Departments of Physiology and Biophysics and Ophthalmology, School of Medicine, State University of New York, Buffalo, New York 14214
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ABSTRACT |
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Shen, Wen and Malcolm M. Slaughter. Internal Calcium Modulates Apparent Affinity of Metabotropic GABA Receptors. J. Neurophysiol. 82: 3298-3306, 1999. The metabotropic GABA receptor (GABABR) regulates calcium influx in neurons. Whole cell voltage-clamp techniques were employed to determine the effects of internal calcium on the activity of GABABRs. GABABR receptor apparent affinity was maximal when bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) maintained internal calcium below 70 nM. Apparent affinity was reduced as internal calcium increased. EGTA did not produce similar effects, suggesting that localized increases in calcium influenced GABABR apparent affinity. Confocal imaging disclosed relatively high internal calcium just below the plasma membrane of isolated neurons. BAPTA, but not EGTA, reduced this ring of high calcium. Heparin, dantrolene, and ryanodine increased GABABR apparent affinity, effects similar to that of BAPTA. Calmodulin inhibitors also increased receptor apparent affinity. These results suggest that internally released calcium activates calmodulin, which reduces GABABR apparent affinity. This identifies a reciprocal system in which the metabotropic GABA receptor can reduce calcium influx, but internal calcium can suppress this receptor pathway. Metabotropic glutamate receptors linked to inositol 1,4,5 trisphosphate (InsP3) raised internal calcium and suppressed the action of GABABRs. Thus negative feedback systems control the balance between excitatory and inhibitory metabotropic receptor pathways in retinal neurons.
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INTRODUCTION |
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The balance between excitatory, glutamatergic synapses, and inhibitory, GABAergic synapses determines the excitability of many neurons in the CNS. Commonly, this interaction can be described by the relative conductances produced by these two transmitters acting at ionotropic receptors. Both transmitter systems also activate metabotropic receptors, yet little is known about interactions through second-messenger systems. Retinal ganglion cells are the final integrators of visual information in the retina, and they perform this function by combining the inputs from a number of receptors, including ionotropic and metabotropic receptors for both GABA and glutamate. Thus they are an ideal substrate for studying interactions between excitatory and inhibitory pathways.
Metabotropic GABA receptors (GABABRs) play a key
role in the regulation of intraneuronal calcium. These receptors
modulate a variety of high-voltage-activated calcium channels, most
prominently the N- and L-types (Dolphin and Scott 1986;
reviewed by Misgeld et al. 1995
). In amphibian retinal
ganglion cells, GABABRs reduce calcium influx by
inhibiting N-type calcium channels (Zhang et al. 1997
).
In contrast, these ganglion cells also possess metabotropic glutamate
receptors that raise internal calcium through activation of inositol
trisphosphate (Akopian and Witkovsky 1996
; Shen
and Slaughter 1998
). Thus these two metabotropic
transmitter systems may have opposing effects on internal calcium.
But calcium is not a passive participant in the actions of guanine
nucleotide binding proteins (G-protein) receptors. Through activation
of calmodulin, calcium can cause a suppression of G-protein-coupled receptor kinases (GRKs) or influence the activity of G-proteins directly (Levay et al. 1998; Liu et al.
1997
; Pronin et al. 1997
). Thus the
regulation of internal calcium by one metabotropic receptor may
influence the properties of heterologous receptors. Therefore the
effects of internal calcium on GABABR function were examined.
The experiments indicate that elevated levels of internal calcium
suppress the action of GABABRs in retinal ganglion cells. This suppression is initiated by local calcium release from inositol trisphosphate and ryanodine-sensitive stores, which stimulates calmodulin. Metabotropic glutamate receptors can generate this release
of internal calcium (Akopian and Witkovsky 1996;
Shen and Slaughter 1998
) and can inhibit the action of
GABABRs. These results indicate that there are reciprocal
negative feedback pathways between excitatory and inhibitory
metabotropic pathways and that the interactions may be critical in
determining the relative weighting of synaptic signals.
