cGMP have been employed to study the
activation properties of native CNG channels in photoreceptors (217, 442) and olfactory neurons (217) and the
heterologously expressed A1 and A2 channels (217; see
sect. VII). Cyclic nucleotides substituted at the C-8 position have proven particularly valuable for cellular studies.
For one reason, a number of substituents at C-8 render
these molecules more membrane permeant than cAMP
and cGMP itself. For example, 8-bromoguanosine 3⬘,5⬘cyclic monophosphate (8-BrcGMP) and 8-(4-chlorophenylthio)guanosine 3⬘,5⬘-cyclic monophosphate (8-pCPTcGMP) are 6- and 90-fold, respectively, more lipophilic
than cGMP (63). Therefore, these compounds readily penetrate membranes providing a simple route for delivery to
the intracellular binding sites. Another favorable feature
of most but not all C-8-substituted derivatives is their high
potency in activating CNG channels (54, 66, 203, 390, 442).
For example, in rod photoreceptors and OSNs, 8-BrcGMP
and 8-pCPT-cGMP activate CNG channels at ⬃10- and
80-fold, respectively, lower concentrations than cGMP
(112, 411, 442). Finally, 8-BrcGMP and 8-pCPT-cGMP are
poor substrates for several PDE isoforms and resist hydrolysis. 8-pCPT-cGMP is not measurably hydrolyzed by
three different PDEs, and the rate of 8-BrcGMP hydrolysis
is 4- to 40-fold lower than that for cGMP (63). The PDE6
of rods and cones hydrolyzes 8-BrcGMP 170- to 500-fold
more slowly than cGMP (19, 442). Thus the triad of lipophilicity, potency of activation, and resistance to hydrolysis makes these C-8-substituted cyclic nucleotides
attractive agents for the study of CNG channels.
Owing to the multiple actions of cyclic nucleotides
inside intact cells, however, results obtained with these
derivatives may sometimes be difficult to interpret. Delivering, for example, a relatively high concentration of cyclic nucleotides to intact cells will eventually activate all
771
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
rods and cones, OSNs, and cell lines (97, 311); 3) the
desensitization of the olfactory CNG channel by Ca2⫹/
calmodulin (CaM) (45); and 4) cyclic nucleotide-stimulated Ca2⫹ entry in mammalian spermatozoa (417).
IV. CELLULAR FUNCTION
1. Structure of caged cGMP and caging groups. Top two panels:
axial and equatorial forms of cGMP. R, caging groups. Bottom panels:
structure of four different caging groups (see text).
FIG.
ideal tools for intracellular studies (Table 1). For
example, [6,7-bis(carboxymethoxy)coumarin-4-yl]methyl
(BCMCM) esters of cAMP and cGMP are highly soluble
(⬎1 mM) (147) and display a high quantum yield (0.1– 0.15,
Ref. 147; Table 1). The (7-diethylaminocoumarin-4-yl)methyl (DEACM) derivatives have an even higher quantum
yield and absorptivity than the BCMCM-caged congeners.
Moreover, MCM-based caged cyclic nucleotides are extremely stable in aqueous solution and react quickly within
a few nanoseconds. The CMCM and BCMCM compounds
are negatively charged, therefore, their high solubility; consequently, they are poorly membrane permeable and must
be introduced into the cell by means of the recording pipette. Recently, bismethoxy and bisethoxy esters of
BCMCM have been synthesized. These compounds are neutral, penetrate cell membranes, and accumulate inside the
cell due to hydrolysis of the ester groups at the coumaryl
moiety (V. Hagen and U. B. Kaupp, unpublished data). They
are compounds of choice for studies on cell suspensions.
Caged cyclic nucleotides have been used to study 1)
the rate of activation of the rod CNG channel in excised
patches (185) and in the whole cell configuration (150,
339); 2) the Ca2⫹ permeability of CNG channels in intact
Physiol Rev • VOL
A. CNG Channels in Vertebrate and
Invertebrate Photoreceptors
Photoreceptors in invertebrates fall into two morphologically distinct subtypes: rhabdomeric photoreceptors with a microvilli-derived photosensitive structure
and ciliary photoreceptors, whereas vertebrate photoreceptors are of the ciliary type. The ciliary-type photoreceptors, whether vertebrate or invertebrate, respond to
light either with a depolarization or hyperpolarization,
whereas the rhabdomere-type invertebrate photoreceptors respond exclusively with a depolarization. Ciliary
photoreceptors, whether depolarizing or hyperpolarizing,
vertebrate or invertebrate, use cGMP-signaling pathways.
The targets of these pathways are CNG channels that
either open or close in response to light. Although the
phototransduction mechanism in depolarizing rhabdomeric photoreceptors is not entirely clear, the light-dependent channels belong to the family of trp/trpl channels
that are targeted by a phosphoinositide pathway (365).
Notwithstanding this well-established pathway, the presence of CNG channels in rhabdomeric photoreceptors has
been considered. We will discuss the function of CNG
channels in 1) the hyperpolarizing rods and cones, 2) the
pinealocytes, 3) the depolarizing photoreceptor in the
parietal eye of some lizards, 4) the hyperpolarizing ciliary
photoreceptor of an invertebrate, and 5) rhabdomeric
photoreceptors of invertebrates.
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The function of CNG channels has been firmly established in rod and cone photoreceptors, in extraretinal
photoreceptors, and in sensory neurons of the olfactory
epithelium. Electrophysiological studies of CNG channels
in these sensory neurons provided a wealth of information regarding their ligand sensitivity, mechanism(s) of
activation, modulation, and ion selectivity. The underlying
channel polypeptides have been identified by molecular
cloning; coexpression of the respective subunits in cell
lines produces channels that recapitulate many, if not all,
properties of the CNG channels in their native membranes. Although CNG channels also exist in other neurons and nonneuronal tissues, their specific functions are
yet to be determined rigorously. Several other ion channels, whose molecular identity is uncertain or not known,
have been speculated to be directly controlled by cyclic
nucleotides.
773
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
TABLE
1. Properties of caged cyclic nucleotides
Solubility, ␮M
␭max, nm
NPE-cGMP
NPE-8-BrcGMPa
DMNB-cGMP
DMNB-8-BrcGMPa
MCM-cGMPa
DEACM-cGMPa
CMCM-cGMPb
BCMCM-cGMPb
225e
35
125e
100
15e
120
⬎1,000
⬎1,000
255
264
347
346
327
403
325
347
NPE-cAMP
NPE-8-BrcAMPa
Desyl-cAMP
DMNB-cAMP
DMNB-8-BrcAMPa
MCM-cAMP
DEACM-cAMPa
CMCM-cAMPa
BCMCM-cAMPb
ND
225
ND
⬎100e
120
25e
135
900
1,000
260
265
320
345
345
325
402
326
347
Absorptivity,
␧[M⫺1 䡠 cm⫺1]
␾
Half-life, h
␶
Reference Nos.
cGMP and derivatives
17,000
19,400
5,500
5,800
13,300
19,300
11,200
11,200
0.25e
0.33
0.004
0.005
0.21
0.25
0.1
0.1
NDc
300
NDc
50
⬎500e
⬎1,000
⬎1,000
⬎1,000
NDd
NDd
300 ␮sg
NDf
Few nse
Few ns
Few ns
Few ns
424
149
303
150
360
147
147
147
0.39g
0.48
0.34
0.05g
0.05
0.10
0.21
0.12
0.08
NDc
600
0.5h
NDc
60
⬎1,000a
⬎1,000
⬎1,000
⬎1,000
230 msg
NDd
Few ns
3 msg
NDf
Few nsi
Few ns
Few ns
Few ns
424
149
123
303
150
116
147
147
147
cAMP and derivatives
a
b
c
␾, Quantum yield; ␶, photochemical reaction time; ␧, extinction coefficient.
Axial isomer.
Equatorial isomer.
Likely to be
d
similar to the respective 8-BrcGMP derivatives.
Likely to be similar to NPE-cAMP (␶ ⫽ ⬃200 ms; J. E. Corrie and D. R. Trentham. Bioorganic
e
f
Photochemistry. Biological Applications of Photochemical Switches. 1993, vol. 2, p. 243–305).
Volker Hagen, unpublished data.
Likely
g
to be similar to DMNB-cGMP.
J. E. Corrie and D. R. Trentham. Bioorganic Photochemistry. Biological Applications of Photochemical
h
i
Switches. 1993, vol. 2, p. 243–305.
T. Furuta, H. Torigai, M. Sugimoto, M. Iwamura. J Org Chem 60: 3953–3956, 1995.
B. Schade, V. Hagen,
R. Schmidt, R. Herbrich, E. Krause, T. Eckardt, and J. Bendig. J Org Chem 64: 9109 –9117, 1999.
1. CNG channels in the outer segment of rod and
cone photoreceptors
Rods respond to a light stimulus with a brief hyperpolarization by closing CNG channels in the surface membrane of the outer segment (for review, see Ref. 431). In
the dark, channels are activated by the binding of cGMP,
allowing a steady cation current (“dark current”) to flow
into the outer segment. Light triggers a sequence of enzymatic reactions that leads to the hydrolysis of cGMP.
When CNG channels close, the inward current ceases and
the cell hyperpolarizes. The enzyme cascade comprises
the photopigment rhodopsin (R), the G protein transducin
(T), and a PDE. Light stimulation decreases the cytoplasmic Ca2⫹ concentration ([Ca2⫹]i), which 1) initiates the
recovery from the light response by enhancing the synthesis of new cGMP molecules and 2) adjusts the sensitivity of the transduction machinery, a process known as
light adaptation (for review, see Ref. 330). The CNG channel is crucially important for the control of [Ca2⫹]i, because it provides the only source for Ca2⫹ influx into the
outer segment. In rods, between 10 and 18% of the dark
current (30 pA) is carried by Ca2⫹ (141, 229, 296, 435).
Ca2⫹ entry through open CNG channels is balanced by
Ca2⫹ extrusion through a Na⫹/Ca2⫹-K⫹ exchange mechanism (reviewed in Refs. 268, 329; Fig. 2). In light, when
CNG channels close but the exchanger continues to clear
Ca2⫹ from the cytosol, the balance is disturbed between
Ca2⫹ entry and Ca2⫹ extrusion. The resulting decline in
Physiol Rev • VOL
[Ca2⫹]i provides a negative feedback mechanism that controls at least three biochemical processes. First, the activity of the guanylyl cyclase (GC) that synthesizes cGMP
is stimulated as Ca2⫹ levels decrease. The Ca2⫹ sensitivity
of the GC is relayed by two small Ca2⫹-binding proteins,
designated GC-activating proteins (GCAP1 and GCAP2).
At rest, when [Ca2⫹]i is ⬃300 –500 nM, the GCAPs prevail
in the inactive form with Ca2⫹ bound. In light, when
[Ca2⫹]i is lowered to 50 –100 nM, Ca2⫹ dissociates from
GCAPs; the Ca2⫹-free form then stimulates GC activity
(for review, see Refs. 202, 313). Second, the lifetime of
active PDE is shortened through the phosphorylation of
light-activated rhodopsin (R*) by the rhodopsin kinase.
This reaction is mediated by another small Ca2⫹-binding
protein, recoverin (for review, see Ref. 202). Finally, the
ligand sensitivity of the CNG channel increases as [Ca2⫹]i
decreases. The regulation of ligand sensitivity by Ca2⫹ is
mediated by a third Ca2⫹-dependent protein, CaM (169,
280). All three reactions by various degrees help to restore the dark state and to adjust the light sensitivity of
the cell (reviewed in Ref. 330).
A similar transduction scheme exists in cones, the
photoreceptors responsible for vision in bright light. The
fundamentally same events underlie phototransduction in
rods and cones, and the two photoreceptor types utilize
similar protein isoforms of the enzyme cascade. However,
the light sensitivity of cones is 30- to 100-fold lower than
that of rods, and cones adapt over a wider range of light
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19,000
19,500
500
5,800
6,000
13,300
18,600
12,500
12,400
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
2⫹
FIG. 2. Ca
feedback mechanism in
rod and cone photoreceptors involving
cyclic nucleotide-gated channels. PDE,
phosphodiesterase; GCAP, guanylyl cyclase-activating protein.
Physiol Rev • VOL
333, 352). In contrast, the range of the CNG channel
modulation in intact cones is much wider than in rods and
is not well mimicked by Ca2⫹/CaM in detached patches
from the outer segment (145). This has led to the hypothesis that an unknown factor, which is lost upon patch
excision, is responsible for the larger range of modulation
of the ligand sensitivity in cones (333). In summary, the
cGMP sensitivity, its modulation by [Ca2⫹]i, and the Ca2⫹
permeation are profoundly different in CNG channels of
rods and cones, supporting the notion that the CNG channel is a pivotal determinant of the dynamics of Ca2⫹
homeostasis in vertebrate photoreceptor cells.
CNG channels in cones serve a second function that
is absent in rods. Light produces a graded hyperpolarization in rods and cones that is up to 35 mV in amplitude.
Not all of this response range is effectively transmitted to
the postsynaptic bipolar and horizontal cells. The highly
nonlinear input-output relation of the rod synapse is
largely accounted for by the voltage dependence of presynaptic Ca2⫹ channels. At the dark resting voltage of
⫺35 mV, a fraction of the Ca2⫹ channel is open, and the
continuous Ca2⫹ entry sustains a tonic release of the
neurotransmitter glutamate from the synaptic terminal.
The Ca2⫹ channels are characterized by an activation
threshold of approximately ⫺45 mV (14). Therefore,
when a rod is hyperpolarized to values more negative than
approximately ⫺45 mV, the Ca2⫹ channels close and synaptic transmission ceases (12, 27). In contrast to rods,
synaptic transmission in cone photoreceptors continues
as the light-induced voltage response grows to ⫺70 mV
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intensities than rods (reviewed in Ref. 330). It has been
suggested that differences in the Ca2⫹ homeostasis underlie the distinct light sensitivity and adaptation range of
the two photoreceptor types. Important elements that
control the dynamics and size of the changes in [Ca2⫹]i
are cell volume, the rate of Ca2⫹ clearance by the Na⫹/
Ca2⫹-K⫹ exchanger, the Ca2⫹-buffering capacity of the
cytoplasm, and Ca2⫹ entry through CNG channels (see
Ref. 274 for a thorough discussion). Several observations
demonstrate that the CNG channels in rods and cones
differ in ion permeation, ligand sensitivity, and modulation by Ca2⫹. The relative ion permeability PCa/PNa of
CNG channels is more than three times larger in cones
than in rods (21.7 and 6.5, respectively; Refs. 322, 414),
and under physiological ionic conditions, the fraction of
the dark current carried by Ca2⫹ is about twofold larger in
cones than in rods (311, 318). The Na⫹/Ca2⫹-K⫹ exchange
current in cones is at least one order of magnitude larger
than that in rods. From these observations it has been
inferred that the light-stimulated changes in [Ca2⫹]i are
far larger and faster in cones compared with rods.
The cGMP sensitivity of the CNG channel and its
modulation by Ca2⫹ is also different in intact outer segments of rods and cones. At elevated [Ca2⫹]i (i.e., in the
dark state), the K1/2 for cGMP can be as large as 550 ␮M
in cones (mean K1/2 ⫽ 335.5 ␮M; Ref. 333) compared with
rods (37.8 – 40 ␮M; Refs. 295, 352). In truncated or electropermeabilized rods, the Ca2⫹-dependent modulation of
the ligand sensitivity is only 1.5- to 2-fold, similar to the
effect of Ca2⫹/CaM on detached membrane patches (295,
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
2. CNG channels in pinealocyte photoreceptors
The pineal regulates various physiological functions
by nocturnal secretion of the hormone melatonin. Light
sensitivity of the pineal has been retained in most vertebrates, except mammals. Pinealocytes, the light-sensitive
cells, display hyperpolarizing responses to brief pulses of
light (328, 398) and express several retinal proteins including arrestin, recoverin, rhodopsin kinase, phosducin,
GC, and a cGMP-specific PDE (for review, see Refs. 212,
250). Dryer and Henderson (94) recorded CNG channel
activity from excised inside-out patches of dissociated
photoreceptors from the chick pineal. These CNG chanPhysiol Rev • VOL
nels in extraretinal photoreceptors feature all the hallmarks of CNG channels in retinal photoreceptors (94, 95).
Activation is half-maximal between 10 and 50 ␮M cGMP.
Even fully activated channels display frequent brief transitions to the closed state. For this reason, the open
probability (Po) becomes not unity at saturating cGMP
concentrations. The brief closing events are more frequent at negative than at positive membrane potentials.
Similar properties have been reported for CNG channels
from retinal photoreceptors (158, 262, 305). Moreover,
expression of several CNG channel subunits in the pineal
has been confirmed by in situ hybridization and immunohistochemistry (see sect. VI). These results collectively
show that the light response in pinealocytes of lower
vertebrates is produced by activation of a cGMP-signaling
pathway, which leads to the closure of cGMP-selective
ion channels. Chick pineal cells display a circadian
rhythm in cGMP concentration (152, 388). It is therefore
conceivable that CNG channels are involved in regulating
the output of the intrinsic circadian oscillator.
3. CNG channels in parietal-eye photoreceptors
Some lizards do have a parietal-eye, or third eye, on
top of their head. The parietal eye seems likely to convey
information about changes in light intensity and spectral
composition during dusk and dawn. The parietal-eye photoreceptors resemble in their morphology rod and cones
of the vertebrate retina, yet they depolarize in response to
a flash of light (377). This suggested that parietal-eye
photoreceptors, like rhabdomeric photoreceptors of the
invertebrate eye, might utilize a phosphoinositide-signaling cascade rather than the cGMP-signaling pathway of
retinal rods and cones. It came as a surprise when Finn et
al. (105) convincingly demonstrated that the outer segment membrane of the parietal-eye photoreceptors harbors a high density of CNG channels with all the hallmarks of CNG channels from rods and cones: the
channels are selectively activated by cGMP, cAMP is
much less effective, the channels are nonselective among
monovalent cations and are permeable to Ca2⫹ ions,
channels are blocked by L-cis-diltiazem, and Ca2⫹/CaM
reduces the cGMP-activated current by reducing the ligand sensitivity.
What type of CNG channel is expressed in the parietal-eye? CNG channels of retinal cones are significantly
more Ca2⫹ permeable than those of rods (113, 146, 155,
322). The relative selectivity for Ca2⫹ over alkali cations
has been determined from reversal potentials (Vrev) under
well-defined ionic conditions in excised patches from rod
and cone (322) and parietal-eye photoreceptors (105). In
cones of striped bass, PCa/PNa ⫽ 21.7; in rods of tiger
salamander, PCa/PNa ⫽ 5.9 (146); and in the parietal-eye,
PCa/PNa ⫽ 8.1–10.3 (105). Thus, at least with respect to the
Ca2⫹ permeability, the CNG channel in parietal-eye pho-
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(23, 114, 308). Whereas the small overlap of the voltage
range of Ca2⫹ channel activation and the voltage range
produced by light can explain signal clipping at the rod
synapse, it fails to explain the broader voltage range over
which synaptic transmission operates in cones. This conundrum has been partially solved by the discovery of
CNG channels in the inner segment and synaptic terminal
of cones (341, 358). The density of CNG channels in the
inner segment is low, whereas in the cone terminal these
channels appear to come in clusters (358). If the clusters
were located near release sites, CNG channels would be
ideally suited to control the Ca2⫹-dependent release of
glutamate. In fact, experimental maneuvers that activate
CNG channels also trigger exocytotic events and release
glutamate from the cone terminal (341, 358). The cGMP
sensitivities measured in patches of membrane excised
either from the outer segment or the axon terminal are
indistinguishable, suggesting that CNG channels from
both locales are built from identical or similar subunits.
The cGMP sensitivity of the CNG channels in the synapse
is as unusually low as that of CNG channels in the cone
outer segment of the fish retina (K1/2 ⫽ 206 and 335.5 ␮M,
respectively; Refs. 333, 358). We note, however, that the
high K1/2 value in fish cones required an intact cone
photoreceptor, whereas the K1/2 of synaptic channels was
determined in excised patches.
CNG channels could serve two different functions in
the cone synapse. First, these channels might extend the
voltage range over which synaptic transmission operates
by providing a sustained Ca2⫹ influx even at very negative
voltages. Second, nitric oxide (NO) is a good candidate to
serve as retrograde neurotransmitter that is released onto
cone terminals from other retinal cells (358). An NO
synthase (NOS) is predominantly found in the inner segment of rods and cones and in processes of bipolar cells
in the outer plexiform layer of the retina (204, 227, 240).
Furthermore, a soluble form of guanylate cyclase (sGC) is
found in the inner segment of cones and is stimulated by
NO (204). Thus CNG channels may play an important role
in the modulation of synaptic transmission by NO in the
axon terminals of cones.
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
4. CNG channels in hyperpolarizing photoreceptors
of invertebrates
The retina of some molluscan eyes is composed of
two layers of photoreceptors: depolarizing rhabdomerictype cells, similar to those found in most other invertebrates, and ciliary photoreceptors that hyperpolarize in
light (269). The mechanism underlying the hyperpolarizing light responses has been studied in two scallop species, Pecten and Lima. Light stimulation under voltage
clamp activates an outward current that is accompanied
by a decrease in the cellular input resistance. The Vrev of
the light-stimulated current lies near the equilibrium potential for K⫹ (EK) (126), demonstrating that the lightdependent channel is highly K⫹ selective and that the
hyperpolarizing light response is brought about by opening K⫹ channels rather than by closing nonselective cation channels, as in retinal rods and cones. In a series of
incisive experiments, Gomez and Nasi (85) convincingly
demonstrated that 1) the inositol trisphosphate (IP3)/
Ca2⫹-signaling pathway is not crucial for phototransduction, 2) the photoreceptors rely on cGMP as the internal
messenger of the transduction cascade, and 3) the lightdependent channel is opened by cGMP. The latter two
observations imply that light elevates cGMP, although it is
unknown whether this involves the inhibition of a PDE or
the stimulation of a GC.
