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J. Biol. Chem., Vol. 281, Issue 34, 24204-24215, August 25, 2006

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The Metabotropic Glutamate G-protein-coupled Receptors mGluR3 and mGluR1a Are Voltage-sensitive^*

From the Department of Neurobiology, the Hebrew University, Jerusalem 91904, Israel

Received for publication, December 19, 2005 , and in revised form, May 22, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
G-protein-coupled receptors play a key role in signal transduction processes. Despite G-protein-coupled receptors being transmembrane proteins, the notion that they exhibit voltage sensitivity is rather novel. Here we examine whether two metabotropic glutamate receptors, mGluR3 and mGluR1a, both involved in fundamental physiological processes, exhibit, by themselves, voltage sensitivity. Measuring mGluR3-induced K⁺ currents and mGluR1a-induced Ca²⁺-activated Cl^– currents in Xenopus oocytes, we show that the apparent affinity toward glutamate decreases (mGluR3) or increases (mGluR1a) upon depolarization. Measurements of binding of [³H]glutamate to oocytes expressing either mGluR3 or mGluR1a corroborated the electrophysiological results. Using the chimeric G subunit, we further show that the voltage sensitivity does not reside in the G-protein. To locate sites within the receptors that are involved in the voltage sensitivity, we used chimeric mGluR1a, where the intracellular loops that couple to the G-protein were replaced by those of mGluR3. The voltage sensitivity of the chimeric mGluR1a resembled that of mGluR3 and not that of the parental mGluR1a. The cumulative results indicate that the voltage sensitivity does not reside downstream to the activation of the receptors but rather in the mGluR3 and mGluR1a themselves. Furthermore, the intracellular loops play a crucial role in relaying changes in membrane potential to changes in the affinity of the receptors toward glutamate.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
G-protein-coupled receptors (GPCRs)² comprise the largest superfamily of proteins in the body (1) and are involved in most signal transduction processes. GPCRs are activated by binding specific agonists, but, being transmembrane proteins, their activity could potentially be modulated also by membrane potential, and this in turn would enlarge the scope of their activity. Indeed, membrane potential was reported to regulate various processes that are mediated by a variety of different subtypes of GPCRs. For example, Ca²⁺ release from intracellular stores induced by a muscarinic receptor (2), a purinergic receptor P2Y (3), or by an adrenergic receptor (4) were all reported to be voltage-dependent. Also, it was reported that the inhibition of P/Q-type Ca²⁺ channels by the metabotropic glutamate receptor of subtype 7 is affected by membrane potential (5).

In all studies mentioned above, as well as in other studies demonstrating a regulatory effect of membrane potential on GPCR-mediated processes (6–8), the step at which the voltage sensitivity resides was not directly discerned. In particular, it was not established whether the voltage sensitivity resides in the GPCR itself or in other components and/or steps involved in the overall process. Recently, however, using the Xenopus oocytes expression system, Ben-Chaim et al. (9) showed that both the M2 and the M1-muscarinic receptors are by themselves voltage-sensitive; their affinity toward ACh depends on membrane potential.

The question that we address here is whether also GPCRs belonging to the mGluRs family exhibit by themselves voltage sensitivity. We selected the mGluRs, because members of this family are involved in most fundamental processes in the central nervous system (10) (i.e. control of release of various neurotransmitters (11), neurotoxicity, and synaptic plasticity (12)). We specifically selected mGluRs that are coupled to different G-proteins: mGluR3 that is coupled to G_i/G_o class (12) and mGluR1a that is coupled to G_q (13). Using Xenopus oocytes, we show that both mGluR3 and mGluR1a are voltage-sensitive.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Preparation of Oocytes and cRNA—Xenopus laevis oocytes were isolated and incubated in NDE96 solution composed of ND96 (96 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM Hepes-NaOH, pH 7.5), with the addition of 2.5 mM Na⁺ pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin (14). A day after their isolation, the oocytes were injected (Picoshpritzer, PLI-100; Medical Systems Corp.) with the relevant cRNAs. In vitro synthesis of RNA transcripts from the cloned cDNA was performed using standard procedures (14). The chimeric receptors R1a/3(i1,i2) and R1a/3(i2,i3), a gift from J. P. Pin (Centre National de la Recherche Scientifique), were described in detail by Gomeza et al. (15). Both chimeras were subcloned into the pGEMHE vector (16). The chimeric G_i3/q was a gift from N. Dascal (Tel-Aviv University, Israel). The amounts of cRNA injected per oocyte were as follows: GIRK1 and GIRK2, 0.2 ng; G_i3 and G_i3/q, 2 ng; mGluR3, 5 ng; mGluR1a, 0.4 ng, and R1a/3(i1,i2) and R1a/3(i2,i3), 4.5 ng. For the [³H]Glu binding experiments, both mGluR1a and mGluR3 were subcloned into the pGEMHE vector, which provides 5'- and 3'-untranslated regions of the Xenopus -globin RNA, ensuring a high level of protein expression in the oocytes (16). The amounts of cRNA injected per oocyte were the same as for the current measurements except for the mGluR1a, which was 5 ng.

