GTP-binding protein βγ subunits mediate presynaptic calcium current inhibition by GABA_B receptor

Abstract

A variety of GTP-binding protein (G protein)-coupled receptors are expressed at the nerve terminals of central synapses and play modulatory roles in transmitter release. At the calyx of Held, a rat auditory brainstem synapse, activation of presynaptic γ-aminobutyric acid type B receptors (GABA_B receptors) or metabotropic glutamate receptors inhibits presynaptic P/Q-type Ca²⁺ channel currents via activation of G proteins, thereby attenuating transmitter release. To identify the heterotrimeric G protein subunits involved in this presynaptic inhibition, we loaded G protein βγ subunits (Gβγ) directly into the calyceal nerve terminal through whole-cell patch pipettes. Gβγ slowed the activation of presynaptic Ca²⁺ currents (I_pCa) and attenuated its amplitude in a manner similar to the externally applied baclofen, a GABA_B receptor agonist. The effects of both Gβγ and baclofen were relieved after strong depolarization of the nerve terminal. In addition, Gβγ partially occluded the inhibitory effect of baclofen on I_pCa. In contrast, guanosine 5′-O-(3-thiotriphosphate)-bound G_oα loaded into the calyx had no effect. Immunocytochemical examination revealed that the subtype of G proteins G_o, but not the G_i, subtype, is expressed in the calyceal nerve terminal. These results suggest that presynaptic inhibition mediated by G protein-coupled receptors occurs primarily by means of the direct interaction of G_o βγ subunits with presynaptic Ca²⁺ channels.

In the central nervous system, synaptic transmission is regulated by presynaptic autoreceptors or heteroreceptors. These receptors are coupled by G proteins to various targets such as Ca²⁺ channels, K⁺ channels, or the exocytotic machinery downstream of Ca²⁺ influx (1). At a rat brainstem auditory synapse formed by a giant nerve terminal, the calyx of Held, metabotropic glutamate receptors (mGluRs), or γ-aminobutyric acid type B receptors (GABA_B receptors) primarily inhibit P/Q-type Ca²⁺ channels (2–4). The inhibitory effect of the GABA_B receptor agonist baclofen on presynaptic Ca²⁺ currents can be blocked by the GDP analog guanosine 5′-O-(2-thiodiphosphate) (GDP[βS]) and occluded by the nonhydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTP[γS]), both loaded directly into the calyx, indicating that G proteins mediate the presynaptic inhibition (3). At the cell soma, heterotrimeric G proteins attenuate Ca²⁺ currents acting either directly via a membrane-delimited pathway involving G protein βγ subunits (Gβγ) or indirectly via second messengers (5, 6). At the nerve terminal, however, it is not known which mechanism underlies this presynaptic inhibition. At the calyx of Held, which can be visually identified in slice, it is possible to load various molecules into the nerve terminal through whole-cell recording pipette (3, 7, 8). Using this technique, we examined the effect of Gβγ on I_pCa. Also, because of its large structure, it was possible to use immunocytochemistry to determine the subtype of G proteins expressed in the calyceal nerve terminal. Our results suggest that the βγ subunits of G_o mediate the presynaptic inhibition at the calyx of Held synapse by directly interacting with presynaptic Ca²⁺ channels.

Methods

Preparations and Solutions.