In contrast to the Ca2⫹-permeable CNG channels of
retinal photoreceptors and OSNs, the Pecten channel is
virtually impermeable to Ca2⫹, and the K1/2 values for
blockage by extracellular Ca2⫹ and Mg2⫹ are 1–2 orders
of magnitude higher (299). The significant K⫹ selectivity,
the lack of Ca2⫹ permeability, and the weak divalent
block suggest that the pore architecture is more like that
of K⫹ channels than that of CNG channels. It is interesting
to note that a cGMP-sensitive K⫹ channel also seems to
underlie the light response of a photosensitive neuron in
the abdominal ganglion of a marine mollusc (136). This
Physiol Rev • VOL
cell generates slow, depolarizing light responses due to
the closure of K⫹ channels that are kept open in the dark
by cGMP. The protein(s) forming the cGMP-dependent
K⫹ channel is unknown. Its molecular identification is
eagerly awaited, as it will certainly further our understanding of the molecular mechanisms that govern ion
selectivity in CNG channels.
5. CNG channels in rhabdomeric photoreceptors
of invertebrates?
During the 1970s and 1980s, when the cGMP-signaling pathway in vertebrate photoreceptors was elucidated,
several groups examined the possibility that light also
regulates the cGMP (or cAMP) concentration in depolarizing rhabdomeric photoreceptors of invertebrates and
that cGMP mediates the light response by opening ion
channels in the microvilli membrane. Injection of cGMP
into Limulus ventral photoreceptors produced a depolarization that mimics the receptor potential (176). Superfusion with cGMP of membrane patches excised from the
light-sensitive lobe of the ventral photoreceptor activated
channels that closely resembled the channels activated by
light in cell-attached patches (13). In contrast, perfusion
of photoreceptors from Drosophila eyes with various
cGMP analogs was without effect (73). The cDNAs of
CNG channel subunits have been cloned from Drosophila
melanogaster and Limulus polyphemus (22, 69, 278). The
precise sites of expression of the two channel subunits
are not known. Further advances with respect to the
function of CNG channels in invertebrate brain will require precise cellular and subcellular localization of the
channel proteins.
B. CNG Channels in Chemosensory Cells
Chemosensory cells of vertebrates can be subdivided
in three major subgroups: olfactory sensory neurons, neurons of the vomeronasal organ, and taste receptor cells.
For each of these different senses, the involvement of
CNG channels in signal transduction has been proposed.
Much less is known about chemosensory transduction in
invertebrates. Recent studies with the nematode C. elegans, however, suggest that CNG channels may play an
important role in chemotaxis to odorants and salt in this
model organism. In this section, we examine the evidence
for the involvement of CNG channels in vertebrate and
invertebrate chemosensation.
1. OSNs
OSNs are embedded in the olfactory epithelium lining the cavity of the nose. OSNs are bipolar neurons; a
single dendrite extends to the apical surface of the neuroepithelium, and a single axon projects to the olfactory
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toreceptors behaves more like the CNG channel of rods
than that of cones. However, permeability ratios of native
CNG channels are not invariant but depend on the cGMP
concentrations (146; see sect. VIII). When comparing relative ion permeabilities, this complication must be kept in
mind.
The depolarizing light response is produced by an
increase in the cytosolic cGMP concentration that is controlled by an unusual cGMP-signaling pathway (426). In
the dark, cGMP is synthesized continuously by GC activity and rapidly degraded by PDE activity. The elevated
PDE activity in the dark seems to rest on a constitutively
active G protein, whereas the mechanism that keeps GC
active in the dark is not known. Light acts by inhibiting
the PDE through another G protein, permitting the cGMP
concentration to rise and CNG channels to open.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
Physiol Rev • VOL
294), and channel activation causes a rapid increase of
[Ca2⫹]i (230, 232). The odorant-stimulated rise of [Ca2⫹]i
plays an important role in both excitation and odor adaptation. It serves as a feedforward signal that enhances the
depolarizing response by activating Ca2⫹-dependent Cl⫺
channels, which, in fact, carry a large fraction of the
receptor current (197, 226). In rat OSNs, as much as 85%
of the olfactory response can be mediated by Cl⫺ channels (251). The increase of [Ca2⫹]i serves also as a delayed
negative feedback signal that reduces the cAMP sensitivity of the CNG channels and stimulates cAMP hydrolysis
by a Ca2⫹-dependent ciliary form of PDE (PDE1C2) (42).
Both processes, channel desensitization and PDE activation, are controlled by Ca2⫹/CaM (42, 72, 248).
In a series of elegant double-pulse experiments using
short puffs of odorants or flashes of ultraviolet light that
rapidly release cAMP from a caged compound, Kurahashi
and Menini (224) show that the principal mechanism underlying odorant adaptation acts at the CNG channel and
that regulatory mechanisms upstream of the channel contribute little to the odorant-induced reduction of the cell’s
sensitivity, at least on a time scale of several tens of
seconds. Additional mechanisms of adaptation that appear to operate on a longer time regime include phosphorylation of odorant receptors (38) and adenylyl cyclase
(412).
B) NO AND CO: GASEOUS MESSENGERS INVOLVED IN ODORANT
SIGNALING? NO is a short-lived molecule capable of diffusing across membranes and reacting with a variety of
targets. It is produced by various isoforms of NOS, some
of which are activated by Ca2⫹/CaM. The physiological
concentrations of NO are in the picomolar range (8). The
most common action of NO involves the activation of
sGC, i.e., NO stimulates the synthesis of cGMP. However,
NO can also regulate the activity of various proteins by
reacting with cysteine sulfhydryls, a covalent modification known as S-nitrosylation (for review, see Ref. 50).
The direct activation by NO was also reported for the
olfactory CNG channel (52). Perfusion of membrane
patches excised from the soma or dendrite of OSNs of
tiger salamander with NO donors like S-nitrosocysteine
(SNC) produced single-channel events similar to those
observed in the presence of cAMP. NO-stimulated channel activity persisted for up to 30 min in the absence of
NO donors and was mimicked by sulfhydryl-modifying
reagents like N-ethylmaleimide (NEM), iodoacetamide
(IAA), and 5,5⬘-dithiobis-(2-nitrobenzoic acid) (DTNB) in
a reversible manner. SNC was also a potent agonist of the
heterologously expressed rat A2 and A2/A4 channels (53).
When C460 in the A2 subunit was mutated to a serine
residue, the activation of the homomeric channel by NO
was completely abolished (51). Mutation of other intracellularly located cysteine residues was without effect.
Moreover, a CNG channel subunit from Drosophila that is
lacking this particular cysteine residue (22) was not NO
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bulb. From the tip of the dendrite ⬃20 –50 thin cilia extend into the layer of mucus that covers the epithelium.
The cilium is the site where the chemoelectrical transduction takes place. It is the functional equivalent of the outer
segment of retinal photoreceptors. It houses all the molecular components to register odorants, to amplify the
signal by a series of enzymatic reactions, and to generate
the electrical response. Like rods, OSNs are exquisitely
sensitive; they can respond to stimulation by a few odorant molecules. This high sensitivity is accompanied by a
rich selectivity. Humans are probably able to discriminate
between more than 10,000 or so different odorous compounds. This enormous achievement is endowed by an
array of several hundreds up to a thousand odorant receptors with overlapping specificity for a few odorants
(for review, see Ref. 327).
Quite remarkably, a cousin of the retinal CNG channels takes center stage in odorant signaling; the vast
majority of OSNs respond to brief pulses of odorants with
a transient receptor current by opening cAMP-gated channels in the ciliary membrane (107, 293). In contrast to the
outer segment of photoreceptors, where the light-sensitive current is solely carried by CNG channels, the receptor current originating in chemosensitive cilia has two
ionic components: an inward cationic component mediated by CNG channels, followed by an inward anionic
component mediated by Ca2⫹-activated Cl⫺ channels. In
addition to cAMP, other signaling molecules have been
implicated in odorant transduction, especially the two
gaseous messengers NO and carbon monoxide (CO). We
will critically examine the experimental foundation for
these hypotheses.
Finally, a cGMP-signaling pathway that is targeting a
highly cGMP-selective CNG channel has been identified in
a small subset of OSNs, whereas components that furnish
the prototypical cAMP-signaling pathway are absent in
those cells. These observations provide compelling evidence that cGMP serves as the principal messenger for
chemosensory signaling in some OSNs.
A) THE PROTOTYPICAL CAMP-SIGNALING PATHWAY. A milestone in olfactory research was the cloning of a family of
odorant receptors by Buck and Axel (61). Since then a
vast number of putative odorant receptors have been
cloned. Although functional expression of these receptors
in heterologous systems has been accomplished in only a
few cases (219, 415), it is taken for granted that these
receptors feed into a common cAMP-signaling pathway.
The principal players of this signaling pathway are shown
in Figure 3, top. The binding of odorants to their cognate
receptors in the membrane of chemosensitive cilia first
activates a G protein (Golf) and then an adenylyl cyclase
(ACIII). The ensuing rise in the concentration of cAMP
opens CNG channels and thereby produces a depolarization of the cell membrane. Like its retinal cousins, the
CNG channel in OSNs is highly Ca2⫹ permeable (97, 113,
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
sensitive (51). These observations collectively support the
notion that NO works through S-nitrosylation. However, it
is noted that CNG channels in rod and cone photoreceptors and from C. elegans do not become activated by
covalent modification with NO (209, 358, 400), although
their respective A subunits carry a cysteine residue at a
homologous position.
Stimulation of OSNs with a short pulse of 500 ␮M
SNC in the whole cell recording configuration elicits a
sizable inward current that, in contrast to the current in
excised patches, returns to baseline within 1.5 s (52). The
authors reasoned that this current was due to covalent
modification by NO, because the recording pipette contained no GTP, which is required for cGMP synthesis. This
observation then indicates that the S-nitrosylation product inside the cell is 100 –1,000 times less stable than in
the excised membrane patch. Although redox reactions
inside the cell might rapidly reverse the S-nitrosylation, it
is conceivable that some endogenous GTP was still available to support cGMP synthesis either because equilibration between the pipette and the cell interior was not
complete and/or because GTP was generated from ATP
by transphosphorylation reactions involving guanylate kinase and nucleotide diphosphate kinase activity (see Ref.
Physiol Rev • VOL
422 and references therein). The chemical nature of the
modification in native channels from OSNs and heterologously expressed A2 subunits seems to be different as
well. The NO-induced channel activity persisted for many
minutes in excised patches of OSNs, but rapidly faded
with a rate constant of 3.8 s⫺1 (␶ ⫽ 260 ms) in patches
with the A2 homomeric channel (51). Such a rapid decay
would seem incompatible with the formation of highmolecular-weight nitrosothiol species, which are stable
on a time scale of several minutes to hours (378). This
raises the intriguing possibility that reversible binding of
either SNC or NO itself gates the channel open.
Lynch (256), working with rat OSNs, was unable to
reproduce the actions of NO donors and sulfhydryl-modifying reagents on the native olfactory CNG channel. In
fact, the NO donors SIN-1 and SNC at moderate concentrations inhibit the cAMP-stimulated currents rather than
activating the unliganded channels. Moreover, the oxidizing agent DTNB caused a permanent inhibition. Inhibition
brought about by either agent was reversed by a 2-min
wash with dithiothreitol. The similar inhibitory effects of
NO and DTNB suggest that two neighboring S-nitrosothiols combine to form a relatively stable disulfide bond.
Because NEM displays no inhibitory action, it can be
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FIG. 3. The cAMP-signaling pathway and the
cGMP-signaling pathway in olfactory sensory neurons
(OSNs). OR, odorant receptor; Golf, G protein
expressed in OSNs; CaM, calmodulin; ClC, Ca2⫹-activated Cl⫺ channel; PDE1C2, CaM-dependent phosphodiesterase; PDE2, cGMP-regulated phosphodiesterase; GC-D, guanylyl cyclase type D; ACIII, Ca2⫹dependent adenylyl cyclase type III. The two
pathways do not exist in one and the same OSN.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
Physiol Rev • VOL
serve as an endogenous gaseous messenger in the nervous system (260). Indeed, OSNs contain high levels of
the CO-producing enzyme heme oxygenase-2 and produce
CO upon stimulation with odorants (173, 174, 404).
How might CO engender odor adaptation? Odor stimulation elicits two kinetically distinct inward currents: a
large and fast transient current mediated by the cAMPsignaling system, followed by a small and persistent current of only a few picoampères in amplitude (444). It is
this “background” current that appears to depend on
cGMP and is underlying long-term adaptation. The authors specifically proposed that the small background
current is flowing through CNG channels and that cGMPdependent Ca2⫹ entry is a crucial step in the development
of long-lasting adaptation. The results are based on an
experimental protocol involving successive puffs of odorant. The first conditioning pulse of odorant provides the
reference cellular response and sets into motion the
adapting processes. The second test pulse probes the
change in odorant sensitivity caused by the first pulse.
Using this experimental design, Kurahashi and Menini
(224) and Reisert and Matthews (335) identified a form of
odorant adaptation that is virtually instantaneous and
operates on a time scale of a few seconds. The amplitude
of the adapted response depends on the time of the
delivery of the test pulse. For short interpulse times
(⬃1–5 s), the response amplitude is significantly reduced;
amplitudes gradually increase with the interpulse time
span (224, 230, 335). The time constant for complete
recovery of the sensitivity is of the order of 3–10 s and
depends on the odorant concentration of the conditioning
pulse (224); intense stimuli require longer times for complete recovery than weaker stimuli. The recovery time
critically depends on the rate of Ca2⫹ clearance from the
cell by a Na⫹/Ca2⫹ exchange mechanism (230, 335), underpinning the idea that Ca2⫹/CaM modulation of the
CNG channel accounts for most odorant adaptation.
The understanding of long-term adaptation is incomplete. For example, does a stimulated cell enter a state of
short-term adaptation first, then recover from stimulation,
and finally transit to a state of long-lasting adaptation? For
obvious reasons, the paired-pulse protocol (pulse every
30 s) designed to study long-term adaptation does not
cover the time regime of short-term adaptation (⬍10 s)
(444). Therefore, it is not clear whether the lower sensitivity revealed by the second pulse reflects a delayed or
incomplete recovery from the first pulse rather than a
slowly progressing adaptation. How does long-term adaptation relate to short-term adaptation if a Ca2⫹-dependent
shift of CNG channel sensitivity is underlying both forms
of adaptation? It is intriguing that cells recover from the
massive Ca2⫹ influx during the cAMP-mediated odorant
response within 5–10 s or so (224, 230, 335), whereas it
takes several minutes to recover from the much smaller
cGMP-mediated response. Does this finding imply that the
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concluded that alkylation of either one or both of the
paired cysteines does not replicate the effect of the disulfide formation.
Future work must establish the physiological function of covalent NO modification of the olfactory CNG
channel. Two questions are particularly pertinent. The
concentration of NO in the olfactory epithelium has not
been determined; therefore, it is not known whether the
concentrations of NO in the ciliary layer reach the high
levels of NO needed to activate the olfactory channel
(125). Second, NO potently activates sGC in cells (EC50
⬃20 –250 nM; Refs. 28, 380). The high sensitivity raises the
question what fraction of CNG channels becomes activated by S-nitrosylation at NO concentrations that fully
activate sGC and thereby cGMP synthesis.
An alternative mechanism of the action of NO in odorant signaling involving sGC and cGMP was proposed by
other groups. These investigations were stimulated by the
fact that the CNG channel of chemosensitive cilia is exquisitely sensitive to both cAMP and cGMP. In fact, the K1/2 of
half-maximal activation is twofold lower for cGMP than for
cAMP (1.0 –2.4 ␮M and 2– 4 ␮M, respectively; Refs. 40, 58, 72,
112, 208, 293). The slightly higher sensitivity for cGMP
spurred several authors to explore a potential physiological
role for channel regulation by cGMP.
Breer and Shepherd (49) proposed that the NO/cGMP
system is involved in both excitation and some form of
olfactory adaptation. What is the experimental evidence
for these hypotheses? Hefty doses of odorant stimulate a
retracted elevation of cGMP concentration in isolated
olfactory cilia or cultured OSNs (48, 220, 404). The response is abolished by NG-nitro-L-arginine, a selective inhibitor of NO formation by NOS. These observations have
been interpreted to indicate that an odorant-induced Ca2⫹
influx activates NOS via Ca2⫹/CaM and, thereby, initiates
NO production. The highly membrane-permeable and diffusible NO might activate sGC in this and in neighboring
cells. Specifically, Breer and Shepherd (49) proposed that
recruitment of adjacent neurons via the NO/cGMP system
could serve as a mechanism to encode very intense stimuli. The NO/cGMP system, however, is unlikely to play
such a role in the adult epithelium, because developing
and regenerating OSNs, but not mature OSNs, contain
NOS activity (47, 196, 221, 345).
A variation of this theme has been proposed by Zufall
and collaborators (234, 235, 444). These authors provide
evidence that the CO/cGMP system might be responsible
for a long-lasting form of odor adaptation. Variants of this
hypothesis can be traced to previous reports showing that
pretreatment of olfactory preparations with membranepermeable cGMP derivatives attenuates the second messenger response to odorant stimuli in rat cilia and the
receptor current in the olfactory epithelium of the bullfrog (319). CO, like NO, stimulates the synthesis of cGMP
by sGC (59, 115, 193); therefore, CO has been proposed to
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2. The vomeronasal organ
The vomeronasal organ (VNO) or Jacobson’s organ is
a chemosensitive organ present in most vertebrates. It is
important for the detection of pheromones that convey
information between individuals of the same species. The
sensory neurons of the VNO have a bipolar organization,
and their axons terminate in a specialized brain region,
the accessory olfactory bulb. In situ hybridization studies
revealed that a modulatory CNG channel subunit that is
also found in OSNs (A4, see nomenclature in sect. V) is
reportedly expressed in these neurons, whereas the principal A2 subunit of OSNs is lacking (30). The A4 subunit
itself does not form functional CNG channels (44, 242),
Physiol Rev • VOL
raising the intriguing possibility that it might coassemble
with members of other channel families.
Alternatively, the A4 subunit might become activated
by another ligand of unknown nature. Indeed, Broillet and
Firestein (53) reported that the A4 subunit, when heterologously expressed, produced NO-activated Ca2⫹-selective
channels and that single NO-activated channels from VNO
neurons have some properties in common with the heterologously expressed channels. The significance of these
findings is unclear, because recent evidence suggests that
signaling in the VNO is mediated by the phospholipase C/IP3
pathway and TRP channels (231, 243, 270, 445).
3. Taste receptor cells
A cyclic nucleotide-sensitive conductance with quite
unique properties has been described in a subset of taste
cells in the frog. Superfusion of excised inside-out
patches from the apical end of frog taste receptor cells
suppressed a current (207) that reversed at about ⫺50 mV
under symmetrical biionic conditions (110 mM intracellular K⫹/110 mM extracellular Na⫹), arguing that the channel is K⫹ selective. Maneuvers intended to raise the intracellular cGMP concentration (perfusion with IBMX or
8-BrcGMP) reduced whole cell inward currents that reverse at ⬃0 mV. The discrepancy between results acquired in the excised-patch and whole cell configuration
raises questions as to the nature of the ionic conductance.
The underlying channels appear to have a ligand sensitivity and selectivity that are both distinctively different
from those of other CNG channels. The action of cAMP
and cGMP on excised patches was exquisitely sensitive
(K1/2 ⫽ 77–160 nM and K1/2 ⫽ 16 –36 nM, respectively).
Moreover, cAMP and cGMP are significantly more potent
than their 8-bromo-substituted analogs, whereas for the
photoreceptor and olfactory CNG channels the opposite
is true. The dependence of current suppression on the
concentration of cAMP, cGMP, and 8-bromo-substituted
congeners is described by a simple binding isotherm (Hill
coefficient n is unity). From the indirect modulation by
cyclic nucleotides of Ca2⫹-activated K⫹ channels in the
same patch, it has been inferred that the cNMP-suppressible conductance is also Ca2⫹ permeable, although this
interpretation has not been substantiated by direct demonstration of Ca2⫹ permeation. The authors propose that
tastants, by binding to G protein-coupled receptors, activate transducin, which is also present in taste cells (351),
which in turn activates a cAMP-specific PDE. The ensuing
drop in cAMP concentration activates the cNMP-suppressible inward current, leading to membrane depolarization and a rise of [Ca2⫹]i. The molecular identity of the
cNMP-suppressible conductance is unknown. The A3 subunit, which is expressed in cone photoreceptors (39, 416)
and a small subpopulation of OSNs (273), has been cloned
from taste buds of rat (277). This CNG channel isoform is
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feedback mechanisms, i.e., hydrolysis of the cyclic nucleotide and extrusion of Ca2⫹, are different for the cAMP
and cGMP response? Because the rate of clearance from
the Ca2⫹ load by the Na⫹/Ca2⫹ exchanger sets the recovery time (335), the long-term effects in some cells might
result from an altered Ca2⫹ homeostasis that is characterized by a slower rate of Ca2⫹ clearance. This interpretation is supported by the observation that only a subpopulation of cells displays long-term adaptation.
C) A CGMP-SELECTIVE CNG CHANNEL IN MAMMALIAN CHEMOSENSORY NEURONS. A small population of OSNs that project to a
group of atypical glomeruli in the main olfactory bulb, the
so-called necklace glomeruli, houses a different repertoire of signaling molecules (178, 273). This subgroup of
OSNs expresses an olfactory-specific guanylyl cyclase
(GC-D), a cGMP-stimulated isoform of PDE (PDE2), and
a cGMP-selective CNG channel (splice variant of the cone
A3). These three proteins are highly enriched in the chemosensitive cilia. Most intriguingly, the characteristic
markers for the enzymatic makeup of the prototypical
cAMP pathway in OSNs, i.e., Golf, ACIII, PDE1C2, and
three distinct CNG channel subunits (A2, A4, and B1b),
are absent from this subset of neurons (see Fig. 3, bottom). These findings rule out the coexistence of the
known cAMP-signaling pathway and this novel cGMPdependent pathway and argue for cGMP as the principal
messenger in this subset of OSNs. GC-D is a member of
the family of receptor-type GCs that become activated by
binding of peptide hormones to the extracellular domain
(111). Although no ligand has yet been identified for GCD, this subgroup of OSNs may not respond to normal
volatile odorants, but, possibly, to ligands that control
some aspects of reproductive behavior. Because very few
OSNs use this cGMP-signaling pathway, it has not been
feasible to electrically record from these cells. It is, however, anticipated that stimulation of this unique cell type
with the cognate ligand of GC-D will produce a depolarizing receptor current and an increase in [Ca2⫹]i by opening cGMP-selective channels.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
activated rather than inactivated by cyclic nucleotides
and, therefore, unlikely to form the cNMP-suppressible
conductance alone.