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METHODS |
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Retinal preparation
Experiments were performed on acutely isolated neurons from the
tiger salamander retina, Ambystoma tigrinum (Kons
Scientific, Germantown, WI) using methods previously described in
detail (Bader et al. 1978). The procedures conformed to
the guidelines set forth by the National Institutes of Health Guide for
the Care and Use of Laboratory Animals. Briefly, the animals were
decapitated and pithed, then the eye was removed and the retina was
isolated. The retina was incubated for ~30-60 min at room
temperature (22°C) in 400 µl of enzyme solution containing papain
(12U/ml papain, Worthington Biochemicals, Freehold, NJ) and 5 mM
L-cysteine in amphibian Ringer solution. Subsequently, the
retina was rinsed five times with amphibian Ringer solution,
transferred to calcium-free Ringer solution, and shaken until the
tissue dissociated. The cells were placed on a lectin-coated cover slip
and stored in Ringer solution at 17°C. Experiments were performed on
acutely isolated cells, usually within 5 h of dissociation.
Retinal cells were superfused at room temperature with amphibian Ringer solution consisting of (in mM) 111 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 dextrose, and 5 HEPES buffered at pH 7.8. To examine calcium channel currents, 10 mM BaCl2 and 40 mM tetraethylammonium chloride (TEA Cl) replaced equimolar NaCl in the Ringer solution after whole cell recordings were initiated. Voltage-activated sodium current was blocked by 1 µM TTX. Ringer and drug containing solutions were applied through a gravity-fed system to a manifold in connection with the tissue chamber. Valves controlled drug application, and there was a delay of ~2-3 s due to exchange time.
Recording pipettes were filled with (in mM) 110 K gluconate, 5 NaCl, 0.1 CaCl2, 1 MgCl2, 5 EGTA, and 5 HEPES and adjusted to pH 7.4 with KOH. The pipette solution also contained an "ATP regenerating cocktail" consisting of 4 mM ATP, 20 mM phosphocreatine, and 50 units/ml creatine phosphokinase. As noted in the text, bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) sometimes replaced EGTA. Impermeant drugs were dialyzed into neurons by including them in the pipette.
Ryanodine, calmidazolium, trifluperazine, heparin, and dantrolene were obtained from Research Biochemicals International (Natick, MA). 1S, 3R trans aminocyclopyridine dicarboxylic acid (ACPD) was purchased from Tocris-Cookson (St. Louis, MO) and calmodulin-binding protein from Calbiochem (San Diego, CA). EGTA-AM, BAPTA-AM, and Fluo-4 were obtained from Molecular Probes (Eugene, OR). Baclofen and CGP35348 were gifts from Novartis Pharma (Basel, Switizerland). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).
Electrophysiological recordings
The whole cell voltage-clamp technique was employed in combination with a List EPC9 amplifier and HEKA Pulse software (ALA Scientific Instruments, Westbury, NY) running on a Macintosh Quadra computer. Igor software (WaveMetrics, Lake Oswego, OR) was used for data analysis, curve fitting, and figure illustration. Data are expressed as means ± SE.
Both voltage steps and ramps were used to monitor calcium channel
currents and, because these channels inactivated slowly, both protocols
gave similar results (Zhang et al. 1997). Neurons were
voltage clamped to
70 mV. In the ramp protocol, the cell was stepped
to
40 mV, then ramped from
40 mV to +40 mV in 50 ms. The step
protocol was a single step from
70 mV to +10 mV for 10-30 ms. This
voltage was chosen because it evoked the largest amplitude inward
current. The inward current was carried by barium and could be blocked
by external cadmium (100 µM), indicative of a calcium channel
current. Filled with the internal solution, electrodes had resistances
of 5 M
. The currents shown are raw data, not corrected for electrode
junctional potentials or access resistance.
Isolated ganglion cells were identified using morphological and physiological criteria, particularly soma diameters over 15 µm and inward sodium currents over 1 nA. These characteristics do not positively exclude amacrine cells, but based on the number of cells studied and the consistency of the results, it is reasonable to conclude the observations are representative of ganglion cell responses.
Calcium imaging
Dissociated cells were loaded for 20 min at room temperature in the dark with 5 µM Fluo-4 AM in Ringer solution containing 0.02% pluronic acid and 0.01% dimethylsulfoxide (DMSO). Then cells were washed with Ringer solution and kept in the dark for 30 min to allow for complete dye deesterification. Fluorescent images were detected by a laser-scanning confocal Biorad MRC-1024 system and upright Nikon Optiphot microscope equipped with a ×63, 1.2 NA water immersion objective. A 488-nm argon laser was used for dye excitation. Emissions were cutoff at 522 nm. Full resolution images (512 × 512 pixels) were collected at 1.4- to 4-s intervals and processed by Confocal Assistant software. When applicable, drugs were applied to the cell chamber for 15 min before images were taken. The stage of the confocal microscope was not equipped with a perfusion or drug application system. Therefore drug application was completed just before confocal imaging except where noted in the text (Fig. 6).
