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The Journal of Neuroscience, April 15, 1999, 19(8):2938-2944
The Metabotropic Receptor mGluR6 May Signal Through
Go, But Not Phosphodiesterase, in Retinal Bipolar Cells
Scott
Nawy
Departments of Ophthalmology and Visual Science, and Neuroscience,
Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
Bipolar cells are retinal interneurons that receive synaptic input
from photoreceptors. Glutamate, the photoreceptor transmitter, hyperpolarizes On bipolar cells by closing nonselective cation channels, an effect mediated by the metabotropic receptor mGluR6. Previous studies of mGluR6 transduction have suggested that the receptor couples to a phosphodiesterase (PDE) that preferentially hydrolyzes cGMP, and that cGMP directly gates the nonselective cation
channel. This hypothesis was tested by dialyzing On bipolar cells with
nonhydrolyzable analogs of cGMP. Whole-cell recordings were obtained
from On bipolar cells in slices of larval tiger salamander retina.
Surprisingly, On bipolar cells dialyzed with 8-(4-chlorophenylthio)-cyclic GMP (8-pCPT-cGMP), or 8-bromo-cyclic GMP
(8-Br-cGMP) responded normally to glutamate or
L-2-amino-4-phosphonobutyrate (L-APB). Response
amplitudes and kinetics were not significantly altered compared
with cells dialyzed with cGMP alone. Comparable results were obtained
with the PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) or with
8-pCPT-cGMP and IBMX together, indicating that PDE is not required for
mGluR6 signal transduction. Addition of the G-protein subunit
Go to the pipette solution suppressed the cation current
and occluded the glutamate response, whereas dialysis with
Gi or with transducin G  had no significant effect
on either the cation current or the response. Dialysis of an antibody
directed against Go also reduced the glutamate response,
indicating a functional role for endogenous Go. These
results indicate that mGluR6 may signal through Go,
rather than a transducin-like G-protein.
Key words:
cGMP; 8-pCPT-cGMP; mGluR6; phosphodiesterase; retinal
bipolar cell; Go; phosphodiesterase
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INTRODUCTION |
The synapse between photoreceptors
and On bipolar cells is the first in the visual system. Glutamate, the
photoreceptor transmitter, hyperpolarizes On bipolar cells via
activation of a G-protein-coupled receptor (Nawy and Jahr, 1990 ;
Shiells and Falk, 1990). This receptor was first cloned in rat and
classified as mGluR6 (Nakajima et al., 1993), one of the group III
metabotropic receptors that are distinguished by their high affinity
for L-2-amino-4-phosphonobutyrate (L-APB)
(Nakanishi, 1994; Pin and Duvoisin, 1995), confirming results of
pharmacological studies in retina (Shiells et al., 1981; Slaughter and
Miller, 1981, 1985). Receptor activation by L-APB,
or the photoreceptor transmitter, suppresses a cation current (Shiells
et al., 1981; Attwell et al., 1987; Nawy and Copenhagen, 1987).
Introduction of cGMP into the cell through a patch pipette increases
the amplitude of this current, leading to the suggestion that the
current is generated by a cyclic nucleotide-gated channel (Nawy and
Jahr, 1990; Shiells and Falk, 1990; de la Villa et al., 1995), although
this has yet to be confirmed by molecular cloning or direct activation
of the channel in isolated patches. The channel may additionally be
regulated by Ca2+/calmodulin-dependent kinase
(Walters et al., 1998).
The mGluR6 receptor is thought to signal through a G-protein to a
cGMP-preferring phosphodiesterase (PDE) (Nawy and Jahr, 1990; Shiells
and Falk, 1990; Thoreson and Miller, 1994). Activation of PDE would
lead to hydrolysis of cGMP and closure of the channel, reminiscent of
the phototransduction cascade. Support for this model comes from the
observation that the G-protein linking the receptor to the effector
enzyme is sensitive to both pertussis and cholera toxin (Shiells and
Falk, 1992a), as is rod transducin, but other studies instead suggest a
role for Go in the mGluR6 cascade. Antibodies directed
against Go, but not transducin, label dendrites of On
bipolar cells (Vardi et al., 1993; Vardi, 1998), and mGluR6 couples
more efficiently to Go than to transducin in transfected
cells (Weng et al., 1997). If Go is part of the cascade,
then there might be an additional step linking Go to PDE,
because Go is not known to couple directly to
phosphodiesterases. Alternatively, PDE may not have any role in the
cascade. The present study was undertaken to distinguish between these possibilities.
