684
Tutorials in Molecular and Cellular Biology
G Protein-Mediated Ion Channel Activation
Gerda E. Breitwieser
Guanine nucleotide binding proteins couple a wide variety of receptors to ion channels via both
"direct" or membrane-delimited and "indirect" second messenger-mediated pathways. This
tutorial summarizes current approaches to denning the mechanisms of guanine nucleotide
binding protein-mediated ion channel activation. Two well-characterized ion channels in the
heart, namely, the 0-adrenergic receptor-activated calcium channel and the muscarinic
receptor-activated potassium channel, are used to illustrate the criteria that can distinguish
between direct and indirect guanine nucleotide binding protein-transduced pathways. (Hypertension 1991;17:684-692)
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T
he initial characterization of ion channels as
voltage-gated pores within biological membranes has crumbled under the new technologies of patch clamp, reconstitution, and molecular
cloning. The vast majority of ion channels are now
known to be modulated by the intracellular and
extracellular environments. The diversity of ion channels contributing to cardiovascular function suggests
that a wide range of modulatory mechanisms will
prevail. Two broad classes of receptor-regulated ion
channels can be distinguished: 1) receptor-gated ion
channels that require receptor activation for channel
opening under physiological conditions, and 2) receptor-modulated ion channels in which a variety of
receptor-mediated pathways may modulate ion channel function but are not responsible for the primary
channel gating event.
I will restrict discussion in this tutorial to receptorgated ion channels since a basic discussion of the
factors regulating the activation of these channels is
also applicable to receptor-mediated secondary modulation of ion channel activity. Receptor-gated ion
channels may either be receptor channels, such as the
nicotinic acetylcholine, GABAA, or NMDA receptor
channels in which receptor and ion channel reside
within the same oligomeric protein complex, or receptor-coupled ion channels such as the muscarinic
acetylcholine receptor-activated K+ channel, the
/3-adrenergic receptor-activated L-type Ca2+ channel, or the GABAg channel, where receptor and ion
channel are distinct proteins, coupled by a variety of
From the Department of Physiology, Johns Hopkins University
School of Medicine, Baltimore, Md.
Supported by grant HL-41972 from the National Heart, Lung,
and Blood Institute. This work was done during the author's
tenure as an Established Investigator of the American Heart
Association.
Address for correspondence: Gerda E. Breitwieser, PhD, Johns
Hopkins University, School of Medicine, Department of Physiology, 725 N. Wolfe St., Baltimore, MD 21205.
possible transducing elements, including guanine
nucleotide binding proteins (G proteins) or cytoplasmic second messengers.
G Protein-Mediated Ion Channel Activation
The majority of receptor-gated ion channels require
coupling between the receptor and ion channel via a
transducing element, which usually involves G proteins
at some stage in the signal transduction cascade. G
proteins consist, in the inactive state, of three subunits:
a • GDP, p, and y. Interaction with receptor induces
release of GDP by the a subunit, binding of GTP, and
dissociation of the complex into an activated a • GTP
and the /3-y subunit complex. The a subunit has an
intrinsic GTPase activity, which hydrolyzes the bound
GTP to GDP. The cycle is completed by reassociation
of a • GDP with /3-y. A recent, comprehensive review by
Birnbaumer et al1 lists at least 80 receptor types (or
subtypes) that couple either directly, via G proteins, or
indirectly, via activation of a number of enzymes that
produce second messengers, to ion channels. Circumstantial evidence for the involvement of G proteins in
the activation of a particular ion channel comes from
the nature of the activating receptor, which may be
known to couple to other effectors via G proteins and
thus may be suspected of coupling to the ion channel
via a G protein-transduced pathway. Unequivocal determination of the nature of the G protein involvement
requires a variety of experimental approaches. The
most commonly used in electrophysiological experiments are 1) modification of the G protein by pertussis
or cholera toxin; 2) direct, receptor-independent activation of the G protein by hydrolysis-resistant GTP
analogues such as GTP-yS [guanosine-5'-O-(3-thiotriphosphate)] or GppNHp (guanylyl-imidodiphosphate); 3) block of the receptor-mediated effects by
GDP£S [guanosine-5'-O-(2-thiodiphosphate)]; and
when available, 4) use of G protein subtype-specific
antibodies to interfere with the signal transduction
Breitwieser
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process; and 5) activation of the ion channel of interest
by application of purified G protein subunits to the
cytoplasmic aspect of the patch.