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RESULTS |
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At a concentration of 10 µM, baclofen (Bowery et al.
1980) potently activated bipolar cell and ganglion cell
GABABRs in amphibian in situ preparations such as
the intact retinal eyecup or the retinal slice (Maguire et al.
1989
; Slaughter and Bai 1989
; Tian and
Slaughter 1994
; Zhang et al. 1997
). This
concentration produced very small effects in isolated cells. But a high
baclofen concentration applied to isolated ganglion cells produced the
same effect as much lower concentrations in the slice preparation,
namely a large reduction in high-voltage-activated calcium channel
current (Zhang et al. 1997
). This was surprising because
isolated cells are usually more sensitive to low drug concentrations,
partly due to the absence of perfusion barriers and uptake mechanisms.
As shown in Fig. 1A, 300 µM
baclofen suppressed 65% of the barium current in an isolated ganglion
cell, whereas 10 µM baclofen reduced <9% of the current. Several
factors indicated that baclofen was acting on the
GABABR, despite the high concentration that was
required. One was that CGP35348, a specific
GABABR antagonist (Bittiger et al.
1990
; Tian and Slaughter 1994
), blocked the
effect of baclofen (Fig. 1B). Another was the voltage
sensitivity of the baclofen effect (Campbell et al.
1993
, 1995
; Grassi and Lux 1989
).
Consistent with this, a prepulse to +100 mV reduced the effect of
baclofen in isolated cells (Fig. 1A) and the peak inward
current evoked by a voltage ramp was shifted to the right in the
presence of baclofen (Fig. 1B). Therefore despite the need
for a significantly higher dose, it appears that baclofen acts
specifically on GABABRs in these isolated cells.
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A possible explanation of this phenomenon came from studies of internal
calcium. Under standard conditions, internal calcium was buffered with
5 mM EGTA. Under these conditions, which are referred to as
"control," 10 µM baclofen had a small effect on inward barium
current and the EC50 for baclofen was 92 µM
(Fig. 2). If the pipette solution
contained 10 mM BAPTA, then 10 µM baclofen produced a 27%
suppression of the barium current, the baclofen dose-response curve was
shifted to the left, and the EC50 was reduced to
28 µM. In five cells buffered with EGTA, 10 µM baclofen suppressed
6 ± 3% of the inward current. In another six cells buffered with
BAPTA, 10 µM baclofen suppressed 23 ± 3% of the current. Based
on calculations using MaxChelator v6.81 (Bers et al.
1994), the change in buffers did not appreciably alter the
overall internal free calcium concentration. A more likely factor is
that BAPTA, a faster buffer than EGTA (Naraghi and Neher
1997
), reduced local calcium near internal release sites. Consequently, BAPTA interfered with calcium-dependent phenomena that
were very localized within the cell.
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Confocal imaging of isolated cells disclosed a ring of relatively high internal calcium near the plasma membrane. Fluo-4 AM was loaded into isolated cells, and then the neurons were scanned. Neurons with long processes were chosen, thus selecting for amacrine and ganglion cells. Although absolute levels of calcium were not determined, the calcium signal near the cell membrane was compared with the fluorescence over the soma. Under control conditions, relatively high levels of fluorescence were observed just beneath the plasma membrane of isolated neurons (Fig. 3A). To contrast the effects of EGTA and BAPTA, cells were loaded with either EGTA (adding 500 µM EGTA-AM to the bath) or BAPTA (adding 500 µM BAPTA AM to the bath) before treatment with Fluo-4 AM. When the EGTA buffer was used, relatively high levels of internal free calcium were still detected around the plasma membrane. But this ring of high calcium was not observed in the presence of BAPTA (Fig. 3B).
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To evaluate the effect of internal free calcium, several concentrations
of BAPTA were included in the pipette solution to control internal free
calcium. Baclofen's dose-response relationship was determined and
fitted to the logistic equation (Fig. 4).