All experiments were performed in accordance with the guidelines of the Physiological Society of Japan. Wistar rats (12–17 days old) were killed by decapitation under halothane anesthesia. The whole brain was immersed in an ice-cold cutting solution containing 250 mM sucrose, 2.5 mM KCl, 26 mM NaHCO₃, 10 mM glucose, 1.25 mM NaH₂PO₄, 1 mM CaCl₂, 4 mM MgCl₂, 0.5 mM myo-inositol, 2 mM sodium pyruvate, and 0.5 mM ascorbate, pH 7.4, when saturated with 95% O₂/5% CO₂. A tissue block containing the brainstem and cerebellum was removed and glued onto the stage of a tissue slicer (Microslicer DTK-1000, Dosaka, Kyoto, Japan) and immersed in the cutting solution. Transverse slices (150 μm in thickness) containing the medial nucleus of the trapezoid body (MNTB) were cut and incubated at 36°C for 1 h in the artificial cerebrospinal fluid containing 125 mM NaCl, 2.5 mM KCl, 26 mM NaHCO₃, 10 mM glucose, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, 0.5 mM myo-inositol, 2 mM sodium pyruvate, 0.5 mM ascorbate, pH 7.4, when saturated with 95% O₂/5% CO₂.

Recording and Data Analysis.

For electrophysiological recordings, one slice was transferred to a superfusing chamber on a stage of upright microscope (Axioskop, Zeiss), and individual calyces in the MNTB were visualized with a 40× water-immersion objective lens (Zeiss) through a charge-coupled device camera. The recording chamber was continuously superfused with the artificial cerebrospinal fluid. Whole-cell presynaptic Ca²⁺ currents were recorded from calyces under voltage clamp being evoked with depolarizing steps from a holding potential of −80 mV. To isolate Ca²⁺ currents, 10 mM tetraethylammonium chloride and 1 μM tetrodotoxin were included in the artificial cerebrospinal fluid, and the presynaptic patch pipette was filled with a solution containing 110 mM CsCl, 40 mM Hepes, 0.5 mM EGTA, 1 mM MgCl₂, 2 mM ATP, 0.5 mM GTP, 12 mM disodium phosphocreatine, and 10 mM tetraethylammonium chloride, with pH adjusted to 7.4 with CsOH. The electrode resistance was 5–10 MΩ, and series resistance was typically 10–30 MΩ and compensated by 70–80%. Whole-cell current recordings were made by using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA). Records were low-pass filtered at 2 kHz and digitized at 5 kHz with a CED 1401 interface (Cambridge Electric Design). Leak currents were subtracted for presynaptic currents by a scaled pulse divided by n (P/N) protocol (3). Statistical significance was evaluated by paired t test unless otherwise noted. The liquid junction potential between the internal and external solutions was not corrected for.

Applications of Drugs and Proteins.

Baclofen was purchased from Research Biochemicals (Natick, MA). Purified bovine brain Gβγ and recombinant myristoylated rat G protein α subunit (G_oα) were from Calbiochem. Gβγ was dissolved at 50 μg/ml in 50 mM Hepes/1 mM DTT/1 mM EDTA/0.1% (1.25 μM) Lubrol, pH 7.6, and stored at −80°C. To inactivate the protein, the stock solution was boiled at 95°C for 10 min. Boiled or nontreated Gβγ was diluted with the pipette solution added with 0.05% or 0.1% CHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate} for the final Gβγ concentrations to be 100 or 200 nM. The stock solution for G_oα (1.7 mg/ml) contained 20 mM Hepes, 100 mM NaCl, 3 mM MgCl₂, and 1 mM EDTA (pH 8.0). To activate G_oα, this stock solution was diluted 10-fold with a solution containing 50 μM GTP[γS], 100 mM NaCl, 25 mM MgCl₂, 1 mM DTT, 1 mM Na₃EDTA, and 20 mM Hepes (pH 8.0) and incubated at 37°C for 2 h. After incubation, unbound GTP[γS] was removed by gel filtration (using Bio-Gel P-6, Bio-Rad). The final effective concentration of G_oα–GTP[γS] assessed by using GTP[γ³⁵S] was 100 nM (total amount of G_oα was 250 nM). The concentration of free GTP[γS] remaining after gel filtration was estimated to be less than 0.12 nM. G protein subunits were applied through whole-cell pipettes into calyces by diffusion. Baclofen (2 or 20 μM) was bath-applied by switching superfusates by using solenoid valves. Experiments were carried out at room temperature (24–27°C).

