J Neurophysiol 105: 321–335, 2011.
First published November 17, 2010; doi:10.1152/jn.00332.2010.
Location of Release Sites and Calcium-Activated Chloride Channels Relative
to Calcium Channels at the Photoreceptor Ribbon Synapse
A. J. Mercer,1,2,* K. Rabl,1,3,* G. E. Riccardi,5 N. C. Brecha,4,5,6,7,8 S. L. Stella Jr,5 and W. B. Thoreson1,2
1
Department of Ophthalmology and Visual Sciences and 2Department of Pharmacology and Experimental Neuroscience, University of
Nebraska Medical Center, Omaha, Nebraska; 3Department of Ophthalmology, University of California San Francisco, San Francisco;
4
Department of Medicine and 5Department of Neurobiology, David Geffen School of Medicine and 6Center for Ulcer Research and
Education/Digestive Diseases Research Center, University of California, Los Angeles; 7Jules Stein Eye Institute; and 8Veterans
Administration Greater Los Angeles Healthcare System, Los Angeles, California
Submitted 9 April 2010; accepted in final form 10 November 2010
INTRODUCTION
Visual responses originating in photoreceptor outer segments are transmitted to the rest of the visual system by
altering synaptic release from the terminals of rods and cones.
Synaptic vesicles are tethered near the active zone at a platelike
structure known as the ribbon (Schmitz 2009). Glutamate
release from photoreceptor synapses requires only submicromolar levels of Ca2⫹, much lower than Ca2⫹ levels typically
required for vesicle release at other synapses (Beutner et al.
* These authors contributed equally to this work.
Address for reprint requests and other correspondence: W. B. Thoreson,
University of Nebraska Medical Center, Department of Ophthalmology and
Visual Sciences, 4050 Durham Research Center, Omaha, NE 68198-5840
(E-mail: [email protected]).
www.jn.org
2001; Bollmann et al. 2000; Heidelberger et al. 1994; Rieke
and Schwartz 1996; Schneggenburger and Neher 2000; Thoreson et al. 2004). Therefore synaptic release from photoreceptors does not necessitate the high levels of Ca2⫹ that are
typically found only in nanodomains immediately adjacent to
Ca2⫹ channels. Nevertheless, L-type Ca2⫹ channels that mediate vesicle release from photoreceptors are clustered in the
terminal (Nachman-Clewner et al. 1999; Morgans 2001; Morgans et al. 2005; Specht et al. 2009; Steele Jr et al. 2005; Xu
and Slaughter 2005) beneath synaptic ribbons (tom Dieck et al.
2005), suggesting that release sites are quite close to Ca2⫹
channels. However, it is also possible that synaptic release
from photoreceptors might occur at ectopic sites located some
distance from the ribbon, as occurs at bipolar cell ribbon
synapses (Midorikawa et al. 2007; Zenisek et al. 2003, 2008).
In addition to stimulating vesicle release, Ca2⫹ influx stimulates Ca2⫹-activated chloride [Cl(Ca)] channels localized to
photoreceptor terminals (Barnes and Hille 1989; Cia et al.
2004; MacLeish and Nurse 2007). In cones, where the reversal
potential of chloride (ECl) is ⫺38 mV (Thoreson and Bryson
2004), activation of Cl(Ca) channels will tend to stabilize the
dark membrane potential. In rods, where ECl is ⫺20 mV
(Thoreson et al. 2002), activation of Cl(Ca) channels promotes
membrane depolarization. Although depolarization enhances
Ca2⫹ channel activity, the dominant effect of this Cl⫺ efflux in
rods appears to be an inhibition of Ca2⫹ channel activity by
actions mediated at intracellular anion binding sites on Ca2⫹
channels (Babai et al. 2010; Thoreson et al. 2000). In this way,
Cl⫺ efflux can act as a negative feedback mechanism to limit
excessive Ca2⫹ entry into rods. The strength of feedback
interactions between Ca2⫹ and Cl(Ca) channels will be influenced by the distance between the two types of channels.
This study analyzed the spatial relationships between Ca2⫹
channels, vesicle release sites, and Cl(Ca) channels in photoreceptors by comparing electrophysiological measurements to
profiles of Ca2⫹ diffusion predicted from a model developed
by Ward and Kenyon (2000). The results indicate an extremely
tight coupling between Ca2⫹ channels and ribbon release sites
in cones, whereas Cl(Ca) channels are more dispersed throughout the synaptic terminals of rods and cones. Consistent with
physiological data, antibodies to the putative Cl(Ca) channel,
TMEM16A, produced more diffuse immunostaining in photoreceptor terminals than antibodies to synaptic Ca2⫹ channels.
Similar to the use of Ca2⫹-activated K⫹ channels as submembrane Ca2⫹ sensors (Burrone et al. 2002; Roberts et al. 1990),
0022-3077/11 Copyright © 2011 The American Physiological Society
321
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Mercer AJ, Rabl K, Riccardi GE, Brecha NC, Stells SL Jr,
Thoreson WB. Location of release sites and calcium-activated chloride channels relative to calcium channels at the photoreceptor ribbon
synapse. J Neurophysiol 105: 321–335, 2011. First published November 17, 2010; doi:10.1152/jn.00332.2010. Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca2⫹ channels,
which are in turn regulated by Cl⫺ moving through calcium-activated
chloride [Cl(Ca)] channels. We assessed the proximity of Ca2⫹
channels to release sites and Cl(Ca) channels in synaptic terminals of
salamander photoreceptors by comparing fast (BAPTA) and slow
(EGTA) intracellular Ca2⫹ buffers. BAPTA did not fully block
synaptic release, indicating some release sites are ⬍100 nm from
Ca2⫹ channels. Comparing Cl(Ca) currents with predicted Ca2⫹
diffusion profiles suggested that Cl(Ca) and Ca2⫹ channels average a
few hundred nanometers apart, but the inability of BAPTA to block
Cl(Ca) currents completely suggested some channels are much closer
together. Diffuse immunolabeling of terminals with an antibody to the
putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca)
channels are dispersed throughout the presynaptic terminal, in contrast
with clustering of Ca2⫹ channels near ribbons. Cl(Ca) currents evoked
by intracellular calcium ion concentration ([Ca2⫹]i) elevation through
flash photolysis of DM-nitrophen exhibited EC50 values of 556 and
377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively.
These relationships were used to estimate average submembrane
[Ca2⫹]i in photoreceptor terminals. Consistent with control of exocytosis by [Ca2⫹] nanodomains near Ca2⫹ channels, average submembrane [Ca2⫹]i remained below the vesicle release threshold (⬃400
nM) over much of the physiological voltage range for cones. Positioning Ca2⫹ channels near release sites may improve fidelity in
converting voltage changes to synaptic release. A diffuse distribution
of Cl(Ca) channels may allow Ca2⫹ influx at one site to influence
relatively distant Ca2⫹ channels.
322
MERCER, RABL, RICCARDI, BRECHA, STELLA JR, AND THORESON
we also measured the Ca2⫹ dependence of Cl(Ca) channels and
used this relationship to convert Cl(Ca) current measurements
into average submembrane Ca2⫹ levels in rod and cone terminals.
METHODS
Retinal tissue preparation
Calculation of free Ca2⫹ profiles
To model profiles of free Ca2⫹, we used an Excel-based macro
program to calculate Ca2⫹ diffusion away from a voltage-gated
L-type Ca2⫹ channel in the presence of diffusible buffers (Ward and
Kenyon 2000; on-line at //www.medicine.nevada.edu/physio/docs/
default.htm). Parameters for the simulation are provided in Table 1.