One prediction of the PDE model is that analogs of cGMP that are
resistant to hydrolysis should be able to support the cation current
but should prevent glutamate from shutting off this current. Here it is
shown that dialysis with 8-bromo-cyclic GMP (8-Br-cGMP) or
8-(4-chlorophenylthio)-cyclic GMP (8-pCPT-cGMP), or with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), does
not significantly inhibit the response to glutamate, compared with
cells dialyzed with cGMP alone. Furthermore, introduction into the cell
of either Go or an antibody directed against
Go interferes with mGluR6 transduction. Thus neither PDE
nor a transducin-like G-protein appear to be essential elements in this
transduction pathway.
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MATERIALS AND METHODS |
Materials. Slices of retina from larval tiger
salamanders (Kons Scientific, Germantown, WI) were prepared as
described previously (Nawy and Jahr, 1990; Walters et al., 1998).
Briefly, salamanders were anesthetized with 3-aminobenzoic acid ethyl
ester and decapitated, and the eyes were enucleated. Whole retinas were
isolated and placed on a 0.65 µm cellulose acetate/nitrate membrane
filter (Millipore, Bedford, MA) that was secured with vacuum grease to a glass slide adjacent to the recording chamber. Slices were then cut
to a thickness of 150-200 µm with a tissue slicer (Stoelting, Wood
Lane, IL), transferred to the recording chamber while remaining submerged, and viewed with a Zeiss (Thornwood, NY) Axioskop equipped with a water-immersion 40× objective with Hoffman modulation contrast (Modulation Contrast, Greenvale, NY). The extracellular solution contained (in mM): 108 NaCl, 2 CaCl2,
2.5 KCl, 10 HEPES, 10 glucose, 0.1 picrotoxin, pH 7.6, and was perfused
continuously through the chamber at ~1 ml/min. The internal solution
was composed of 75 KH2PO4, 10 KCl, 10 HEPES, 10 EGTA, 4 MgATP, 1 cGMP, and 0.5 NaGTP, pH 7.4 with KOH. In the
experiments summarized in Figure 6, K+-gluconate (90 mM), was used instead of KH2PO4. To
block K+ currents during measurements of
I-V plots, 20 mM tetraethylammonium (TEA) Cl
was substituted for NaCl in the extracellular solution, and for
KH2PO4 in the pipette solution on an equimolar
basis. ATP, GTP, cGMP, 8-Br-cGMP, and 8-pCPT-cGMP were dissolved in
water as 100× stocks, aliquoted for single experiments, and stored
frozen. A concentrated stock (200-500 mM in DMSO) of IBMX
was stored frozen and added to the internal or external solution on the
day of the experiment. Cyclosporin A was stored at 4°C in ethanol as
a 2 mM stock. Go and Gi were
aliquoted and stored in buffer at  80°C. G was stored at
20°C in 10 mM HEPES, pH 7.0, 2 mM
MgCl2, 1 mM -mercaptoethanol, and
50% glycerol, and was added to the pipette solution at a dilution of
1:400. Anti-Go (1 mg/ml) was stored at 4°C and diluted
to a final concentration of 30 µg/ml in pipette solution immediately
before use. All compounds listed above were obtained from Sigma (St.
Louis, MO) except 8-Br-cGMP and 8-pCPT-cGMP (Biolog, La Jolla, CA),
Go and Gi (Calbiochem, San Diego, CA), transducin G (a gift of Dr. Thomas Sakmar, Rockefeller
University), and anti-Go (Chemicon, Temecula CA).