The use of pertussis or cholera toxin to ADPribosylate G, and Go or Gs a subunits, respectively,
has allowed both demonstration of a functional alteration in the signal transduction pathway via physiological experiments and identification, in some
cases, of the particular G protein involved in the
signal transduction pathway via biochemical approaches. A lack of toxin effect on receptor-ion
channel coupling does not necessarily rule out a G
protein-mediated step in the signal transduction
pathway. Some G proteins are not sensitive to toxin
modification2-3 or are not modified in every cell type
in which the pathway is functional.4-5 Biochemical
studies of toxin modification reactions suggest that
the degree of G protein precoupling to unoccupied
receptors may influence the ability of toxin to act.6 In
addition, GTP-dependent protein cofactors are required for cholera toxin-mediated ADP-ribosylation
of G,.7-8 Although the requirement for protein cofactors has not been demonstrated for pertussis toxinmediated labeling of G; or Go, the extent of labeling
can be affected by the presence of /3y subunits.9
Thus, if cholera or pertussis toxin cannot be shown to
alter channel activation, other approaches may be
needed to demonstrate G protein involvement.
Hydrolysis-resistant guanine nucleotide analogues (GXP), such as GTPyS and GppNHp, are
able to activate G proteins in the absence of
receptor stimulation,10-15 since G proteins release
GDP at a finite rate, potentially catalyzed by G
protein interaction with unoccupied receptors.116"18 GXP competes with endogenous cellular
GTP for the unoccupied nucleotide binding site,
resulting in the slow accumulation of activated,
GXP-bound G protein. The buildup of activated G
protein is reflected in the activation of effectors in
the cell, including ion channels. It should be noted
that all of the G proteins in the cell will be activated
under these conditions and that care must be
exerted to isolate the particular pathway of interest.
For example, activation of the cardiac muscarinic
acetylcholine receptor K+ channel I^AQ] (acetylcholine) by Gk (=G,3) in the presence of high
concentrations of GTP-yS is accompanied by activation of all of the other G proteins present in the
cell, including G5, Go, Gp, and the other subtypes of
Q. This may result in not only activation of IjqACh]
via the primary signal transduction pathway but also
production of a variety of secondary metabolites. In
addition, a large pool of fiy subunits will be generated (contributed by all of the activated G proteins
in the cell). If, however, low concentrations of
hydrolysis-resistant GTP analogues are included in
the pipette solution, it is possible to irreversibly
activate I^ACH) only after addition of ACh, since the
competition between cellular GTP and hydrolysisresistant analogues precludes significant activation
of the G protein in the absence of receptor-en-
G Protein-Mediated Ion Channel Activation
685
hanced GDP release.19 Similar approaches can be
used to selectively activate other G protein-transduced pathways culminating in ion channel activation. A final caveat to the use of hydrolysis-resistant
GTP analogues is that these compounds are not
enzymatically inert but are capable of being converted via transphosphorylation into a variety of
other compounds with distinct biological effects,
either in their own right or as competitors of either
GTP or GXP.20-22
Activation of ion channels in the presence of high
concentrations of GXP confirms that a G protein
resides in the signal transduction pathway. The "uncoupling" of the physiologically relevant receptor,
however, presents difficulties in the interpretation of
the mechanism of activation, if the primary signal
transduction pathway is also modulated by additional
receptor-G protein cascades, since these pathways
will also be activated by high concentrations of GXP.