Immunocytochemistry.

Wistar rats (13–15 days old) were anesthetized with Nembutal and transcardially perfused with a fixative (4% paraformaldehyde and 0.2% picric acid in 0.1 M sodium phosphate, pH 7.4). After fixation, rats were killed by decapitation, and a tissue block of brainstem including the MNTB region was removed for postfixation for 1–2 days at 4°C. The fixed tissue was cryoprotected at 4°C in 0.1 M sodium phosphate with sucrose of graded concentrations: in 4% sucrose for 30 min, 10% for 2 h, 15% for 2 h, and 20% overnight. Transverse slices (25 μm in thickness) were cut with a cryostat (CM3050, Leica, Nussloch, Germany) at −21°C. The sections were then processed for immunocytochemistry as follows: (i) blocking and permeabilization in phosphate-buffer saline solution containing 4% skim milk and 0.3% (wt/vol) Triton X-100 for 5 h; (ii) application of primary antibodies in PBS containing 0.5% (wt/vol) BSA and 0.05% Triton X-100 for 2 days at 4°C; (iii) application of secondary antibodies in the same buffer as primary antibodies for overnight at 4°C; and (iv) mounting with ProLong antifade kit (Molecular Probes). The primary antibodies were rabbit polyclonal anti-G_oα subunit, anti-G_oα and G_iα3 subunits, anti-G_iα1 and G_iα2 subunits, anti-G_iα3 subunit (Calbiochem), and mouse monoclonal anti-synapsin I (Chemicon) diluted 1:100. The secondary antibodies were anti-mouse IgG conjugated with Alexa fluor 568 and anti-rabbit IgG conjugated with Alexa fluor 488 (Molecular Probes, diluted 1:200). Stained sections were viewed with a 100× oil-immersion objective (numerical aperture 1.35) or 100× water-immersion objective (numerical aperture 1.0) using a confocal laser scanning microscope (Fluoview FV300, Olympus Optical, Tokyo). Excitation wavelengths were 488 nm (argon laser) and 543 nm (He/Ne laser). Emission wavelengths were 510–530 nm (for green) and >610 nm (for red). All of the immunocytochemical procedures were carried out at room temperature (22–27°C) unless otherwise noted.

Results

Inhibitory Effect of Gβγ on Presynaptic Ca²⁺ Currents.

To examine the effect of Gβγ on I_pCa, we loaded Gβγ (100 nM) directly into the calyceal nerve terminal through a whole-cell patch pipette by passive diffusion. After rupture of patch membrane, I_pCa was evoked by a depolarizing pulse (10 ms) from a holding potential of −80 mV (Fig. 1). At the calyx of Held at this postnatal age (12–17 days old), I_pCa has been pharmacologically characterized as the P/Q type (9, 10). After rupture with a pipette containing Gβγ, the activation rate of I_pCa gradually became slower and its amplitude smaller (measured 2.5 ms after the onset of pulse). The slowness of the effects of Gβγ might, at least in part, be due to a slow rate of diffusion of Gβγ (about 40 kDa) into the calyx. The mean amplitude of I_pCa 25 min after rupture was 65.4 ± 3.1% (n = 3) of the amplitude measured 5 min after rupture. In contrast, no appreciable change was observed for I_pCa when Gβγ inactivated by boiling (100 nM) was loaded into calyces (96.2 ± 4.0%, n = 3). Thus, Gβγ specifically and significantly inhibited I_pCa (P < 0.005, unpaired t test). This effect of Gβγ is similar to that obtained by externally applying the GABA_B agonist baclofen (3, 4), the mGluR agonist L-2-amino-4-phosphonobutyrate (2) or by intracellularly infusing GTP[γS] (3, 7).

Figure 1.