Flash photolysis of DM-nitrophen
Flash photolysis of the photolysable Ca2⫹ chelator, DM-nitrophen
(Invitrogen, Carlsbad, CA), produces rapid and spatially homogeneous increases in intracellular calcium ion concentration ([Ca2⫹]i)
throughout the terminal. [Ca2⫹]i measured at the center of the terminal therefore provides a measure of the change in submembrane
[Ca2⫹]. The pipette solution for photolysis experiments consisted of
(in mM): 10 DM-nitrophen, 5 CaCl2, 4 diethylenetriaminepentaacetic
acid, 4 MgCl2, 26 Cs-gluconate, 78 HEPES, 6.5 TEA-Cl, 11 Na2ATP,
0.5 GTP (pH 7.2). DM-nitrophen was photolyzed by flashes of UV
light derived from a xenon arc flash lamp (Rapp Optoelectronic,
Hamburg, Germany). Oregon Green BAPTA-6F (OGB-6F, 0.5 mM,
Kd ⫽ 3 ␮M; Invitrogen) was also included in the pipette solution to
measure [Ca2⫹]i. Confocal Ca2⫹ measurements were made using a
laser confocal scanhead (UltraVIEW Live Cell Imaging System;
PerkinElmer, Waltham, MA), equipped with a cooled charge-coupled
device camera (Orca ER, Hamamatsu, Hamamatsu City, Japan)
mounted onto a fixed-stage upright microscope (E600 FN). Images
were acquired at 60- to 150-ms intervals with single-frame durations
of 48 –145 ms and pixel values were binned 2 ⫻ 2. To convert
OGB-6F fluorescence measurements into Ca2⫹ levels, we used the
following formula (Helmchen 2000)
⌬[Ca2⫹]i ⫽
兵[Ca2⫹]rest ⫹ Kd(⌬F ⁄ F) ⁄ (⌬F ⁄ Fmax)其
[1 ⫺ (⌬F ⁄ F) ⁄ (⌬F ⁄ F)max]
Electrophysiology
Patch pipettes were pulled on a PP-830 vertical puller (Narishige
USA, East Meadow, NY) from borosilicate glass pipettes (OD: 1.2
mm; ID: 0.9 mm, with internal filament; World Precision Instruments,
Sarasota, FL) and had tips ⬍2 ␮m in diameter with resistance values
ranging between 12 and 18 M⍀. For Ca2⫹ buffering experiments, we
used a pipette solution containing (in mM): 42 CsCl, 48 Cs-gluconate,
1.9 MgCl2, 32.9 HEPES, 9.4 tetraethylammonium chloride (TEA-Cl),
9.4 magnesium adenosine triphosphate (MgATP), 0.5 guanosine
triphosphate (GTP), plus 0.5 ethylene glycol tetraacetic acid (EGTA),
5 EGTA, or 5 1,2-bis(o-aminophenoxy)ethane,N,N,N=,N=-tetraacetic
acid (BAPTA) (pH 7.2). For measurements of depolarization-evoked
tail current amplitudes illustrated in Figs. 8 and 9, we used a pipette
solution with a predicted ECl of ⫺39 mV that consisted of (in mM):
11.3 CsCl, 75 Cs-gluconate, 1.9 MgCl2, 32.9 HEPES, 9.4 TEA-Cl, 9.4
MgATP, 0.5 GTP, and 5 EGTA (pH 7.2). We adjusted membrane
potentials with this solution for a measured liquid junction potential of
⫺7 mV.
TABLE
(1)
where ⌬F/F represents the fractional change in fluorescence resulting
from stimulation. (⌬F/F)max was determined from the maximal fluorescence change produced by application of 500-ms depolarizing
steps to ⫺10 mV. We used the Kd of 3 ␮M for OGB-6F provided by
Invitrogen. The resting Ca2⫹ concentration ([Ca2⫹]rest) for each
solution was determined ratiometrically using 0.2 mM Fura-2 as
described previously (Thoreson et al. 2004). As a test of Ca2⫹
measurements obtained with Eq. 1, we compared OGB-6F measurements with measurements made using a higher-affinity dye, OGB- 1
(Kd ⫽ 0.17 ␮M). We evoked intraterminal calcium changes by
applying depolarizing test steps (⫺70 to ⫺10 mV) of varying duration
(50 –500 ms). Despite large differences in the Kd, the same depolarizing stimuli yielded similar intraterminal [Ca2⫹] measurements with
the two different dyes (Choi et al. 2008).
The increase in Cl(Ca) current as a function of increasing Ca2⫹ was
fit with a sigmoidal binding curve
I⫽
1. Parameters to determine free Ca2⫹ around a Ca2⫹
Imax
兵1⫹10 关(log EC50 ⫺ log [Ca2⫹]) ⴱ h兴其
^
(2)
where h is the slope factor and Imax is the top of the sigmoidal curve.
The bottom value was constrained to zero.
channel in the presence of a diffusible chelator
Parameter
Value
Reference
DCa
ICa
EGTA Kd
EGTA Kon
BAPTA Kd
BAPTA Kon
0.2 ␮m2/ms
0.25 pA
1.58 ⫻ 10⫺7 M
2.7 ⫻ 106 M⫺1 · S⫺1
1.8 ⫻ 10⫺7 M
4.5 ⫻ 108 M⫺1 · S⫺1
Neher 1986
Church and Stanley 1996
Naraghi 1997
Naraghi 1997
Naraghi 1997
Naraghi 1997
J Neurophysiol • VOL
Immunohistochemistry
Salamanders were placed in an induction tank (⬃10 L) containing
MS-222 (2 g/L). Following induction, the animal was removed and
decapitated with large shears, after which the brain and spinal cord
were rapidly pithed. After enucleation, each eye was opened along the
ora serrata; the cornea, lens, and vitreous body were then removed and
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Experiments were performed using aquatic-phase tiger salamanders
(Ambystoma tigrinum, 18 –25 cm; Kons Direct, Germantown, WI or
Charles Sullivan, Nashville, TN). Care, handling, and experimentation
procedures were approved by the University of Nebraska Medical
Center Institute for Animal Care and Use Committee or the UCLA
Animal Research Committee. Animals were decapitated and pithed
and each eye was enucleated. After surgical removal of the cornea,
iris, and lens, the resultant eyecup was quartered. Eyecup pieces were
placed vitreous side down onto filter paper (2 ⫻ 5 mm, type AAWP;
0.8 ␮m pores; Millipore, Bedford, MA) and the retina was isolated in
a cold amphibian saline solution containing (in mM): 111 NaCl, 2.5
KCl, 1.8 CaCl2, 0.5 MgCl2, 10 HEPES, and 5 glucose (pH 7.8).
Tissue was sliced into 125-␮m-thick sections using a razorblade
(#121-6; Ted Pella, Redding, CA) tissue slicer (Stoelting, Wood Dale,
IL). Slices were placed in a recording chamber for viewing on an
upright fixed-stage microscope (E600FN; Nikon, Tokyo), fitted with a
⫻60, 1.0 numerical aperture (NA) water-immersion objective (Nikon).
Recording electrodes were positioned with Huxley–Wall micromanipulators (Sutter Instrument, Novato, CA) and visualized through the
eyepieces or with a video camera (Watec 502H; Rock House Products
International, Middletown, NY) mounted on the microscope. Photoreceptors were voltage clamped using a Multiclamp or Axopatch
200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA).
Currents were acquired using a Digidata 1322 interface and pCLAMP
9.2 software (Molecular Devices). In all experiments, retinal slices
were superfused with the amphibian saline solution described earlier
bubbled with 100% O2.
CALCIUM CHANNELS AT THE PHOTORECEPTOR RIBBON SYNAPSE
Antibodies
A rabbit polyclonal antibody to TMEM16A raised against a 620
amino acid peptide was used at a dilution of 1:500 (ab53212; Abcam,
Cambridge, MA). This antibody has been shown to react with both
human and rodent sequences (manufacturer’s data sheet). We also
used another rabbit polyclonal antibody raised against a 17 amino acid
segment on the N terminus (1:100, SIG5419; Zyagen, San Diego,
CA). A mouse monoclonal antibody against glial fibillary acidic
protein (GFAP) was used at a dilution of 1:1,000 (catalog no. CH
22102; Neuromics, Edina, MN) to identify Müller cells in the
J Neurophysiol • VOL
salamander retina (Sassoè Pognetto et al. 1992). A sheep polyclonal
antibody raised against amino acids 712 to 730 of the human ␣1F
calcium channel pore-forming subunit (a generous gift from Dr.
Catherine Morgans, OHSU, Portland, OR) was used at a dilution of
1:100 to label calcium channels on terminals of photoreceptors
(Morgans 2001). Staining in the retina with this antibody was
blocked by the peptide used to develop the antibody (Morgans
2001). A monoclonal antibody raised against the synaptic vesicle
protein, SV2 (Developmental Studies Hybridoma Bank at the
University of Iowa, Iowa City, IA), was used at a dilution of
1:2,000 to label synaptic terminals of photoreceptors in the tiger
salamander retina (Mandell et al. 1990; Yang et al. 2002; Zhang
and Wu 2009).