Several approaches can be taken to sort out the
confusion. Although activation of the secondary signal transduction pathways cannot be prevented in the
presence of GTPyS, potential second messengers can
be eliminated by including in the pipette or bath
solutions specific blockers such as protein kinase
inhibitors (e.g., Walsh inhibitor,23 protein kinase C
inhibitor peptide,24 calmodulin binding domain25),
Ca2+ chelators (BAPTA or EGTA), phospholipase
A2 (PLA2) inhibitors (bromophenacylbromide, mepacrine)26 or inhibitors of arachidonic acid metabolism
(ETYA [eicosatetraynoic acid], NDGA [nordihydroguaiaretic acid], indomethacin, salicylic acid).27 It
should be noted that most of the blockers mentioned
may have additional specific and nonspecific effects
on other cell enzymes, and thus a variety of blockers
must be tested before conclusions can be drawn.
An alternate approach to the question of whether
secondary G protein-mediated pathways modulate
the primary receptor-G protein-ion channel pathway
is to test the effect of various second messengers
under conditions in which only the primary pathway
has been activated (e.g., by activation with agonist in
the presence of "physiological" pipette solutions).
Specific second messengers, such as Ca2+, cyclic AMP
(cAMP), inositol triphosphate, diacyl glycerol, and
arachidonic acid can be added to either bath or
pipette solution. The ability to modify the intracellular milieu of cells with second messengers or blockers
will vary greatly depending on the cell type and the
agents being introduced.28-30 A variety of approaches
providing independent confirmation of a suspected
second messenger "modulatory" pathway are usually
required.
A third criterion for G protein involvement in the
signal transduction pathways resulting in ion channel
activation is the ability of GDP/3S to block receptormediated activation. Although GDP)3S competes
with endogenous GTP for the nucleotide binding site
on the relevant G protein,31-33 GDP/3S may also
retard production of GTP via a number of GTP
synthetic pathways. In addition, as with the use of
hydrolysis-resistant GTP analogues, the effects of
GDP0S will be mediated via both the primary signal
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transduction pathway as well as any secondary
modulatory pathways (as a result of GDP/3S-mediated block of all of the G proteins in the cell).
GDP0S conversion to an active triphosphate form,
presumably by transphosphorylation, has also been
observed.34-35
The last two criteria for establishing that G proteins are intimately involved in the ion channel
activation process have proven most powerful but will
be considered here only briefly since they will be
discussed in greater detail in the examples below.
Both the use of G protein subtype-specific antibodies
and the patch "reconstitution" of ion channel activation with isolated G protein subunits have been
instrumental in denning a "direct" or membranedelimited1 role for G proteins in ion channel activation. The only drawback to such an approach is the
obvious requirement for a ready source of either the
antibodies or purified subunits. One can only hope
that the expansion of research using these approaches will entice commercial suppliers.
Direct Versus Indirect G Protein-Mediated Ion
Channel Activation
The distinction between direct and indirect activation of ion channels by G proteins has evolved as a
central issue in the study of G protein-mediated
signal transduction.36-37 Definitive proof of direct G
protein-ion channel interaction will require co-reconstitution of both cloned G protein subunits and
cloned ion channels in a defined membrane. Because
this has not yet been demonstrated for any ion
channel, certain functional criteria have been used in
vivo to distinguish between direct and indirect G
protein-mediated activation of ion channels.
The operational criteria that have been used to
define a direct interaction of activated G protein
subunits with ion channels include 1) the speed of
current activation on agonist binding to receptor
(although this can vary greatly as a function of
receptor, G protein, and effector densities, for example, IK|AOI] activation T=650 msec, while 1^ activation
38
T = 1 5 0 msec ); 2) the absence of identifiable second
messengers (although this does not rule out second
messengers, either soluble or membrane-delimited,
which have not yet been characterized); 3) the inability of bath-applied agonist to activate single channels
within an on-cell patch (although the production of
membrane-delimited, localized second messengers is
not ruled out); and 4) most importantly, the ability to
reconstitute channel activity by addition of isolated G
protein subunits to excised membrane patches
(which may also be plagued by the localized, G
protein-induced production of membrane-delimited
second messengers). Clearly, the assignment of a
direct G protein-ion channel interaction must make
use of both the above criteria as well as the ultimate
purification and reconstitution of the system. Direct
interaction of G protein with ion channel would
suggest that the G protein interaction sites of the ion
channel protein may have structural homology with
analogous sites on other G protein effectors, such as
adenylyl cyclase and cyclic GMP (cGMP) phosphodiesterase. The cloning of a number of effector molecule types, including ion channels, may result in the
localization of a consensus sequence for G protein
interaction.