The effect of Gβγ on I_pCa. Untreated (traces a and b) or boiled (traces c and d) Gβγ (each 100 nM) was applied to calyceal nerve terminals by means of whole-cell pipettes. Here and in Fig. 3, abscissae indicate the time after the patch membrane rupture. I_pCa was evoked by a 10-ms depolarizing pulse stepped from −80 mV to 10 mV every 10 s. Ordinates indicate the mean amplitude of I_pCa (derived from three cells each) and normalized to the mean of the first five data points. The mean amplitude of I_pCa was measured 2.5 ms after the command pulse onset shown here and in Figs. 2 and 3. Error bars represent SEM here and in Fig. 2. Sample records on top row indicate I_pCa in calyces loaded with Gβγ (a and b) or boiled Gβγ (c and d), 5 min (a and c) and 25 min (b and d) after ruptures, respectively (superimposed).

Depolarization-Induced Unblock of I_pCa Inhibitions by Gβγ and Baclofen.

Inhibition of voltage-dependent Ca²⁺ channels by G_i or G_o takes place via a membrane-delimited pathway and is transiently disinhibited after strong depolarization (11, 12). We examined whether this disinhibition might be observed for the effects of Gβγ on I_pCa (Fig. 2). First, we evoked a series of I_pCa by using 10-mV incremental depolarizing step pulses of a 10-ms duration to formulate a current–voltage relationship (9). We then applied a large conditioning depolarizing pulse (to +100 mV, 10-ms duration) and followed this with the same series of test pulses. When Gβγ (200 nM) was present in the recording pipette, the conditioning pulse increased the activation rate and amplitude of I_pCa for a wide range of test potentials (Fig. 2A, significant differences at −20 mV and −10 mV, P < 0.02, n = 6 cells). The peak of I_pCa in the current–voltage relationship showed a concomitant shift by about −10 mV, indicating that gating of the Ca²⁺ channels had shifted toward the “willing” state (13). Similar effects were observed when baclofen (20 μM) was present in the superfusate (Fig. 2C), as previously reported (4). In contrast, no such disinhibition was observed when boiled Gβγ (200 nM) was present in the recording pipette (Fig. 2B, n = 8) or before application of baclofen (not shown). Thus, Gβγ mimics the inhibitory effect of baclofen.

Figure 2.

Depolarization-induced disinhibition of I_pCa. I_pCa was evoked by a depolarizing test pulse from a holding potential of −80 mV to different membrane potentials by 10-mV steps with (□) or without (○) a prior conditioning depolarizing pulse (to +100 mV, duration 10 ms). (Left) Experimental paradigm (Top) and sample records of I_pCa evoked by depolarizing steps to −10 mV. Calibrations are 5 ms and 0.5 nA. (Right) In the current–voltage relationship, the I_pCa amplitude was normalized to that at −10 mV without conditioning. Recording pipettes contained Gβγ (A) or boiled Gβγ (B) (200 nM each), or none of them (C). In C, baclofen (20 μM) was present in superfusates. Mean amplitudes and SEM were derived from 6 (A), 8 (B), and 4 (C) calyces, respectively. Asterisks indicate data points of significant difference (P < 0.02).

Occlusion of Gβγ with Baclofen in the Inhibitory Effect on I_pCa.

If Gβγ mediates the baclofen-induced inhibition of I_pCa, intracellular loading of Gβγ would be expected to occlude the effect of baclofen. In the presence of Gβγ in the presynaptic pipette (200 nM), the first application of baclofen (2 μM) just after rupture (<6 min) clearly inhibited I_pCa (Fig. 3A). However, the second application of baclofen 15–20 min later inhibited I_pCa amplitude to a significantly lesser extent (66 ± 0.4%, n = 8, P < 0.00002). In contrast, when heat-inactivated Gβγ was included in the presynaptic patch pipette, the second application of baclofen inhibited I_pCa to a similar extent as the first application (Fig. 2B, 94 ± 3%, n = 7, P > 0.1). Thus, Gβγ occluded the inhibitory effect of baclofen on I_pCa. The occluding effect of Gβγ on baclofen-induced inhibition of I_pCa was weaker than that obtained with GTP[γS] (3). This may be due to insufficient presynaptic concentrations of Gβγ because of its slow diffusion, inadequate conditions for its full potency, or the involvement of additional subunits or second messengers.