4. Chemosensation in invertebrates
C. CNG Channels in the Brain
1. cGMP-sensitive currents in retinal neurons and
glia cells
Bipolar cells are retinal interneurons that receive
synaptic input from photoreceptors. Glutamate, released
Physiol Rev • VOL
from the synaptic terminal in the dark, hyperpolarizes
ON-bipolar cells (301, 375) through activation of a
metabotropic glutamate receptor (mGluR6; Ref. 292) and
subsequent suppression of a nonselective cation current.
When glutamate release ceases in the light, the bipolar
cells depolarize (ON-response). The nonselective cation
current is enhanced when cGMP was introduced into the
cell through a patch pipette, indicating that the current
might flow through CNG channels. The mGluR6 receptor
is thought to signal through a G protein to a PDE. Activation of the PDE would stimulate cGMP hydrolysis,
thereby closing the CNG channel. This led to the concept
that a cGMP-signaling pathway similar to that in the photoreceptor outer segment operates in ON-bipolar cells.
However, subsequent experiments did not support this
concept (300). When intracellularly perfused with the hydrolysis-resistant analogs 8-pCPT-cGMP and 8-BrcGMP or
the PDE inhibitor IBMX, bipolar cells continue to respond
to glutamate, and no difference was observed in the kinetics and the amplitude of the response (300), whereas
similar treatments of rods result in profoundly altered
kinetics and size of light responses (355, 442). These
results argue against regulation of the glutamate-sensitive
current by PDE activity. Moreover, the properties of the
presumptive cGMP-sensitive channel do not match those
of known CNG channels. For example, the cGMP-sensitive current in bipolar cells is not blocked by extracellular
Ca2⫹ (376). Finally, several different antibodies against
known subunits of retinal CNG channels (A1, A2, A3, and
B1a, see sect. V) do not label ON-bipolar cells (408; F.
Müller, unpublished observations). These results argue
against the presence of a photoreceptor-type CNG channel in ON-bipolar cells.
There is also some suggestion of a rodlike CNG channel in retinal ganglion cells (2, 190). About one-half of the
ganglion cells in the rat retina respond to stimulation by
NO with an inward current (190). This current is mimicked by either intracellular perfusion with cGMP or extracellular application of the membrane-permeable analogs 8-BrcGMP and 8-pCPT-cGMP, suggesting that the
cGMP-sensitive current flows through CNG channels.
This conclusion appears to be supported by in situ hybridization and PCR that detect transcripts coding for an
A1 subunit in ganglion cells (2).
However, an electrophysiological study in salamander
(164) found no evidence that treatment with membranepermeant cGMP analogs, IBMX, or NO donors led to the
activation of CNG channels. Moreover, the cGMP-sensitive
current displays properties that do not match the properties
of known CNG channels. First, the blockage of the current
by divalent cations is weak and does not result in a strong
outward rectification, i.e., the voltage dependence of blockage is weak. Second, the current in ganglion cells reverses at
roughly 0 mV under biionic conditions (Cs⫹ inside, Na⫹
outside), whereas CNG channels would display a distinc-
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A precedent for chemosensory signaling using cGMPselective CNG channels exists in the nematode C. elegans
(74, 210). The tax-2 and tax-4 genes of C. elegans encode
two distinct CNG channel subunits required for chemosensation and thermosensation. Tax-2 and tax-4 mutants
display defects in the chemotaxis to volatile compounds
and salts, as well as in thermotaxis. Mutations in either
gene affect similar behavioral responses, and tax-2 and
tax-4 are expressed in the same olfactory, gustatory, and
thermosensory neurons, suggesting that the native channel is composed of these two subunits.
The ceB (Tax-2) and ceA (Tax-4) proteins have been
localized to sensory neurons that also express some of the
29 different receptor GC genes of C. elegans. The homomeric ceA channel and the heteromeric ceA/ceB channel
highly prefer cGMP over cAMP. Both observations suggest
that the CNG channels mediate an electrical response that
relies on a cGMP- rather than a cAMP-signaling pathway.
In addition to their function in sensory transduction,
both the tax-2 and tax-4 genes play a role in the guidance
of sensory axon outgrowth during development and the
maintenance of normal axon morphology throughout the
adult stage (74, 75). In mammals, CNG channels may
serve a similar function because CNG channel activity is
present in growth cones of cultured OSNs (181), and the
A4 subunit was immunohistochemically localized to
growing fibers of cultured hippocampal neurons (46).
The congruence of developmental and behavioral defects in C. elegans mutants might be explained by either a
primary defect in neuronal connectivity that perturbs normal behavior or by the fact that the correlated neuronal
activity is required to refine synaptic connections during
development like in sensory systems of vertebrates (75).
This latter hypothesis was specifically tested in mice lacking
a functional cAMP-sensitive CNG channel due to targeted
disruption of the A2 subunit gene. Two groups reported that
the peripheral olfactory projections are in part influenced by
neuronal activity (438, 439), whereas another group concluded that the olfactory CNG channel, and by inference
chemosensory activity, is not required for generating synaptic specificity in the olfactory bulb (244).
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
2. Cyclic nucleotide-sensitive currents
in hippocampal neurons
A variety of different transcripts encoding CNG channel subunits have been detected in several brain areas by
in situ hybridization, cloning of cDNA, and PCR (see sect.
VI); however, studies on the functional characterization of
neuronal CNG channels in situ are sparse. Two laudable
examples are the work by Leinders-Zufall et al. (233) and
Bradley et al. (46). At rest (⫺80 mV), a whole cell inward
current is evoked in hippocampal neurons upon superfusion with 8-BrcGMP. Although this basic observation is
shared by both reports, the underlying currents appear to
be different.
In one study, the I-Vm relation of the 8-BrcGMPactivated current was almost linear in the presence of 2.5
mM Ca2⫹ and 1 mM Mg2⫹ in the bath (233). The current
was 1.5- to 2-fold larger in the absence of divalent cations,
but the shape of the I-Vm relation was largely unchanged.
This result contrasts with the strong blockage of known
CNG channels by extracellular divalent cations, whether
Ca2⫹ or Mg2⫹, which produces a pronounced outward
rectification of currents in photoreceptors and OSNs. FurPhysiol Rev • VOL
thermore, the I-Vm relation has been recorded with 140
mM Cs⫹ in the pipette and 140 mM Na⫹ in the bath. Under
these biionic conditions, known CNG channels show a
distinctively positive Vrev (⬃20 mV), and the I-Vm relation
is slightly inwardly rectifying (112, 255, 271, 309, 416); in
contrast, the currents in hippocampal neurons reverse
either at ⬃0 mV or at ⫺40 mV (Figs. 2C and 3C, respectively, of Ref. 233) and rectify slightly in the outward
direction.
Leinders-Zufall et al. (233) also attempted to study
Ca2⫹ permeability by estimating the Ca2⫹ current flowing
through this channel relative to the Na⫹ current. To this
end, the authors compared the current amplitudes in Na⫹
and in various choline/Ca2⫹ mixtures. At 30, 100, and
1,000 ␮M Ca2⫹, the current ratio ICa/INa was roughly 0.25,
0.5, and 0.7, respectively, implying that Ca2⫹ carries almost as much current as Na⫹ at more than 100-fold lower
concentrations. Similar experiments with CNG channels
in rods and heterologously expressed subunits yielded
entirely different results. At 70 –100 mM external Ca2⫹, ICa
amounts to only 1–2% of INa in the native rod CNG channel, to roughly 10% in the A2 homomeric channel, and to
14% in the Drosophila channel (22, 65, 97). In conclusion,
the currents recorded from hippocampal neurons (233)
do not match the properties of currents flowing through
known CNG channels.
The I-Vm relation reported by Bradley et al. (46) is
slightly outwardly rectifying in the absence of external
divalent cations, and 1 mM Mg2⫹ blocked the inward
current at ⫺80 mV almost completely. Although the currents have not been further characterized, this piece of
evidence is consistent with the idea that the current is
carried by CNG channels.
3. cAMP-sensitive currents in invertebrate neurons
A cAMP-activated cation current is widely distributed among central molluscan neurons (for references,
see Refs. 191, 192, 382). In neurons of Helix, Aplysia, and
Pleurobranchaea, the current persists in the presence of
inhibitors of PKA (165, 192, 382), arguing that the cAMP
action on channels is direct and does not involve phosphorylation. In excised membrane patches, perfusion
with 1 mM cAMP produces single-channel events of ⬃40
pS (382). These two observations have been taken as
evidence that the underlying channels belong to the class
of CNG channels. The unit conductance of 40 pS was
determined in the presence of 60 mM divalent cations,
and the maximum Po at saturating cAMP concentrations
was only 6.3% (at 0 mV; Ref. 382). Known CNG channels,
whether vertebrate or invertebrate, are strongly blocked
by 60 mM divalents, and no single-channel recordings
would be feasible.
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tively more positive Vrev under similar ionic conditions (see
sect. VIII on ion selectivity). Third, under these ionic conditions, the current (I)-voltage (Vm) relations of CNG channels
display a distinctive inward rectification, due to the smaller
conductance of CNG channels for Cs⫹ compared with Na⫹
(see, for example, Refs. 112, 255, 271, 309, 416), whereas the
cGMP-sensitive current in ganglion cells is rectifying in the
outward direction (2). Moreover, antibodies against known
CNG channel subunits do not label ganglion cells in the
mammalian retina (408; Müller, unpublished observations).
With the proviso that CNG channels in photoreceptors and
ganglion cells are antigenically similar, a low channel density cannot explain this failure. As an example, in the rod
outer segment, 1–2% of open CNG channels sustain a current of 30 pA (297, 434), whereas the cGMP-sensitive conductance in ganglion cells carries currents up to 500 pA.
8-BrcGMP and NO donors also enhance whole cell
currents in acutely dissociated or cultured retinal Müller
cells and stimulate Ca2⫹ influx (228), whereas 8-BrcAMP
has no effect on whole cell currents. A fraction of the
cGMP-sensitive current seemed to be carried by a Ca2⫹activated K⫹ channels and another fraction by a nonselective cation channel. Transcripts encoding a CNG channel subunit have been amplified from cultured human
Müller cells using primers specific for the A1 subunit of
the cGMP-gated channel of rod photoreceptors. These
pieces of evidence have been interpreted to indicate that
Müller cells express a rodlike CNG channel. Again, immunohistochemical studies do not support the presence of a
rodlike CNG channel in retinal glia cells (408; Müller,
unpublished observations).
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
D. CNG Channels in Spermatozoa
Physiol Rev • VOL
by brief flashes of ultraviolet light. Photolysis of both
caged compounds evokes a Ca2⫹ influx into sperm; the
respective derivatives of cAMP are much less effective.
The Ca2⫹ influx depends on the presence of extracellular
Ca2⫹ and was greatly reduced at high extracellular Mg2⫹
concentrations.
Because knock-out mice lacking the A3 subunit are
fertile (33), at this point we can only speculate about a
functional role of cGMP-selective CNG channels in sperm.
CNG channels might be involved in some aspect of motility control or, more specifically, in chemotactic swimming behavior or in the process of capacitation or acrosomal exocytosis. Another concern is the failure so far to
identify a receptor-type GC in mammalian sperm, leaving
alone its cognate ligand. There is also no evidence in
sperm for the presence of the Ca2⫹-regulated GCs (GC-E
and GC-F) known from retinal photoreceptors.
Mouse sperm express two other channels (CatSper1
and CatSper2) that bear similarity with a single repeat of
the four-repeat structure of voltage-activated Ca2⫹ channels (332, 336). Targeted disruption of the CatSper1 gene
results in male sterility; moreover, the cAMP-induced
Ca2⫹ influx is abolished in mutant mice. The CatSper
channels are unrelated to CNG or HCN channels; in particular, they are lacking in a cAMP/cGMP-binding domain.
The cloned CatSper channel genes so far have resisted
functional expression, suggesting that they might require
additional subunits to become functional. An intriguing
possibility is that CNG and CatSper subunits coassemble
to form Ca2⫹-permeable and cyclic nucleotide-sensitive
ion channels.
E. Miscellaneous Nonneuronal Tissues
Cyclic nucleotide-sensitive channels have been reported to exist in several nonneuronal cells. In this section we discuss some of the available evidence.
1. Airway epithelial cells
Xu et al. (427) report the expression of transcripts
coding for the CNG channel from rod photoreceptors (A1
and B1 subunits) in a human alveolar cell line (A549).
Furthermore, the authors compared whole cell currents
recorded from the alveolar cell line to whole cell currents
from A1-transfected HEK293 cells and from untransfected
controls in the presence and absence of 8-BrcGMP. The
averaged current amplitude recorded from one set of
alveolar cells in the presence of 8-BrcGMP was about
twofold larger than the averaged amplitudes recorded
from another set of alveolar cells without 8-BrcGMP. A
problem with this approach is that it does not allow
correcting for leak currents in one and the same cell.
Moreover, when working with transfected cell lines, the
fluorescence of the cotransfected green fluorescent pro-
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Cyclic nucleotides are key elements of cellular signaling in sperm of both vertebrates and invertebrates (for
reviews, see Refs. 118, 406). cAMP and cGMP mediate
several cellular responses, including acrosomal exocytosis, swimming behavior, and chemoattraction. The swimming behavior of sperm is controlled by factors, usually
short peptides, that are secreted by the egg or cellular
structures of the oviduct. In a few species, primarily
marine invertebrates with external fertilization, the amino
acid sequence of the peptides and their cognate membrane receptors on the surface of the sperm have been
identified (119, 275, 406). For example, speract, a short
peptide from the sea urchin Strongylocentrotus purpuratus, activates a receptor-type GC and thereby stimulates a
rise of the intracellular cGMP concentration (118, 406).
Speract also gives rise to an increase of [Ca2⫹]i. These
observations suggest that cGMP activates a Ca2⫹ conductance, although a direct causal relationship has not been
established rigorously. Owing to their high Ca2⫹ permeability, CNG channels are among the prime candidates for
the Ca2⫹-entry pathway. Unfortunately, in sea urchin,
where their potential function would have been so obvious, no CNG channels have been detected by homology
screening (R. Gauss and U. B. Kaupp, unpublished observations). Instead, Gauss et al. (120) identified the first
member of the family of pacemaker channels (or HCN
channels), which are controlled by both cyclic nucleotides and voltage. The HCN channel in S. purpuratus is
highly selective for cAMP and is not Ca2⫹ permeable. Its
function in spermatozoa is not known.
The search for CNG channels was more successful in
mammalian sperm. The testicular expression of several
CNG channel subunits (A3, B1, and B3) has been suggested by cloning of cDNA from testis libraries or by
Northern analysis (34, 35, 122, 416, 417). Antibodies specific for the A3 and B1 subunits labeled the flagellum of
mature sperm and precursor cells in cross-sections of
seminiferous tubules (417). Heterologous expression of
the A3 subunit cloned from testis produces channels that
are cGMP sensitive and cGMP selective: the K1/2 for cAMP
is ⬃200-fold higher than the K1/2 for cGMP (8.3 and 1,720
␮M, respectively; Ref. 416). Therefore, these channels
might be involved in a cGMP-stimulated Ca2⫹ influx into
intact sperm (417). cGMP-stimulated channel activity was
detected in small vesicles that might have been derived
from cytoplasmic droplets and in patches excised from
osmotically swollen sperm. The low success rate of establishing a giga-seal resistance and of detecting channel
activity prevented a more thorough characterization of
the native channel in situ. Cyclic nucleotide-mediated
Ca2⫹ influx into sperm was studied by confocal laser
scanning microscopy (417). The 8-bromo- and 8-pCPTanalogs of cGMP were delivered from caged compounds
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
2. Gonadotropin-releasing hormone-secreting neuronal
cell line
Vitalis et al. (405) report the identification by PCR of
transcripts for the CNG channel subunits A2, A4, and B1
in a neuronal cell line (GT1) secreting the gonadotropinreleasing hormone (GnRH). These three subunits make
up the native CNG channels in chemosensitive cilia of
OSNs (40, 357). Perfusion of inside-out patches excised
from GT1 neurons with 200 ␮M cAMP produced singlechannel events with a unit conductance of ⬃60 pS in 140
mM symmetrical Na⫹ and in the presence of 4 mM divalent cations on both sides of the membrane. Under these
conditions, all known CNG channels are severely
blocked, and single-channel events cannot be resolved.
For example, in high extracellular Ca2⫹, the unit conductance of CNG channels in rods and OSNs has been estimated from noise analysis to be significantly smaller than
1 pS (37, 87, 140, 199). It, therefore, seems unlikely that
the 60-pS channel is produced by the CNG channel subunits detected by PCR.
3. Endothelial cells of pulmonary artery
Wu et al. (425) sought to identify CNG channels in
endothelial cells by recording I-Vm relations in the whole
cell configuration under various conditions. The ionic
selectivity, the Ca2⫹ and Mg2⫹ dependence of blockage,
and the rectification behavior of the endothelial cell currents are incompatible with the properties of known CNG
channels. Therefore, the authors’ claim is equivocal that
the currents are carried by a CNG channel comprising the
A2 subunit.
4. Kidney
cGMP-sensitive channels have been identified in cells
of the renal inner medullary collecting duct (IMCD cells;
Ref. 241). These channels exhibit a complex pattern of
regulatory mechanisms. The constitutively active chanPhysiol Rev • VOL
nels appear to be under the dual control of cGMP and
PKG. In excised patches, cGMP reduced the Po directly by
39% through a phosphorylation-independent mechanism.
cGMP also inhibited the channel by 96.1% through a phosphorylation-dependent mechanism involving PKG. When
guanosine 5⬘-O-(3-thiotriphosphate), an activator of G
proteins, was present, PKG had no effect. The authors
suggested that channel activity is controlled by the complex interaction between PKG and a Gi protein. In a
subsequent report from the same laboratory (401), a
seemingly related channel was studied in a cell line derived from the inner medullary collecting duct. The authors argue that this channel exhibits a cation selectivity,
unit conductance, Ca2⫹ permeability, and pharmacology
similar to the classic CNG channels and support their
notion by cloning of cDNA (182) that displays 99% nucleotide sequence identity to the respective cDNA of the A1
subunit cloned from the mouse retina (324). The authors
also suggest that the differences between the retinal and
renal cDNA sequence may account for the functional
differences. We take issue with this view.
First, the 20- and 28-pS channel activity in IMCD cells
was recorded in the presence of millimolar concentrations of Ca2⫹ and Mg2⫹ in the extracellular medium.
Under these ionic conditions, classic CNG channels are
severely blocked and display a strong outward rectification, and single-channel recordings are not feasible (see
above). Second, CNG channels are not constitutively active in the absence of cGMP or cAMP (see, however, sect.
VII). Third, CNG channels are neither inhibited by cGMP
nor downregulated by PKG activity or activated by Gi
proteins. Fourth, the diltiazem sensitivity of the renal
channel is almost two orders of magnitude lower than
that of retinal CNG channels. The authors probably used
the racemate of diltiazem for their blocking experiments.
However, only the L-cis-form of the diastereomeric diltiazem blocks CNG channels at micromolar concentrations
(203).
F. Conclusions
The function of CNG channels in photoreceptors and
OSNs has been established beyond reasonable doubt,
whereas for other cell types either their expression is
uncertain or their function is not fully understood. Most
needed is a convincing demonstration of cyclic nucleotide-sensitive currents in excised patches and quantitative
comparison with the properties of known CNG channels.
A panel of caged cyclic nucleotides now provides powerful tools to record CNG channel activity in the whole cell
configuration or by Ca2⫹ imaging. We trust that these
techniques will clarify some of the issues addressed in
this section.
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tein (GFP) does not always correlate with channel activity. Therefore, the contribution of the cGMP-activated
current to the total whole cell current is not known.
Moreover, the blockage of CNG channels by L-cis-diltiazem in rods and cones and OSNs is strongly voltage
dependent (112, 154, 266), whereas the blockage in A549
cells is not. This study would have been more convincing
had the cGMP-sensitive currents been unequivocally identified in excised membrane patches or in the whole cell
configuration by infusion of cGMP from the recording
pipette. CNG channels have also been involved in liquid
homeostasis of lung in 6-mo-old sheep but not in 6-wk-old
sheep on the basis of a pharmacological study using dichlorobenzil, amiloride, and pimozide, which, among
other channels, also block CNG channels (179, 180)
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
V. MOLECULAR DIVERSITY OF CYCLIC
NUCLEOTIDE-GATED CHANNEL SUBUNITS
A. Diversity of Subunit Genes
TABLE
2. Nomenclature of CNG channel subunits
Original
Cloning
Subunits
Reported Names
CNGA1
Rod CNG channel
CNG1, CNG␣1, RCNC1
Olfactory CNG channel
CNG2, CNG␣3, OCNC1
Cone CNG channel
CNG3, CNG␣2, CCNC1
Second/modulatory subunit of olfactory
CNG channel
CNG5, CNGB2, CNG␣4, OCNC2
Second/modulatory subunit of rod
CNG channel
CNG4, CNG␤1
Second/modulatory subunit of cone
CNG channel
CNG6, CNG␤3
CNGA2
CNGA3
CNGA4
CNGB1
CNGB3
188
90, 254
39, 416
44, 242
214
122
CNG, cyclic nucleotide gated.
Physiol Rev • VOL
isons, CNG channel genes fall into different subfamilies.
In mammals, two gene subfamilies can be distinguished.
Homologous members of these two subfamilies are found
in the genomes of species as distant as human, C. elegans,
and Drosophila. The encoded polypeptides of the two
subfamilies are referred to as A and B subunits. Roman
letters have been previously used to classify the subunits
of several families of ligand-gated channels. Members of a
subfamily are numbered (A1, A2, etc.). Second, a designation used in the past for one gene cannot be used for
another gene even if the former usage has been discontinued. This rule prevented the numbering of A and B
subunits based on functional relatedness or functional
context rather than on the order of molecular cloning or
baptizing. Moreover, this rule created gaps in the numbering system. For example, one subunit of olfactory CNG
channels has been previously thought to be a B subunit
(formerly CNGB2). This subunit is now designated
CNGA4. As a consequence, the designation CNGB2 is
excluded from future use, and one of the other B subunits
cannot be renamed but must keep its original designation
(CNGB3).