Unless otherwise specified, chemicals were obtained from SigmaAldrich. The criterion for statistical significance was chosen to be P ⬍
0.05 and evaluated using Student’s t-test or one-way ANOVA and
GraphPad Prism 4.0. Variability is reported as ⫾SE.
RESULTS
Distance from Ca2⫹ channels to release sites
Ca2⫹ channels are clustered in photoreceptor synaptic terminals (Morgans 2001; Morgans et al. 2005; NachmanClewner et al. 1999; Specht et al. 2009; Xu and Slaughter
2005) at the base of the ribbon (tom Dieck et al. 2005),
suggesting that release sites are likely to be very close to Ca2⫹
channels. We analyzed the distance between Ca2⫹ channels
and vesicle release sites by comparing the effects of different diffusible buffers (EGTA or BAPTA) on synaptic release from cones. BAPTA and EGTA have a similar high
affinity for Ca2⫹ but BAPTA has a much faster Kon rate, so
it chelates Ca2⫹ very rapidly near the mouth of a Ca2⫹
channel. Buffers were introduced into voltage-clamped
cones through the patch pipette. To examine effects of the
different Ca2⫹ buffers on synaptic release, cones were
depolarized from ⫺77 to ⫺17 mV for 500 ms and postsynaptic currents (PSCs) were recorded simultaneously from
voltage-clamped horizontal or OFF bipolar cells. Recordings
were conducted in the presence of cyclothiazide (0.1 mM) to
block ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) receptor desensitization. We waited ⱖ10 min
to allow time for diffusion of the Ca2⫹ buffer to the
photoreceptor terminal. Effects of the different buffers on
cone-driven PSCs averaged from multiple cell pairs are
illustrated in Fig. 1A (0.5 mM EGTA, n ⫽ 10; 5 mM EGTA,
n ⫽ 12; 5 mM BAPTA, n ⫽ 7). Cone-driven PSC waveforms were essentially unchanged by elevating the EGTA
concentration from 0.5 (dashed line) to 5 mM (black solid
line) and diminished, but not fully blocked, by the use of 5
mM BAPTA (gray solid line). Excitatory postsynaptic currents (EPSCs) evoked with shorter 25-ms test pulses were
also not blocked by BAPTA (comparison of charge transfer
during EPSCs with 0.5 EGTA, 5 EGTA, and 5 BAPTA: P ⫽
0.25, one-way ANOVA), indicating that the persistence of
synaptic responses was probably not due to saturation of
BAPTA during the 500-ms depolarizing step. Sustained
components of the cone-driven PSC were also not blocked
by BAPTA, consistent with the hypothesis that both transient and sustained release from cones occurs principally at
the ribbon (Jackman et al. 2009).
We also tested the effects of these chelators in rods. Like
cones, BAPTA did not block PSCs evoked in horizontal cells
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the eyecups were immersion-fixed in 4% paraformaldehyde (PFA) in
0.1 M phosphate buffer (PB; pH 7.4) for 15 min at room temperature.
The eyecups were then cryoprotected in 25% sucrose overnight at
4°C. Prior to cutting the tissue with a cryostat the retina was washed
with 0.1 M PB, embedded in Tissue-Tek OCT compound (Sakura
Finetek, Torrance, CA) and rapidly frozen with dry ice or liquid
nitrogen. Cryostat sections of the retina were cut at 12–15 ␮m,
mounted onto gelatin-coated slides, air dried, and stored at ⫺20°C.
All tissue was labeled using the indirect immunofluorescence technique (Stella Jr et al. 2007). Briefly, retinal sections were warmed for
10 min at 37°C. Tissue was preincubated in a 0.1 M PB mixture
containing 10% normal goat serum (NGS) or donkey serum (DS;
Invitrogen), 1% bovine serum albumin (BSA; Sigma-Aldrich, St.
Louis, MO), and 0.5% Triton-X 100 (Sigma-Aldrich) for 1 h. The
sections were then incubated in primary antibodies overnight at 4°C,
which were all diluted in 0.1 M PB (pH 7.4) containing 3% NGS or
DS, 1% BSA, and 0.5% Triton-X 100. The primary antibody/antigen
complex was detected using secondary antibodies conjugated to either
Alexa 488 or Alexa 568 (Invitrogen). Following incubation with the
antibody, retinal sections were washed three times for 10 min with 0.1
M PB to remove any unbound primary or secondary antibody. For
double-labeling experiments tissue sections were incubated in a
mixture of primary antibodies followed by a mixture of secondary
antibodies. All slides were allowed to air dry in the dark at room
temperature and coverslipped with Prolong Gold anti-fade (Invitrogen).
Images of retinal sections were acquired using a Zeiss Laser
Scanning Microscope 510 META (Zeiss, Thornwood, NY) with a
C-Apochromat ⫻40 1.2 NA water objective. To identify fluorescent
signals, different lasers were used for excitation: for Alexa 488 the
488-nm argon laser line was used and for Alexa 568 the 543-nm HeNe
laser line was used. Little or no staining was observed when using the
secondary antibodies alone. During acquisition of signals from double-labeled specimens, scans were collected sequentially to prevent
spectral bleed-through. Specific band-pass filters were used to achieve
proper separation of signals (for double labeling 488/505–530, 543/
560LP). In some scans, fluorescent emissions were further separated
using a linear unmixing algorithm (Zeiss LSM ver. 4.2). To validate
this process and reduce any potential mismatch or bleed-through of
fluorescence among different channels, separation of fluorescent
emissions was achieved by processing reference samples that were
immunostained with a single label to visualize their entire emission
spectra and localization within the retina. Most images were
acquired at a 12-bit resolution of 2,048 ⫻ 2,048 and, in some cases,
1,024 ⫻ 1,024. To increase signal-to-noise ratio, images were
averaged on-line (e.g., n ⫽ 4) and the scan speed and photo
multiplier detector gain were decreased. The digital fluorescent
images were single confocal scans taken in the same planes as
corresponding differential interference contrast (DIC) images.
Most digital images were acquired at an approximate optical
thickness of 0.5 ␮m or 1.0 Airy units. Digital images were saved
as Zeiss .LSM files and final publication quality images were
exported in .TIFF format at 300 dpi. Images were processed and
adjusted for brightness and contrast using Adobe Photoshop CS4
Extended (Adobe Systems, Mountain View, CA).
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MERCER, RABL, RICCARDI, BRECHA, STELLA JR, AND THORESON
Distance from Ca2⫹ channels to Cl(Ca) channels
2⫹
FIG. 1. Effects of the diffusible Ca
buffers EGTA and BAPTA on EPSCs
evoked in horizontal or OFF bipolar cells by depolarizing steps (⫺77 to ⫺17 mV
for 500 ms) applied to voltage-clamped cones. Records in A show the average
postsynaptic current (PSC) from multiple cone/horizontal cell and cone/OFF bipolar
cell pairs obtained after waiting ⬎10 min for diffusion of 5 mM BAPTA (n ⫽ 7,
solid gray line), 5 mM EGTA (n ⫽ 12, solid black line), or 0.5 mM EGTA (n ⫽
10, dashed line) to the cone terminal. Cyclothiazide (0.1 mM) was present in these
experiments to prevent AMPA receptor desensitization. EPSCs were diminished
but not fully blocked by the addition of 5 mM BAPTA to the patch pipette
solution. B: decline in [Ca2⫹] as a function of distance from an open Ca2⫹ channel
predicted from the “Pore” macro (Ward and Kenyon 2000) with Ca2⫹ buffering by
5 mM BAPTA (solid gray line), 5 mM EGTA (solid black line), or 0.5 mM EGTA
(dashed black line). Model parameters are provided in Table 1. AMPA, ␣-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid; BAPTA, 1,2-bis(o-aminophenoxy)ethane,N,N,N=,N=-tetraacetic acid; EGTA, ethylene glycol tetraacetic acid; EPSC,
excitatory postsynaptic current.
by test steps applied to voltage-clamped rods (data not shown).