Electrophysiology. Patch pipettes were fabricated from
borosilicate glass (WPI, Sarasota, FL) using a two-stage vertical
puller (Narishige, Sea Cliff, NY) and were fire-polished to resistances of 2-3 M . Whole-cell recordings were obtained with an Axopatch 200A
amplifier (Axon Instruments, Foster City, CA), and had input and series
resistance values of ~1 G and 10-19 M, respectively. Cells
were discarded if the series resistance exceeded 20 M, if the
holding current changed suddenly, or if the holding current during the
first application of agonist exceeded 20 pA (i.e., current measured
while the sustained inward current was suppressed). Drugs were applied
via two polymer-coated fused silica tubes (outer diameter 350 µm,
inner diameter 250 µm; Polymicro Technologies, Phoenix, AZ)
containing external control solution, or agonist, usually glutamate (1 mM), or L-APB (2 µM). The tubes
were mounted to a computer-controlled piezo-bimorph (Morgan-Matroc,
Bedford, OH). Both barrels of the apparatus were supplied by two
separate reservoirs, each with its own control valve, allowing
application of IBMX with a delay of ~15 sec, without repositioning
the barrels. After establishment of whole-cell recording, cells were
typically voltage clamped at 40 or 30 mV and immediately perfused
with control solution. Agonist was applied every 30 sec for a duration of 5 sec beginning 30 sec after breaking into the cell. Data were acquired with Axobasic software and the Digidata 1200 interface (Axon
Instruments) and analyzed with Kaleidagraph (Synergy Software, Reading, PA).
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RESULTS |
Both cGMP and nonhydrolyzable analogs of cGMP potentiate the
response to glutamate
On bipolar cells, identified by their characteristic outward
responses to glutamate and L-APB, were recorded in
solutions that contained ATP and GTP but no cyclic nucleotides. Under
these conditions, there was a small outward response to glutamate that did not change appreciably during the first several minutes after the
starting of the recording (Fig.
1A). The glutamate
response results from the suppression of a sustained cation current
(Nawy and Jahr, 1990; Shiells and Falk, 1990; de la Villa et al., 1995) and is mediated by mGluR6 (Nomura et al., 1994). Addition of cGMP to
the pipette solution enhanced the cation current and increased the
amplitude of the glutamate response (Fig. 1B),
suggesting that cGMP either modulates or gates the underlying channels
directly. Glutamate may suppress the cation current by decreasing cGMP
levels through the activation of a cGMP-hydrolyzing PDE (Nawy and Jahr, 1990, 1991; Shiells and Falk, 1990; Shiells and Falk, 1992a; Thoreson and Miller, 1994; de la Villa et al., 1995). Substitution of cGMP with
nonhydrolyzable cGMP analogs would be expected to eliminate or reduce
suppression by agonist. Accordingly, two 8-substituted cGMP analogs,
8-Br-cGMP and 8-pCPT-cGMP, were added to the pipette in separate
experiments. 8-Br-cGMP is poorly hydrolyzed by PDE and has a higher
affinity for cGMP-gated channels in rods than cGMP (Zimmerman et al.,
1985), whereas 8-pCPT-cGMP is virtually nonhydrolyzable and has an even
higher affinity for the rod channel (Kramer and Tibbs, 1996). Neither
8-pCPT-cGMP (Fig. 1C) nor 8-Br-cGMP (Fig.
1D) prevented suppression by glutamate. Instead, both
increased the glutamate response by potentiating the cation current, as would be expected if the analogs mimic the effect of cGMP.
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Figure 1.
The glutamate response is potentiated by cGMP even
when hydrolysis of cGMP is inhibited. A, Whole-cell
recording of an On bipolar cell in the tiger salamander retinal slice.
The pipette solution contains ATP and GTP but not cGMP. The amplitude
of the response to 1 mM glutamate is unchanged during the
first 3 min of recording. Thick trace is 30 sec
response; thin trace is 3 min response.