Current Measurements—The currents were measured 3–5 days after cRNA injection and were recorded using the standard two-electrode voltage clamp technique (Axoclamp 2B amplifier, Axon Instruments, Foster City, CA). The oocyte was impaled with two electrodes pulled from 1.5-mm Clark capillaries (CEI, Pangbourne, UK). Both electrodes were filled with 100 mM KCl solution in order to prevent elevation of [K⁺]_in. The recording and the current-passing electrode resistances were 15 and 1 megaohms, respectively. pCLAMP8 software (Axon Instruments) was used for data acquisition and analysis.

mGluR3-induced GIRK Currents—For measurements of mGluR3-induced GIRK currents (17), oocytes injected with cRNA of GIRK1, GIRK2, G_i3, and mGluR3 were voltage-clamped at either –80 mV or +40 mV in ND96 solution (see example in Fig. 1B). cRNA of G_i3 was injected in order to decrease the basal GIRK current (I_K) that is produced by free endogenous G (18) and hence to improve the relative activation of the GIRK channels by the agonist (19). To measure K⁺ currents, ND96 was replaced by 24 mM K⁺ solution (similar to ND96 but with 72 mM NaCl, 24 mM KCl, and pH adjusted with KOH), and I_K appeared (20). Then four Glu concentrations were applied in an increasing order, without washout between applications, and the mGluR3-induced GIRK currents (I_Glu) appeared. I_Glu was terminated upon Glu washout (see example in Fig. 1A). After a 10-min washout in ND96, the oocyte was voltage-clamped to the next holding potential, and the procedure was repeated. The order of the holding potentials was randomly selected. Such a continuous exposure to Glu (3 min) may distort the true DR curve if the receptor undergoes desensitization. mGluR3 does not exhibit desensitization in a time scale of seconds (17) (see Fig. 1B), yet it might undergo desensitization with a slow time constant in the range of minutes. To check for this possibility, the following experiment was conducted (see one example in Fig. 1A). Four concentrations of Glu (30, 80, 300, and 20,000 nM) were applied sequentially. After 10 min of washout, only 30 and 20,000 nM Glu were reapplied. The ratio between I_Glu induced by 30 nM and I_Glu induced by 20,000 nM was measured under the two experimental protocols. The ratio of I_{Glu (30 nM)}/I_{Glu (20,000 nM)} was found to be 0.21 at the first application and 0.23 at the second application. In a total of five experiments, the average ratio was found to be 0.25 ± 0.59 when four concentrations of Glu were applied and 0.25 ± 0.77 when only 30 and 20,000 nM were applied. These results indicate that the mGluR3 did not desensitize in the course of the 3-min experiment. The results further show that the 10-min washout between the measurements in the two holding potentials does not affect the reliability of the DR curves.

Chimeric R1a/3(i1,i2)- and R1a/3(i2,i3)-induced GIRK Currents—The activity of R1a/3(i1,i2) and R1a/3(i2,i3) was measured as described above for the mGluR3 in oocytes injected with cRNA of GIRK1, GIRK2, G_i3, and R1a/3(i1,i2), or R1a/3(i2,i3). A control experiment similar to that described in Fig. 1A was repeated for the chimeric receptors, with similar results (data not shown).