The NCBI database contains the sequences of six
different human genes encoding CNG channels. These
genes have been known already from either cloning of
human cDNA or cloning of orthologs from other vertebrates including bovine, mouse, rabbit, rat, and chick. The
Drosophila and C. elegans genomes harbor, respectively,
four and six different CNG channel genes. The mammalian CNG channel genes fall into two different gene subfamilies (see phylogenetic tree in Fig. 4). Members of one
subfamily represent the principal subunits that, except
one (CNGA4), form functional channels on their own and
are responsible for several key properties of CNG channels. This subfamily consists of four members, designated
here CNGA1, CNGA2, CNGA3, and CNGA4. For brevity,
we refer to these subunits as A1 to A4. The A1 subunit is
the founding member, first identified in retinal rods (188).
The principal subunit identified in OSNs (90, 254) is designated A2, and the principal subunit identified in cones
(39) is designated A3 (see Table 2). The sequence similarity among A1 to A3 is high, ranging from 75.1 to 78.9%
(Table 3).
The A4 subunit, first identified in rat OSNs (44, 242),
is set apart from A1 to A3. It seems to fall into a subfamily
of its own, except it would represent the only member of
that subfamily. Only one other ortholog in humans is
known. At the time when this subunit was cloned, it was
believed that the olfactory CNG channel, like the channel
of retinal rods, is built from two different subunits. Moreover, like the CNGB1 and CNGB3 subunits described
below, A4 does not form functional channels on its own.
Therefore, A4 has been frequently referred to as the “␤
subunit” or second subunit of the olfactory CNG channel.
However, based on the overall sequence similarity and
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The advent of molecular cloning has uncovered a
bewildering number of genes from various species encoding different CNG channel subunits. The chase for new
CNG channel genes has come to a preliminary end by the
recent release of the genomic sequences of several species, including human, D. melanogaster, C. elegans, and
Arabidopsis thaliana. The CNG channel genes and subunits have received a bewildering variety of different
names, which inevitably caused some confusion. Subunits
that have initially been cloned from either rod, cone, or
OSN have been named according to their cellular origin.
When it was realized that CNG channels form heteromultimers, the previously cloned subunits have been designated “first” or ␣-subunit and the novel subunits have
been designated “second” or ␤-subunit. However, subunits are not specific for rod, cone, and OSN but are
expressed in other cells as well. Therefore, classification
of channel subunits based on the tissue of original identification became inappropriate. Moreover, it was recognized that the so-called ␣-subunits form functional channels on their own and, therefore, have been also referred
to as “principal” subunits, whereas the ␤-subunits are
unable to form functional channels and, therefore, have
been designated “modulatory” subunits. A comprehensive
list of the various designations across subunits, tissues,
and species is given by Richards and Gordon (340).
Throughout this review, for the mammalian genes we
use the new nomenclature that most laboratories working
with CNG channels have agreed on (Table 2; Ref. 43). It
follows two guidelines. First, based on sequence compar-
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
functional sequence motifs, it is more akin to A1 to A3
than to the two B subunits (186). For example, A4 shares
49.2–51.7% sequence identity with A1 to A3, but only 24.9
and 16.3% identity with B1 and B3, respectively. Therefore, it seems appropriate to classify this subunit as an A
subunit.
The second subfamily comprises two members, designated CNGB1 and CNGB3, which have been first identified in rod and cone photoreceptors, respectively (71,
122, 214). These subunits are often referred to as modulatory subunits, because, unlike the A1 to A3 subunits,
they do not form functional channels on their own. However, when coexpressed with A subunits in a heterologous
cell system, the resulting heteromeric channels display
TABLE 3. Sequence identity and similarity
between subunits
A1
A1
A2
A3
A4
B1
B3
A2
A3
A4
B1
B3
60.1 (75.3)
59.9 (75.1)
61.9 (78.9)
49.2 (69.4)
51.3 (71.6)
51.7 (70.8)
30.3 (52.8)
29.4 (55.2)
31.8 (55.1)
24.9 (51.5)
27.7 (59.8)
30.0 (53.0)
22.4 (44.8)
16.3 (38.0)
48.1 (67.8)
Numbers are given in %. Similarity is given in parentheses; it is
defined as the percentage of identical and conserved residues.
Physiol Rev • VOL
properties that are characteristically different from the
homomeric A channels with respect to ligand sensitivity
and selectivity, gating, pharmacology, ion selectivity, and
modulation by Ca2⫹/CaM. The B1 and B3 subunits share
48.1% sequence identity with each other, but only between 22.4 and 31.8% with A1 to A3 (Table 3).
Genes encoding CNG channel subunits have been also
identified from invertebrates, notably C. elegans, D. melanogaster, and L. polyphemus. The cDNAs of two different
genes have been cloned from C. elegans, tax-2 and tax-4
(74, 210), and an additional four genes have been identified
by the C. elegans Genome Project. How do these subunits
relate to their mammalian cousins? Tax-4 is closely related
to the mammalian A subunits and produces functional channels when heterologously expressed. Tax-2 is phylogenetically related to the mammalian B subunits and does not
form functional channels on its own. We will refer to these
subunits as ceCNGA (Tax-4) and ceCNGB (Tax-2). The
other four C. elegans genes seem to fall into a subfamily on
its own. For the time being, we will refer to these subunits as
ceCNG3 to ceCNG6.
Two genes encoding CNG channel subunits have
been cloned from D. melanogaster (22, 278). Furthermore, the sequences of two other subunits have been
determined by the Drosophila Genome Project. One subunit displays significant sequence similarity with the
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FIG. 4. Phylogenetic tree of CNG channel subunits from
rat (A1-A4 and B1), mouse (B3), Caenorhabditis elegans
(ceA, ceB, ce3-ce6), and Drosophila melanogoster (dmA,
dmB, dm3, dm4).
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
B. Functional Domains
The major structural sequence motifs and functional
domains of CNG channels are depicted in Figure 6 and are
discussed below.
1. Transmembrane topology
The current model for the membrane topology of the
A and B subunits of CNG channels is illustrated in Figure
Physiol Rev • VOL
6. The core structural unit consists of six membranespanning segments, designated S1-S6, followed by a
cNMP binding domain near the COOH terminus. A pore
region of ⬃20 –30 amino acids is located between S5 and
S6. The S4 segment in CNG channels resembles the voltage-sensor motif found in the S4 segment of voltage-gated
K⫹, Na⫹, and Ca2⫹ channels. Because the voltage-sensor
motif, the six transmembrane segments, and the pore
region are also characteristic features of voltage-gated
channels, it has been suggested that CNG channels and
voltage-gated channels are members of a gene superfamily of cation channels, which evolved from a common
primordial channel (160, 175).
Experimental evidence in support of the topological
model was first obtained from immunogold labeling for
electron microscopy of the CNG channel from rod photoreceptors. In these studies, both the NH2 and COOH
termini were localized to the cytoplasmic side of the ROS
plasma membrane (282), and a glycosylated segment connecting S5 to the pore region was localized to the extracellular side (420). The topological model for this and
other A subunits received further support from a gene
fusion approach using enzyme reporters (161). Although
similar studies are lacking for the B subunits, because of
the significant sequence similarity and similar structural
features, most likely A and B subunits share a common
transmembrane topology.
2. Voltage-sensor motif
The S4 segment of all CNG channels comprises a
charged sequence motif that is reminiscent of the “voltage-sensing” S4 segment of voltage-gated channels. This
segment, in voltage-gated channels, has been proposed to
serve as sensor of membrane voltage that upon depolarization moves toward the extracellular surface and thus
opens the channel. The S4 segment of K⫹ channels is
characterized by five to seven positively charged residues
(Arg or Lys) at every third position interspersed with
predominantly hydrophobic residues (Fig. 7A). It is sufficiently long to be able to span the membrane as an
amphipathic ␣-helix. Studies using site-directed mutagenesis
and state-dependent labeling of residues with fluorescent
dyes or with antibodies supported the voltage-sensor hypothesis (218, 259, 353). Indeed “gating currents” were
observed directly demonstrating that charges of S4 were
transferred during voltage changes (11). Recent evidence
suggests that the S4 helix, upon depolarization, undergoes
a 180° rotation with a slight tilt (68, 124); this movement
is sufficiently large to transfer positive charges from an
intracellular to an extracellular environment.
The core of the S4 segment in CNG channels displays
significant sequence similarity to the respective region of
K⫹ channels, although it includes only three or four regularly spaced Arg or Lys residues (Fig. 7A). The functional
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mammalian A1 to A3 subunits and produces functional
channels in a heterologous cell system (22). We will refer
to this subunit as dmCNGA. Another subunit (accession
number AAF46757) clearly falls into the subfamily of
mammalian B subunits; we will refer to this subunit as
dmCNGB. The other two subunits of Drosophila, like four
of the C. elegans genes, are not as easily categorized as
the others and will be provisionally designated dmCNG3
and dmCNG4.
The subunit cloned from L. polyphemus is significantly longer than its mammalian counterparts and may
represent an A subunit (69).
The subdivision into A and B subunits is also supported by a comparison of the genomic structure of CNG
channel genes. The NH2-terminal region up to S2 of the
mammalian A1 to A3 subunits is encoded by several small
exons, whereas the region from S2 to the COOH terminus
is encoded by a single large exon. This exon structure is
conserved among A1 to A3 and from chick to human (41,
89, 419) (Fig. 4). The exon structure between B1 and B3 is
also conserved, but entirely different from that of A1 to
A3. The human B1 gene is composed of 33 small exons (9,
10); there appears to be no conservation of splice sites
between A and B subunit genes. Most notably, the region
from S2 up to the COOH terminus is encoded by 12 exons
in the B subunits, but only by a single exon in A1 to A3.
The exon structure of the NH2-terminal region up to S2 is
largely conserved between A4 and A1 to A3, whereas that
of the COOH-terminal region is not conserved, arguing
that the A4 gene falls into a subfamily of its own.
Several splice variants have been identified of the A3
and the B1 subunits (36, 40, 41, 71, 214, 273, 276, 395, 417).
The various splice forms of A3 differ in their NH2-terminal
region (Fig. 5). Why cone photoreceptors, pinealocytes,
sperm, and a subpopulation of OSNs express different
splice forms of the A3 gene is not known, and the electrophysiological properties of the various A3 forms have
not been systematically compared.
The two most important splice forms of the B1 gene
are B1a and B1b, expressed in rods and OSNs, respectively. The B1b subunit is a short form of B1a, which is
lacking the glutamic acid-rich part (see sect. VB). Several
B1 splice forms that differ only in their very NH2-terminal
region are expressed in testis (36, 417).
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
significance of the S4 segment is, however, still elusive, as
CNG channels show very little voltage dependence. A few
studies have been carried out to address this question.
Transfer of the S4 segment from the rat A2 subunit to the
voltage-gated dmEAG channel produced a chimeric channel that is activated by depolarization, suggesting that a
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FIG. 5. Genomic structure of cyclic nucleotide-gated (CNG) channel genes. A: exon structure of the six human CNG
channel genes. Red lines demarcate exon boundaries. B: splice variants of the A3 gene cloned from various tissues and
species. The numbering of exons is according to Reference 41.
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CYCLIC NUCLEOTIDE-GATED ION CHANNELS
789
CNG channel S4 segment is capable of serving as a voltage sensor (391). However, when the extracellular S3-S4
loop was transferred from A2 to dmEAG, the channel
chimera apparently became constitutively active. The authors went on to show that the midpoint potential of
activation (V1/2) of this chimera was shifted to such large
negative values that, within the physiological range of
membrane voltage, channels are fully activated and
thereby became voltage insensitive. A mechanism that
might explain this result is that hydrophilic amino acids
Physiol Rev • VOL
within the S3-S4 loop of CNG channels interfere with the
gating movements by “locking” the S4 segment and
thereby the channel in the open state. A Glu residue
present in the S3-S4 loop (E246) appears to be important;
exchange of the respective position of dmEAG (A345E)
shifted the V1/2 by 50 mV to more negative values. In
addition, it was shown that the sign of the charge is not
important; Arg at this position was as effective as Glu in
shifting V1/2 (391).
A similar mechanism is conceivable for CNG chan-
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FIG. 6. Organization of functional domains. Transmembrane segments are indicated by numbers 1– 6. CaM, calmodulin-binding site; cNMP, binding site for
cyclic nucleotides; P, pore region; Glu,
glutamic acid-rich part; R1-R4, proteinprotein interaction domains in GARPs.
Bottom left: similarity dot matrix between the B1a subunit of chick and bovine. Bottom right: sequence alignment
of the four repeats R1-R4.
790
U. BENJAMIN KAUPP AND REINHARD SEIFERT
nels. The S4 charges might be “locked” in a fixed conformation by hydrophilic residues in the S3-S4 loop, and
gating of these channels, despite the S4 motif, is solely
promoted by the binding of cyclic nucleotides. Unfortu-
nately, to date, it has not been possible to test this hypothesis by “unlocking” the S4 segment to produce a
voltage-activated CNG channel mutant. Moreover, sequence alignment of the S3-S4 loop reveals only little
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FIG. 7. Sequence motifs of CNG subunits. A: alignment of the S4 regions of
vertebrate and invertebrate CNG channels, HCN channels, and voltage-dependent K⫹ channels. Hydrophobic amino
acids are highlighted by a yellow backgound; positively and negatively charged
amino acids are highlighted by a blue and
red background, respectively. RatA1 (91),
ratA2 (90), ratA3 (356), ratA4 (44, 242),
dmA (22), ceA (210), ratB1 (356), mouseB3
(122), ceB (74), dmB (accession
AAF46757), dm3 (278), dm4 (accession
BAA89278), ce3 (accession T20936),
ce4 (accession T33125), ce5 (accession
AAF36062), ce6 (accession T21969),
HCN1 (354), dmEAG (407), and shaker
(326) are shown. B, top: alignment of the
pore regions of the bacterial KcsA K⫹
channel and the ratA1 CNG channel.
Identical or conserved residues are
shown with a black or gray background,
respectively. Middle: side view of the
structure of the pore region of KcsA
(left). Top view of the structure of the
pore region of KcsA (right). Bottom:
alignment of the pore region of vertebrate and invertebrate CNG channels.
The rat A1 subunit was taken as a reference. Identical or conserved residues are
shown with a black or grey background,
respectively. KcsA (363), ratA1 (91),
ratA2 (90), ratA3 (356), ratA4 (44, 242),
dmA (22), ceA (210), ratB1 (356),
mouseB3 (122), ceB (74), dmB (accession AAF46757), dm3 (278), dm4 (accession BAA89278), ce3 (accession T20936),
ce4 (accession T33125), ce5 (accession
AAF36062), and ce6 (accession T21969)
are shown.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
similarity among CNG channels (Fig. 7A). Most importantly, E246 is only found in A1 to A3 subunits. The S3-S4
loop of other CNG channels, for example, dmA, is less
hydrophilic than that of rat A2, yet the voltage dependence of dmA channels is not enhanced compared with
A2. Clearly, more work is needed to eventually understand the role of the S4 segment of CNG channels.
3. P region
Physiol Rev • VOL
a cysteine side chain can be demonstrated in a functional
assay, the assignment of its position within the pore is not
straightforward. Third, to avoid misinterpretations, it is
necessary to account for the membrane permeability of
the reactive compounds.
The SCAM method applied to K⫹ channels revealed
an accessibility map that is quite consistent with the
high-resolution structure of KcsA (252). Similar studies on
CNG channel pores yielded conflicting results. The study
by Sun et al. (385) suggested that the structure of the CNG
channel pore is significantly different from that of the K⫹
channel because several residues seemed to be accessible
from both sides of the membrane. The authors proposed
that the P loop forms a thin blade that extends almost
parallel to the surface of the membrane toward the central axis and that this irislike structure both serves as gate
and selectivity filter. Other studies observed only slight
differences in the accessibility map between pores of K⫹
channels and CNG channels (25, 26). None of the cysteine
residues in the pore region was accessible from both sides
of the membrane. In addition, the authors demonstrate
that each of the cysteine mutants displayed distinctively
altered gating behavior (24 –26), whereas Sun and collaborators (385) state that the gating of the same mutants
were “near normal.” A large number of the cysteine-substituted P-region mutants do not produce functional channels. To overcome this limitation and to increase the
number of functional P mutants, 9 of the 12 nonfunctional
mutants were rescued by using tandem dimers that contain one wild-type pore and one mutated pore (247).
Differences in the pattern of reactivity of cysteine-substituted residues between the open and closed states of the
channel indicate that a conformational change takes
place in or around the pore loop. Previous studies have
also concluded that the pore region at or near the negatively charged residue E363 is involved in channel gating
(24, 62, 121).
4. Cyclic nucleotide-binding domain
All CNG channel subunits contain a COOH-terminal region (comprising ⬃80 –100 amino acid residues)
that exhibits significant sequence similarity to the
cNMP-binding domains of PKA and PKG and the catabolite activator protein of Escherichia coli (CAP). The
three-dimensional structure of CAP has been determined by X-ray crystallography (265). The cAMP-binding site of CAP, comprising three ␣-helices (A, B, and
C) and eight ␤-strands (␤1 to ␤8), served as template
for cNMP-binding domains of other proteins (Fig. 8).
The ␤-strands form a flattened ␤-barrel consisting of
two antiparallel ␤-sheets, each with four strands, connected in a jelly-roll topography. The three helices are
connected to the ends of the ␤-barrel. The A helix is
NH2 terminal of the ␤-barrel, whereas the B helix,
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For K⫹ channels, the view of the P region and the
question how selectivity is produced was taken into a new
era when the crystal structure of the bacterial KcsA K⫹
channel was solved (93). The pore region of KcsA comprises roughly 25 amino acids that form an inwardly directed helix and the selectivity filter, which consists of the
GYG signature sequence of K⫹ channels (Fig. 7B). The
Tyr residue of the GYG motif interacts specifically with
two Trp residues of the pore helix to form a sheet of
aromatic amino acids (a total of 12 in a tetramer) that is
positioned like a cuff around the selectivity filter and is
thought to behave like a layer of strings stretched radially
outward to hold the pore open at its proper diameter (93).
Can we envision a similar P region architecture for
CNG channels? Sequence conservation among K⫹ channels and CNG channels in the P region is quite remarkable
(Fig. 7B). Within the pore helix, many residues are identical or at least conserved, arguing for a common pore
architecture of K⫹ channels and CNG channels. However,
the sequence similarity diverges exactly at the selectivity
filter. At the respective position of the Tyr residue in the
K⫹ channel, a negatively charged (Glu, Asp) or at least a
hydrophilic residue exists in most CNG channels (Fig.
7B). The structural features of a selectivity filter made of
up to four negative charges in tetrameric channels are
difficult to predict because the electrostatic interaction of
the carboxylic side chains strongly depends on their mean
charge and distance. To date, we rely on several indirect
methods that have been employed to study pore structures. For example, several studies employed the substituted cysteine accessibility method (SCAM) (5): each
amino acid within the P region is consecutively replaced
by a cysteine, and the accessibility of the reactive thiol
group is tested from either side of the membrane with
thiol-specific reagents {i.e., (2-sulfonatoethyl)methane
thiosulfonate (MTSES), [2-(trimethylammonium)ethyl]methane thiosulfonate (MTSET), or (2-aminoethyl)methane thiosulfonate (MTSEA)}. From these results an
accessibility map of the pore region can be constructed.
Although this method is very attractive in principle, caution has to be taken to appreciate the results. First, it
needs to be tested whether the gating and permeation
behaviors of the mutant channels are unchanged to ascertain that the mutation itself does not grossly perturb the
structure of the pore. Second, even if the accessibility of
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
immediately followed by the C helix, is COOH terminal
of the ␤-barrel. The cyclic nucleotide is bound within
the binding site through a network of polar and nonpolar interactions. The phosphate and the ribose ring of
cAMP interact with the protein through several hydrogen bonds and through electrostatic contacts. These
interactions involve residues in the loop linking ␤6 and
␤7. In contrast, the adenine ring interacts primarily
through hydrophobic and stacking interactions with
residues in and near the C helix. The location of
␤-strands and ␣-helices in the cNMP-binding domain of
CNG channels, HCN channels, protein kinases, EPAC,
and CAP is shown in Figure 9.
5. The glutamic acid-rich part of the B1a subunit
The B1a subunit of the CNG channel of rod photoreceptors features a unique bipartite structure (214). The
membrane-spanning so-called ␤⬘ part is homologous to A
subunits, whereas the large cytoplasmic NH2-terminal domain is lacking in all other CNG channel subunits. Even
more intriguing, except for a few amino acid residues, this
NH2-terminal domain is identical with two soluble glutamic
acid-rich proteins (GARPs) (78, 213, 383), which seem to be
specifically expressed in rod photoreceptors. We refer to the
Physiol Rev • VOL
soluble proteins as GARP1 and GARP2 and to the respective
channel domain as GARP-part (213). GARP1 is twice as
large as GARP2 and, therefore, these two proteins have also
been designated full GARP and truncated GARP (f-GARP
and t-GARP, respectively; Ref. 78). The B1a subunit and
GARP1/GARP2 are derived from two different genes. The
two soluble GARP1/GARP2 probably represent alternatively
spliced forms (9, 10). Splice variants of the rod B1a lacking
the GARP-part are found in OSNs (40, 357) and testis (417).
The presumptive B3 subunit expressed in cone photoreceptors is much shorter than B1a (122) and has no GARPrelated sequences, suggesting that GARPs serve a rod-specific function. In fact, specific anti-GARP antibodies stain the
outer segment of rods but not of cones (213).
The common NH2-terminal region of GARPs carries
four short proline-rich repeats R1 to R4 (⬃15 amino acids)
that are highly conserved among each other and that represent the most conserved structural elements between
GARPs from different species (see dot blot in Fig. 6).