Activation of ion channels by indirect G proteinmediated pathways with known second messengers
are most easily tested. The ability to activate the ion
channel by direct addition of all putative second
messengers (under conditions in which activation of
the G protein is prevented) presents good evidence
that the G protein mediates production of the second
messenger. Care must be taken, however, to determine whether all of the properties of the channel
activated directly by the second messenger resemble
those observed on activation of the channel via
agonist binding to the receptor.
Our evolving understanding of the roles for G
proteins in the activation of two well-characterized
ion channels of the cardiovascular system illustrates
the complexities of G protein-transduced systems.
The £-adrenergic receptor-activated Ca2+ channel in
heart is a prototypical example of a mechanism that
ascribes an indirect role to G proteins, whereas the
muscarinic receptor-activated K+ channel exemplifies a putative direct role for G proteins in channel
activation. Recent experiments have suggested that
neither classification is exclusive and that both ion
channels are activated or modulated by direct and
indirect G protein-mediated mechanisms.
P-Adrenergic Receptor Activation of Ca2+ Channels
in Heart
The elucidation of the mechanism of /3-adrenergic
receptor-mediated activation of 1^ has evolved from
a series of elegant experiments in a number of
laboratories. The links between /J-adrenergic receptor activation, increases in cellular cAMP, and alterations in the properties of the cardiac action potential were made before the discovery of the
importance of G proteins in the signal transduction
process.3940 Developments in the areas of biochemistry, molecular biology, and electrophysiology have
combined to provide a detailed understanding of the
linkage between the £-adrenergic receptor and the
slow inward Ca2+ current 1^.
Several features of the 0-adrenergic receptormediated increase in 1^ suggested that the primary
response might be mediated by cytoplasmic second
messengers: there is a considerable latency (1-3
seconds) between agonist binding to the 0-adrenergic
receptor and activation of 1^; direct, receptor-independent perturbations of the cAMP pool result in an
increase in 1^, hinting at a role for cAMP-dependent
phosphorylation (for review, see Reference 41); and
fluoride ions have a positive inotropic effect,42 presaging the involvement of G proteins.
Breitwieser G Protein-Mediated Ion Channel Activation
687
AA
I
PKA
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FIGURE 1. Schematic drawing of major G protein-mediated
pathways for IA activation and inhibition. Stimulation of I^
results from both Grmediated activation of adenyfyl cyclase
and by direct interaction of a, with the Ca2+ channel protein.
Inhibition is through inhibitory receptor-mediated (e.g.,
mAChR or adenosine) activation of G,, and inhibition of
adenytyl cyclase. It is not yet clear whether aj directly inhibits
adenyfyl cyclase, or whether /3y subunits shift the equilibrium
of G, activation. Further details are presented in the text. fiR,
^-adrenergic receptor; PKA, cyclic AMP-dependent protein
kinase; R, regulatory subunit of PKA; PKA^, catalytic subunit
of PKA; mAChR, muscarinic acetylcholine receptor, Iti, calcium current; a, and as, a subunits of stimulatory and
inhibitory GTP binding proteins, respectivefy; fiy, complex of ft
and y subunits that are interchangeable among a subunit types
and combine with the a subunits to form the holo-GTP
binding protein; AC, adenyfyl cyclase.
The advent of the patch clamp technique as applied to isolated cardiac myocytes allowed a rapid
dissection of the steps in the pathway from /J-adrenergic receptor to the calcium channel. Figure 1
illustrates the various receptor-gated pathways for 1^
activation/modulation and the involvement of G proteins in the process.