When internal calcium was buffered to 70 nM, the baclofen
EC50 was 28 µM. Increasing the pipette's BAPTA
concentration did not lower the EC50. For
example, when BAPTA was increased to a level where the calculated free
calcium approached 1 pM, the dose response curve was almost identical
to the curve generated for 70 nM internal calcium. The
Kd of BAPTA is ~200 nM (Tsien
1980), and buffers are less effective at more than one order of
magnitude away from their Kd values.
Nevertheless, it is reasonable to conclude that an internal free
calcium concentration of ~70 nM yielded maximal receptor apparent
affinity. When internal free calcium was buffered with BAPTA to 200 nM,
it produced a dose-response curve that was very similar to control
(EGTA buffering) conditions. Although it is not shown in Fig. 4, BAPTA
was used to buffer calcium at several concentrations between 70 and 200 nM. Each of these concentrations produced a parallel shift in the dose
response curve. The curves fit between the two illustrated (70 and 200 nM), with higher calcium levels shifting the curve to the right. Thus
within this range, the baclofen EC50 increased
monotonically with calcium concentration. When internal calcium was
buffered to 1 µM, the dose-response curve became compressed, but the
EC50 increased only slightly to 100 µM.
Therefore moderate concentrations of internal calcium produced a
competitive suppression of the baclofen effect while high
concentrations of calcium produced a noncompetitive inhibition of
baclofen's action. Because the GABABR acts
through a second-messenger pathway, the site of inhibition may not be
at the receptor, but could be at any of the steps along the
transduction cascade.
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Although this ambiguity exists for agonists, it may not be true of receptor antagonists because they act directly at the receptor and do not involve the transduction pathway. Consequently, the influence of internal calcium on the IC50 of CGP-35348, a competitive GABABR antagonist, was explored. The effects of internal buffering with 5 mM EGTA and 10 mM BAPTA were compared. For each condition, the concentration of antagonist was varied while the concentration of agonist was kept constant. Because baclofen was more potent when cells were buffered with BAPTA, we used equivalent effective concentrations (but different absolute concentrations) of baclofen. Based on EC50 values calculated from the dose-response curves shown in Fig. 1, 100 µM baclofen was used when buffering with EGTA, whereas 30 µM baclofen was used when buffering with BAPTA. Experiments revealed that BAPTA shifted the IC50 of CGP35348 to the left (Fig. 5), an effect that was qualitatively similar to the effect of this buffer on baclofen's EC50. In seven cells buffered with 5 mM EGTA, the IC50 of CGP35348 was 47 µM. In a different set of five cells, which were buffered with 10 mM BAPTA, the IC50 was 20 µM. This confirms that internal calcium alters GABABR apparent affinity.
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The source of internal calcium could be a standing high level of bulk calcium, but this is unlikely because of the differing effects of EGTA and BAPTA. An explanation consonant with the different actions of these two buffers is that there is a local source of internal calcium. This local source could be very close to its binding site, so that it could produce a rapid effect that EGTA was unable to buffer. One potential source is the membrane calcium channel, either one regulated by the GABABR or another type of calcium channel. Thus divalent ion influx might act to reduce the affinity of the receptor. Although this might influence the baclofen response, it is unlikely to account for our observations. Baclofen acts at much lower doses in the slice and intact retina, where a similar inward calcium current has been observed. It seems more likely that dissociated neurons have altered internal calcium dynamics, perhaps weaker calcium buffering or greater release from internal stores. Intracellular sources of calcium could produce localized calcium increases that might be EGTA insensitive. Two internal sources were investigated: the InsP3-sensitive and ryanodine-sensitive stores.
Heparin was included in the pipette solution to block
InsP3-stimulated calcium release (Hill et
al. 1987). The baclofen dose-response curve in the presence of
heparin was determined in seven cells and compared with the set of five
control neurons shown in Fig. 1. All experiments were performed with 5 mM EGTA in the pipette. Heparin produced a parallel, leftward shift in
the dose-response curve (Fig.
6B). The
EC50 was reduced from 92 to 41 µM. This
suggests that calcium release from
InsP3-sensitive stores was responsible, at least
in part, for the lowered affinity of the GABABR
in isolated cells. Because heparin is membrane permeable, imaging
experiments were performed to test the effect on internal calcium
distribution. Confocal calcium imaging demonstrated that heparin
suppressed the ring of high calcium under the plasma membrane that was
observed in control cells. These imaging experiments were performed in two ways. In one protocol, cells were superfused with heparin (Fig.