B, Recording of another On bipolar cell in which the
pipette solution contains 1 mM cGMP. The amplitude of the
glutamate response is potentiated. C, D, The glutamate
response was similarly potentiated when the pipette contained either
250 µM 8-pCPT-cGMP or 1 mM 8-Br-cGMP. In
B and D the 3 min traces were shifted
slightly for comparison with the 30 sec traces. Holding potential for
all cells was 40 mV. E, Summary of the effects of cGMP
analogs on the agonist response 30 sec after breaking into the cell
(black bars) and after 3 min of dialysis (striped
bars). Error bars represent SEM in this and the following
figures. Asterisks indicate significance between each
condition and control at p < 0.01 level after 3 min (unpaired t test). Numbers in
parentheses indicate the number of cells in each group. Data
obtained with 1 mM glutamate and with L-APB
(2-5 µM) have been pooled because no differences were
observed.
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These data are summarized in Figure 1E. The black
bars are the mean amplitudes of the glutamate response soon after
beginning of the recording with the indicated solution. As early as 30 sec there was a slight potentiation of the response with all three analogs, compared with control (no cGMP). After ~3 min (striped bars), the responses in cGMP or its analogs were not significantly different from each other, but all three differed significantly from
control (p < 0.01).
It is possible that the concentrations of the analogs, which are both
membrane-permeant, are insufficient to reach the dendrites where the
channels are presumably located, although it is clear that the levels
are sufficient to enhance the cation current. To rule this out,
8-pCPT-cGMP (100 µM) was added to the control and
glutamate flowpipes as well as the pipette solution (250 µM). No inhibition of the agonist response was observed
(n = 3 cells; data not shown).
Suppression of the cation current is not prevented by the PDE
inhibitor IBMX
Further evidence that PDE is not required for current suppression
was obtained by dialyzing cells with IBMX along with cGMP. IBMX did not
prevent cells from responding to glutamate or prevent cGMP from
increasing the amplitude of the response (Fig.
2A). In separate
experiments, IBMX was applied to the outside of On bipolar cells so
that its effect on cation current and agonist responses could be
compared within a single cell. An example of the effects of
extracellular application of IBMX is illustrated in Figure
2B, which is a composite of nine trials, each lasting 15 sec and separated by an additional 15 sec period. At the time indicated by the solid line, IBMX was applied by switching the flowpipes from reservoirs containing control and glutamate solution to
reservoirs containing IBMX and IBMX with glutamate. Rather than
inhibiting the glutamate response, IBMX increased its amplitude as it
augmented the cation current, as indicated by the dashed line. The
effects of IBMX on inward current and on the glutamate response in four
cells are summarized in the right panel of Figure 2B.
These findings indicate the presence of basal PDE activity, whose
inhibition increases the effective cGMP concentration but whose
activity is not required for suppression of the inward current.
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Figure 2.
Inhibition of PDE does not prevent suppression of
the cation current by glutamate. A, Left,
Glutamate responses at 30 sec (thick trace) and 3 min
(thin trace) after breaking into an On bipolar cell
dialyzed with 500 µM GTP, 1 mM cGMP, and 250 µM IBMX. IBMX did not inhibit the response to glutamate.
Right, Summary of the effect of dialysis of IBMX (250 or
500 µM) on the agonist response 30 sec after breaking
into the cell (black bars) and after 3 min of dialysis
(striped bars). Control and cGMP data are from Figure 1.
B, The glutamate response persists during extracellular
application. Left, Response of an On bipolar cell to
external application of 1 mM IBMX. At the time indicated by
the solid line, the flowpipes were switched to
reservoirs containing IBMX, and IBMX + glutamate. Each record is 15 sec
long and is separated from the next record by an additional 15 sec.
Right, Summary of the increase in holding current and
the agonist response produced by IBMX (n = 4 cells). C, Recording of an On bipolar cell dialyzed with
500 mM IBMX and externally perfused with 1 mM
IBMX for the duration of the recording. The response to glutamate is
unaffected. Similar results were obtained in two other cells. Holding
potential for all three cells illustrated in A-C was
40 mV.