The major pathway for In activation (defined as the
pathway producing the largest increase in Iri) is via
phosphorylation by cAMP-dependent protein kinase.
Iu can be activated under whole cell recording conditions by direct application of the catalytic subunit
of cAMP-dependent protein kinase (PKA),43-45 by
intracellular application of cAMP or extracellular
application of membrane-permeable forms of cAMP
(which induce dissociation of PKA into regulatory
and catalytic subunits),394446 by phosphodiesterase
inhibitors such as isobutylmethylxanthine (which increase cell cAMP),3940'47-48 by forskolin (which directly activates adenylyl cyclase),49-si and by intracellular application of hydrolysis-resistant guanine
nucleotide analogues or A1F" (which activate G,),19-53
and by extracellular application of isoproterenol or
epinephrine (which activate the entire signal transduction pathway). Cholera toxin activates the entire
pathway in an irreversible manner in the absence of
/3-adrenergic receptor stimulation by causing irreversible dissociation and activation of G,.54 ATP-yS
can irreversibly phosphorylate and hence activate !„
AA
metabolites
FIGURE 2. Schematic drawing of major G protein-mediated
pathways for IKIAOI activation and inhibition. Muscarinic (or
adenosine) receptor stimulation produces activation of several
G protein types, including Gk (defined as the G protein that
activates the K* channel) and Gp (which activates phospholipase C), to produce active OQ, subunits, a common pool of fiy
subunits, and active a,,. Further details are presented in the
text. mAChR, muscarinic acetylcholine receptor; PLA2, phospholipase A2; AA, arachidonic acid; Gk, pertussis toxinsensitive G protein responsible for IK/ACH/ activation; Gp, G
protein responsible for phospholipase C activation; PLC,
phospholipase C; DG lipase, diacylgfycerol lipase; Ix/AChf,
muscarinic receptor-activated K+ current; OQ,, a subunit ofGk;
fiy, complex of fi and y subunits that are interchangeable
among a subunit types and combine with the a subunits to
form the holo-GTP binding protein; a^ a subunit of Gp.
because the resulting thiophosphorylated channel is
not susceptible to phosphatase-mediated inactivation.55 Other receptors (e.g., glucagon and histamine), which activate G, and increase cellular cAMP,
also activate I^.41 Thus, a great deal of evidence
supports the activation of 1^ via G,-mediated increases in cellular cAMP and the ultimate phosphorylation of Ij,.
Several results suggest that the story is not, however, complete. First and foremost is the inability to
maintain 1^ in excised patches despite inclusion in the
bath solution of the activated catalytic subunit of
PKA plus ATP.56 In addition, rundown of 1^ under
whole cell recording conditions occurs in the presence of both PKA and ATP in the pipette solution.57-58 Other processes are clearly acting to limit I,,
activation.
Attempts to stabilize 1^ in excised patches led
Yatani and coworkers38'56 to suggest a second regulatory role of G proteins in the activation of 1^.
GTP-yS, purified G, plus GTP or a* • GTP-yS caused a
slowing of the rundown of 1^ above that achieved with
PKA catalytic subunit plus ATP, in both excised
membrane patches from guinea pig atrial cells and in
bovine sarcolemmal vesicles incorporated into phospholipid bilayers. This suggests either that
a, • GTP-yS interacts directly with the channel or that
it can block a membrane-delimited pathway responsible for la rundown. In support of a role for GTP-yS
in directly activating 1^ are experiments performed
under whole cell recording conditions in which it was
possible to "jump" isoproterenol concentrations rap-
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idly (response time less than 10 msec). Experimental
conditions that promoted G, activation resulted in a
rapid (r=150 msec) activation of 1^ followed by a
slower phase ( T = 3 6 seconds), which corresponded to
the cAMP-dependent phosphorylation pathway.