mGluR1a-mediated Cl^– Currents—For measurements of mGluR1a-induced Ca²⁺-activated Cl^– currents (I_Cl(Ca)), oocytes injected with cRNA of mGluR1a were placed in ND96 solution and voltage-clamped to either –80 or +40 mV. Then a specific Glu concentration was applied, and I_Cl(Ca) appeared (see example in Fig. 3A). The Cl^– channel undergoes inactivation (see Fig. 3A), and tens of minutes are required for full recovery (21). We thus examined the duration of washout needed under our experimental conditions for full recovery of the I_Cl(Ca). To do so, a saturating concentration of Glu (200 µM) was applied, and the induced I_Cl(Ca) was measured. Then, after various times of Glu washout, the same Glu concentration was reapplied, and the ratio of I_Cl(Ca) before and after the washout was determined. We found that the shortest time required for full recovery of I_Cl(Ca) is 20 min. In a total of three experiments, the average ratio between I_Cl(Ca) before and after the 20-min washout was 1.06 ± 0.05 (data not shown).

mGluR1a-mediated GIRK Currents via Coupling to Chimeric G_i3/q—The mGluR1a-induced GIRK currents were measured in oocytes injected with cRNAs of GIRK1, GIRK2, mGluR1a, and chimeric G_i3/q. 30 minutes before the recordings, the oocytes were injected with 25 nl of 50 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and 600 µM U-73122 (an inhibitor of phosphlipase C) and incubated in 10 µM U-73122 (17). I_Glu was measured as described for mGluR3.

Radioligand Binding Experiments—Binding of radiolabeled ligand was performed by a procedure developed specifically for single Xenopus oocytes (9). We measured binding of L-[G-³H]glutamic acid (50 Ci/mmol; Amersham Biosciences) or [³H]LY341495 (34.61 Ci/mmol; Tockris Cookson, Bristol, UK) to oocytes injected with cRNA of mGluR3 or mGluR1a or R1a/3(i2,i3), at two membrane potentials. Oocytes were also injected with cRNA of GIRK1 and GIRK2 and also with G_i3 in the case of mGluR3 and R1a/3(i2,i3). Variations in membrane potential were achieved by changing the [K⁺]_out while osmolarity and ionic strength were kept constant by replacing NaCl with KCl. GIRK channels were expressed in order to amplify the difference between the membrane potentials thus achieved. The membrane potential of the oocytes at the different K⁺ solutions was measured with a standard intracellular electrode. Oocytes not injected with the GIRK channel exhibited at ND96 solution a resting potential of –31.8 ± 3 mV (n = 6), whereas the resting potential of oocytes injected with the GIRK channel shifted to –82 ± 2.6 mV (n = 9). In high K⁺ solution (similar to 24 mM K⁺ solution but with 2 mM NaCl and 96 mM KCl), the oocytes, either injected or uninjected with GIRK channels, were depolarized to +3 ± 0.75 mV (n = 6). Single oocytes were incubated for 90 s with the indicated concentrations of radioligand, in either ND96 or high K⁺ solution. Each oocyte was rapidly dropped into a washing chamber filled with 4 ml of ice-cold ligand-free ND96 or high K⁺ solution. In less than 1 s, the oocyte reached the bottom of the washing chamber and was collected by a pipette into a vial containing 3 ml of scintillation liquid. Nonspecific binding was measured similarly but in the presence of saturating concentrations of a selective nonradioactive ligand in the incubation solution: 5 µM LY-379268 (a generous gift of Lilly Research Laboratories) for mGluR3 and 0.3 mM quisqualate (Tockris Cookson) for mGluR1a and R1a/3(i2,i3). It should be noted that nonspecific binding varied between oocytes from different donors. Specific binding was calculated for each experiment by subtraction of the average nonspecific binding at each radioligand concentration and each membrane potential (5–10 oocytes), from the total binding to individual oocytes. Binding of [³H]Glu, as described above, was also measured in control oocytes injected only with GIRK subunits.

Binding of [³H]Glu was also measured, as above, in perforated oocytes. The oocytes were gently impaled with a standard recording electrode several times (but remained intact), and their resting potential was measured to be –26 ± 1.6 mV at ND96 and +2 ± 0.9 mV (n = 36) at high K⁺ solution.

Data Analysis—The electrophysiological DR curves as well as the radioligand binding data were fitted with Equation 1, a Michaelis-Menten type equation assuming two agonist binding sites.

(Eq. 1)

In the case of the DR curves, Y is the fractional amplitude of the current at any agonist concentration, B_max is the response to saturating concentration of agonist defined as 100%, X is the concentration of the agonist (Glu), and K_d denotes the dissociation constant. In the case of the binding data, Y is the specific binding, B_max is the maximal binding, and X and K_d are as above. The DR curves of mGluR3-induced GIRK currents were also fitted with Equation 2, a Michaelis-Menten type equation that assumes the existence of two populations of receptors and two agonist binding sites.