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To insure that IBMX reached the distal dendrites where the transduction
machinery is presumably located, IBMX was added to the pipette and the
extracellular solution. In three of three cells tested, one of which is
illustrated in Figure 2C, IBMX did not inhibit the response
to glutamate.
After 10-15 min of recording under a number of conditions that are
highly unfavorable for the hydrolysis of cGMP, responses were still not
diminished compared with cells dialyzed in cGMP (summarized in Fig.
3A). Furthermore, at the level
of resolution afforded by the drug application system, there was
essentially no change in the response rise time throughout the duration
of the recording in cells dialyzed with either 8-pCPT-cGMP or IBMX (Fig. 3B). Taken together, these results provide strong
evidence that hydrolysis of cGMP is not an obligatory step in the
mGluR6 transduction pathway.
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Figure 3.
Prolonged inhibition of phosphodiesterases does
not block or alter the kinetics of the agonist response.
A, Comparison of the amplitude of the agonist
(L-APB or glutamate) after 10 min of dialysis with no cGMP,
with cGMP, or with solutions designed to inhibit cGMP
phosphodiesterase. Responses under all conditions continued to be
significantly greater than control (p < 0.01, unpaired t test). B, Glutamate
responses after 30 sec and 15 min of recording in On bipolar cells
dialyzed with 500 µM GTP, 1 mM cGMP, and 250 µM IBMX (left) and in another cell
dialyzed with 500 µM GTP and 250 µM
8-pCPT-cGMP (right). Glutamate was applied, after a 2 sec delay, for a period of 5 sec (indicated by the continuous
line). In each panel the amplitude of the response after 15 min
(thin line traces) was scaled to the response after 30 sec of dialysis (thick line trace). The kinetics of the
agonist response, at this degree of temporal resolution, are unchanged
by prolonged dialysis with either IBMX or 8-pCPT-cGMP.
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Evidence that Go is involved in the
transduction pathway
Antibodies directed against Go label On bipolar cell
dendrites, and the labeling is colocalized with mGluR6 antibody
labeling (Vardi et al., 1993; Vardi, 1998). Furthermore, mGluR6 couples much more efficiently to Go than to rod transducin in
transfected cells (Weng et al., 1997). To test for a functional role of
Go in mGluR6 transduction, cells were dialyzed with cGMP
and the Go subunit. After 3 min of whole-cell recording,
the agonist response had increased, as expected in the presence of
cGMP. Soon after, the response began to decline, and after 10 min, the
response was smaller than it was initially (Fig.
4A). The averaged time course of the response of cells dialyzed with cGMP alone (control) and
those dialyzed with cGMP and Go is summarized in Figure
4B. Initially the average response size in both
groups is nearly the same. After a delay of several minutes, the
response in cells dialyzed with the Go was significantly
depressed compared with control, although the control cells did run
down slightly as well. This delay is presumably caused by the
relatively slow rate of diffusion of the 35 kDa subunit out into
the distal dendrites.
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Figure 4.
Go inhibits the glutamate response.
A, Example of the effect of dialysis of
Go on an On bipolar cell. Cell was dialyzed with 64 nM Go along with 1 mM cGMP added
to the normal internal solution. After 3 min the response is enhanced,
because of the presence of cGMP (compare left and
center panels). Later, after allowing for diffusion of
Go into the cell, the response is significantly
depressed (right panel). B,
Comparison of the time course of inhibition in cells dialyzed with cGMP
( ) or with cGMP and 42 to 62 nM Go ( ).
C, Summary of responses in cells dialyzed for 10 min
with Go, G (60 nM), or
Gi (61 nM) along with cGMP. Neither G
nor Gi had any significant effect compared with cGMP
alone.
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Studies of mGluR6 in expression systems have suggested that mGluR6 can
couple negatively to adenylate cyclase through Gi (Nakajima et al., 1993; Nakanishi, 1994; Laurie et al., 1997). However, dialysis of Gi had no significant effect on the response
to glutamate (Fig. 4C), suggesting that mGluR6 does not
signal through this pathway in the native cell.