However, the persistence of rod-driven PSCs is more difficult
to interpret since rod-driven PSCs are at least partly due to
synaptic output from neighboring rods coupled to the voltageclamped cell through gap junctions (Attwell et al. 1984; Cadetti et al. 2005; Zhang and Wu 2004).
We confirmed that Ca2⫹ buffers could diffuse to the
synaptic terminal by comparing intraterminal Ca2⫹ levels
measured with OGB-6F fluorescence in rods 5–10 min after
patch rupture (Supplemental Fig. S1).1 The rise in intraterminal Ca2⫹ evoked by a 100-ms depolarization from ⫺77 to
⫺17 mV declined after the step much more rapidly when the
patch pipette contained 5 mM EGTA (␶ ⫽ 0.22 s, n ⫽ 6) or
BAPTA (␶ ⫽ 0.19 s, n ⫽ 5) than with 0.5 mM EGTA (␶ ⫽
51 s, n ⫽ 6). Although the time course of decline is
influenced by extrusion, it is dominated by Ca2⫹ binding to
1
The online version of this article contains supplemental data.
J Neurophysiol • VOL
Depolarizing test steps (⫺77 to ⫺17 mV) applied to rods
and cones activate L-type ICa, voltage-dependent K⫹, Ca2⫹activated K⫹, and Cl(Ca) currents (Attwell and Wilson 1980;
Bader et al. 1982; Barnes and Hille 1989). Whole cell currents
evoked by depolarizing test steps (⫺70 to ⫺10 mV, 500 ms) were
typically dominated by outward currents (Fig. 2). Using a pipette
solution in which ECl ⫽ ⫺20 mV, long-lasting inward tail
currents were observed after termination of the test step
(Fig. 2). The duration of Cl(Ca) tail currents lasts ⱕ12 s and
exhibits considerable variability, declining in direct proportion to the decline in intracellular Ca2⫹ levels (MacLeish
and Nurse 2007). Tail currents could be inhibited by the Cl⫺
channel blocker niflumic acid (0.1 mM) and reversed around ECl
(n ⫽ 11 for rods and n ⫽ 8 for cones; data not shown), consistent
with other studies indicating that tail currents reflect calciumdependent chloride current [ICl(Ca)] activity (Barnes and Deschenes 1992; MacLeish and Nurse 2007; Thoreson et al. 2003).
Tail currents were not significantly inhibited by bath application
of the vesicular glutamate uptake inhibitor D-threo-␤-benzyloxyaspartate (TBOA, 0.1 mM), indicating little contribution from Cl⫺ efflux through excitatory amino acid transporter anion channels (rods: 5 mM BAPTA, P ⫽ 0.70,
paired t-test, n ⫽ 12; 5 mM EGTA, P ⫽ 0.70, n ⫽ 3; 0.5
mM EGTA, P ⫽ 0.64, n ⫽ 3; cones: 5 mM BAPTA, P ⫽
0.71, n ⫽ 7; 5 mM EGTA, P ⫽ 0.97, n ⫽ 3; 0.5 mM EGTA,
P ⫽ 0.54, n ⫽ 3).
Like Ca2⫹ channels, Cl(Ca) channels are located almost
entirely in the synaptic terminals of photoreceptors (MacLeish
and Nurse 2007). To analyze the distance between Ca2⫹ and
Cl(Ca) channels in rods and cones, Cl(Ca) tail currents in rods
and cones were measured after introducing different concentrations of Ca2⫹ buffers (0.5 mM EGTA, 5 mM EGTA, and 5
mM BAPTA) into the photoreceptor through the recording
pipette. We waited ⱖ10 min for buffers to diffuse to the
synaptic terminal. Figure 2 illustrates recordings obtained from
rods and cones using the different buffers. The bar graphs in
Fig. 2, G and H show the average amplitude of tail currents
measured 15 ms after termination of the test step with the
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the buffer so these results show that buffers can diffuse
through the axon to the rod terminal. Cone pedicles do not
possess a thin axon and are thus more accessible to buffers
introduced through a patch pipette on the cell body (Mariani
1986; Sherry et al. 1998).
The profiles of [Ca2⫹] surrounding individual open Ca2⫹
channels predicted for different buffering conditions were
simulated using a model developed by Ward and Kenyon
(2000). Parameters for [Ca2⫹]i simulations under each buffering condition are provided in Table 1 and the predicted Ca2⫹
profiles are illustrated in Fig. 1B. With 5 mM BAPTA, free
Ca2⫹ surrounding a Ca2⫹ channel should decline below the
release threshold of 400 nM (Duncan et al. 2010; Thoreson et
al. 2004) within 50 nm. Even if BAPTA levels attained a
concentration at the synapse of only 0.5 mM, 10-fold lower
than the pipette concentration, then Ca2⫹ levels should still fall
below release threshold within 100 nm of a Ca2⫹ channel.
Thus conservatively, the persistence of depolarization-evoked
PSCs in the presence of BAPTA indicates that Ca2⫹ channels
in cones are ⬍100 nm from synaptic release sites.
CALCIUM CHANNELS AT THE PHOTORECEPTOR RIBBON SYNAPSE
325
different buffers. In both rods and cones, tail currents were
significantly diminished by increasing the EGTA concentration
from 0.5 to 5 mM (t-test, rods, P ⫽ 0.003; cones P ⫽ 0.002,
Fig. 2, G–H). BAPTA caused a further reduction in tail
currents but did not block them completely. Although the
persistence of Cl(Ca) tail currents in the presence of 5 mM
BAPTA may be partly due to partial saturation of BAPTA
during the lengthy depolarizing step, it also suggests that a
small population of Cl(Ca) channels is located within 100 nm
of Ca2⫹ channels.
To estimate the average distance between Ca2⫹ and
Cl(Ca) channels, we compared buffer effects on tail currents
with profiles of free Ca2⫹ predicted for the three buffering
conditions (Fig. 1B; Naraghi 1997; Ward and Kenyon 2000).
In rods, increasing EGTA from 0.5 to 5 mM reduced tail
currents by 82% relative to the greater reduction produced
by 5 mM BAPTA. By comparison, the predicted Ca2⫹
profiles suggest that increasing EGTA from 0.5 to 5 mM
should reduce [Ca2⫹] by 82% relative to the levels predicted
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for 5 mM BAPTA at a distance of 395 nm from the Ca2⫹
channel (Fig. 1B). In cones, increasing EGTA from 0.5 to 5
mM reduced tail currents by 89% relative to the reduction
produced by 5 mM BAPTA. The same buffer-induced
change in Ca2⫹ levels should occur at a distance of 490 nm
from the Ca2⫹ channel (Fig. 1B). Measurements of the Ca2⫹
dependence of Cl(Ca) channels described later suggested
that their activation is likely to require the binding of two
Ca2⫹ ions. If so, the decline in [Ca2⫹] should cause a
squared decline in Cl(Ca) activation, which would in turn
suggest distances of 206 and 263 nm between Ca2⫹ and
Cl(Ca) channels in rods and cones, respectively.
In another approach to assess the average distance between Ca2⫹ channels and Cl(Ca) channels, we used the
calcium dependence of ICl(Ca) to estimate [Ca2⫹]i from
ICl(Ca) amplitude. Sigmoidal concentration–response curves
were generated using the half-maximal effective concentration (EC50) and Hill slope values for Cl(Ca) channels,
determined from caged Ca2⫹ experiments described in the
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FIG. 2. Effects of the diffusible Ca
buffers EGTA and
BAPTA on depolarization-evoked Ca2⫹-activated chloride
[Cl(Ca)] tail currents in rods and cones. Rod and cone
photoreceptors were depolarized by application of a test
step from ⫺77 to ⫺17 mV for 500 ms. Peak calciumdependent chloride current [ICl(Ca)] activation was measured
15 ms after the depolarizing step to avoid recording contamination from the capacitative transient. Depolarizationevoked tail currents arising from the activation of ICl(Ca)
persisted in both rods and cones in the presence of 0.5 mM
EGTA (A and B), 5 mM EGTA (C and D), and 5 mM
BAPTA (E and F). G: mean tail current amplitude measured in rods with Ca2⫹ buffering provided by 0.5 mM
EGTA (316.0 ⫾ 35.0 pA, n ⫽ 24), 5 mM EGTA (120.7 ⫾
21.4 pA, n ⫽ 14), and 5 mM BAPTA (78.7 ⫾ 14.6 pA,
n ⫽ 23). Tail currents were measured 15 ms after the end of
the step. Measurements were made ⱖ10 min after patch
rupture. H: tail current amplitudes in cones with Ca2⫹
buffering provided by 0.5 mM EGTA (123.3 ⫾ 12.4 pA,
n ⫽ 17), 5 mM EGTA (38.1 ⫾ 8.64 pA, n ⫽ 8), and 5 mM
BAPTA (27.0 ⫾ 6.70 pA, n ⫽ 14).