Conditions that bypassed G, (such as forskolin or
dibutyryl-cAMP) induced only the slow phase of
activation. Shuba et al59 have demonstrated whole
cell Iti activation by hydrolysis-resistant GTP analogues in the presence of phosphorylation inhibitors,
also suggesting that direct activation of Ifi by
a, • GTP-yS is likely. Further experiments are required to distinguish the relative importance of the
membrane-delimited and cytoplasmic pathways for Iri
activation, as well as the effects of the two pathways
on the single channel properties of Lj.
Isoproterenol-induced increases in I5i can be
blocked by41: intracellular application of GDP/JS
(which blocks G, activation60), RpCAMPS (Rp-adenosine 3',5'-monothionophosphate, which inhibits
PKA activation6061), the Walsh inhibitor (which
blocks the PKA catalytic subunit23), and by AppNHp
(adenylylimidodiphosphate, which competes with
ATP to inhibit phosphorylation44). Inhibition of Lj is
also mediated by G proteins. A variety of receptors,
such as muscarinic and adenosine, inhibit 1^ by
interaction with two systems that decrease cellular
cAMP: direct Gj-mediated inhibition of adenylyl
cyclase62 and activation of a cGMP-stimulated cyclic
nucleotide phosphodiesterase.48-63"65 The G,-mediated pathways predominate under conditions in
which all of the G proteins in the cell are activated by
hydrolysis-resistant GTP analogues, although under
some experimental conditions, GTPyS-dependent
activation of Lj can be observed.19-41
A full synthesis of the biochemical modulation of In
activation in cardiac cells has yet to be achieved. Roles
for both membrane-delimited direct and indirect second messenger-mediated G protein pathways have
been demonstrated, although stable recording of Lj in
excised membrane patches from cardiac sarcolemma
have not yet been reported. Since biochemically purified L-type Ca2+ channels produce stable currents in
bilayers,66-67 several additional regulatory pathways that
do not use G proteins may also be operating. It has
been demonstrated that a Ca2+-activated protease,
calpain, promotes Ca2+ channel inactivation in vivo,
suggesting that conditions which promote Lj activation
(and thus increase intracellular Ca2+) are also those
which promote Lj rundown.41 •57-58 Application of cytosol to excised patches or addition of calpstatin inhibits
the action of calpain and prolongs Ca2+ channel activity.57 Thus, activation of G,, phosphorylation of the
Ca2+ channel, and inhibition of calpain, may all be
required before stable L-type Ca2+ channel activity may
be achieved in excised patches. The amino acid sequence of the cloned dihydropyridine receptor68-69 has
consensus sequences for phosphorylation sites for protein kinase C, cGMP-dependent protein kinase, and
the Ca2+ calmodulin-dependent protein kinase, although functional modulation of 1^ by some of these
kinases has yet to be demonstrated. Protein kinase C
activation by phorbol esters has been shown to have a
transient, voltage-dependent stimulatory effect on Lj.70
The physiological role of this mechanism is unclear
since protein kinase C activation can be one of the
results of muscarinic receptor stimulation,71 which also
has an inhibitory effect on Lj through adenylyl cyclase
inhibition (as discussed above). Whatever their specific
role in modulating Lj activity, the activation of these
kinases is probably also triggered by G protein-transduced receptor pathways.