Peptide affinity chromatography suggested that these
repeats are involved in protein-protein interaction between GARP and the PDE, the GC, and the retina-specific
ATP-binding cassette (ABCR) transporter (213). The
ABCR transporter (6, 172) probably translocates all-trans
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FIG. 8. A RasMol ribbon drawing of the
CAP dimer with one cAMP molecule bound per
monomer.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
retinal complexed together with phosphatidylethanolamine as a Schiff’s base (384). The ABCR is better known
as “rim” protein because it is confined to the rim of the
disk membrane (172, 315). Reportedly, the GC is also
located near the disk margin, although the electron mi-
793
croscopic evidence is less compelling than for ABCR
(249). The soluble GARPs are tightly associated with the
margin of the disk, probably by binding to one or several
of the rim-confined proteins. The interaction between
GARPs and ABCR and GC has recently been called into
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FIG. 9. Alignment of cNMP-binding sites of vertebrate and invertebrate CNG channels, HCN channels, cyclic
nucleotide-dependent kinases, EPAC, and the catabolite gene activator protein CAP. The ratA1 subunit was taken as a
reference. Residues identical or similar to the respective amino acid in the ratA1 subunit are shown with a black or gray
background, respectively. RatA1 (91), ratA2 (90), ratA3 (356), ratA4 (44, 242), dmA (22), ceA (210), ratB1 (356), mouseB3
(122), ceB (74), dmB (accession AAF46757), dm3 (278), dm4 (accession BAA89278), ce3 (accession T20936), ce4
(accession T33125), ce5 (accession AAF36062), ce6 (accession T21969), HCN1 (354), PKA1 (cAMP-binding site 1 of PKA)
(396), PKG1 (cGMP binding site 1 of PKG) (389), EPAC (86), and CAP (4) are shown.
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
question (325). Using immunoprecipitation techniques,
Poetsch et al. (325) showed that both ABCR and GC
cannot be pulled down with GARP-specific antibodies, no
matter whether the potential binding partners have been
covalently attached to each other by cross-linking reagents. Instead, peripherin, another protein located at the
disK rim, seems to interact with GARP2 and the GARP⬘part of the B1a channel subunit. The interaction between
B1b subunit and proteins at the disK margin is expected
to force the CNG channels to align in circles that are
stacked along the length of the outer segment (Fig. 10).
Such a nonuniform distribution might account for the
high variability of channel density in the rod outer segments determined by patch-clamp recording (184), although the authors do not favor this explanation.
GARP2, the most abundant GARP species, has been
proposed to interact with the PDE (213). In fact, recombinant GARP2 inhibits light-activated PDE with a K1/2 of
⬃10 nM. We have reexamined the control of PDE activity
by using purified native GARP2 and observed only a weak
inhibition of PDE activity, whereas recombinant GARP2
was a potent inhibitor (H. G. Körschen, unpublished results). The difference between native and recombinant
Physiol Rev • VOL
GARP2 suggests that the small fusion part of the recombinant GARP2 (57 amino acid residues) might be responsible for the inhibitory action.
The CNG channel also undergoes an interaction with
the Na⫹/Ca2⫹-K⫹ exchanger that involves the A rather
than the B subunit (21, 281, 325, 364). The molar ratio of
exchanger to channel is about two (21, 334), implying that
two exchanger molecules are bound per channel. The
juxtaposition of the channel, which promotes Ca2⫹ influx
and the Na⫹/Ca2⫹-K⫹ exchanger, which promotes Ca2⫹
efflux, suggests that Ca2⫹ dynamics inside the cell are
localized to microdomains in the vicinity of the channel.
The high density of negatively charged Glu residues may
serve as a low-affinity Ca2⫹ buffer that kinetically impedes the diffusion of Ca2⫹ away from the site of entry.
C. Posttranslational Modifications
1. Proteolytic processing
Molecular characterization of CNG channel proteins
was initiated when Kaupp et al. (188) cloned the cDNA for
the A subunit of the CNG channel from bovine rods. The
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⫹
FIG. 10. Interaction of the rod CNG channel with other proteins. Left: interaction with peripherin and the Na /
Ca2⫹-K⫹ exchanger. Right: predicted ringlike distribution of CNG channels in the outer segment membrane of rods.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
2. Glycosylation
Enzymatic deglycosylation indicated that the 63-kDa
A1 subunit but not the 240-kDa B1a subunit contains an
N-linked oligosaccharide chain (421). The A1 subunit of
the bovine rod CNG channel is N-glycosylated at residue
N327 (420). The glycosylated and deglycosylated forms of
A1 differ in molecular mass by 2 kDa. Glycosylation has
not been studied in the A3 subunit.
The olfactory A2 subunit is heavily glycosylated. In
Western blots of isolated cilia, an A2-specific antibody recognizes two bands: a sharp but faint ⬃75-kDa band and a
prominent diffuse band at 110–145 kDa (40). Treatment of
cilia preparations with glycosidase abolished the diffuse
band entirely and at the same time strongly enhanced the
75-kDa signal, demonstrating that the vast majority of the A2
subunit exists in a highly glycosylated form. It seems likely
that the glycosylated 110- to 145-kDa form is specifically
targeted to cilia, whereas the non- or low glycosylated 75kDa form might be expressed elsewhere in OSNs, for example, the soma or dendrite. The A4 and B1b subunits of the
olfactory CNG channel are not glycosylated (40).
The roles for proteolytic processing and N-glycosylPhysiol Rev • VOL
ation are not known at the present time, but both processes may be required for targeting of channels to the
outer segment and the chemosensitive cilia.
VI. MOLECULAR COMPOSITION AND
TISSUE DISTRIBUTION
The list of tissues in which CNG channel subunits
are reportedly expressed has been rapidly expanding. It
includes nonneuronal tissue such as heart, lung, kidney, pancreas, liver, spleen, testis (Table 5), and various brain areas such as cortex, cerebellum, hippocampus, thalamus, hypothalamus, retinal olfactory bulb,
gustatory and olfactory epithelium, VNO, pineal gland,
and pituitary (Table 4). Although high expression levels
of CNG channels allowed a comprehensive characterization in photoreceptors and OSNs, their study in
other tissues, because of the much lower abundance, is
technically demanding and hence much less advanced.
A comprehensive list of the expression pattern of CNG
channel subunits in various species is given by Richards and Gordon (340).
A. Molecular Composition of CNG Channels
in Photoreceptors and Olfactory Neurons
The first indication that CNG channels are built from
several distinct subunits came from the functional characterization of the heterologously expressed A1 subunit
of rod photoreceptors. The recombinant A1 channel recapitulates several of the key properties of the CNG channel
in intact rods, but also deviated in some aspects (188).
Furthermore, the channel protein purified by either immunoaffinity or CaM-affinity chromatography consists of
two prominent polypeptides that migrate on SDS-polyacrylamide gels with apparent molecular masses of 63 and
240 kDa, respectively. Initially, it was thought that only
the 63-kDa polypeptide comprised the channel itself and
the 240-kDa component represented a spectrinlike cytoskeletal protein that was tightly associated with the
CNG channel (79, 279). Subsequent studies have revealed
that the 240-kDa polypeptide is the second subunit of the
channel (214). It is now generally believed that CNG
channels in various tissues form heteromeric complexes
consisting of distinct subunits. Because A and B subunits
from nematode to human functionally coassemble with
each other (104), a large number of distinct channels can,
in principle, be combinatorially generated from the six
subunit types. Whether the promiscous assembly of diverse subunits is a general strategy to generate CNG
channels that suit a particular cellular function is not
known and deserves further examination. It is also not
known whether in some tissues CNG channels exist as a
homomeric complex.
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full-length cDNA codes for a polypeptide of 690 amino
acids with a predicted molecular mass of 79.6 kDa. This
size is in good agreement with the molecular mass (⬃78
kDa) of the heterologously expressed polypeptide measured by SDS gel electrophoresis, but considerably larger
than the apparent molecular mass of 63 kDa observed in
bovine ROS and purified channel preparations (282). Immunocytochemical labeling studies of retinal tissue has
firmly established that the 63-kDa subunit is the predominant form of the A subunit in rods. The discrepancy in
size is primarily due to the absence of the NH2-terminal 92
amino acid residues. The truncated A subunit is also
present in ROS from human, mouse, rat, guinea pig, and
pig retinas (283). Shortened forms of the rod and cone A
subunits have also been detected in chicken retinal extracts (39). These results led to the view that the NH2
terminus of the A subunits undergoes photoreceptor-specific posttranslational cleavage. The role for this proteolytic processing is not known at the present time, but it
may be required for targeting of the channel to the plasma
membrane of the outer segment or for interaction with
other photoreceptor proteins.
Proteolytic processing seems to be absent in the A2
subunit of the rat olfactory CNG channel, because the
heterologously expressed and the deglycosylated native
subunit display a similar molecular mass of ⬃75 kDa,
which reasonably agrees with the predicted molecular
mass of 76 kDa for the cloned subunit (40). For the same
reasons, the B1a and B1b subunits of the rod and olfactory CNG channels, respectively, do not seem to undergo
posttranslational processing (40, 214).
795
796
U. BENJAMIN KAUPP AND REINHARD SEIFERT
TABLE
4. Subunit distribution in brain
Region
RT-PCR
Northern/RPA
In Situ
Western
Immunohistochemistry
Reference Nos.
A1
Rod photoreceptors
Retinal ganglion cells/retina
Whole brain
Cortex
Hippocampus
Pinealocytes/pineal organ
Cerebellum
Anterior pituitary
GnRH-secreting cell line (GT1)
OSNs
Cortex
Hippocampus
Cerebellum
Olfactory bulb
Thalamic and hypothalamic nuclei
Pineal
Anterior pituitary
Cone photoreceptors
Subpopulation OSNs
NA
⫹
ND
⫾
⫹
⫹
⫾
⫹
⫹
NA
⫾
⫾
⫹
⫹
⫺
ND
⫺
NA
⫹
OE
⫹
⫹
⫹
ND
⫺
NA
⫺
⫹
⫹
⫹?
⫹
ND
NA
⫹
⫹
ND
NA
NA
⫾
⫹
⫹
ND
ND
ND
ND
NA
ND
ND
ND
⫹
ND
ND
ND
NA
NA
⫹
⫹
NA
⫹
⫾
⫹
⫹
ND
ND
⫹
⫹
⫹
⫹
⫹
⫹
⫹
ND
ND
ND
⫹
ND
NA
ND
ND
ND
ND
ND
ND
⫹
ND
⫹
ND
ND
ND
ND
ND
⫹
NA
⫹
⫺
NA
ND
ND
ND
ND
ND
ND
⫹
ND
⫹
ND
ND
ND
ND
ND
⫹
⫹
2, 39
2, 408
34, 102
46, 91, 346
46, 91, 194
42, 91
46, 91
397
405
40, 46
46, 99, 195, 346, 381
46, 194, 381
46, 195, 381
46, 99, 195
99, 195, 381
381
99, 397
39, 416
273
ND
NA
ND
⫺
ND
NA
ND
ND
ND
ND
ND
⫹
⫹
ND
⫹
⫺
⫹
ND
⫹
ND
ND
⫹
ND
⫹
ND
ND
ND
ND
⫹
ND
⫹
ND
ND
NA
ND
ND
ND
⫹
ND
⫹
ND
ND
ND
⫹
⫹
ND
ND
ND
ND
⫹
ND
ND
ND
ND
ND
⫹
ND
ND
ND
⫹
⫹
ND
ND
ND
395
41
346
34
397
40, 44, 46, 242
46
46
46
46
405
214, 357
40, 357
405
122
122
A2
A3
A4
B1
B3
Embryonic chick brain
Pinealocytes
Cortex
Cerebellum
Anterior pituitary
OSNs
Cortex
Hippocampus
Olfactory bulb
Cerebellum
GnRH-secreting cell line (GT1)
Rod photoreceptors or retina
OSNs
GnRH-secreting cell line (GT1)
Retina
Total brain
⫹, Positive results; ⫺, negative results; ⫾, positive and negative results; ND, not determined; NA, not applicable.
The native CNG channel in rods consists of two types
of subunits, A1 and B1a (Fig. 11). Purified channel preparations do only contain these two polypeptides, and
coexpression of the A1 and B1a subunits produces channels that recapitulate most, if not all, of the key properties
of the native channel from rods (70, 71, 214). Notably, the
rapid flickery gating that is so characteristic of the native
channel (156, 392, 399, 441) and the high sensitivity to
blockage by L-cis-diltiazem (203, 379) require the coexpression of the A1 and B1a subunits (71, 214).
Although the CNG channel from cones has not been
as extensively studied as that in rods, it likely is also
composed of A and B subunits (A3 and B3) (39, 122, 416).
Similar to the rod CNG channel, the channel from cones
displays flickery activity and is blocked by micromolar
concentrations of L-cis-diltiazem in a voltage-dependent
manner (154, 158). Coexpression of A3 and B3 produces
channels that display flickery gating behavior and are
blocked by L-cis-diltiazem (122).
The CNG channels in the chemosensitive cilia of
OSNs unlike those in photoreceptors consists of three
Physiol Rev • VOL
different subunits: A2, A4, and B1b (40). The mRNAs of all
three subunits are expressed in OSNs, and the three subunit polypeptides are colocalized in the cilia. Furthermore, the properties of native channels and various combinations of heterologously expressed subunits were
compared using five functional criteria: 1) the sensitivity
for activation by cAMP, 2) the discrimination between
Na⫹ and K⫹, 3) the single-channel conductance, 4) the
kinetics of open-closed transitions, and 5) the ability to
adopt a subconductance state. The A2A4B1b channel resembles the native channel in each of the five critera,
whereas other combinations (e.g., A2A4 or A2B1b) or
homomeric A2 channels display clear differences in all of
the criteria with respect to the native channel (40, 44, 242,
357). However, subtle differences between native and
A2A4B1b channels were noted that may either result from
different glycosylation patterns of the native and heterologously expressed A2 subunit or from the expression
system. In fact, two studies have shown that the kinetic
properties of A2A4 channels depend on the expression
system. The channels partially desensitize when ex-
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Subunit
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
pressed in Xenopus oocytes (242), but do not do so in
HEK293 cells (44).
The strategy taken to examine the stoichiometry and
arrangement of subunits has been to express wild-type
and mutant subunits. The mutants are expected to change
the channel properties in characteristic ways and thereby
“report” the number and arrangement of individual subunits in the channel complex. For example, Liu et al. (246)
coexpressed the wild-type A1 subunit with a chimeric A1
subunit in which the pore region is replaced with that of
the A2 subunit. The wild-type and chimeric subunits,
when expressed alone, display a single-channel conductance of 30 and 85 pS, respectively. Upon coexpression,
four additional intermediate conductances are observed
that, in principle, could arise from the four possible combinations of two subunits forming a pentameric channel.
With the use of homo- and heterodimer constructs, some
evidence was provided that two of the intermediate conductances might arise from different arrangements of
wild-type and chimeric constructs in a tetrameric channel, i.e., the same subunits can be adjacent or opposite to
each other (246). Experimental evidence in favor of the
tetrameric complex was also provided by Ni2⫹ potentiation studies (135).
More recently, the Ni2⫹ technique has been extended
Physiol Rev • VOL
to heteromeric rod CNG channels (A1B1b) (159, 372),
unfortunately with conflicting results. Shammat and Gordon (372) coexpressed A and B subunits with either constructed or mutated Ni2⫹ potentiation sites and concluded that the native rod channel exists in an AABB
arrangement. These authors assumed that a channel with
engineered C-linker histidines in the B subunit (N546H)
but mutated C-linker histidines in the A subunit (H420Q)
can only be potentiated by Ni2⫹ when two B subunits are
localized adjacent to each other. This view was challenged by He et al. (159), who could show that Ni2⫹
potentiation is mediated by the wild-type B subunit in the
absence of A subunit histidines. Potentiation even persisted when residues in the C-linker of the B subunit that
possibly bound Ni2⫹ were mutated, suggesting that regions other than the C-linker might be responsible for
Ni2⫹ potentiation in the heteromeric channel. In contrast
to Shammat and Gordon (372), He et al. (159) proposed a
diagonal ABAB stoichiometry. However, their experiments using the blockage by L-cis-diltiazem and the cAMP
activation properties of coexpressed tandem constructs
provided no evidence against an AAAB or BBBA arrangement, which they explicitly stated in their discussion.
Therefore, it seems important to rigorously determine the subunit stoichiometry and arrangement of native
channnels by other methods. Indeed, a biochemical approach based on cross-linking experiments of native rod
channels favors an AAAB stoichiometry (Weitz, D., N.
Ficek, E. Kremmer, and U. B. Kaupp, unpublished data).
Nevertheless, direct structural techniques such as singleparticle electron microscopy or biophysical methods able
to detect each subunit in the oligomeric channel complex
would be highly desirable.
B. Subunit Distribution in the Brain and
Nonneuronal Tissues
The distribution of CNG channel subunits in the brain
has been examined by various techniques (Table 4).
These studies have produced conflicting results. The need
for extensive PCR amplification and long development
times for the in situ hybridization suggests that the levels
of mRNAs encoding channel subunits are very low and, in
combination with the use of different primers or cDNA
probes, amplification protocols, and hybridization conditions, may account for the variable results. Finally, expression of the A1, A2, and A3 subunits in the cortex is
each developmentally regulated in a specific temporospatial pattern (346), which further enhances the experimental complexity. For example, the level of the A1 transcript
in rat cortical areas is high at birth (postnatal day 0) and
gradually decays with time until it becomes practically
nondetectable at postnatal day 55, whereas a similar developmental regulation was not observed in the hippocampal region (346).
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FIG. 11. Top: subunit stoichiometry of the CNG channels from rods,
cones, and the two populations of OSNs. Bottom: amino acid residues
forming the intrapore ion binding site in different CNG channels.
797
798
U. BENJAMIN KAUPP AND REINHARD SEIFERT
TABLE
Subunit
A1
A2
A4
B1
B3
Region
RT-PCR
Northern
In Situ
Western
Immunohistochemistry
Reference Nos.
Heart
Kidney
Liver
Lung
Testis
Spleen
Pancreas
Colon
Keratinocyte
Muscle
Vascular endothelium
M-1 cell line
Heart
Kidney
Adrenal gland
Liver
Lung
Skeletal muscle
Testis
Pancreas
Colon
Heart
Kidney
Liver
Lung
Testis
Pancreas
Colon
Heart
Liver
Heart
Kidney
Testis
Heart
Kidney
Liver
Lung
Spleen
Skeletal muscle
Testis
⫾
⫹
⫾
⫾
⫾
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫾
⫺
⫺
⫺
⫹
ND
⫺
⫺
⫺
⫾
⫾
?
⫹
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫹
ND
ND
ND
ND
ND
ND
ND
⫾
⫾
⫾
⫾
⫾
⫾
⫺
ND
⫹
⫾
⫹
⫹
⫺
⫺
⫺
⫺
ND
⫺
ND
⫺
ND
⫹
⫾
⫺
ND
⫹
⫺
⫹
ND
ND
ND
ND
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
ND
ND
⫹
ND
⫹
ND
ND
ND
ND
⫹
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
⫹
⫺
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
⫹
ND
ND
ND
ND
ND
ND
⫹
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
⫹
⫺
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
⫹
ND
ND
ND
ND
ND
ND
⫹
ND
ND
ND
ND
ND
ND
ND
3, 46, 91, 102, 437
3, 91, 102, 182, 437
3, 46, 91, 102, 437
91, 102, 427, 437
1, 3, 102
91, 102, 437
310
429
310
3, 91, 102
429
1
31, 32, 46, 351
137
350
46, 137
427
137
31, 92
92
34, 92
34, 92
427
34, 416, 417
92
92
46
46
36
417
122
⫹, Positive results; ⫺, negative results; ⫾, positive and negative results; ND, not determined; NA, not applicable.
The expression of CNG channel subunits in nonneuronal tissue has been studied by various techniques (Table 5).
Except for testis and spermatozoa, the experimental evidence for expression of any of the known six CNG channel
genes in nonneuronal tissue is weak. In testis, the expression of mRNA encoding A3, B1, and B3 has been shown by
cloning of cDNAs and Northern analysis (34, 122, 416, 417).
With the use of several polyclonal antibodies, the A3 and B1
subunits have been localized immunhistochemically to the
flagellum of ejaculated sperm and precursor cells in crosssections of seminiferous tubules. Cloning identified one
short and three long transcripts of the B1 subunit (417). The
highly abundant short transcript would give rise to a
polypeptide that begins shortly before S1 (molecular mass
74.3 kDa). The three longer transcripts differ in their 5⬘terminal region, are much less abundant, and would give rise
to polypeptides with a molecular mass of 104–106 kDa.
Western analysis of cauda epididymal and ejaculated sperm
Physiol Rev • VOL
detected only a 80-kDa polypeptide but provided no evidence for polypeptides of higher molecular mass (417).
Therefore, the long B1 subunits must be expressed at rather
low levels, if at all.
The presence of CNG channel transcripts in nonneuronal tissues other than testis has been studied by Northern
analysis. Biel et al. (34) demonstrated expression of A3
transcripts in renal cortex and renal medulla and a weak
signal in cardiac atrium and ventricle. Ahmad et al. (3)
detected the presence of A1 in heart and kidney of rat,
whereas in canine, Zhang et al. (437) were unable to detect
A1 transcripts in liver, spleen, lung, heart, and kidney (437).
No signals using probes for A1-A3 were detected in liver of
rabbit; adrenal gland and diencephalon of bovine; and brain,
kidney, and pancreas of rat (92). The A1 transcript was also
detected in keratinocytes (310). Ruiz et al. (350) could not
detect by Northern analysis the A2 message in heart using
several specific probes; however, using RNase protection
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A3
5. Distribution of subunits in nonneuronal tissues
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
VII. LIGAND SENSITIVITY AND SELECTIVITY
The ligand sensitivity and selectivity of native and
exogenously expressed CNG channels have been studied
by measuring the currents activated in excised inside-out
membrane patches upon superfusion with various concentrations of ligand. All known native CNG channels
respond to both cAMP and cGMP, but lower concentrations of cGMP than cAMP are required to open the chanPhysiol Rev • VOL
FIG. 12. Ligand sensitivity of the native rod photoreceptor CNG
channel and the native CNG channel from OSNs. Shown are normalized
currents versus cNMP concentration. Concentrations for half-maximal
activation are as follows: cGMP, 50 ␮M and 1.6 ␮M; cAMP, 1,500 ␮M and
3 ␮M for the CNG channels from rod photoreceptor and OSNs,
respectively.
nels. In rods and cones, CNG channels sharply discriminate between cGMP and cAMP, whereas channels in
OSNs respond equally well to both ligands, i.e., are much
less selective (Fig. 12). Moreover, CNG channels from
OSNs have a much higher sensitivity for both cAMP and
cGMP than photoreceptor CNG channels. Figure 12 illustrates yet another important difference between CNG
channels from photoreceptors and OSNs: cAMP is a full
agonist of the olfactory channel but only a partial agonist
of the rod channel.