It is possible that mGluR6 signals through Go, but it is
G that provides the relevant signal in this cascade. Addition of
exogenous Go might prevent signaling by buffering
endogenous G subunits. To test this possibility, cells were
dialyzed with transducin (kindly provided by Dr. Thomas Sakmar).
At three times the concentration necessary to modulate
Ca2+ currents (Diverse-Pierluissi et al., 1995;
Zamponi et al., 1997), G had no effect on the glutamate response
(Fig. 4C). However, other isoforms will need to be
tested to rule out a role for transduction in this pathway.
Inhibition of the glutamate response by Go would result
if the subunit either prevented suppression of the cation current or
shut off the current, essentially occluding the glutamate response. To
distinguish between these two possibilities, current-voltage (I-V) measurements were obtained immediately
after starting the whole-cell recording, and 10 min later, when the
glutamate response was diminished and the subunit presumably had
reached the dendrites. In this experiment, the pipette and bath
solutions contained 20 mM TEA to reduce
K+ currents. The holding potential was stepped from
70 to +20 mV for 500 msec, as shown in Figure
5A, and the holding current
obtained at the end of the step was plotted (Fig. 5C).
Glutamate was applied to the cell, and the procedure was repeated.
Glutamate dramatically reduced membrane conductance, the small residual
current most likely consisting of a mixture of Cl
and nonspecific leak. The reversal potential for the current suppressed
by glutamate was 5.4 mV in this cell, in good agreement with previous
measurements (Attwell et al., 1987; Nawy and Jahr, 1991; Yamashita and
Wassle, 1991; de la Villa et al., 1995). After 10 min of dialysis,
membrane conductance was dramatically reduced. Application of glutamate
further decreased membrane conductance, but only minimally (Fig.
5B,D). The currents suppressed by dialysis with
Go (Fig. 5E, closed symbols) and
by glutamate (open triangles) had similar reversal
potentials. In all cells, the mean reversal potentials of the current
suppressed by Go (mean: 6.2 ± 2) and by
glutamate (mean: 4.3 ± 2.5 mV; n = 10) were
nearly identical, providing additional evidence that both acted
downstream to close the same population of channels.
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Figure 5.
Go and glutamate may close the same
population of channels. A, Internal and external
solutions throughout this figure contained 20 mM TEA to
reduce K+ conductances. Immediately after breaking
into an On bipolar cell with a pipette solution containing 64 nM Go and 1 mM cGMP, the holding
potential was stepped from 70 to +20 mV in 10 mV increments for 500 msec. Each step was separated by a period of 2 sec, during which the
cell was held at 30 mV. Glutamate was then applied continuously to
the cell, and the step protocol was repeated. B, After
10 min of dialysis, the procedure was repeated, except that the cell
was held at 40 between steps. C, Plot of the data
shown in A. Continuous lines are the
least-squares fit to the data with an inverse slope of 268 M (30 sec
control) and 826 M (glutamate). D, Plot of the data
shown in B. Continuous lines are the
least-squares fit to the data with an inverse slope of 690 M (10 min
perfusion with Go) and 847 M (glutamate).
E, Plots of the current suppressed by glutamate after 30 sec ( ) and after dialysis with Go for 10 min ( ).
Plots were obtained by taking the difference between the glutamate and
control data points in C and D. Dialysis
with Go decreased the slope conductance of the
glutamate-suppressed current from 2.52 to 0.27 nS. The current
suppressed by Go is also shown ( ). Plot is the
subtraction of the 10 min from the 30 sec control
I-V.
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To examine the possibility that the endogenous G-protein is related to
Go, cells were dialyzed with a monoclonal antibody that has previously been shown to inhibit Go function
(Kim et al., 1998). This antibody (clone 2A), raised against partially purified bovine brain Go, reacts with Go
but not with Gi or Gs (Li et al., 1995).