Figure 3.

Gβγ occluded the baclofen-induced inhibition of I_pCa, whereas G_oα–GTP[γS] did not. Whole-cell recording pipettes contained Gβγ (A) or boiled Gβγ (B) (200 nM each). Baclofen (2 μM) applied 3–5 min after rupture inhibited the I_pCa amplitude by 43.1 ± 2.2% (n = 8). Baclofen-induced I_pCa inhibition was attenuated by Gβγ in the second application after a 15- to 20-min interval (A). Boiled Gβγ had no effect (B). (C) G_oα–GTP[γS] included in the presynaptic pipette (100 nM; total G_oα, 250 nM) had no effect on the baclofen-induced I_pCa inhibition.

Effects of G_oα on I_pCa.

We examined whether G_oα subunits are also involved in the baclofen-induced presynaptic inhibition by loading calyces with constitutively active G_oα containing bound GTP[γS]. In the presence of G_oα–GTP[γS] in the presynaptic whole-cell pipette (100 nM), I_pCa remained unchanged for at least 40 min after rupture, and the first and second applications of baclofen (2 μM), separated by 15- to 20-min intervals, inhibited I_pCa to similar extents (Fig. 3C, 103 ± 4.8%, n = 5, P > 0.5). G_oα–GTP[γS] has been reported to be effective in attenuating Ca²⁺ currents in chick dorsal rot ganglia in culture at lower concentrations (<20 nM) (14). Thus, G_oα does not seem to mediate baclofen-induced I_pCa inhibition.

Effects of Cyclic Nucleotides on the Baclofen-Induced I_pCa Inhibition.

Our present results are consistent with a membrane-delimited, G protein-dependent mechanism for the baclofen effect. However, it has been reported that cyclic (c) GMP is responsible for mGluR-mediated presynaptic inhibition (15) and somatostatin-induced inhibition of Ca²⁺ currents (16). We examined further the possible contribution of adenylyl cyclase or guanylyl cyclase to presynaptic inhibition by loading calyces with cAMP or cGMP at high concentrations (200 μM). This concentration of cyclic nucleotides is three orders of magnitude higher than their resting concentrations (17). In the presence of 200 μM cAMP, baclofen (20 μm) attenuated I_pCa by 37.7 ± 3.5% (n = 4). Similarly, in the presence of 200 μM cGMP, baclofen attenuated I_pCa by 39.5 ± 2.8% (n = 5). The magnitudes of inhibition of I_pCa by baclofen are comparable with that observed in their absence (38.0 ± 3.8%, n = 6). These results suggest that the presynaptic inhibitory effect of baclofen is mediated mainly by a membrane-delimited pathway, rather than via intracellular cyclic nucleotides.

Effects of Botulinum Toxin C1 on the Baclofen-Induced I_pCa Inhibition.

It has been reported that cleaving syntaxin 1A with botulinum toxin C1 blocks (18) or attenuates (19) G protein modulation of N-type Ca²⁺ channels. We examined whether botulinum toxin C1 might affect the baclofen-induced inhibition of I_pCa. Preincubation of slices with botulinum toxin C1 (332 nM) for 3–4 h had no significant effect on the magnitude of inhibition of I_pCa by baclofen (20 μM, 31.8 ± 5.6%, n = 6, data not shown). When botulinum toxin C1 was included in whole-cell pipette (286 nM), no appreciable change was observed for the amplitude and kinetics of I_pCa, and baclofen inhibited I_pCa to a similar extent for at least 40 min after rupture. Thus, it remains undetermined whether syntaxin plays a role in G protein modulation of presynaptic P/Q-type Ca²⁺ channels.