The analysis of dose-response relations showed that
channel activation is steeply dependent on cGMP concentration. In double-logarithmic plots (log I/Io vs. log
[cGMP]), the limiting slopes ranged between ⬃1.5 and
⬃3.5. The shape of the dose-response relation and the
limiting slope was interpreted to indicate that several
cGMP molecules bind to the channel in a cooperative
manner. Because each subunit harbors a single cNMPbinding site (188) and because homo- and heteromeric
channels most likely form tetrameric complexes (246),
maximally four ligand molecules bind to the channel.
Numerous studies examined the molecular basis of
ligand sensitivity and selectivity and cooperativy. Selectivity can be achieved either by differential control of the
affinity for binding of the ligand, efficacy of gating, or a
combination of both. Binding affinity is a measure of how
tightly cyclic nucleotides bind to the channel; it has not
been determined yet for any CNG channel by a direct
binding assay but has been inferred from models that
describe the activation of the channel (see below). Efficacy refers to the ability of the ligand to gate the channel
open once it has been seated in the binding cavity. Although the distinction between binding and gating is a
useful concept, in practice it has been proven difficult to
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assay, they were able to detect the A2 transcript in mouse
heart and brain, but not in liver.
Ding et al. (91) studied the expression pattern of A1
in rat by four different techniques. These authors detected
transcripts in total RNA or poly(A)⫹ RNA from pineal and
pituitary gland, retina, adrenal gland, and in testis with a
size of 3.1–3.5 kb, consistent with the size of cloned
cDNA. Transcripts of 1.8 kb and of ⬃6 kb were detected
in kidney and spleen, respectively. No signals were detected in poly(A)⫹ RNA of heart, brain, lung, liver, skeletal muscle, and kidney. The RNase protection assay suggested the presence of A1 transcripts in retina, brain,
lung, and spleen, but not in liver, heart, testis, and kidney.
A third detection method, in situ hybridization, suggested
the presence of A1 transcripts in alveolar tissue of lung,
the endothelial layer of aorta (but not in smooth muscle
layers), thymus, and spleen. These authors also reported
intense staining in hippocampus and cerebellum and
weaker staining in the cortex. Finally, using RT-PCR, A1
fragments could be amplified from all tissues tested.
The expression pattern has been also studied by
RT-PCR, again with mixed results (Table 5). For example,
A2 seems to be expressed in heart of rabbit, but not in rat
and bovine, whereas A3 was detected in bovine heart, but
not in rat and rabbit (92).
In summary, although many of these studies are suggestive, none of them conclusively demonstrates the presence, leaving alone the function of CNG channel polypeptides in an identified nonsensory cell, perhaps with the
exception of testis and spermatozoa. In the few studies,
where cyclic nucleotide-sensitive currents have been recorded by electrophysiological techniques, the properties do
not match those carried by the well-characterized CNG
channels in photoreceptors and OSNs. Finally, targeted disruption of the A2 and A3 genes in mice revealed no obvious
phenotypes other than anosmia and loss of cone function,
respectively (33, 60). Future studies in this field will require
the unequivocal demonstration of CNG currents in excised
membrane patches or in the whole cell configuration using
caged cyclic nucleotides and the corraboration of subunit
expression on the single-cell level using the panel of sensitive and well-characterized polyclonal and monoclonal antibodies used for the study of sensory cells and testis (see, for
example, Refs. 40, 214, 273, 283).
799
800
U. BENJAMIN KAUPP AND REINHARD SEIFERT
A. cNMP-Binding Site
Molecular modeling and mutagenesis studies predicted 10 different interactions between cGMP and the
binding pocket involving 8 amino acid residues, 5 with the
purine ring and 5 with the ribofuranose (7, 366, 368, 369,
394, 402) (Fig. 13). It is tacitly assumed that interactions
with the common ribofuranose moiety are largely similar
for all cyclic nucleotides, whereas differential interaction
with the purine ring controls ligand selectivity. The location of these residues in the cNMP-binding fold is shown
in Figure 9. The five interactions with the ribofuranose
include hydrogen bonding between T560 (numbers refer
to bovine A1) and O6⬘ of the diester group, between G543
und O2⬘ of the ribose moiety, and between I545 and O2⬘ or
O3⬘. A salt bridge is formed between R559 and the negatively charged phosphate group. Finally, an ionic hydrogen bond is established between E544 and O2⬘. The five
interactions with the purine ring include a hydrogen bond
between the hydroxyl group of T560 and N2C. This interaction has been proposed to be mediated by an intercalated water molecule (367). Residue F533 is predicted to
Physiol Rev • VOL
FIG. 13. Interaction of amino acid residues of the cNMP-binding site
with cGMP. Numbering of residues refers to the bovine A1 subunit (188)
(see text for explanation).
undergo an aromatic-aromatic interaction with the purine
ring. Finally, residue D604 either shares a single hydrogen
bond with N1 (367) or two hydrogen bonds with N1 and
N2C of cGMP (402). cGMP and cAMP differ in their
structure only at C6-N1-C2. cAMP cannot form a hydrogen
bond with either N1 or N2C, resulting in an unfavorable
electrostatic interaction between the negatively charged
carboxylate side chain of D604 and the free electron pair
at N1 of cAMP (402).
Differential interaction of cAMP and cGMP with
D604 in the C helix of the cNMP-binding domain has been
proposed to underlie ligand discrimination in CNG channels (402). At the equivalent position in CAP, residue T127
in one subunit and S128 from the opposite subunit of the
CAP dimer form hydrogen bonds with the N2C amino
group of adenine (409) (Fig. 8). In agreement with this
prediction, the efficacy of gating by cAMP is enhanced 1)
in A1 mutants with a neutral residue at position 604
(D604Q and D604M) (402); 2) at low pH, when D604 is
presumably protonated (131); and 3) in channels that
include A4, B1, and B3, i.e., subunits, which carry a neutral or positively charged amino acid at the respective
position (44, 122, 131, 242).
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experimentally dissect binding from gating, because both
processes are inextricably coupled to each other (for a
brilliant account, see Ref. 77). However, in some instances, efficacy can be gleaned from the relative maximal open probability [relative Pmax ⫽ Pmax(cAMP)/Pmax(cGMP)] obtained at saturating ligand concentrations. For
example, cAMP is a poor agonist for the native CNG
channel from rods (390) (Pmax in the presence of saturating cAMP concentrations is ⬃20% of that in the presence
of cGMP) or for the homomeric A1 channels, where relative Pmax is only a few percent (7, 134), whereas for A2
channels, cAMP is able to fully open the channel (relative
Pmax reaches nearly unity).
Several important questions were addressed by a
multitude of studies. Which amino acid residues interact
with the ligand, and what is the molecular nature of these
interactions? Which of these interactions are state dependent, i.e., occur either in the open or the closed state, and
which are state independent? What is the molecular basis
for ligand sensitivity and discrimination? Which part of
the channel is involved in gating, i.e., the transition from
the closed to the open state of the channel and which part
is involved in binding?
The search for key residues involved in binding and
gating have been guided by previous work on ligand binding and activation mechanism in PKA and PKG and the
CAP protein. Several regions determine the binding and
the activation process, including the cNMP-binding domain itself, the linker region connecting S6 and the cNMPbinding domain (“C-linker”), the NH2-terminal domain,
and the pore region.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
Physiol Rev • VOL
CNG channel, this binding domain mediates full channel
activation by cGMP (full agonist), but only poor activation
by cAMP (partial agonist), illustrating the profound impact of protein environment on the properties of a functional domain in an allosteric protein.
B. C-Linker Region
Low concentrations of transition metal ions, including Ni2⫹, Zn2⫹, Cd2⫹, Co2⫹, and Mn2⫹, potentiate the
response of the rod CNG channel to subsaturating cGMP
concentrations but have little effect on the maximal response at saturating cGMP concentrations (171, 183).
Ni2⫹ also increased the potency of cAMP to activate
homomeric A1 channels by almost 50-fold (133). Subsequent studies identified H420 in A1 as part of the Ni2⫹binding site responsible for the potentiation (133). Nickel
potentiation in homomeric A1 channels involves the interaction of at least two H420 residues localized on adjacent subunits (135), which presumably coordinate the
transition metal ion. However, a H420Q mutant of A1
when coexpressed with wild-type B1 forms heteromeric
channels, whose activity can be potentiated by Ni2⫹, suggesting that potentiation of the native channel involves
residues from both A1 and B1 subunits (159). In contrast,
Ni2⫹ inhibited the activation of the olfactory A2 channel.
This action is mediated by another histidine residue
(H396) (134). The two His residues are each located in the
C-linker region of the respective channel protein, suggesting that this region participates in the conformational
changes leading to channel opening (134).
Swapping experiments with the C-linker region between A1 and ceA (Tax-4) channels (314) and between A2
and A3 channels (443) also suggest that this domain somehow relays the binding event to the gating machinery near
or inside the pore. Three amino acid residues in the C
linker seem to account for most of the difference in
efficacy of activation by cAMP between mouse A3 and
rabbit A2 (443). When the residues I439, D481, and D494
of rat A3 are replaced by the respective amino acids found
in rabbit A2 (I439V, D481A, and D494S), the efficacy of
activation by cAMP is significantly enhanced. Quite sadly,
the A2 ortholog from catfish features the identical residues at these positions as bovine A3, yet with respect to
efficacy of gating, this subunit behaves like all other A2
orthologs and not like an A3 subunit (137). Furthermore,
three other residues were identified in the C linker of A1
that when changed to the respective residues in ceA
(Tax-4) improved the efficacy of gating (314).
C. NH2-Terminal Region
The involvement of the NH2-terminal region in channel activation was suggested by studies using chimeras
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One would have hoped that this model explains the
higher gating efficacy by cAMP in olfactory (A2) channels
compared with rod (A1) channels. Unfortunately, several
lines of evidence suggest that various CNG channel subtypes
may employ distinct amino acid residues and mechanisms
to achieve ligand selectivity. For example, A2 orthologs from
mammals and catfish (90, 137, 254) have a glutamate or
glutamine, respectively, at this position, yet these subunits
form channels that are almost indistinguishable with respect
to the efficacy of gating by cAMP. Furthermore, A1 and A3
from bovine, mouse, and rat all carry an aspartate residue at
the equivalent position, yet the gating efficacy by cAMP
relative to that by cGMP varies from ⬃0.02 (bovine A1; Ref.
133) to 0.14 (mouse A3; Ref. 443) to almost 1.0 (rat A3; Ref.
273). Another caveat of these mutagenesis studies is that
contacts formed by residues F533, K596, D604, and T560
with the purine ring are not independent from each other
(369, 402) and, consequently, it is difficult to assign the
selectivity switch to any one of these residues alone. Finally,
the model requires cGMP to bind in the anti conformation,
whereas other models propose the syn conformation (369).
The interaction of the cyclic nucleotide with the
␤-roll of the cNMP-binding domain was probed with phosphorothioate derivatives of cAMP and cGMP (Rp-cAMP,
Rp-cGMP, Sp-cAMP, and Sp-cGMP; Refs. 217, 442). Although these compounds bind to the different CNG channels, their mode of action is quite diverse. Both Rp- and
Sp-derivatives of cGMP are full agonists of native rod
(442) and homomeric A1 channels (217), but only SpcGMP is an agonist of homomeric A2 channels, whereas
Rp-cGMP is a competitive antagonist (217). Rp-cAMP is
an antagonist of the rod CNG channel, but a partial agonist of the olfactory CNG channel. In contrast, Sp-cAMP
fully activates the olfactory CNG channel (217). Similarly,
Rp- and Sp-cAMP are both antagonists of the homomeric
A1 channel, whereas A2 channels discriminate between
the two analogs: Sp-cAMP is an agonist, whereas RpcAMP is an antagonist. These results indicate that the
interaction of ␤-roll residues of the cNMP-binding domain
with the cyclic nucleotide is different for various channels
and that one has to be cautious to generalize results
obtained from one channel type.
Recently, a chimera between the cyclic nucleotidebinding domain of A1 and the DNA-binding domain of
CAP has been crystallized (370). At low resolution (7 Å),
the overall structure of this chimera is the same as that of
CAP. This seems to be a promising avenue toward a
three-dimensional structure at high resolution, and it
might be a unique opportunity to compare the cNMPbinding domains of different A and B subunits. However,
some caution must be taken in the interpretation of the
results. In fact, the A1-CAP chimera binds both cAMP and
cGMP with equal affinity, whereas DNA binding is promoted by cAMP only and not by cGMP, like in the wildtype CAP protein. However, within the context of the
801
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
between A1 and A2 subunits (139). The NH2 terminus in
A2 interacts by means of a CaM-binding site (248) with the
cNMP-binding domain in the COOH-terminal region (403).
This interaction lowers the change in free energy between
the unliganded open and closed states. Binding of CaM
interferes with this interaction and thereby increases the
K1/2 of activation. This allosteric control seems to be less
important for the A1 channel.
ity filter itself is the location of the gate. SCAM studies of
the state dependence of the accessibility of pore residues
of A1 indicate that the pore helix undergoes a 100 –180°
rotation around its long axis during channel activation.
Possibly, this conformational change either occurs in parallel with or precedes the movement of the gate. Future
work needs to unravel at higher structural resolution how
movements of the cNMP-binding domain, the C linker,
and the pore helix are interconnected.
D. Pore
F. Kinetic Models
E. Conformational Changes of Protein Domains
During Activation
Inspired by the crystal structures of KcsA and CAP,
recent studies focused on the nature of the conformational
rearrangements that led to CNG channel opening. Important
insight into the secondary structure of subdomains as well
as into their movement was obtained by applying SCAM (5)
to residues of the cNMP-binding site (263), the putative pore
helix (247), and S6 (108), but also by applying a histidine
scan to residues in the C linker (177).
The ligand initially binds to the closed channel involving interactions between the ␤-roll structure of the
cNMP-binding site and the ribofuranose moiety of the
cyclic nucleotide. Subsequently, a movement of the C
helix relative to the ␤-roll structure occurs (263). This
movement is somehow transduced into a rotation of the
␣-helical C linker (177), which connects the transmembrane segment S6 with the cNMP-binding site. Finally, the
rotation around the central axis of the C-linker helix is
likely to become transmitted to the S6 region. This step
seems to be important for channel activation. The region
located COOH terminally of S6 seems to serve different
functions in Shaker K⫹ channels and CNG channels. In
Shaker K⫹ channels, this region mechanically occludes
the channel entrance (84) and harbors the activation gate
(168). In CNG channels, ions can enter the inner vestibule
in both the open and closed state, arguing against an
intracellular location of the activation gate (108).
Instead, some experiments suggest that the selectivPhysiol Rev • VOL
It is useful to discuss channel activation within the
framework of linear state or sequential models and cyclic
allosteric models (for an insightful discussion of different
models, see Ref. 239). The sequential model involves binding of n molecules of cGMP to the closed channel, followed by a gating conformational change of the fully
liganded channel to the open configuration (see Fig. 14).
The binding of ligand to each subunit is described by the
binding constant KT and the open-closed transition by the
equilibrium constant L.
The four-site sequential model would be most appropriate for CNG channels because of their tetrameric structure. This model predicts a limiting slope of the doseresponse relation of four; however, in most studies slopes
between two and three were obtained. A neat explanation
for this discrepancy is provided by Ruiz et al. (347). They
found that the slope of dose-response relation constructed from single-channel recordings is consistently
steeper (Hill coefficient 3.0) than the slope of the doseresponse relation deduced from macroscopic currents
(Hill coefficient ⬍2.0). However, large variations in K1/2
were observed in multichannel patches. The authors suggest that the sum of channels with high Hill coefficients
yet variable K1/2 values will give rise to artifactually shallow dose-response relations. Notwithstanding this complication, Karpen et al. (185) used a three-site sequential
model to describe the kinetics of channel activation using
jumps in cGMP concentration (caged cGMP) and voltage.
A two-site sequential model has been used to describe
some of the differences in ligand sensitivity and selectivity between CNG channel subfamilies (134, 402). The
analysis of dose-response relations suggested that the
allosteric transition from the closed to the open configuration was energetically more favorable when cGMP was
bound to homomeric A1 and A2 channels than when
cAMP was bound, i.e., the difference in the activation by
cGMP and cAMP was primarily attributed to differences
in the free energy of the allosteric transition (⌬GL ),
whereas changes in KT alone could not account for the
behavior of the cAMP as partial agonist of the rod channel
(134). Similarly, the difference between activation of A2
channels compared with A1 channels could be accounted
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Many pore mutants show alterations in channel gating
(24, 25, 62, 121, 343). Because of the principle of reciprocity,
changes in gating are likely to affect binding as well. In fact,
several mutations at or near E363 in A1 and the corresponding position E340 in A2 demonstrated that gating defects in
the mutant channel are associated with an increase in the
K1/2 of activation (62, 121). Likewise, an A3 pore mutant
(T369S) found in patients with deficient color vision shows
dramatically altered gating kinetics, a lower maximal Po, and
a 10-fold increase of the K1/2 of activation (Tränkner, D., H.
Jägle, S. Kohl, R. Seifert, L. T. Sharpe, U. B. Kaupp, and B.
Wissinger, unpublished data).
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
803
for by varying only the equilibrium constant L, implying
that the initial binding of cAMP and cGMP, characterized
by the binding constant KT, is similar (134). However, in
another study by Zagotta and co-workers group (402), a
10-fold lower affinity was obtained for binding to the rod
channel of cAMP compared with cGMP [KT(cAMP)
⫽ 2,020 ␮M; KT(cGMP) ⫽ 226 ␮M]. The problem is this:
“high efficacy” ligands, characterized by L values ⱖ20 are
indistinguishable from each other, because they yield
maximal Poⱖ0.95 at saturating ligand concentrations (see
Ref. 77). This problem was overcome by Li and Lester
(238). Using single-channel kinetics of the olfactory A2
subunit, these authors showed that cGMP binds with
much higher affinity than cAMP (30-fold difference in KT)
and that the equilibrium constant L that governs the openclosed transitions is largely similar for cAMP and cGMP.
In the linear state models, the unliganded and partially liganded states do not lead to the open configuration. The cyclic allosteric model proposed by Monod et al.
(288) is a more general form of sequential models that
permits this transition (Fig. 14). The allosteric model
assumes that the binding sites are equivalent but that the
ligand affinity is higher in the open than in the closed
states. The opening of the unliganded channel, characterized by the equilibrium constant L0, is usually low (L0
⬍1). Because the open channels bind ligands more tightly
than the closed channels, the stability of the open configPhysiol Rev • VOL
uration is enhanced by a factor f each time an additional
ligand molecule binds to the channel.
Three predictions can be derived from the model of
Monod et al. (288): 1) channels should show openings
independent from the ligand, 2) each liganded state
should be associated with a unique open state, and 3) the
equilibrium constant L and, therefore, the Po should increase by a constant factor f for each ligand that binds.
One of the three predictions of the model of Monod et al.
(288) is satisfied by experiment, and two are not. In fact,
CNG channels show ligand-independent openings (200,
323, 348, 349, 393). The spontaneous open probability
(Psp) is significantly lower for A1 than for A2 channels
[Psp ⫽ 1.25 ⫻ 10⫺4 for a chimeric channel comprising the
A1 sequence and an olfactory A2 pore, Psp ⫽ 1.5 ⫻ 10⫺5
for the A1 subunit (348), and Psp ⫽ 2.25 ⫻ 10⫺3 for A2
(393)], indicating that the free energy of gating is smaller
in olfactory channels than that in rod channels. Transplantation of the NH2-terminal domain from A2 subunit to
A1 produces chimeric channels whose Psp is nearly identical to that of A2 (393). These and previous results show
that the NH2-terminal domain determines differences in
both ligand gating and spontaneous openings between
rod and olfactory CNG channels and that channel activation occurs by a cyclic allosteric mechanism. The analysis
of the allosteric mechanism was taken one significant step
further by Ruiz and Karpen (349). They succeeded to
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FIG. 14. Three different models of channel activation. Left: sequential model (red) and cyclic
allosteric model (red and blue). Right: coupleddimer model. Subunits can exist in the inactive
(“tense”) conformation (T, squares) or the active
(“relaxed”) conformation (R, circles). KT and KR
are the equilibrium constants for binding of the
agonist A to the T and R states, respectively; L0,
equilibrium gating constant between the unliganded
T and R states; L, equilibrium gating constant between T4A and R4A in the sequential model (red); f is
a multiplicative factor, f ⫽ KR/KT. [Modified from Li
et al. (239) and Richards and Gordon (340).]
804
U. BENJAMIN KAUPP AND REINHARD SEIFERT
Physiol Rev • VOL
VIII. ION SELECTIVITY
CNG channels strongly select cations over anions,
discriminate poorly among monovalent cations, and are
also permeable to divalent cations, in particular Ca2⫹. In
physiological ionic solutions, therefore, both monovalent
and divalent cations permeate CNG channels, and any one
ion species carries only a fraction of the total current. The
Ca2⫹ permeability of CNG channels is a crucially important part of their cellular function. Changes in CNG channel activity are inevitably associated with changes in
[Ca2⫹]i that initiate various excitatory or adapting cellular
processes.
A. Selectivity for Alkali Ions
The selectivity of CNG channels for monovalent cations was determined from relative ion permeabilities recorded under biionic conditions in excised patches. Ion
selectivity of native CNG channels has been first examined in intact cells by changing ionic solutions at the
extracellular side of the cell (67, 166, 167, 222, 223, 272,
297, 432). Although these early studies were complicated
by the lack of control over the intracellular concentrations of ions, they revealed that CNG channels from rod
and cone photoreceptors and OSNs do not select strongly
among alkali cations.
With the use of the excised inside-out patch configuration, the following sequence of permeability ratios was
obtained for the channel from salamander rods: Li⫹
⬎ Na⫹ ⬃ K⫹ ⬎ Rb⫹ ⬎ Cs⫹ ⫽ 1.14:1:0.98:0.84:0.58 (271).