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MERCER, RABL, RICCARDI, BRECHA, STELLA JR, AND THORESON
Immunohistochemical localization of Cl(Ca) channels
Based on expression cloning and physiological data,
TMEM16A has been implicated as a Cl(Ca) channel (Caputo et al. 2008; Hartzell et al. 2009; Rock et al. 2009;
Romanenko et al. 2010; Schroeder et al. 2008; Yang et al.
2008). TMEM16A mRNA is present in the outer retina
(Gritli-Linde et al. 2009) and the related protein TMEM16B
also appears to form Ca2⫹-activated chloride channels localized to mammalian photoreceptor terminals (Stöhr et al.
2009). We tested whether TMEM16A might contribute to
Cl(Ca) channel activity in photoreceptors. Consistent with
the presence of TMEM16A protein in photoreceptor terminals, indirect immunofluorescence with an antibody to
TMEM16A produced labeling of the outer plexiform layer
(OPL) in the tiger salamander retina (Fig. 3A). In addition,
TMEM16A labeled cells in the inner nuclear layer (INL)
that could also be immunolabeled with antibodies for glial
fibrillary acidic protein (GFAP) to label Müller cells (Fig. 3,
A–D). This is consistent with electrophysiological evidence
for Cl(Ca) channels in salamander Müller cells (Welch et al.
2006). Higher magnification of the OPL suggests that
TMEM16A immunolabels photoreceptor terminals (arrows
in Fig. 3 indicate rod and cone terminals).
To test whether TMEM16A is expressed in photoreceptor
terminals, retinal sections and isolated photoreceptors were
double-immunolabeled with the TMEM16A antibody and an
antibody to the synaptic vesicle protein, SV2 (Zhang and
Wu 2009). TMEM16A expression overlapped with and was
limited to SV2 expression in both vertical retinal sections
FIG. 3. TMEM16A is expressed in both the outer plexiform layer (OPL) and Müller cell bodies, which span the entire retina. A: retinal section immunolabeled
for TMEM16A (red). B: Müller cells immunolabeled in a retinal section with glial fibillary acidic protein (GFAP, green). C: merged fluorescent image of
TMEM16A and GFAP. D: merged TMEM16A (red) and GFAP (green) immunofluorescence with the differential interference contrast (DIC) brightfield image.
Arrows in C indicate labeling of TMEM16A in photoreceptor terminals. E: high-magnification zoom region in the OPL illustrating TMEM16A immunolabeling
in the synaptic terminals of photoreceptors (red). F: GFAP labeling of the same high-magnification region (green). G: merged fluorescent high-magnification
image of TMEM16A and GFAP. H: merged TMEM16A (red) and GFAP (green) with the differential interference contrast (DIC) brightfield image. Arrows in
G indicate labeling of TMEM16A in individual photoreceptor terminals. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL,
ganglion cell layer. Scale bars ⫽ 10 ␮m.
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following text. Peak current amplitudes were assumed to be
slightly larger than the average currents recorded with 0.5
mM EGTA (130 and 330 pA for cones and rods, respectively). Using these curves, we converted tail current measurements into [Ca2⫹]i and then matched these Ca2⫹ levels
to the predicted decline in Ca2⫹. For 5 mM EGTA, this
yielded estimated distances of 358 and 478 nm in rods and
cones, respectively. Estimates with 0.5 mM EGTA are more
sensitive to values chosen for the top of the sigmoidal curve,
but repeating this procedure with data for 0.5 mM EGTA
yielded estimates of 296 and 365 nm for rods and cones,
respectively. Distance estimates also depend on parameters
used for simulation of the Ca2⫹ decline. For example, using
slightly slower on rates for Ca2⫹ binding to EGTA from
Stern (1992; 1.5 ⫻ 106 M⫺1 · S⫺1), we obtained distances
with 5 mM EGTA of 454 and 596 nm in rods and cones,
respectively. Although the precise values differ somewhat,
results of these different analyses all agree that the distance
between Ca2⫹ and Cl(Ca) channels averages a few hundred
nanometers. The inability of BAPTA to block tail currents
completely in rods and cones indicates that there is also a
subpopulation of Cl(Ca) channels that are very close to
Ca2⫹ channels. Due to the limited spatial spread of Ca2⫹
when using BAPTA as a buffer, the amplitude of tail
currents measured in the presence of 5 mM BAPTA suggested that Ca2⫹ and Cl(Ca) channels are separated by only
about 60 nm. Together, these results suggest that Cl(Ca)
channels are dispersed throughout the synaptic terminal,
with some channels quite close to Ca2⫹ channels and others
further away.
CALCIUM CHANNELS AT THE PHOTORECEPTOR RIBBON SYNAPSE
(Fig. 4, A–C) and within the synaptic terminals of isolated
photoreceptors (arrows, Fig. 4, D–I), indicating that
TMEM16A is localized to the synaptic terminals of photoreceptors. The overlap between TMEM16A and SV2 expression is also consistent with electrophysiological results,
suggesting that Cl(Ca) channels are dispersed throughout
the presynaptic terminal.
327
The retina-specific protein CaV1.4 (␣1F) has been implicated
as the pore-forming subunit of the Ca2⫹ channel underlying
transmitter release from photoreceptors (Bech-Hansen et al.
1998; Firth et al. 2001; Morgans 2001). Similar to the pattern
observed in other species (Firth et al. 2001; Morgans 2001),
antibodies to CaV1.4 labeled the OPL, photoreceptor inner
segments, and IPL of salamander retina (Fig. 5B). CaV1.4
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FIG. 4. TMEM16A and SV2 labeling colocalize at photoreceptor terminals. A: immunolabeling of TMEM16A (green) in a
vertical retinal section. B: immunolabeling
of the same retinal section with a synaptic
vesicle marker, SV2 (red). Autofluorescence
in the outer segments was observed without
secondary antibodies. C: merged fluorescent
and DIC image of TMEM16A and SV2.
D: brightfield DIC image of a rod photoreceptor lacking its outer segment immunolabeled with TMEM16 (green). E: brightfield
DIC image of the same rod immunolabeled
with SV2 (red). F: merged brightfield DIC
image with both TMEM16A and SV2. G–
I: magnified images of the same rod terminal
showing that TMEM16A overlaps completely with SV2. J–L: brightfield DIC images of a small single cone photoreceptor
immunolabeled with TMEM16A (green, J),
SV2 (red, K), or both (L). M–O: magnified
images of the same cone terminal showing
that TMEM16A overlaps completely with
SV2. OPL, outer plexiform layer; INL, inner
nuclear layer; IPL, inner plexiform layer;
GCL, ganglion cell layer. Synaptic terminal
regions are indicated by the arrows in D and
J. Scale bar is 10 ␮m.
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MERCER, RABL, RICCARDI, BRECHA, STELLA JR, AND THORESON
labeling overlapped with TMEM16A in both the OPL and
terminals of isolated photoreceptors (Fig. 5). High-magnification images showed punctate labeling of CaV1.4 in rod and
cone terminals (Fig. 5, G–I and M–O). The TMEM16A antibody produced more diffuse labeling of photoreceptor terminals.