Presence of G Proteins at the Calyx of Held Nerve Terminal.

Baclofen-induced presynaptic inhibition can be blocked by externally applied N-ethylmaleimide (4). When N-ethylmaleimide (1 mM) was included in pipettes, baclofen (20 μm) only marginally inhibited I_pCa (by 5.2 ± 1.2%, n = 4, data not shown). These suggest that the G_i or G_o mediates the presynaptic inhibitory effect of baclofen. However, it is not known which type of G protein is expressed at the nerve terminal. The large size of this nerve terminal is advantageous for the immunocytochemical identification of the presynaptic molecules expressed there. We stained the calyces with antibodies specific for different G protein subtypes together with antibody against synapsin I as a nerve terminal marker. As shown in Fig. 4A, an antibody that binds both G_oα and G_iα3 stained calyces and postsynaptic MNTB cells, with its immunofluorescence substantially overlapped with that of an anti-synapsin I antibody. By contrast, an antibody specific for G_iα3 stained mainly postsynaptic MNTB cells, with little overlap with the synapsin immunoreactivity (Fig. 4B). Similarly, antibody that recognizes both G_iα1 and G_iα2 stained only the postsynaptic cells sparing calyces (Fig. 4C). In contrast, an antibody specific for G_oα clearly stained both calyces and postsynaptic cells (Fig. 4D). These results indicate that the main G_i family protein expressed at the calyx of Held nerve terminal is the G_o subtype, whereas both G_i and G_o are expressed at the postsynaptic MNTB cell.

Figure 4.

Immunocytochemical localization of G_i family at the calyx of Held nerve terminal. Distributions of immunoreactivities for G_i and/or G_o subtypes (labeled green with Alexa fluor 488) and the presynaptic protein synapsin (labeled red with Alexa fluor 568) and overlaid (yellow). Immunofluorescence stainings were made with anti-[G_oα + G_iα3] (A), anti-G_iα3 (B), anti-[G_iα1 + α2] (C), and anti-G_oα (D) antibodies.

Discussion

The observation that GABA depresses Ca²⁺ currents in chick dorsal root ganglion cells suggested that a similar mechanism might operate at the nerve terminal (20). Subsequent studies have been carried out on transmitter-induced modulation of Ca²⁺ channels expressed in the cell soma. In soma of sympathetic neurons, a membrane-delimited mechanism was shown to be the major pathway for the inhibition of N-type Ca²⁺ channel currents by heterotrimeric G proteins (5). In experiments using overexpressed Gβγ (21–24), Gβγ-binding peptide (23, 25), or Gα (26) in culture cells, this mechanism has been shown to involve Gβγ subunits. Similarly, in culture cells, recombinant P/Q-type Ca²⁺ channel currents can be attenuated by injection or overexpression of Gβγ subunits (21), and their inhibition by GTP[γS] can be reversed by Gβγ-binding peptides (27). Despite the wealth of information accumulated on somatic Ca²⁺ currents, there had been no direct evidence indicating that subunits of heterotrimeric G proteins attenuate presynaptic Ca²⁺ currents. In the present study, we loaded heterotrimeric G protein subunits directly into a mammalian giant nerve terminal, the calyx of Held, and demonstrated that Gβγ attenuates presynaptic Ca²⁺ currents. GTP[γS]-bound G_oα subunit similarly loaded into the nerve terminal had no effect. This effect of Gβγ mimicked and occluded the effects of the GABA_B receptor agonist baclofen. At this synapse, baclofen inhibits presynaptic Ca²⁺ currents (3, 4), and this effect fully explains its inhibitory effect on transmitter release (3), despite other mechanisms postulated at other synapses (1). Our present results indicate that presynaptic inhibition via GABA_B receptor is mediated mainly by Gβγ at this mammalian central synapse.