6A), and the fluorescence of these cells was compared with control cells (e.g., Fig. 3A). Alternatively, cells were
observed under the confocal microscope while heparin was added to the
solution bathing the cells. This permitted observation of the same
cells before and during heparin treatment, but a perfusion system was not available, so there was imprecise control of drug application (Fig.
6B). Both protocols demonstrated that heparin reduced the ring of high fluorescence. Again, this suggests a link between this
ring of high calcium and the reduction in apparent affinity of the
GABABR.
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The ryanodine-sensitive store was also investigated. Two blockers of
the ryanodine receptor were tested: dantrolene (Ohta et al.
1990) and high concentrations of ryanodine (Buck
et al. 1992
). Both had very similar effects (Fig.
7, A-C). In the presence of these blockers, 10 µM baclofen significantly suppressed the barium
current, and both blockers shifted the baclofen dose-response curve to
the left, reducing the EC50 to 27 µM. The effect of each antagonist was almost identical to the maximum effect of internal BAPTA
buffering.
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The effect of ryanodine receptor stimulation was examined by applying baclofen alone or in the presence of caffeine (Fig. 7D). Internal calcium was buffered with 5 mM EGTA. The inward barium current was partially suppressed by baclofen alone. Application of 10 mM caffeine rapidly reduced the inward current and almost totally blocked baclofen's effect. This could be interpreted to indicate that caffeine increased internal calcium to a range where it produced the noncompetitive inhibition of the GABABR described in Fig. 4. Calcium imaging of isolated cells showed that caffeine increased internal levels of free calcium (not shown). Although supporting the data obtained with ryanodine receptor antagonists, it is also possible that caffeine closed the same calcium channels that were modulated by baclofen, and thus simply occluded the baclofen effect. An important outcome of the caffeine experiments was that the calcium stores in isolated cells were far from depleted, despite the presence of elevated free calcium under the plasma membrane.
Calcium could have a direct action on the GABABR, or might
stimulate an intracellular pathway. Because calmodulin is a ubiquitous detector of internal calcium, we examined the influence of three calmodulin antagonists: trifluperazine, calmidazolium, and the calcium
binding domain peptide (CaM kinase II 290-309) (James et al.
1995; Van Belle 1981
; Vandonselaar et al.
1994
). All three agents produced a parallel, leftward shift of
the baclofen dose-response curve (Fig.
8). Calmidazolium and trifluoperazine had
similar effects, producing a shift of baclofen EC50 to 36 µM. The effect of the calmodulin binding domain was slightly more
pronounced, shifting the EC50 to 20 µM.
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Metabotropic glutamate receptors stimulate InsP3-mediated
increases in internal calcium in amphibian ganglion cells in
Xenopus (Akopian and Witkovsky 1996) and
salamander (Shen and Slaughter 1998
). This suggests that
glutamate could reduce the effectiveness of the GABABR. To
test this, baclofen's effect on barium current was measured before and
after activation of metabotropic receptors. The metabotropic glutamate
receptors were stimulated with ACPD. Because metabotropic glutamate
receptors suppress L-type calcium channels while baclofen inhibits
N-type channels (Shen and Slaughter 1998
; Zhang
et al. 1997
), all the experiments were done in the presence of
50 µM nifedipine. ACPD could still activate the InsP3 system in the presence of nifedipine, but did not produce an effect on
calcium channels that might be confused with the action of baclofen. In
the presence of nifedipine, 100 µM baclofen was applied and produced
a suppression of the inward current (Fig.
9). However, after application of ACPD,
the suppressive effect of baclofen was significantly reduced. Inward
current was 47 ± 4% suppressed by 100 µM baclofen alone, but
baclofen in the presence of ACPD suppressed 29 ± 3% of the
current (n = 4).
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DISCUSSION |
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Regulation of the metabotropic GABA receptor
These experiments indicate that modulation of calcium release from internal stores influences the affinity of the GABABR. This modulation can be very significant if synaptically released GABA is acting on the steep portion of the dose-response curve. For example, the equivalent of 50 µM baclofen could produce 75% of its peak inhibition if the internal calcium is 70 nM, whereas a modest increase to 200 nM internal calcium would reduce this inhibition to ~30%. An additional increase in internal calcium to 1 µM would reduce the effect of 50 µM baclofen to only 10%. These levels of internal calcium are very likely to be within the normal physiological range of excitable cells.