Similar permeability sequences have been reported for
CNG channels from frog rods (103), mammalian rods
(255), and depolarizing photoreceptors of the lizard parietal eye (105, 106). The permeability of the CNG channel
from salamander rods was also probed with several large
organic cations (320). From space-filling models of the
tested cations, the narrowest part of the pore is estimated
to be at least a 3.8 Å ⫻ 5 Å rectangle.
The sequence of ion selectivities and permeability
ratios in native CNG channels of rods and recombinant A1
subunits are rather similar, except for Li⫹, which is more
permeant than Na⫹ in rod CNG channels, but less permeant in A1 homomeric channels (188, 255). Coexpression of A1 and B1a subunits yielded channels with permeability ratios similar if not identical to native channels
(214).
The ion selectivity of the CNG channel of cones is
similar to that of rods (321): K⫹ ⬎ Na⫹ ⬃ Li⫹ ⬃ Rb⫹
⬎ Cs⫹ ⫽ 1.11:1.0:0.99:0.96:0.8.
A small difference, however, is noticeable. In cones,
Li⫹ is equally or less permeable than Na⫹, whereas in rods
it is more permeable. The ionic selectivities of recombinant A1 and A3 homomeric channels from chick are by
and large identical (39).
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covalently tether a cGMP photoaffinity analog to the A1
channel and thereby permanently activate the channel
(55). This technique allows to lock exactly one, two,
three, or four cGMP molecules onto their binding sites
and, in combination with single-channel recordings, to
observe the gating of single channels with fixed ligand
occupancies (348). Channels occupied by four ligands
stay open most of the time; unliganded channels and
channels with one ligand open with a Po of 10⫺5. When
two and three ligands were locked in place, Po is 0.01 and
0.33, respectively. These Po values are not in the ratios
predicted by the model of Monod et al. (288). More interestingly, when less than four ligands are bound, the opening events show frequently smaller subconductance
states, as if the flipping of each subunit opens the channel
wider, and the probability distribution of subconducting
states depends on the number of ligands bound. These
observations are inconsistent with the simple model of
Monod et al. (288) as outlined in Figure 14.
As elegantly as these experiments are conceived,
they are not entirely unequivocal. The issue of multiple
conducting states is unresolved. Several authors do not
find subconductance states or as many as four (29, 170,
245, 348, 349, 386).
An alternative route to test the model of Monod et al.
(288) has been taken by Liu et al. (245). To dissect the
impact of each individual binding event on channel activation, these authors constructed channels with a constrained number of functional cNMP-binding sites. This
goal was achieved by coexpressing subunits with wildtype binding sites and mutant subunits with disabled binding sites. The binding of the ligand was abolished by
mutations of R559 and T560; these residues have previously been shown to decrease ligand sensitivity by several
orders of magnitude (7, 394). The results of Liu et al. (245)
and Ruiz and Karpen (348, 349) qualitatively agree with
each other in as much as both show a progressive increase in Po with increasing numbers of bound ligands.
However, the two studies significantly differ in the factor
by which each bound ligand increases Po. For example,
binding of one ligand does not change Po in the tethered
ligand approach, whereas Po was 0.017 in the disabled
binding-site approach. Whatever mechanism might cause
this discrepancy, both studies show that the conventional
model of Monod et al. (288) is inadequate to fully comprehend the complexity of CNG channel activation. Liu et
al. (245) therefore proposed an interesting variation of the
model of Monod et al. (288) in which the channel is
composed of a pair of dimers [coupled-dimer (CD) model,
see Fig. 14]. In the CD model, each subunit in a dimer
binds the ligand independently, yet the two subunits undergo a concerted allosteric conformational change to
open. Moreover, each of the two dimers makes this transition independently.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
The selectivity sequence of the native CNG channel
from OSNs is qualitatively similar to that of rods and
cones (112): Na⫹ ⬎ K⫹ ⬎ Li⫹ ⬎ Rb⫹ ⬎ Cs⫹ ⫽ 1.0:0.81:
0.74:0.60:0.52 and similar to that of A2 homomeric channels of catfish OSNs (138). The diameter of the channel
pore probed by the permeability of large organic cations
was 5.8 –5.9 Å for the A1 homomeric channel and 6.3– 6.4
Å for the A3 the homomeric channel (138). Similar experiments with the native CNG channel from rat OSNs
yielded slightly larger pore dimensions (6.5 Å ⫻ 6.5 Å)
(16).
In conclucion, selectivity for alkali ions is similar in
various isoforms of CNG channels and across species.
To permeate, Ca2⫹ must bind to a site inside the pore
and thereby block the current carried by monovalent
cations. The characteristic features of the blocking action
of extracellular Ca2⫹ are summarized in Figure 15. The
current recorded from an excised patch in the outside-out
configuration is progressively suppressed by increasing
concentrations of Ca2⫹ (Fig. 15A). The blockage is
strongly voltage dependent: inward currents are more
effectively suppressed than outward currents (Fig. 15A,
red trace). The voltage dependence of Ca2⫹ blockage is
characteristic of a positively charged and permeable
blocking molecule. This is illustrated by plotting the fraction of unblocked current against Vm (Fig. 15B). Starting
from ⫹80 mV, blocking efficacy increased continuously
when Vm is made less positive. At around ⫺40 mV, blockage is maximal, although not complete, and is relieved
again at more negative values of Vm. The relief of blockage at negative Vm has been interpreted as facilitation of
Ca2⫹ permeation (76). The voltage dependence of the
Ca2⫹ blockage has been analyzed (371) using the
Woodhull model that originally has been used to describe
the proton block of Na⫹ channels (423). One parameter in
the Woodhull model, the “effective valence” z represents
the number of blocking charges. In the three channel
types tested, z adopts values of ⬃2, supporting the notion
that a single Ca2⫹ blocks the ionic pathway. Another
parameter that characterizes the fraction of the membrane voltage acting at the blocking site, which is often
equalled to the position of the ion binding site, adopts
values of ⬃0.4, indicating that the site is located in the
membrane approximately one-third of its thickness (98).
The Ca2⫹ affinity of the intrapore binding site has
been determined by measuring the Ca2⫹ (and Mg2⫹) dependence of the blockage of monovalent currents (113,
371). The dose-response relation between normalized current and extracellular Ca2⫹ (Fig. 15C) over the entire
voltage range is well described by a simple binding isotherm, supporting the notion that CNG channels carry a
Physiol Rev • VOL
single binding site inside the pore and that the occupancy
by a single Ca2⫹ is sufficient to occlude the channel (371).
A glutamate (Glu) residue in the P loop of the CNG
channel A1 to A3 subunits has been identified as an
important structural element of Ca2⫹ binding. When this
Glu residue is replaced by a neutral amino acid, Ca2⫹
blockage is almost entirely abolished (98, 121, 317, 343)
(Fig. 15E). Because a Glu residue is provided by each A
subunit, in homotetrameric channels, a set of four glutamate residues is expected to form the high-affinity intrapore Ca2⫹-binding site. The net negative charge of the
Glu residues appears to be an important determinant of
the Ca2⫹ affinity. Experimental maneuvers that either
enhance or diminish the negative charge at this site also
decrease or increase, respectively, the K1/2 of blockage.
First, when the Glu residue is replaced by various neutral
residues, in each case, the high-affinity Ca2⫹-binding site
is abolished, whereas substitution of Glu with Asp, another negatively charged amino acid, enhances rather
than impairs the Ca2⫹-binding affinity (121, 317, 371).
Second, protonation of the Glu residues also weakens the
Ca2⫹ affinity. At low external pH, a higher extracellular
Ca2⫹ is required to produce the same fraction of blockage
than at high pH (338, 371) (Fig. 15F). The four Glu carboxylates form two identical noninteracting protonatable
sites (pKa of 7.6) in channels composed of the catfish A2
subunit (344). Protonation of these sites produces two
substates of smaller conductance. Third, reduction of the
negative charge in heteroligomeric channels consisting of
A and B subunits (with a Gly residue at the respective
position in the pore loop) also results in a lower Ca2⫹
affinity (214) (Fig. 15C). Thus experimental measures expected to either reduce or eliminate the negative charge at
the Ca2⫹-binding site by three independent mechanisms
also reduce or eliminate Ca2⫹ binding. These results underscore the importance of negative charges for binding
of Ca2⫹ inside the pore vestibule.
Various homomeric CNG channels formed by either
A1, A2, A3, or dmA distinctively differ in their sensitivity
to blockage by extracellular Ca2⫹ (113, 371), implying that
the sites inside the pore bind Ca2⫹ with different affinity
(Fig. 15D). However, the pore region itself, including the
key Glu residue, is highly conserved among A1-A3 subunits and dmA (Fig. 15G), suggesting that residues outside
the pore region either participate directly in the binding of
Ca2⫹ or modify the geometric arrangement of glutamates.
By an extensive mutagenesis study, Seifert et al. (371)
showed that all aspects of Ca2⫹ blockage are transferred
from one channel type to another when the S5-P-S6 region is
exchanged between different channels, whereas swapping
of domains outside the S5-P-S6 region has no or only small
effects. The S5-P-S6 region serves as a basic pore module
that governs both blockage and ion permeation in CNG
channels. These authors also showed that several amino
acid residues at the extracellular ends of the S5 and S6
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B. Blockage by Divalent Cations
805
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
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2⫹
FIG. 15. Ca
blockage of CNG channels. A: current-voltage relations of heterologously expressed homomeric A1
channels in the presence of various concentrations (see labels in micromolar) of extracellular Ca2⫹. Currents carried by
potassium are strongly blocked by Ca2⫹ in a dose-dependent fashion. [Data from Seifert et al. (371).] B: voltage
dependence of the fraction of unblocked currents (see text). [Data from Seifert et al. (371).] C: dose-response relation
of Ca2⫹ blockage at ⫺60 mV of the heterologously expressed A1 channel (black circle) compared with the coexpressed
A1B1 channel (blue circle). Coexpression of the A1 and B1 subunits leads to a reduction in the apparent affinity of the
intrapore binding site for Ca2⫹. [Data from Körschen et al. (214).] D: dose-response relation of Ca2⫹ blockage for
different heterologously expressed A subunits. The apparent affinity of the intrapore binding site differs among channels.
[Data from Baumann et al. (22) and Frings et al. (113).] E: dose-response relation of Ca2⫹ blockage of heterologously
expressed A1 channels (solid circles) and the E363Q mutant channel (open squares). The apparent affinity of the
intrapore binding site in the mutant is reduced by about three orders of magnitude. [Data from Eismann et al. (98).] F:
dose-response relation of Ca2⫹ blockage of heterologously expressed A channels at different pH values. The apparent
affinity of the intrapore binding site is reduced by protonation of pore glutamates. [Calculated from data in Seifert et al.
(371).] G: alignment of the pore region of A1, A2, A3, dmA, and B1.
segments located near the membrane water interface, as well
as the linkers that connect these segments with the pore loop,
determine how strongly Ca2⫹ interacts with the pore.
Physiol Rev • VOL
CNG channels not only differ in their interaction with
Ca2⫹, but also in their pH sensitivity. The reduction of
monovalent currents by external protons in the A1 and A2
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CYCLIC NUCLEOTIDE-GATED ION CHANNELS
channels seems to be controlled by acidic groups with a
pKa of ⬃6.5 and 7.6, respectively (344, 371). As transplantation of the S5-P-S6 region from one channel type to
another transfers both the extracellular pH sensitivity and
the sensitivity to Ca2⫹ blockage, it has been suggested
that different protonation patterns of the Glu residues
underlie the different Ca2⫹ sensitivities (371).
C. Ca2ⴙ Permeation
1. Relative permeabilities PCa / PM
All CNG channels are more permeable to Ca2⫹ than
to Na⫹ (Table 6). Relative ion permeabilities PCa/PNa are
6.5 and 10.3 in salamander rods and lizard parietal eye,
respectively, but 21.7 in cones of striped bass (105, 146,
322). The difference in PCa/PNa between CNG channels of
rods and cones is also observed in recombinant channels
formed from the respective A1 and A3 subunits alone
(113).
The interaction between photoreceptor CNG channels and divalent cations depends on the cGMP concentration. Torre and co-workers (64, 67) were the first to
notice that elevating the level of cGMP modifies the ionic
selectivity for divalent cations of the rod CNG channel.
For example, Mn2⫹, which under normal conditions does
not appreciably permeate the channel, becomes permeable at high cGMP concentrations. This effect has now
been examined in more detail by measuring the dependence of PCa/PNa on the cGMP concentration (146). In
TABLE
6. Relative ion permeabilities for divalent cations
Rod
Parietal eye
A1 homomer
Cone
A3 homomer
OSNs
A2 homomer
dmA1 homomer
PCa/PM*
PMg/PM
Reference
Nos.
6.5†
10.3
3.1†
21.7†
10.9
6.5
6.8†
34.9†
ND
ND
3.3†
ND
9.3†
ND
11.9†
11.2†
146, 322
105
113
146
113
225
113
22
* Some authors used the Fatt and Ginsborg (101) approximation to
calculate PCa/PM; others used the equation derived by Lewis (237)†.
Some authors used ion concentrations, instead of activities. To make
figures comparable all data have been calculated using the equation
given by Lewis and using ion activities.
Physiol Rev • VOL
rods, PCa/PNa changes 3.4-fold between its minimum and
maximum value (1.9 and 6.5) at low and high cGMP
concentrations, respectively. A similar effect is present in
CNG channels of cones: PCa/PNa increases from 13.9 to
21.2 in going from low to high concentrations of cGMP.
The modulation of ionic selectivity by cGMP is specific for
divalent cations and is not observed for alkali ions (112,
146, 271). A likely mechanism explaining this effect might
be that CNG channels exist in two or more conductance
states, each of different ion selectivity, and that the distribution of these states changes with the cGMP concentration. At low cGMP, the prevalent state is predicted to
display a lower PCa/PNa value than the state prevalent at
high cGMP concentrations. In fact, multiple conductances
in CNG channels have been recognized ever since the first
single-channel recordings (156, 170, 441). The quantitative
analysis of single-channel events, in particular, the incidence of subconductance levels, is seriously hampered in
native channels due to their rapid “flickery” gating. However, in homomeric A1 channels, gating is significantly
slower and allows quantification of single-channel events.
Ruiz and Karpen (348, 349) provide evidence that partially
liganded channels (A1 homomers) display subconductance states in addition to the fully open state and that the
probability to occupy the largest conductance state is
highest for the fully liganded channel, i.e., at high cGMP
concentration. Unfortunately, the change in ionic selectivity with cGMP levels is observed in native channels and
recombinant heteromeric channels (A1 and B1a) only but
is absent in homomeric channels built of A1 subunits
alone (146). Either subconductance levels in A1 channels
are rare events and, therefore, do not contribute to the
PCa/PNa value, or all subconductance states display the
same ionic selectivity.
2. Fractional Ca2⫹ currents
An important measure of Ca2⫹ permeability is the
fraction of the total current (It) that is carried by Ca2⫹
under physiological conditions. It is also referred to as
fractional Ca2⫹ permeability (Pf) (361). Pf adopts values
between 0 and 1. The fraction of the dark current carried
by Ca2⫹ in intact rods has originally been estimated using
an indirect method. This method relies on the measurement of the current generated by the electrogenic transport of cations by the Na⫹/Ca2⫹-K⫹ exchanger and comparison with the changing current through the lightdependent CNG channel. This method consistently
provided estimates that 10 –18% of the dark current is
carried by Ca2⫹ (141, 229, 297, 435). The electrogenic
Na⫹/Ca2⫹-K⫹ exchange current in cones is at least 10-fold
larger than in rods (163, 298, 318), and hence, separating
the electrogenic from the photocurrent is fraught with
error (J. Korenbrot, unpublished observations). Therefore, the analysis of the fractional Ca2⫹ current in cones is
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Ca2⫹ selectivity of CNG channels has been probed by
two independent experimental measures. First, the permeability for Ca2⫹ relative to that for monovalent cations,
PCa/PM, has been determined from Vrev measured under
various biionic conditions. Second, the fraction of the
total ionic current carried by Ca2⫹ was determined under
physiological conditions.
807
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
Physiol Rev • VOL
dependence of Pf. The difference has been characterized
by the constant KPf, i.e., the extracellular Ca2⫹ concentration at which one-half of the current is carried by Ca2⫹
(Pf ⫽ 0.5) (97). KPf has a characteristic value for each A
isoform. The rod A1 channels displays the lowest KPf (0.4
mM), and Pf is essentially unity when Ca2⫹ is above 1 mM,
whereas KPf ⫽ 4.2 mM for the dmA subunit and extracellular Ca2⫹ values ⬎10 mM are required to achieve a Pf
⫽ 1. The KPf value for the A2 and A3 channels are similar
to each other and intermediate between A1 and dmA.
Two striking differences exist between the native
CNG channels from rod and cones and the corresponding
A homomeric channels. First, the KPf of the recombinant
channels is lower in rods than in cones, whereas in the
native channels this is reversed. As a consequence, the Pf
value at 1 mM Ca2⫹ in cone channels is about twofold
higher than that in rod channels, whereas for recombinant
channels it is about three times lower. Second, the Ca2⫹
dependence of Pf is extremely steep in the recombinant
A1 channel but remarkably shallow in the native rod
channel; even when extracellular Ca2⫹ is 10 mM, Pf is still
only 0.5. The mechanisms responsible for these functional
differences are not known. One possibility is that the B
subunits, which carry a Gly instead of a Glu residue in the
pore loop, create pores that are distinctively different
from those in A homomeric channels. A second possibility
is that the arrangement and dielectric environment of the
Ca2⫹-coordinating residues are changed by Mg2⫹. The Pf
values in the native CNG channels were all measured in
the presence of external Mg2⫹, whereas the studies in A
homomeric channels were conducted without Mg2⫹. Because Mg2⫹ binds to (113) and permeates (433) CNG
channels, the presence of Mg2⫹ might interfere with the
interaction between Ca2⫹ and the pore.
D. Comparison of CNG Channels and
Voltage-Gated Ca2ⴙ Channels
Comparison of similarities and differences between
CNG channels and voltage-gated Ca2⫹ channels can help
to understand the permeation mechanism in CNG channels. In homomeric CNG channels and in voltage-gated
Ca2⫹ channels, a set of four glutamate residues forms the
cation-binding site in the pore (100, 428). Both channels
conduct monovalent cations in the absence of Ca2⫹,
showing values of single-channel conductance in the
range of 20 – 80 pS in Ca2⫹-free solution. Micromolar concentrations of extracellular Ca2⫹ block monovalent currents in both channel types, but voltage-gated Ca2⫹ channels show higher Ca2⫹ affinity (Ki ⬃ 1 ␮M) compared with
CNG channels (Ki between 3 and 500 ␮M). Finally, current recordings at high Ca2⫹ concentrations have revealed a similar conductance of Ca2⫹ relative to that of
Na⫹. The single-channel conductance of voltage-gated
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less reliable than that in rods. Notwithstanding these
experimental difficulties, Perry and McNaughton (318)
reported a roughly twofold larger Ca2⫹ current in
salamander cones than in rods (21 and 10%, respectively).
Neher and collaborators (361, 440) developed a
method to measure Pf values in glutamate and acetylcholine receptor channels. The method requires the simultaneous measurement of total membrane current It by
means of the patch-clamp technique and Ca2⫹ flux
through spectroscopic techniques using fluorescent Ca2⫹
indicators (reviewed in Ref. 302). Frings and Korenbrot
and their collaborators applied this method to study fractional Ca2⫹ currents in recombinant CNG channels (97,
113) and native CNG channels of intact rods and cones
(311). An important prerequisite of the accurate determination of Pf is the fast and synchronous activation of CNG
channels. This goal was achieved by using caged cyclic
nucleotides. These photolabile substances release the ligand upon irradiation with a flash of ultraviolet light,
thereby activating CNG channels within a few milliseconds (150, 185, 187).
With the use of this direct method, Pf values of 14 and
21% were determined for the percentage of current carried by Ca2⫹ in rods of catfish and salamander (311),
respectively, at 1 mM extracellular Ca2⫹. These results are
similar to those that are based on the analysis of the
Na⫹/Ca2⫹-K⫹ exchange current. In cones of striped bass
and catfish, the fractional Ca2⫹ currents are more than
twofold larger than in rods (33 and 34%, respectively)
(311). These results also demonstrate that the differences
in fractional Ca2⫹ current are maintained across several
species. The differences in Pf between CNG channels of
rods and cones in combination with the known difference
in the clearance rate of Ca2⫹ from the outer segment
suggests that changes in [Ca2⫹]i will be larger and faster
in cones than in rods. A difference in Ca2⫹ balance may
underlie the dissimilarity in the light sensitivity, the kinetics of the light response, and characteristics of the light
and dark adaptation of the two types of photoreceptors.
The small size of the chemosensitive cilia makes
measurements of Pf in intact OSNs inpracticable. A value
of Pf has been estimated though in HEK293 cells by
coexpression of the A2, A4, and B1b subunits that produce channels with properties that are largely identical to
those of native channels (40). At 2 mM external Ca2⫹, 41%
of the current are carried by Ca2⫹ (97). Given the high
[Ca2⫹] of 3–7 mM (83, 337) in the olfactory mucus that
covers the cilia, we expect that most, if not all, of the
current passing the olfactory CNG channel is carried by
Ca2⫹.
The relationship between Ca2⫹ concentration and Pf
has been systematically analyzed in recombinant channels formed from A subunits alone (97, 113). While all A
isoforms carry a pure Ca2⫹ current at [Ca2⫹] ⱖ10 mM (Pf
⬃ 1), they are set apart from each other by the Ca2⫹
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
IX. CHANNEL MODULATION
CNG channels, unlike other ligand-gated channels,
do not desensitize in the continued presence of the ligand.
However, the activity of CNG channels appears to be
modulated by Ca2⫹-binding proteins and phosphorylation/dephosphorylation. In this section we discuss the
underlying mechanisms and the physiological significance
of this modulation.
A. Phosphorylation/Dephosphorylation
Repeated measurement of the dose-response curve
of channel activation in excised patches of ROS membranes disclosed a slow increase in cGMP sensitivity over
time (128). The decrease in K1/2 was usually 2- to 3-fold
but could be as large as 10-fold. The enhancement of
ligand sensitivity was slowed down by both ATP and
inhibitors of Ser/Thr phosphatases and was accelerated
Physiol Rev • VOL
by purified type 1 phosphatase, suggesting that phosphorylation might control the conversion of channels between
states of high and low ligand sensitivity. When the membrane patch was excised into a Ca2⫹-free medium (containing 1–2 mM EGTA), the time-dependent decrease of
K1/2 was irreversibly abolished. This observation suggests
that an unknown factor, possibly a Ca2⫹-dependent phosphatase tightly adhering to the membrane patch, is permanently inactivated or lost in Ca2⫹-free medium.