It was not practical to synthesize an antigenic peptide for the
Abcam antibody that was raised against a 620 amino acid
peptide, so to assess the specificity of labeling in the OPL and
photoreceptor terminals, we tested another antibody raised
against a different epitope of TMEM16A (Zyagen, San Diego,
J Neurophysiol • VOL
CA). This antibody showed a similar labeling pattern with
immunofluorescence visible in the OPL and IPL, along with
diffuse labeling of the terminals of isolated rods and cones
(Supplemental Figs. S2 and S3). Immunolabeling was abolished by preincubation with the peptide used to develop the
antibody (Supplemental Fig. S2B). We also examined methanol-fixed cells and found that, consistent with results from
paraformaldehyde-fixed cells, TMEM16A antibodies produced
diffuse staining of the terminal and surrounding regions,
whereas CaV1.4 antibodies showed a more concentrated distribution in the synaptic terminals of rods and cones (Supple-
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FIG. 5. TMEM16A and the Ca
channel CaV1.4 are
both expressed at photoreceptor terminals. A: TMEM16A
labeling (green) in a vertical retinal section. B: CaV1.4
labeling (red) in the same retinal section. C: merged fluorescent and DIC image of TMEM16A and CaV1.4. D–
F: brightfield DIC images of a rod photoreceptor immunolabeled with TMEM16A (green, D), CaV1.4 (red, E), or
both (F). Autofluorescence in the inner segment region
with the green channel was observed without the secondary antibody and is likely due to the presence of NADH
and NADPH in the mitochondrial-rich ellipsoid. G–I: magnified images of the same rod terminal illustrate the more
punctate expression of CaV1.4 (H) compared with the
diffuse labeling of TMEM16A (G) throughout the terminal
region. J–L: brightfield DIC images of a large single cone
photoreceptor immunolabeled with TMEM16A (green, J),
CaV1.4 (red, H), or both (I). M–O: magnified images of the
same cone terminal illustrate the more punctate expression
of CaV1.4 (N) compared with the diffuse expression of
TMEM16A (M) throughout the terminal region. OPL,
outer plexiform layer; INL, inner nuclear layer; IPL, inner
plexiform layer; GCL, ganglion cell layer. Synaptic terminal regions are indicated by the arrows in D and J. Scale
bar is 10 ␮m.
CALCIUM CHANNELS AT THE PHOTORECEPTOR RIBBON SYNAPSE
mental Fig. S3). Together, these anatomical findings are consistent with physiological results, suggesting that Cl(Ca) channels are distributed more diffusely than Ca2⫹ channels.
Ca2⫹ dependence of Cl(Ca) channels
intrinsic difference between rod and cone currents since ICl(Ca)
activated at a significantly faster rate in rods (␶ ⫽ 3.1 ⫾ 0.25
ms) than that in cones (␶ ⫽ 4.0 ⫾ 0.30 ms; P ⫽ 0.049) when
compared over a similar range of Ca2⫹ levels (rods: 2.62 ⫾
0.49 ␮M; cones: 2.60 ⫾ 0.50 ␮M; P ⫽ 0.98).
Peak current amplitudes from rods and cones were plotted as
a function of intraterminal [Ca2⫹]i (Fig. 7, A and B). The
relationships between amplitude and log [Ca2⫹]i were assumed
to obey standard receptor binding kinetics and therefore fit with
sigmoidal binding curves (see METHODS). In most experiments,
we obtained only a single response per cell. Cell-to-cell variability in current amplitude produced scatter in the data. In a
few recordings, we held individual cells long enough for
[Ca2⫹]i to recover to basal levels between uncaging flashes,
allowing us to make more than one measurement. Figure 7, C
and D illustrates recordings from a rod and cone, respectively,
in which multiple uncaging flashes were applied. As illustrated
by these examples, repeated measurements in individual cells
yielded a Ca2⫹ dependence similar to that in the overall
sample. The best-fit sigmoid to the overall sample from rods
exhibited an EC50 of 556 nM, with a slope factor of 1.8 (n ⫽
88, Fig. 7A). Data from cones yielded an EC50 of 377 nM, with
a slope factor of 2.4 (n ⫽ 34, Fig. 7B). Comparisons of the 95%
confidence intervals showed no significant differences between
the best-fit parameters in rods and cones.
Average submembrane [Ca2⫹]
Similar to the use of Ca2⫹-activated K⫹ channels as submembrane Ca2⫹ sensors (Burrone et al. 2002; Roberts et al.
1990), we used the empirically determined Ca2⫹ dependence
of ICl(Ca) to assess levels of submembrane Ca2⫹ attained at
Cl(Ca) channels in the presynaptic terminal. We chose 5 mM
EGTA as the Ca2⫹ buffer for these experiments because tail
2⫹
FIG. 6. Flash photolysis of caged Ca
in
photoreceptor terminals instantaneously ele2⫹
vated intraterminal [Ca ] and stimulated
inward Cl(Ca) currents. A: grayscale images
of Oregon Green BAPTA-6F (OGB-6F) fluorescence in a single confocal section from a
rod loaded with the reagent DM-nitrophen.
Images were obtained prior to flash photolysis (left) and immediately after flash photolysis (right). Intracellular calcium ion concentration ([Ca2⫹]i) was measured from a
region of interest placed in the synaptic terminal (arrows). Fluorescence is brighter in
the soma due to the presence of more dye.
B: grayscale images of OGB-6F fluorescence in a single confocal section from a
cone before (left) and after (right) flash photolysis of DM-nitrophen. C and D show
intraterminal [Ca2⫹] measured at 60-ms intervals in the rod (C) and cone (D). E and F
show inward Cl(Ca) currents evoked in the
same rod (E) and cone (F). Cells were voltage-clamped at ⫺77 mV. Ca2⫹-activated K⫹
currents were inhibited by Cs⫹ and tetraethylammonium (TEA) in the pipette solution and
reversal potential of chloride (ECl) ⫽ ⫺39 mV.
Scale bar ⫽ 5 ␮m.
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We measured the Ca2⫹ dependence of Cl(Ca) channels in
rods and cones by abruptly elevating submembrane Ca2⫹
levels in photoreceptor terminals via flash photolysis of the
caged Ca2⫹ compound, DM-nitrophen (10 mM). After introduction of DM-nitrophen into the photoreceptor through the patch
pipette, flash photolysis produces an instantaneous and homogeneous rise in intraterminal [Ca2⫹] that can be quantified from the
change in fluorescent intensity of OGB-6F (Fig. 6). Rods and
cones were voltage-clamped at a membrane potential of ⫺77
mV (illustrated in the left panels of Fig. 6, A and B), below the
estimated value of ECl ⫽ ⫺39 mV. The activation of Cl(Ca)
channels by the increase in [Ca2⫹]i generated inward currents
in both rods and cones (Fig. 6). The amplitude of inward
currents evoked in rods and cones by flash photolysis of caged
Ca2⫹ did not differ significantly (P ⫽ 0.8, rods; P ⫽ 0.54,
cones) between trials in which flash photolysis evoked detectable glutamate release (rods, n ⫽ 19; cones, n ⫽ 14) and trials
that failed to evoke a postsynaptic response in simultaneously
voltage-clamped horizontal or OFF bipolar cells (rods, n ⫽ 69;
cones, n ⫽ 20). This is consistent with effects of TBOA
described earlier and suggests a minimal contribution from
glutamate transporter currents to the inward currents evoked in
photoreceptors by flash photolysis of caged Ca2⫹.
In the examples shown in Fig. 6, ICl(Ca) increased more
rapidly in the rod than in the cone. This faster rate is not readily
explained by the higher Ca2⫹ level attained after flash photolysis in the rod since time constants for the rise in ICl(Ca) did not
show a clear Ca2⫹ dependence. This may instead reflect an
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MERCER, RABL, RICCARDI, BRECHA, STELLA JR, AND THORESON
currents and postsynaptic responses exhibited smaller changes
over time following patch rupture when using 5 mM EGTA in
the pipette solution compared with experiments with 0.5 mM
EGTA or 5 mM BAPTA. This suggests that 5 mM EGTA is
closer to the endogenous buffering capacity. ECl in the pipette
solution (⫺39 mV) was matched to the ECl in the DM-
nitrophen pipette solution used to measure Ca2⫹ dependence.
We stimulated rods and cones with a series of voltage steps
from ⫺57 to ⫺17 mV, ranging in duration from 50 ms to 1 s.