The metabotropic GABA receptor appears very sensitive to modulation by
internal calcium. By comparison, the
K1/2 of the photoreceptor cyclic
nucleotide gated channel, which is regulated by internal calcium during
dark adaptation, changes from 86 µM in the presence of 20 µM
calcium to 59 µM in a calcium-free environment (Hackos and
Korenbrot 1997). The GABABR goes through
a larger change in apparent affinity when internal calcium changes by
<100 nM.
Internal calcium can regulate ionotropic, as well as metabotropic, GABA
receptors. Rapid influx of calcium through voltage-regulated calcium
channels produced a significant reduction in the apparent affinity of
the GABAA receptor (Inoue et al.
1986). Other studies have shown that calcium release from
internal stores can suppress (Brussaard et al. 1996
;
Desaulles et al. 1991
), enhance (Llano et al.
1991
), or produce a biphasic effect (Taleb et al.
1987
) on the GABAA receptor current. In a
preliminary report, Akopian and Witkovsky (1997)
found
that internal calcium suppressed the GABAA
receptor in turtle ganglion cells and identified a link between
internal calcium and calmodulin. Therefore both metabotropic and
ionotropic GABA receptors may be concomitantly modulated by changes in
internal calcium.
Apparent or real affinity?
It is difficult to determine whether calcium is truly modulating
receptor affinity, or whether it is having an effect downstream of the
receptor. To some extent this ambiguity is resolved by the observation
that calcium shifts the IC50 of
GABABR antagonists as well as receptor agonists.
The logic is that antagonists only interact with the receptor and are
independent of the downstream cascade. However,
IC50 measurements clearly require agonist action. Based on published reports, at least one plausible mechanism that might
act downstream of the receptor yet appear to affect affinity is the
action of calmodulin on G-proteins. Calmodulin binds to the
subunit of G-proteins (Liu et al. 1997
).
GABABRs suppress calcium channels by promoting
binding to the calcium channel. Thus calmodulin may inhibit this
interaction. If only submaximal stimulation of
GABABRs is normally required for maximal
suppression of calcium channels (spare receptor model), then
calmodulin inactivation of
subunits could shift the
dose-response curves for both agonists and antagonists. This would
appear to be a change in receptor affinity.
Calcium-activated second-messenger pathway
The experiments link internal calcium release with
GABABR apparent affinity. BAPTA, but not EGTA,
affected receptor apparent affinity. This suggests that local regions
of high intracellular calcium influence receptor apparent affinity.
Based on their distinct on-rates, it has been estimated that neither
EGTA or BAPTA can alter the free calcium concentration within 20 nm of
a calcium release site and that both buffers are equally effective a
distances >200 nm from the release site (Augustine et al.
1991). The distinct effects of BAPTA and EGTA suggest that
calmodulin binding occurs within ~200 nm of the calcium release site.
Confocal images of isolated cells suggest that these release sites are
just below the plasma membrane, although these neurons have very large
nuclei resulting in a restriction of the cytoplasmic compartment.
The calcium that regulates apparent receptor affinity comes from internal stores, not from extracellular sources. Heparin, a blocker of InsP3-sensitive stores, shifts the affinity of the receptor to almost the same extent as dantrolene and ryanodine, which are ryanodine receptor blockers. This suggests that both release sites are important in controlling intracellular calcium in isolated cells.
The effectiveness of trifluperazine, calmidazolium, and calmodulin-binding peptide suggests that calcium release from internal stores stimulates calmodulin, leading to a reduction of GABABR apparent affinity. Thus the simplest interpretation of the experimental data is that isolated retinal neurons possess leaky InsP3 receptors that produce small, localized increases in internal calcium. This calcium signal could be amplified by ryanodine receptors, detected by calmodulin, leading to a reduced affinity of the GABABR. The calcium release must be close to a source of calmodulin, although not necessarily near the GABABR.