The A1 subunit of the rod CNG channel, when heterologously expressed in Xenopus oocytes, displays a seemingly similar decrease of K1/2 after patch excision (287).
Unlike in the native rod CNG channel, inhibitors of Ser/
Thr phosphatases are without effect, whereas orthovanadate and pervanadate, inhibitors of phosphotyrosine
phosphatases (PTPs), slow down the progressive sensitivity enhancement (285, 287). The effect is reduced but
not entirely abolished by the mutation Y498F in the ␤1strand of the cNMP-binding site, consistent with the idea
that this residue in oocytes is phosphorylated by a protein
tyrosine kinase (PTK). In Ca2⫹-free medium, the timedependent effect of PTPs on A1 subunits in oocyte membranes persists, whereas in native ROS membranes no
change in ligand sensitivity occurs (128, 287). Genistein, a
PTK inhibitor, slows dramatically channel activation and
reduces maximal currents by twofold (284, 286). These
effects occur in the absence of ATP and were taken as
evidence that genistein, in addition to inhibiting tyrosine
phosphorylation, also promotes an allosteric inhibitory
interaction between the PTK and the channel that does
not involve phosphorylation (284, 286). The action of
genistein was also observed in native channels of rods,
cones, and OSNs, raising the possibility that CNG channels are part of a regulatory complex that contains PTKs
(286).
The K1/2 value of the A3 subunit of the cone CNG
channel in HEK293 cells shifts from 19 to 56 ␮M upon
treatment with phorbol esters (290). The change in ligand
sensitvity involves phosphorylation of two serine residues
(S577 and S579) in the cNMP-binding domain. The ␦-isoform
of PKC is specifically expressed in cone outer segments and
might mediate the phosphorylation. Phosphorylation and its
effect on ligand sensitivity have not been studied in the
native CNG channel of cone photoreceptors.
In the chick, the cGMP sensitivity of the cone CNG
channel is under the control of a circadian rhythm (201).
During the subjective night, the sensitivity is approximately twofold higher than during the subjective day.
Circadian modulation of ligand sensitivity is driven, at
least in part, by rhythms in the activities of two protein
kinases: the Erk form of mitogen-activated protein kinase
and the Ca2⫹/CaM-dependent protein kinase II (CaMKII).
Perturbation of these signaling pathways causes phasedependent changes in the cGMP sensitivity of the cone
CNG channel. The mechanism of the sensitivity regulation
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Ca2⫹ channels from ventricular heart cells is 85 pS with
150 mM Na⫹ and 9 pS with 110 mM Ca2⫹ (162). A relative
Ca2⫹/Na⫹ conductance of ⬃0.1 is also observed for native
olfactory CNG channels and homomeric dmA channels
(22, 97), although the absolute single-channel conductance for Ca2⫹ is likely to be at least 10-fold smaller in
CNG channels than in Ca2⫹ channels. While these similarities in pore structure and conducting properties point
to a common mechanism for ion permeation, some dissimilarities illustrate the different tasks the two channel
types fulfill under physiological conditions. The higher
Ca2⫹ affinity of voltage-gated Ca2⫹ channels causes a
virtually complete suppression of monovalent currents
and produces pure Ca2⫹ currents at 1–2 mM extracellular
Ca2⫹, whereas the known native CNG channels conduct
mixed cation currents at this extracellular Ca2⫹ concentration (297, 318). Despite their high Ca2⫹ affinity, voltagegated Ca2⫹ channels conduct substantial Ca2⫹ currents,
possibly sustained by electrostatic repulsion of two Ca2⫹
that can occupy the pore at the same time. Such a mechanism appears to be much less effective in CNG channels,
although double occcupancy may also play a role. Indeed,
the Ca2⫹ dependence of Pf for various A subunits is
significantly steeper than predicted from the GoldmanHodgkin-Katz equation (97). This result was interpreted to
indicate that more than one Ca2⫹ can occupy the CNG
channel pore. Nevertheless, high Ca2⫹ affinity in CNG
channels is clearly correlated with a low Ca2⫹ conductance, and the most substantial Ca2⫹ influx is mediated by
low-affinity channels. In conclusion, several structural
features common to both channel types give rise to pure
Ca2⫹ currents in voltage-gated Ca2⫹ channels and to
mixed cation currents with a high Ca2⫹ fraction in CNG
channels.
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
B. Ca2ⴙ-Binding Proteins
A variety of ion channels, notably Ca2⫹-permeable
channels, are modulated by Ca2⫹/CaM (236). Often Ca2⫹/
CaM downregulates channel activity and, thereby, provides a mechanism that interrupts Ca2⫹ entry and terminates the cellular response. CNG channels are no
exception to this rule. The ligand sensitivity of CNG channels is modulated by binding of CaM or by as yet unidentified Ca2⫹-binding proteins (for review, see Ref. 280).
CaM lowers the ligand sensitivity of CNG channels from
rod, cone, and OSN to different extents and by way of
different subunits.
1. Olfactory channel
Ca2⫹/CaM increases the K1/2 (i.e., lowers the ligand
sensitivity) of the native olfactory CNG channel for cAMP
up to 50-fold (17, 72). The change in K1/2 is thought to be
the main adaptive feedback mechanism in OSNs (224)
(see sect. IVB). CaM binds to a short segment in the
NH2-terminal region of the A2 subunit in a Ca2⫹-dependent fashion (248, 413). The K1/2 of CaM binding to the
target peptide (4 –12 nM; Refs. 248, 413) and the EC50 of
the Ca2⫹/CaM action on channel activity (21 nM; Ref. 72)
are of similar magnitude, suggesting that this target sequence is a major determinant of the CaM action. The
amino acid sequence of the target peptide belongs to a
common form of CaM-binding sites. These sites are characterized by aromatic residues at positions 1 and 14 of the
motif and additional hydrophobic residues at positions 5
and 8. Several positively charged residues are interspersed between the hydrophobic residues. In the ␣-helical conformation, basic and hydrophobic residues bePhysiol Rev • VOL
come located on opposite sides of an amphipathic helix
(82, 312).
2. Rod channel
The native CNG channel of rod photoreceptors is
exquisitely sensitive to regulation by Ca2⫹/CaM [K1/2
(CaM) of 1–2 nM; Refs. 20, 169, 413], yet the decrease of
cGMP sensitivity, by comparison with the olfactory channel, is modest (maximal 2-fold increase of K1/2 for cGMP
activation; Refs. 20, 130, 169, 295, 413). The Ca2⫹ dependence of the modulation of the native channel [K1/2(Ca2⫹)
⫽ 48 nM; Ref. 295] and the binding of CaM to the target
peptide [K1/2(Ca2⫹) ⫽ 117 nM; Ref. 413] are similar and
well within the range of Ca2⫹ concentrations during the
light response (141, 211, 229, 264). In contrast to the
olfactory A2 subunit, the A1 subunit of rod CNG channels
does not bind CaM, and homomeric channels composed
of A1 subunits are not modulated by CaM (70, 169, 214).
CaM sensitivity is conferred to heteromeric rod channels
by an unconventional CaM-binding site in the NH2-terminal region of the B1a subunit (143, 413).
Although CaM modulation of the rod CNG channel is
well established in vitro, its significance as an adaptive
mechanism has been questioned on various grounds (141,
215, 295, 352; for review, see Ref. 280). Two of the most
nagging problems have been that the less than twofold
decrease in cGMP sensitivity of the native rod channel by
CaM seems small compared with the large changes in the
cell’s sensitivity during light adaptation, and efforts to
demonstrate in vivo that a living rod is using that mechanism yielded mixed results (88, 142, 216, 280, 295, 352).
Even an unidentified endogenous factor has been implied
because exogenous CaM does not always fully recapitulate the change in the Ca2⫹ dependence of the cGMP
sensitivity that is observed after patch excision or after
truncation of the ROS (130, 352). It is unlikely that this
factor is another Ca2⫹-binding protein. Removal of endogenous CaM from the cytosol of ROS by immunoprecipitation also removes all Ca2⫹-dependent modulatory activity (R. S. Molday, personal communication).
3. Cone channel
Although cone CNG channels in excised patches
from some species are weakly modulated by CaM [heterologously expressed chicken A3 (41), native cone CNG
channels of striped bass (145)], cones from other species
were found to be insensitive to CaM [native catfish (157),
human and bovine A3 (41, 144, 436)]. These observations
would not support a role of CaM in sensitivity regulation
of the cone’s light response, but matters seem to be more
complex. Surprisingly, the A3 subunit of the cone CNG
channel, like the A2 subunit of OSNs, comprises in its
NH2-terminal region a fairly conserved CaM target motif
that, in various binding assays, shows CaM binding (41,
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has not been determined. It may involve phosphorylation
of A or B subunits by Erk or CaMKII or phosphorylation
of other signaling molecules that interact with the channels and lead to modulation of their sensitivity.
The cGMP sensitivity of the A2 subunit of the olfactory CNG channel is greatly enhanced (10-fold decrease of
K1/2) by PKC-mediated phosphorylation of a serine residue (S93) adjacent to a CaM-binding site. However, the
shift of K1/2 is not observed in native CNG channels from
phorbol ester-treated OSNs (289).
In conclusion, the physiological implications of phosphorylation of CNG channels, whether in rods, cones, or
OSNs or whether by PKC, PTK, Erk, or CaMKII, are not
completely understood and deserve further study in intact
cells. It seems necessary 1) to show that CNG channels in
fact become phosphorylated in situ, 2) to identify the
respective protein kinases and phosphatases along with
their regulatory mechanisms, 3) to identify the channel
subunits and amino acid residues involved, and 4) to
characterize the cellular function of this modulation.
CYCLIC NUCLEOTIDE-GATED ION CHANNELS
Physiol Rev • VOL
functionally less important or is nonexistent (see also Ref.
57 for discussion).
The dissociation of CaM from the native rod channel
is distinctively faster at high (large Po) than at low cGMP
concentration (low Po) (130). This observation suggests
that Ca2⫹/CaM binds more tightly to the closed than to the
open state of the channel, i.e., binding is state dependent.
For the rod channel, which spends most of the time in the
closed state (i.e., low Po) (430), such a mechanism is
deemed suggestive. Gordon (127) proposed that Ca2⫹/
CaM also preferentially binds to the closed state of the
olfactory CNG channel, which is closed at rest and opens
upon stimulation of OSNs with odorants. However, for the
negative-feedback modulation to produce rapid adaptation, Ca2⫹/CaM must bind effectively and rapidly to the
open state. In a series of elegant experiments, Bradley et
al. (45) addressed this issue. Using caged cyclic nucleotides, they measured the decline of the cAMP- or cGMPinduced currents due to the action of Ca2⫹/CaM. In native
channels from OSNs, the current decline was complete
within 0.5 s, whereas for heterologously expressed A2, the
decline proceeded ⬃100 times more slowly. Coexpression
of combinations of A2, A4, and B1b subunits demonstrated that only the A2A4B1b channel species matched
the rapid kinetics of the native channel, whereas A2A4
and A2B1b displayed distinctively slower current declines. The slower time course resulted from a 200-fold
smaller rate constant kon of Ca2⫹/CaM binding to the A2
channel compared with the A2A4B1b or native channel.
More importantly, the kon for A2 channels decreased by
10-fold going from high to low values of Po, suggesting
that binding of Ca2⫹/CaM in fact is state dependent. In
contrast, kon for the binding of Ca2⫹/CaM to the native or
A2A4B1b channel was independent of Po. The rate of
dissociation of Ca2⫹/CaM from the channel was too slow
to be measured and, therefore, koff could not be determined. The dissociation constant KD depends on the ratio
koff/kon of the respective rate constants. Thus these results
do not allow a conclusion as to which channel state, open
or closed, Ca2⫹/CaM preferentially binds to. In line with
these in vitro studies, CNGA4 null mice displayed a pronounced defect in odor adaptation (291). In a paired-pulse
paradigm, the amplitude of the electro-olfactogram
(EOG) response to the second pulse was about one-half
that of the first EOG response in wild-type mice exposed
to cineole, whereas in CNGA4 mice, the first and second
peak responses were nearly identical.
Many mechanisms of CNG channel function have
been studied in homomeric channels (A1 to A3). The new
works by Bradley and Munger and collaborators (45, 291)
and previous works by Bönigk, Dzeja, Körschen, Chen,
and collaborators (40, 71, 97, 214) give a reminder that all
aspects of channel activation, gating, ion selectivity, and
modulation are finely tuned by the subunit composition of
CNG channels. This work also cautions against rash gen-
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144, 290). These seemingly paradoxical findings might be
reconciled by the idea that the cGMP sensitivity of the
CNG channel of cones is controlled by another Ca2⫹binding protein and that CaM can act as a weak partial
agonist in some but not all species. Experimental support
for this hypothesis has been provided by Rebrik and
Korenbrot (333). In electropermeabilized cones, the K1/2
(cGMP) increased from 84.3 ␮M in the absence of Ca2⫹ to
335 ␮M in the presence of 20 ␮M Ca2⫹ (the range is
67–550 ␮M). The Ca2⫹-dependent modulation of K1/2 progressively and irreversibly vanished during recording of
ligand sensitivity in low Ca2⫹ medium. In membrane
patches detached from cone outer segments, the K1/2 is
reduced only ⬃1.5-fold in a solution free of Ca2⫹, and the
modulation is quickly lost after exposure of the patch to
the Ca2⫹-free solution (145). Finally, CaM does not mimick in detached membrane patches the effect of the diffusible factor. These results argue that a soluble factor,
which is lost during perfusion, reversibly interacts with
the CNG channel in a Ca2⫹-dependent manner. In electropermeabilized and truncated rods, the extent of Ca2⫹dependent modulation of K1/2 is significantly smaller than
in cones, and it is essentially recapitulated by CaM in
excised patches (295, 333, 352).
How does binding of CaM bring about a decrease in
apparent ligand affinity? The mechanism(s) has mostly
been studied using the olfactory A2 subunit. The NH2- and
COOH-terminal regions of A2 interact with each other
(403). The CaM target motif participates in this interdomain interaction; deletion of this segment disrupts the
CaM sensitivity of the channel (248) and the interdomain
binding (403) and increases the K1/2 value of channel
activation. The interaction between NH2 and COOH terminus leads to an energetically more favorable conformation stabilizing the open state of the channel. This interaction is antagonized by binding of CaM, which results in
an increase in K1/2 of channel activation. In this respect,
CaM affects gating rather than the strength of ligand
binding (248, 403).
How general is this domain interaction among CNG
channel subunits and does this mechanism also hold for
the native channel? Interaction between NH2- and COOHterminal regions of the A1 subunit of the rod channel has
also been proposed (132), although this subunit does not
carry a CaM-binding site. The evidence was mostly based
on overlay assays of Western blots. However, using a
different binding assay (pull-down of NH2- and COOHterminal fusion constructs), Varnum and Zagotta (403)
reported no interaction between NH2- and COOH-terminal
regions of A1. In fact, the NH2- and COOH-terminal constructs of A1 were used as negative controls to emphasize
the specificity of the interaction in A2, begging the question of how specific these assays are. We suggest that this
interaction in the rod CNG channel is either weak and
811
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U. BENJAMIN KAUPP AND REINHARD SEIFERT
eralizations; a mechanim established for homomeric A
subunits may be entirely different in another setting of A
and B subunits.
a Ki of 100 nM. The peptide is several orders of magnitude
less effective in blocking the heteromeric channels.
XI. CHANNELOPATHIES AND
KNOCK-OUT MODELS
C. Miscellaneous
X. PHARMACOLOGY
The pharmacology of CNG channels is poorly understood, partly for lack of suitable compounds with which
to probe CNG-mediated currents. A number of organic
compounds have been reported to block current through
CNG channels. The list of blockers includes L-cis-diltiazem (71, 106, 112, 122, 154, 203, 214, 266, 267, 331, 342,
379); pimozide (306); amiloride and its derivative (112,
307); tetracaine (109, 110, 362); polyamines (253, 257,
304); W-7, a calmodulin inhibitor (198, 444); H-8, a PKA/
PKG inhibitor (410); and ruthenium red and neomycin
(258). Except for pseudechetoxin, a peptide venom of the
Australian King Brown snake (56), most inhibitors block
the channel at micromolar concentrations.
L-Cis-diltiazem has been studied most extensively. It
blocks the CNG channels of rods and cones and OSNs in
a voltage-dependent fashion (154, 266, 331). The drug
exerts its effect from the cytoplasmic face of the channel;
extracellular application is much less effective (342, 379).
In the fish, L-cis-diltiazem blocks the rod CNG channel at
10-fold lower concentrations than the cone CNG channel.
The Michaelis constants of inhibition were 0.9 and 8.7 ␮M
at ⫹30 mV for rod and cone, respectively (154). For both
rod and cone CNG channels, the sensitivity to L-cis-diltiazem is conferred upon the heteromeric channels by the
B subunits (71, 122). Intriguingly, the L-cis-diltiazem sensitivity is lost upon solubilization and subsequent functional reconstitution of the rod CNG channel, although A
and B subunits are still present (79, 80).
The most potent blocking agent for CNG channels is
pseudechetoxin (56). It inhibits the homomeric A2 channel with a Ki of 5 nM and the homomeric A1 channel with
Physiol Rev • VOL
Mutations in the genes encoding the A1 subunit cause
a rare autosomal recessive form of retinitis pigmentosa
(RP) (96), a genetically heterogeneous group of diseases
that are characterized by a progressive degeneration of
the rod and cone photoreceptors ultimately leading to
blindness. Three of the five subunit alleles are null mutants because they would encode proteins that are lacking
in most or all of the functional domains (Fig. 16). The
other two alleles (S316F and R654 1bp deletion) encode
channels that, while functional, mostly fail to reach the
plasma membrane when heterologously expressed in
HEK293 cells, suggesting that photoreceptor degeneration is due to the paucity or entire lack of the rod CNG
channel. Another form of autosomal recessive RP is
caused by mutation of the CNGB1 gene encoding the B1
subunit of the rod CNG channel (18).
Mutations in either the CNGA3 or the CNGB3 genes,
encoding, respectively, the A3 and B3 subunits of the cone
photoreceptors, cause achromatopsia (or total color
blindness) (205, 206, 387), a rare autosomal recessive
disorder characterized by the total loss of color discrimination, by photophobia, nystagmus, and severely reduced
visual acuity (for review, see Refs. 373, 374). Kohl and
Wissinger and collaborators (206, 418) identified 46 mutations in the CNGA3 gene of families originating from
Germany, Norway, and the United States of America.
Most mutations (39 of 46) represent amino acid substitutions. Four mutations (R277C, R283W, R436W, and
F547L) account for almost 42% of all mutant CNGA3
alleles. The affected residues are located in the NH2terminal domain near S1, in the S4 motif, the linker region, and the cGMP-binding site (Fig. 16).
In the CNGB3 gene encoding the B3 subunit, three
missense mutations have been identified in the Pingelapese islanders of Micronesia (387). Six different mutations have been identified in caucasian families originating from Germany, Italy, and the United States (205),
including a missense mutation, two stop-codon mutations, a 1- and a 8-bp deletion, and a putative splice-site
mutation of intron 13 (Fig. 16). None of the mutations has
been functionally characterized; the disruption of cone
photoreceptor function in achromatopsia, however, suggests that these mutations cause a total or at least severe
impairment of CNG channel function.
The function of CNG channels has been also studied
by targeted disruption of genes encoding the A2, the A3,
and the A4 subunit (15, 33, 60, 291, 316). A2 ⫺/⫺ mice
lacking functional cAMP-sensitive olfactory channels ex-
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Short-chain analogs of diacylglycerol (DAG) reduced
the cGMP-induced current in native CNG channels from
rods (81, 129). Because full-length DAG has a stimulatory
rather than inhibitory effect (422), the functional implications are unclear.
The rod CNG channel is inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2) (422). The inhibitory action of PIP2 is strongest in heteromeric A1B1 channels,
less strong in A1 monomers, and intermediate in membrane patches from rod outer segments. The olfactory
CNG channel is not inhibited. The functional significance
of a regulatory role of PIP2 for CNG channel activity in
rod photoreceptors at present is not known.
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FIG. 16. Mutations in the human subunits
A1, A2, and B1a causing achromatopsia or retinal degeneration. X, stop codon mutation; fs,
frame-shift mutation.
814
U. BENJAMIN KAUPP AND REINHARD SEIFERT
XII. OUTLOOK
Ion channels that are directly gated by cyclic nucleotides play important roles in vision and olfaction. Although many important questions regarding CNG channel
function have been adequately answered, future studies
need to address the following issues.
1) The stoichiometry and arrangement of subunits in
the native channel complex are either not known or controversially discussed. Most of the elegant biophysical
studies on channel gating and modulation have been using homomeric A subunits. Whether the models and
Physiol Rev • VOL
mechanisms derived from these studies also hold for the
heteromeric channels deserves further work.
2) As with other ion channels, the structure at low
and high resolution is not known. Low-resolution images
could shed light on the quaternary structure and arrangement of subunits. A high-resolution structure could provide information on the molecular mechanism of ion
selectivity, permeation, channel gating, and ligand selectivity.
3) The physiological function of CNG channels in
cells other than sensory neurons is ill-defined. The available tools now allow us to address this issue more rigorously than has been possible in the past.
4) More than 40 mutations in CNG channel genes
have been identified that give rise to various forms of
achromatopsia. The mutant channels provide a rich
source for the study of the functional significance of
individual amino acid residues.
5) CNG channels carry up to three identified CaMbinding sites (e.g., the olfactory channel). The function of
each of these sites and their interaction is not completely
understood. Moreover, other as yet unidentified Ca2⫹binding sites or other factors are probably involved in the
regulation of CNG channels.
Address for reprint requests and other correspondence:
U. B. Kaupp, Institut für Biologische Informationsverarbeitung,
Forschungszentrum Jülich, 52425 Jülich, Germany (E-mail:
[email protected]).
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