Muscarinic Receptor-Mediated Activation of K+
Channels in Heart
The muscarinic receptor-activated K+ channel was
initially assumed to be a receptor-gated channel, in
direct analogy with the nicotinic acetylcholine receptor channel.72 A significant difference, however, was
the slow time course of IKJAOI] activation, suggesting a
rate-limiting step beyond acetylcholine binding, compatible with activation by a soluble second messenger.73-75 Various attempts to demonstrate modulation or receptor-independent activation of IKJAOII by a
host of potential second messengers were, however,
consistently unsuccessful. The involvement of pertussis toxin-sensitive G proteins in the coupling of
muscarinic receptors to adenylyl cyclase inhibition
prompted experiments designed to test whether G
proteins were also involved in the coupling of muscarinic receptors to K+ channels. Reductions in cellular GTP levels76 or incubation with pertussis toxin76"79 blocked muscarinic receptor-mediated I(qACh]
activation, while hydrolysis-resistant GTP analogues
produced a persistently activated I^ACS] that was
uncoupled from further interventions at the muscarinic receptor.19 These results strongly implicated a
pertussis toxin-sensitive G protein in the coupling
between muscarinic receptor and IKJAQIThe notion of a direct interaction of activated G
protein with the I^AQ] channel protein developed
from two key observations. First, single channel
IiqACb] could only be elicited in on-cell patches by
acetylcholine in the patch pipette solution, and not
by acetylcholine applied to the cell via the bath.80
This argued against production of a cytoplasmic
second messenger that would activate all of the
channels in the cell. This is in contrast to the
activation of Lj, which does occur in the on-cell patch
configuration when isoproterenol is added to the
bath.81 Second, also in contrast to IB (which runs
down rapidly in excised patches55), I^ACD] can be
stably activated in inside-out excised patches by a
number of conditions, including agonist (pipette)
plus GTP (bath), bath-applied GTP-yS, or activated
subunits of Gi.82-84
A significant controversy developed from the excised patch reconstitution experiments with purified
G, subunits: both o^ • GTPyS85-87 and /3-y subunits83
were found to activate single channel IKJAOI]- Although the controversy has yet to be completely
resolved, it is clear that activation of the a subunit is
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a prerequisite for IKJAQ] activation. IKJAQ] can be
activated in excised patches by carbachol plus GTP
or by GTP-yS, which liberates equal amounts of a and
/3-y subunits.1-83-84 Exogenously applied holo G protein (afiy • GDP) 84 or biochemically purified
Oj • GTPyS,85-87 or bacterially cloned a, subunits1-88-89
are all capable of activating I^ACH] when applied to
excised patches. Activation of IKJACU] can be inhibited
by a-subunit-specific antibodies.90 Although there
are three distinct oj genes, producing o,i, a^, and a^,
neither the biochemically purified subtypes nor their
bacterially cloned counterparts show any selectivity
in activating IiqAch] (although the bacterially cloned
versions are 30-50-fold less potent than the biochemically purified subtypes, presumably because of
missing posttranslational modifications).188-89 Birnbaumer et al1 have thus suggested that all of the
subtypes of a, can be considered isoforms with respect to IKJAOI] activation (although it is still possible
that there is differential activation by muscarinic
acetylcholine receptor [mAChR] in vivo). Thus, the
initial assumption that the number of distinct a,
subtypes formed the basis for signal transduction
specificity has not held up on closer examination. The
cellular mechanisms controlling G protein-mediated
signal transduction specificity remain one of the
major unanswered questions in this intensely studied
area.
The physiological role, if any, for fiy subunits has
been a matter of some dispute. Four fi and two y
subunits have been cloned, and there is some evidence for tissue-specific differences in their levels of
expression.1-89 Since fiy subunit complexes cannot be
dissociated under physiological conditions, at least
six distinct complexes are possible. There have, however, been no reports of functional differences among
the various complexes, nor is there evidence of
preferential association of one type of /3y complex
with a particular a type. All /3y complexes (except
transducin fiy) are hydrophobic, and thus detergents
are included in solutions of purified /3-y subunits. In
addition, /3y subunits have no intrinsic activity (such
as binding GTPyS), and it is thus difficult to assess
the viability of the /3-y preparation. Reconstitution
experiments using /3-y subunits must therefore contend with both uncertainty about the viability of the
preparation and possible interference by the solubilizing detergent. Opponents of the notion of /3ymediated I^AQ] activation have suggested that all of
the observed effects are likely to be due to either
contamination of the fiy subunit preparation by <%, or
to the detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate) used to solubilize the /3y subunits.18991 CHAPS has been shown
to activate I^ACHI under some conditions87-92-93 and
not in others.83-94'95 /3-y Subunits have been shown to
inhibit GTP-dependent I^AQ] activation when applied to excised patches but to have no effect on
GTP-yS or a, • GTPyS-activated I^ACh].89-93 although
the physiological significance of this effect is uncertain since in vivo Oj • GTP and /3-y subunits are
689
released simultaneously in equimolar amounts, and
the net effect under these conditions is stimulation of
Proponents of a role for fiy subunits in the activation of IKJAOI] have suggested that PLA2 may be the
effector for fiy.96-98 /Jy-Mediated activation of
PLA2""101 results in the localized release of arachidonic acid and production of a host of lipoxygenase
and cyclooxygenase products that can have profound
physiological effects. The activation of I^ACH] by exogenously applied fiy subunits was blocked by antibodies to PLA297 and by blockers of arachidonic acid
metabolism, suggesting that /3-y subunits are able to
activate/modulate IKJAOI] through PLA2- Although the
effects of exogenously applied /3-y subunits do not
require GTP, the effects of a variety of exogenously
applied arachidonic acid metabolites do,96 again suggesting that fiy subunits may not be working solely
through activation of PLA2.