However, alternative interpretations remain possible. There are some
reports that heparin blocks ryanodine receptors and others, indicating
that there is an internal calcium pool that is sensitive to both
ryanodine and InsP3 receptor agonists (Stauderman
et al. 1991; Zacchetti et al. 1991
). Thus the experiments strongly
suggest that internal calcium release is a key step in regulating
apparent affinity; the evidence is less compelling that both release
sites contribute to this mechanism. Similarly, the identification of a
calmodulin-dependent step depends on the specificity of calmidazolium, trifluperizine, and calmodulin-binding protein. The latter may be the
most specific, but there is a report that phospholipase C delta
contains a sequence that is very similar to calmodulin and that the
phospholipase C could be blocked by an inhibitory calmodulin peptide
(Richard et al. 1997
).
Transmembrane calcium flux may regulate the GABAA
receptor (Inoue et al. 1986). A similar mechanism could
operate on metabotropic GABA receptors. The
GABABR regulates the N-type calcium channel by a
direct G-protein interaction, implying that the channel and receptor
are close to each other (Campbell et al. 1993
; reviewed by Misgeld et al. 1995
). If calmodulin was present near
the mouth of the calcium channel, it might mediate this negative
feedback. Barium was used in our experiments to avoid this complicating factor. However, in a few experiments where calcium was used as the
current carrier, there was no apparent decrease in the effect of
baclofen during the course of calcium influx. Therefore it appears that
this calmodulin regulatory system was not near the calcium channel.
However, this regulation might not have been detected in our
experiments if it occurred very quickly (less than a few milliseconds)
or very slowly (longer than our 30-ms voltage step). If calcium influx
regulates GABABRs in retinal ganglion cells, it
functions in parallel with the regulatory mechanism dependent on
internal calcium release. The same may be true of GABAARs. Although Inoue et al.
(1986)
suggested that voltage-gated calcium channels could
modulate GABAA receptor affinity, other studies
have suggested GABAA responses are suppressed by
internal calcium release and not by influx across the plasma membrane
(Desaulles et al. 1991
).
Physiological significance
At this point, we can only speculate about the functional
significance of the interaction between metabotropic glutamate and GABA
receptors. There are several sites in the retina where this interaction
might occur. One is the ganglion cell, where
GABABRs suppress a calcium-activated potassium
conductance (Zhang et al. 1997). When this conductance
is suppressed, bipolar cell glutamatergic input can produce a larger
depolarization of the ganglion cell (Zhang et al. 1998
).
This added depolarization could move the ganglion cell to a voltage
that produces inactivation of the sodium channels. That is, counter
intuitively, the absence of the calcium-activated potassium current may
reduce the spike output of the ganglion cell. Under these conditions, a
suppression of the GABABR might be advantageous
because it would restore the calcium-activated potassium current,
allowing for a repolarization that removes sodium channel inactivation.
Therefore ganglion cell responsivity might be improved if glutamate
release from bipolar cells activated both ionotropic and metabotropic
ganglion cell glutamate receptors. Strong ionotropic glutamate receptor
activation would be accompanied by a metabotropic glutamate receptor
stimulation that inhibited the GABABR. This might
increase the dynamic range of a ganglion cell, permitting high spike
rates during strong excitatory inputs.
Glutamate, acting through metabotropic receptors to lower the affinity
of the GABAB receptor, might also play a key role
at the bipolar cell synaptic terminal. The bipolar presynaptic terminal releases glutamate (Tachibana and Okada 1991) and may
have GABABRs (Maguire et al. 1989
;
but see Lukasiewicz and Werblin 1994
). These bipolar
cell terminals also contain metabotropic glutamate receptors (Brandstätter et al. 1996
) and high levels of
InsP3 (Peng et al. 1991
). (These
properties are cumulative data from different species and may not be
found in combination in a single species, but serves as a heuristic
model.) When GABABRs at the terminal are active,
this would reduce transmitter release during bipolar cell excitation
(Maguire et al. 1989
). However, if the released glutamate fed back onto high affinity,
InsP3-linked metabotropic glutamate receptors,
this would suppress the GABABR inhibition and
enhance glutamate release at the synapse. The net effect would be that
metabotropic glutamate autoreceptors stimulate a positive feedback
system that produces synaptic facilitation. This is opposite to the
conventional model of autoreceptors as a negative feedback system that
reduce transmitter release at the synapse.
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ACKNOWLEDGMENTS |
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This work was supported by National Eye Institute Grant EY-05725.
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FOOTNOTES |
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Address for reprint requests: W. Shen, State University of New York, Dept. of Physiology and Biophysics, 124 Sherman Hall, Buffalo, NY 14214.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 May 1999; accepted in final form 24 August 1999.
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