At the calyx-type nerve terminal in chick ciliary ganglia, both the G_o and G_i subclasses are expressed together with other G proteins (28). Surprisingly, however, the calyx of Held terminal expressed only G_o, whereas the postsynaptic cell expressed both G_o and G_i. These results suggest that GABA_B receptors are coupled with G_oα (but not G_iα) and that its partner, G_oβγ, inhibits presynaptic P/Q-type Ca²⁺ channels at this synapse. This is consistent with the hypothesis that G_o-type G proteins (but not G_i-type) are involved in the membrane-delimited inhibition of Ca²⁺ currents (29–31). It has been reported that G_iα (not G_oα) inhibits adenylyl cyclase (6). Our results also indicate that cyclic nucleotides are not involved in the baclofen-induced inhibition of presynaptic Ca²⁺ currents. It has previously been reported that G_i (but not G_o) activates the G protein-activated inwardly rectifying potassium current (GIRK) (32). At the calyx of Held synapse, baclofen does not activate GIRK, but photo-released GTP[γS] from caged compound can activate it (3). It is possible that G_oα fully activated by GTP[γS] might activate GIRK. It is also possible that G_iα is expressed to a small extent at the calyx terminal and can activate GIRK after strong stimulation by GTP[γS], although it cannot couple GABA_B receptors with GIRK.

An involvement of a phospholipase C-dependent pathway has been suggested for the inhibition of P/Q-type Ca²⁺ currents by mGluR7 in cerebellar granule cell soma (33). The calyx of Held terminal expresses mGluR4 and also mGluR7 at later stages of development (34), and presynaptic inhibition by an mGluR agonist is mediated by inhibition of presynaptic P/Q-type Ca²⁺ currents (2). This effect of mGluR agonist can be completely occluded by baclofen (Y.K. and T.T., unpublished observation), suggesting that the presynaptic inhibitory mechanism via mGluRs and GABA_B receptors may be identical. Also, at this synapse, activation of protein kinase C by phorbol ester has no effect on presynaptic Ca²⁺ currents (8). Thus, the G_oβγ seems to play essential roles in autoreceptor- and heteroreceptor-mediated presynaptic inhibition at the calyx of Held synapse. At this synapse, a general block of G protein activity using GDP[βS] slows the rate of recovery from activity-dependent synaptic depression by blocking vesicle trafficking (7). It remains to be seen whether G_oβγ is involved in the vesicular replenishment at this mammalian nerve terminal.

Inhibition of Ca²⁺ currents by G proteins is characterized by a slowing in the rate of activation and the relief of inhibition following large depolarization, which has been explained by proposing a shift of Ca²⁺ channel gating between “reluctant” and “willing” states (13). These properties were also observed for the inhibitory effects of Gβγ and baclofen on presynaptic P/Q-type Ca²⁺ currents. It is possible that presynaptic inhibition by means of G protein-coupled receptors is relieved during high-frequency transmission because of presynaptic action potentials (35). At the calyx of Held synapse, presynaptic Ca²⁺ currents undergo facilitation during repetitive activation (36, 37). This facilitation depends on Ca²⁺ influx and can be induced by a 1-ms depolarizing conditioning pulse from −80 mV to −10 mV but is not affected by GDP[βS] or GTP[γS], excluding an involvement of G proteins in its mechanism (36). In fact, much stronger depolarization is required for relieving I_pCa from G protein-dependent inhibition. Thus, the physiological significance of the depolarization-induced relief of Ca²⁺ currents from G protein-dependent inhibition remains to be determined.

Abbreviations

GABA_B receptor

γ-aminobutyric acid type B receptor

GDP[γS]

guanosine 5′-O-(2-thiodiphosphate)

GTP[γS]

guanosine 5′-O-(3-thiotriphosphate)

Gβγ

G protein βγ subunits

MNTB

medial nucleus of the trapezoid body

GIRK

G protein-activated inwardly rectifying potassium current

mGluR

metabotropic glutamate receptor