We subtracted capacitative and leak curents using a P/8 protocol. Figure 8A shows a series of traces illustrating the
increase in tail currents evoked in a rod with increasing
FIG. 8. Tail currents evoked by depolarizing test steps over a range of voltages
(⫺57 to ⫺17 mV) and durations (50 ms to 1
s). A and B each show the currents evoked in
a rod (A) and cone (B) by changes in the
amplitude of a 1-s test step from ⫺57 to ⫺17
mV (10-mV increments). Small inward ICa is
detectable during steps to ⫺57 mV and
above. Cl(Ca) tail currents become visible
with steps to ⫺47 mV and above. C and D
each illustrate currents evoked by changing
the duration of a strong test step (⫺77 to
⫺17 mV) from 50 ms to 1 s in a rod (C) and
cone (D). Inward ICa values were evoked by
the step and Cl(Ca) tail currents were observed following termination of the test step
with pulses of ⱖ200 ms in these examples.
Passive membrane resistance (Rm) and membrane capacitance (Cm) were subtracted using a P/8 protocol. ECl ⫽ ⫺39 mV.
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2⫹
FIG. 7. Ca
dependence of ICl(Ca) in
rods and cones. The amplitudes of inward
Cl(Ca) currents evoked by flash photolysis
of caged Ca2⫹ were plotted as a function of
intraterminal [Ca2⫹]. Data were fit with sigmoidal concentration–response curves (see
METHODS). The best fit for data from rods (A)
was obtained with a half-maximal effective
concentration (EC50) of 556 nM and slope
factor of 1.76 (n ⫽ 88). The best fit for data
from cones (B) was obtained with an EC50 of
377 nM and slope factor of 2.44 (n ⫽ 34).
Repeated measurements in an individual rod
(C) and cone (D) yielded a similar sigmoidal
relationship. For these 2 cells, the best fits
were obtained with EC50 values of 675 and
262 nM and slope factors of 2.56 and 2.44,
respectively.
CALCIUM CHANNELS AT THE PHOTORECEPTOR RIBBON SYNAPSE
brane [Ca2⫹] to rise to 2 ␮M in both rods and cones, consistent
with estimates obtained using Ca2⫹-sensitive dyes (Choi et al.
2008; Steele Jr et al. 2005; Szikra and Krizaj 2006). [Ca2⫹]
attained during 1-s steps to ⫺27 and ⫺17 mV may exceed
estimated levels since tail currents evoked by these stimuli
often exceeded 125 pA in rods or 153 pA in cones.
DISCUSSION
Release sites are ⬍100 nm from Ca2⫹ channels in
photoreceptor terminals
The persistence of cone-driven PSCs in the presence of
BAPTA indicates that a number of release sites are ⬍100 nm
from L-type Ca2⫹ channels in cone terminals. These dimensions are consistent with ultrastructural observations that ribbon-associated synaptic vesicles contact the plasma membrane
along the flanks of the synaptic ridge at a distance of about 50
nm from the edge of the ribbon (Lasansky 1973; Raviola and
Gilula 1975). This close proximity between Ca2⫹ channels and
release sites is similar to the squid giant synapse (Adler et al.
1991), GABAergic basket cells in the hippocampus (Bucurenciu et al. 2008), mature calyx of Held neurons (Fedchyshyn and
Wang 2005), and retinal bipolar cells (Jarsky et al. 2010).
However, it differs from many other CNS synapses, where
EGTA can significantly depress release, indicating that Ca2⫹
channels are much further away from release sites. These
include immature calyx of Held (Borst and Sakmann 1996;
Meinrenken et al. 2002), cortical pyramidal cells (Ohana and
Sakmann 1998; Rozov et al. 2001), and hair cells (Moser and
Beutner 2000). In capacitance recordings from isolated goldfish bipolar cell terminals, 5 mM EGTA diminished vesicle
release (Mennerick and Matthews 1996), whereas introduction
of 10 mM EGTA into mouse bipolar cell somas did not
diminish synaptic release (Singer and Diamond 2003). The
FIG. 9. Conversion of the amplitude of
depolarization-evoked Cl(Ca) tail currents
into submembrane [Ca2⫹] for different test
step voltages and durations. ICl(Ca) (A, rods;
B, cones) amplitude was converted into
[Ca2⫹]i (C, rods; D, cones) attained at
Cl(Ca) channels using the sigmoidal curves
fit to the data in Fig. 7. Currents exceeding
153 pA in cones or 134 pA in rods were
assigned a concentration value of 10 ␮M.
Stimuli that failed to evoke inward tail currents were assigned a concentration value of
31 nM.
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depolarization from ⫺57 to ⫺17 mV using test steps of 1-s
duration. Figure 8B shows the same measurements performed
in a cone. Inward currents due to activation of L-type ICa were
observed during the test step and inward ICl(Ca) tail currents
were observed after termination of the test step. Consistent
with ICl(Ca) being driven by Ca2⫹ influx through L-type Ca2⫹
channels (Barnes and Hille 1989; MacLeish and Nurse 2007),
both ICa and tail currents increased with depolarization above
⫺57 mV. Figure 8, C and D illustrates the effects of increasing
test step duration when using a step to ⫺17 mV in a rod and
cone, respectively. Tail currents were accentuated by the use of
longer test steps, consistent with an increase in [Ca2⫹]i exceeding the local buffering capacity. The large increase in ICl(Ca)
observed in the rod with test steps of 500 ms and 1 s may
reflect contributions of Ca2⫹-induced release of Ca2⫹ from
intracellular stores (Cadetti et al. 2006).
Using the sigmoidal curves fit to rod and cone data in Fig. 7,
we converted ICl(Ca) amplitude into [Ca2⫹]i attained at Cl(Ca)
channels. Experiments on Cl(Ca) channel localization suggested that Cl(Ca) channels are dispersed throughout the synaptic terminal. This, in turn, suggests that Ca2⫹ levels attained
at Cl(Ca) channels should provide an estimate of the average
submembrane [Ca2⫹]. Inward Cl(Ca) tail currents exceeding
limits of the sigmoidal curve (⬎153 pA in cones and ⬎125 pA
in rods) were assigned a concentration value of 10 ␮M. Stimuli
that failed to evoke inward tail currents were assigned a
concentration value of 31 nM. The results of this conversion
are shown in Fig. 9, C and D for rods and cones. Consistent
with Fura2 measurements (Choi et al. 2008; Steele Jr et al.
2005; Szikra and Krizaj 2006), tail current measurements
suggest basal Ca2⫹ levels (assessed from 50- to 200-ms steps
to ⫺57 mV) slightly below 100 nM. Ca2⫹ levels rose steeply
as the membrane potential approached the dark resting potential of ⫺40 mV. Application of a 1-s depolarization to ⫺37
mV, slightly above the average dark potential, caused submem-
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MERCER, RABL, RICCARDI, BRECHA, STELLA JR, AND THORESON
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different output modes to improve release precision under
light-adapted conditions when the occurrence of Ca2⫹ channel
openings is closely synchronized to the appearance of a stimulus but reduce synaptic noise in darkness when Ca2⫹ channel
openings occur more randomly.
The distance between Ca2⫹ and Cl(Ca) channels averages a
few hundred nanometers
In addition to stimulating synaptic release, Ca2⫹ influx
through Ca2⫹ channels activates Cl(Ca) channels. The finding
that 5 mM BAPTA did not fully eliminate ICl(Ca) in rods or
cones suggests that some Cl(Ca) channels are quite close
(⬍100 nm) to Ca2⫹ channels. However, other Cl(Ca) channels
are further away since the distance between Ca2⫹ channels and
Cl(Ca) channels averaged 300 – 600 nm in cones and 200 – 450
nm in rods. By comparison, the distance between individual
ribbons in published electron micrographs of salamander retina
is often a micron or more (Lasansky 1978; Pang et al. 2008;
Townes-Anderson et al. 1985), consistent with the hypothesis
that Cl(Ca) channels are dispersed between ribbons.
Until the identity of the channel(s) contributing to Cl(Ca)
currents is fully characterized, measurements of these spatial
relationships are necessarily limited to physiological approaches.
The putative Cl(Ca) channel, TMEM16B, has been shown to be
localized to photoreceptor terminals (Schroeder et al. 2008; Stöhr
et al. 2009). The present immunohistochemical results indicate
that the related isoform, TMEM16A, is also present at photoreceptor synaptic terminals, consistent with the presence of
mRNA for the TMEM16A gene in outer retinal layers (GritliLinde et al. 2009; Fig. 3). TMEM16A may therefore participate with TMEM16B in generating ICl(Ca) in photoreceptors.