Figure 6 illustrates the effect of
dialysis with 30 µg/ml anti-Go on the agonist
response. The left panel is the average of four trials recorded
immediately after breaking into an On bipolar cell. The right panel is
the average of four traces acquired after 20 min of dialysis with the
antibody. The amplitude of the response was reduced, although the
baseline current was relatively unchanged, indicating that dialysis of
the antibody inhibited G-protein-mediated suppression of the cation
current but alone did not directly alter or occlude the current.
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Figure 6.
An antibody to Go reduces function
of the endogenous G-protein. All data in this figure were obtained
using the gluconate-based solution (see Materials and Methods)
supplemented with 1 µM Cyclosporin A, which slowed
rundown of the response. A, This cell was dialyzed with
30 µg/ml anti-Go (clone 2A, obtained from Chemicon).
Each panel is the average of four consecutive trials. The
continuous line indicates the timing of the application
of 1 mM glutamate. Left panel, The first of
the four averaged traces was obtained ~30 sec after establishing
whole-cell access. Right panel, The first trace was
obtained after 19 min of recording. B, Mean and SE of
responses to glutamate in control cells (n = 10)
and cells dialyzed with antibody (n = 9).
C, Summary of the mean responses after 20 min of
recording in control (internal solution alone) cells, cells dialyzed
with antibody, and cells dialyzed with antibody that had been
inactivated by heating at 70°C for 4 min. Asterisk
indicates significance of p < 0.01 compared with
control.
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In 9 of 10 cells dialyzed with anti-Go, there was a
pronounced time-dependent depression of the agonist response compared with cells that were recorded with the same gluconate-based internal solution but with no antibody (Fig. 6B). On average,
the antibody reduced the responses to ~50% of control. As a further
control, cells were dialyzed with antibody that had been
heat-inactivated (70°C for 4 min). In these cells, the size of the
response after 20 min was not significantly different from control
cells (Fig. 6C). These results support the idea that an
endogenous Go-like G-protein mediates suppression of
cation current by mGluR6.
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DISCUSSION |
Evaluation of the existing model of mGluR6 transduction
According to the existing model of mGluR6 transduction, the
postsynaptic channel is gated by cGMP, and mGluR6 activation lowers cGMP levels by activating a cGMP-hydrolyzing PDE, closing the synaptic
channel and hyperpolarizing the dendrites. Evidence presented in this
study strongly suggests that PDE does not have an obligatory role in
the mGluR6 transduction cascade of On bipolar cells. Bipolar cells
continued to respond to glutamate when dialyzed with nonhydrolyzable analogs of cGMP, with the PDE inhibitor IBMX, or with both. When compared with cells dialyzed only with cGMP, there was no difference in
the amplitude or the kinetics of the response throughout the recording.
In contrast, dialysis of these and similar compounds into
photoreceptors profoundly alters the amplitude and kinetics of light
responses (Zimmerman et al., 1985; Sather and Detwiler, 1987).
Because bipolar cells continue to respond to glutamate when
intracellular cGMP levels are fixed, it seems clear that a change in
cGMP is not the signal for channel closure. Does this mean that cGMP
does not gate the channel? One possibility is that the channel may
require cGMP to open but is closed through a separate, cGMP-independent
process, such as a direct interaction with Go. Alternatively, the channel may be modulated, but not gated by cGMP, and
might more closely resemble the recently cloned family of
hyperpolarization-activated cation channels, which also show modulation
by cyclic nucleotides (Gauss et al., 1998; Ludwig et al., 1998; Santoro
et al., 1998), than cyclic nucleotide-gated (CNG) channels. It is clear
that this cation channel has properties that are functionally distinct
from known CNG channels. The On bipolar cell cation channel is
not blocked by physiological concentrations of Ca2+
(Shiells and Falk, 1992b) as CNG channels are (Frings et al., 1995). As
a result, the open probability of the On bipolar cell cation channel is
thought to exceed 90% in the presence of cGMP and physiological
concentrations of Ca2+ (de la Villa et al., 1995),
whereas CNG channels in rods spend ~1% of the time in the open state
(Yau and Baylor, 1989). Furthermore, antibodies against known CNG
channels do not label On bipolar cells (Wassle et al., 1992). However,
none of these studies directly address the issue of channel gating, and
questions about the relative homology of this channel with CNG or
hyperpolarization-activated channels can only be answered when more
detailed and quantitative information about the properties of the
channel becomes available.