Whether fiy subunits are the physiological activators of PLA2> it is clear that arachidonic acid metabolites can affect activation of I^ACh]- Indeed, CHAPS
may be mediating some of its activating effects on
93
IKIAQ] through activation of PLA2.
In addition,
because GTP is required, but pertussis toxin does not
block the effects of arachidonic acid metabolites,
Kurachi et al96 have suggested that arachidonic acid
metabolites may induce IiqAct] activation by enhancing the GTP-GDP exchange activity of the a subunit.
The effects of arachidonic acid and metabolites on
GTP-GDP exchange can be tested explicitly by examining the rate of whole cell I^ACI] activation in the
presence of saturating ratios of GXP/GTP.102 Under
these specific conditions, the rate of appearance of
I^ACh] is thought to reflect the rate of GDP release
from the inactive, GDP-bound G protein. Arachidonic acid, its metabolites, and blockers of arachidonic acid metabolism all modulate the rate of IKJAQ]
activation103: the IKJAQ] activation rate is enhanced by
lipoxygenase products such as LTC4, whereas cyclooxygenase products slow the IKJACS] activation rate.
The interaction of a, • GTP-yS with the channel also
appears to be affected by arachidonic acid metabolites since LTC4 can increase the magnitude of
steady-state GTPyS-activated I^ACh]- Th e ability of
arachidonic acid metabolites to modulate IiqACh) after
activation with GTP-yS suggests that the metabolites
are not acting through a classic receptor-G protein
pathway since ctj • GTP-yS should be uncoupled from
all receptors under these conditions. These results
strongly suggest that arachidonic acid metabolites
can play a dynamic role in modulating the Oj-channel
interaction. If, as has been suggested, activation of
PLA2 is the result of interaction with fiy, arachidonic
acid and its metabolites may be produced concomitant with mAChR activation. Further work is required to define the roles for /3-y and arachidonic acid
metabolites in the activation of I^AO.] under physiological conditions.
The discovery of a membrane-delimited second messenger pathway in excised patches sends a strong
690
Hypertension
Vol 17, No 5 May 1991
cautionary signal against an overzealous interpretation
of patch reconstitution experiments. Final verification
of direct G protein-ion channel interactions must come
from true reconstitution experiments, in which the G
protein subunits and the ion channel are the only
proteins present, in a defined lipid environment.
In summary, this is a truly exciting time in the study
of the mechanisms of ion channel activation. The
convergence of patch clamp techniques with the
sophisticated tools of biochemistry and molecular
biology allow the probing of major and minor pathways of ion channel regulation with a detail hitherto
not possible. The complexities and interdependence
of the emerging pathways suggest that the study of
ion channel activation or modulation by G proteinmediated pathways may provide a prototype for the
study of the regulation of a variety of receptoractivated, membrane-associated enzymes.
Acknowledgments
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Thanks to R.W. Scherer for helpful discussions
and L. Hamosh for technical support.
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KEY WORDS • G proteins • ion channels • heart •
channels • K+ channels
Ca2+
G protein-mediated ion channel activation.
G E Breitwieser
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Hypertension. 1991;17:684-692
doi: 10.1161/01.HYP.17.5.684
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