Staining for TMEM16A antibodies produced labeling of rod
and cone terminals that overlapped extensively with labeling
for the synaptic vesicle protein SV2, but was more diffuse than
the punctate staining observed with antibodies to CaV1.4 (␣1F)
Ca2⫹ channels. Punctate labeling of rod terminals by L-type
Ca2⫹ channel antibodies was also observed by NachmanClewner et al. (1999). Although uncertainties remain about the
molecular identity of the Cl(Ca) channel, these results are
consistent with electrophysiological results, suggesting that
Cl(Ca) channels are not clustered as tightly near synaptic
ribbons as Ca2⫹ channels, but more dispersed throughout the
synaptic terminals of rods and cones.
Ca2⫹ dependence of Cl(Ca) channels in photoreceptors
Using flash photolysis of DM-nitrophen to elevate submembrane [Ca2⫹] instantaneously, we found that ICl(Ca) could be
activated by submicromolar [Ca2⫹] levels in photoreceptors.
The slopes of sigmoidal fits to Ca2⫹ dependence suggest that
binding of two or more Ca2⫹ ions is required for activation.
This is consistent with TMEM16 expression studies and studies on Cl(Ca) channels in other tissues that show Hill coefficients ⱖ2 (Arreola et al. 1996; Evans and Marty 1986; Giovannucci et al. 2002; Kuruma and Hartzell 2000; Pifferi et al.
2009; Reisert et al. 2003; Yang et al. 2008). The EC50 values
of 556 nM in rods and 377 nM in cones are similar to the Kd
of ICl(Ca) in acinar cells (Arreola et al. 1996; Giovannucci et al.
2002) but lower than Kd values obtained from excised patches
containing TMEM16A channels, TMEM16B channels, Cl(Ca)
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more pronounced effects of EGTA observed in the former
study may be due to differences in the preparation and cellular
access: Mennerick and Matthews (1996) recorded directly
from large goldfish bipolar cell terminals, whereas Singer and
Diamond (2003) recorded from rat bipolar cell somas that are
separated from the terminal by a long, thin axon. The finding
that release sites are situated extremely close to Ca2⫹ channels
in photoreceptors is particularly surprising given the high
affinity for Ca2⫹ exhibited by the release mechanism, which
requires only submicromolar levels to stimulate exocytosis
(Duncan et al. 2010; Rieke and Schwartz 1996; Sheng et al.
2007; Thoreson et al. 2004). The presence of highly localized
Ca2⫹ nanodomains suggests strong endogenous Ca2⫹ buffering in photoreceptor terminals.
One consequence of a tight coupling between Ca2⫹ channels
and release sites may be a lower likelihood of ectopic release
(i.e., release at sites other than the active zone). In addition to
Ca2⫹ channels in the synaptic terminals of photoreceptors,
immunohistochemical studies show faint labeling of the soma
and inner segments, suggesting some channels may be located
at nonsynaptic sites (Morgans 2001; Morgans et al. 2005;
Nachman-Clewner et al. 1999; tom Dieck et al. 2005). However, electrophysiological measurements suggest that 95% of
the Ca2⫹ channels are localized to the synaptic terminal in rods
(Xu and Slaughter 2005) and high-resolution confocal microscopy and immunoelectron micrographs show that Ca2⫹ channels in the terminal are mainly located beneath the arciform
density at the base of the ribbon (Specht et al. 2009; tom Dieck
et al. 2005). The finding that both transient and sustained
components of synaptic release involve release sites ⬍100 nm
from Ca2⫹ channels is thus consistent with the hypothesis that
release occurs predominantly at the ribbon in cone photoreceptors (Jackman et al. 2009). However, our data are also consistent with the possibility of nonribbon release sites located very
close to nonribbon Ca2⫹ channels. Like cones, rod-driven
EPSCs were not blocked by BAPTA in the patch pipette, but
interpretation of these responses is complicated by the presence
of synaptic output from neighboring, gap-junctionally coupled
rods (Cadetti et al. 2005).
Ca2⫹ levels rise to micromolar levels beneath the ribbon
when photoreceptors are maintained in darkness (Choi et al.
2009; Jackman et al. 2009; Steele Jr et al. 2005; Szikra and
Krizaj 2006; present study, Fig. 9). Micromolar Ca2⫹ levels are
sufficient to release many of the vesicles in the readily releasable pool at the base of the synaptic ribbon (Jackman et al.
2009). When maintained in darkness, the sustained release rate
is thus determined by the rate at which vesicles replenish the
readily releasable pool, not the frequency of individual Ca2⫹
channel openings (Jackman et al. 2009). Linking the rate of
release to replenishment rather than individual channel openings may reduce the synaptic noise that can result from stochastic channel openings. By contrast, when photoreceptors are
strongly hyperpolarized in bright light, Ca2⫹ channel openings
occur principally when the cell depolarizes in response to a
light decrement. In this situation, positioning Ca2⫹ channels
close to release sites may allow fusion to be closely synchronized with Ca2⫹ channel opening, thus enhancing fidelity and
temporal precision in the conversion of membrane potential
changes to synaptic release. The combination of high Ca2⫹
affinity in the release mechanism with a close proximity to
Ca2⫹ channels may allow the synapse to switch between
CALCIUM CHANNELS AT THE PHOTORECEPTOR RIBBON SYNAPSE
Significance of Ca2⫹/Cl(Ca) channel relationships
Ca2⫹ influx through voltage-gated Ca2⫹ channels activates
Cl(Ca) channels and activation of Cl(Ca) channels can in turn
influence Ca2⫹ channel activity (Thoreson et al. 1997, 2000).
In rods, ECl averages ⫺20 mV, so activation of ICl(Ca) results in
a Cl⫺ efflux. Although the membrane depolarization that
results from Cl⫺ efflux stimulates ICa, this stimulatory effect is
countered by a simultaneous strong inhibitory influence of Cl⫺
efflux whereby reductions in intracellular Cl⫺ act at anion
binding sites on individual Ca2⫹ channels to directly inhibit
channel open probability (Babai et al. 2010; Thoreson et al.
2000). CaV1.4 Ca2⫹ channels, which are the principal subtype
in rods, exhibit slow calcium-dependent inactivation (Baumann et al. 2004; Koschak et al. 2003; McRory et al. 2004).
Inhibition of ICa by negative feedback from ICl(Ca) activity may
provide an alternative mechanism to limit excessive Ca2⫹
entry.
ECl in cones averages ⫺38 mV, close to the dark resting
potential (Thoreson and Bryson 2004). ICl(Ca) evoked by strong
depolarizing steps was smaller in cones than that in rods,
suggesting a lower Cl(Ca) current density. Thus Cl(Ca) channel activation is likely to produce only small changes in
intracellular Cl⫺ levels that will have only a small effect on
Ca2⫹ channel open probability. The activation of Cl(Ca) channels in cones may instead be more important for stabilizing the
membrane potential near its dark resting value (Maricq and
Korenbrot 1988).
The present results suggest that Ca2⫹ influx through an
individual Ca2⫹ channel could potentially influence the activity
of Cl(Ca) channels hundreds of nanometers away and thereby
indirectly influence the activity of relatively distant Ca2⫹
J Neurophysiol • VOL
channels. Such long-range interactions may be boosted by
calcium-induced calcium release in rod terminals (Babai et al.
2010; Cadetti et al. 2006; Krizaj et al. 1999, 2003; Suryanarayan and Slaughter 2006). Regulation of relatively distant
Ca2⫹ channels by the activation of Cl(Ca) channels may help
to ensure similarity in the behavior of Ca2⫹ channels located at
different positions along the nearly 1 ␮m long synaptic ridge in
rods. In amphibian rods with multiple ribbons, long-range
interactions might also help to reduce functional heterogeneity
between channels at neighboring synaptic ribbons (Frank et al.
2009; Meyer et al. 2009).
GRANTS
This work was supported by Research to Prevent Blindness, National Eye
Institute Grants EY-10542 to W. B. Thoreson and EY-15573 to N. C. Brecha,
National Institutes of Health American Recovery and Reinvestment Act award
to support G. E. Riccardi (to N. C. Brecha), a Senior Career Scientist Award
from the Department of Veterans Affairs to N. C. Brecha, Fight for Sight grant
to K. Rabl, and a University of Nebraska Medical Center Graduate Student
Fellowship to A. Mercer.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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