The evidence for PDE as the effector enzyme in the On bipolar cell
pathway was based primarily on the observation that IBMX increases the
size of the cation current (Nawy and Jahr, 1990; Shiells and Falk,
1990), and that IBMX, when applied together with agonist, appeared to
inhibit responses to low but not high concentrations of agonist (Nawy
and Jahr, 1990). An alternative hypothesis is that basal PDE activity
is present, that inhibition of this PDE augments the size of the
current, and that a saturating concentration of agonist can suppress
this added current, as was demonstrated in this study. Application of
subsaturating concentrations of agonist, followed by agonist and IBMX,
would increase the inward current. Superimposed on the agonist
response, this might be interpreted as a decrease in the agonist response.
An alternative model: transduction via Go
Two lines of evidence suggest that mGluR6 may couple through
Go. First, comparison of the I-V relations
obtained at the start of recording, during application of glutamate,
and after 10 min of dialysis suggests that Go may close the
same population of cation channels that are closed by the endogenous
G-protein during the agonist response. No effect on these channels was
observed during dialysis of Gi or transducin G.
Second, dialysis of an antibody that blocks function of
Go inhibits the agonist response. The simplest model of
mGluR6 transduction that can account for these observations is that
mGluR6 couples to Go, a finding that is consistent
with recent studies showing that antibodies directed against
Go label On bipolar cell dendrites, and the labeling is
colocalized with mGluR6 antibody labeling (Vardi et al., 1993; Vardi,
1998). Recently, mGluR6 has been shown to couple much more efficiently
to Go than to rod transducin in transfected cells (Weng et
al., 1997). Cation currents in various cells are modulated by
activation of metabotropic (Crepel et al., 1994; Guerineau et al.,
1995; Batchelor and Garthwaite, 1997; Congar et al., 1997; Tempia et
al., 1998) and muscarinic (Inoue and Isenberg, 1990; Kim et al., 1998)
receptors. In many cases there is evidence for the involvement of a
pertussis-sensitive G-protein.
The mechanism by which Go closes the cation channel remains
to be elucidated. It is well established that Go can
inhibit ion channels via a membrane-delimited pathway (Hille, 1994;
Dolphin, 1998). Recent studies of G-protein regulation of N-type
Ca2+ channels (Herlitze et al., 1996; Ikeda, 1996)
and rectifying K+ channels (Logothetis et al., 1987;
Wickman et al., 1994) indicate that many, if not all, of these
membrane-delimited effects of Go are mediated by G
rather than Go. In the present study, injection of
transducin was unable to mimic the effect of glutamate. It might
have been anticipated that exogenous G would have buffered endogenous Go and prevented signaling, but this was not
observed. The reason for this is unclear but may be caused by a surplus of endogenous Go. It will be necessary to test other subunits before a role for G in mGluR6 signaling can be defined.
The simplest model that can account for the present results is a direct interaction of the channel with Go or perhaps G,
but these results do not rule out the possibility that there are
additional steps in the cascade.
| |
FOOTNOTES |
Received Aug. 31, 1998; revised Jan. 13, 1999; accepted Feb. 1, 1999.
This work was supported by National Institutes of Health Grant EY10254,
and by an unrestricted grant from Research to Prevent Blindness
Inc. I thank Drs. Alejandro Peinado and Eric Wexler for helpful discussions.
Correspondence should be addressed to Dr. Scott Nawy, Kennedy Center
Room 525, Albert Einstein College of Medicine, 1410 Pelham Parkway
South, Bronx, NY 10461.
| |
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ABSTRACT
INTRODUCTION
Compound via MeSH