(Eq. 2)

K^H_d denotes the K_d of the high affinity population, K^L_d denotes the K_d of the low affinity population, B^H_max is the fraction of the high affinity population, and B^L_max is the fraction of the low affinity population. The sum of the two fractions is set to be 100%. Y and X are the same as in Equation 1. For all of the curves, the data were best fitted with the assumption of two agonist binding sites, in particular in the low Glu concentrations. This is in agreement with the dimerization of mGluRs and with the reports that full activation requires binding of Glu to both subunits in the homodimer (22, 23).

The decay time constants of GIRK currents (Figs. 4D and 6) were analyzed by fitting a single exponent to the falling phase of the current trace. In all fittings, the least-square method was employed. Least-square fitting and t test statistical analysis were performed with Prism software (GraphPad). Statistical results are presented as mean ± S.E. Chemicals were purchased from Sigma Israel (Rehovot, Israel), unless otherwise stated.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Membrane Potential Affects the Dependence of mGluR3-mediated K⁺ Currents on Glu Concentration—To assay the activity of mGluR3, we used the mGluR3-induced GIRK currents (17). The GIRK channel undergoes inward rectification that occurs at potentials more positive than –50 mV (24) (see Fig. 2A). In order to unravel whether membrane potential exerts, in addition to its effect on the GIRK channel, also an effect on mGluR3 itself, we compared the dependence of I_Glu on Glu concentration, DR, at two holding potentials of –80 and +40 mV.

The protocol for the DR curves construction is described in the legend to Fig. 1B and under "Experimental Procedures." To evaluate I_Glu, we notice that the amplitude of I_K declines with time (broken line in Fig. 1A; see Ref. 20). Therefore, the magnitude of I_Glu (dotted line) was calculated by subtraction of the extrapolated I_K from the total current. The DR curves were constructed by normalizing I_Glu to the response obtained with the saturating concentration of Glu (20,000 nM) at the same holding potential. Normalization is required to compensate for the intrinsically different I_K obtained at the two holding potentials of –80 and +40 mV in a single oocyte and to be able to compare between oocytes varying in their I-V relation.

Fig. 1C shows a clear shift to the right of the DR curve at +40 mV. Fitting the data with Equation 1 (solid lines) indicates that depolarization shifted the apparent K_d from 38 ± 2.4 nM at –80 mV to 136 ± 9 nM at +40 mV (p < 0.001, n = 28) (i.e. a 3.5-fold increase). Concerning GPCRs, it was shown that they exist in two affinity states toward their agonist: high affinity when coupled to the G-protein and low affinity while free (25). This suggests that the data of Fig. 1C may also be fitted by an equation assuming the existence of two populations of receptors. Indeed, the data could be fitted with Equation 2 (broken lines), showing that at –80 mV a high affinity population prevails (K_d = 38 nM, 98%), whereas at +40 mV the high affinity population accounts for only 10% of the receptors, and 90% of the receptors are at a low affinity state (K_d = 160 nM). However, since both equations fit the data equally well, we cannot conclude at this stage whether depolarization affects the K_d itself or reduces the high affinity population.

The observed shift in the DR curve may be caused by cellular changes due to the long depolarization rather than by a reversible specific effect of depolarization on the mGluR3. To check for this possibility, after measuring a DR curve at –80 mV, the holding potential was switched to +40 mV for a period of 10 min and then switched back to –80 mV, and a DR curve was remeasured. Fig. 1D rules out this possibility, since the DR curves established at –80 mV before and after the depolarization period were practically identical.

Some of the oocytes contain voltage-dependent Na⁺ channels (26) that could potentially distort the DR curves. This possibility can be ruled out, since the results obtained in oocytes that did not contain voltage-dependent Na⁺ channels (Fig. 1C, empty symbols) are indistinguishable from the rest of the data.

Application of Glu may activate endogenous Glu receptors or channels other than GIRK. This possibility is ruled out, since uninjected oocytes, as well as oocytes injected with cRNA of mGluR3 but without the GIRK channels, did not show any current in response to Glu application at both holding potentials of –80 and +40 mV (data not shown).

The Voltage Sensitivity Does Not Reside in the Activation of the GIRK Channel—The shift in the DR curve seen in Fig. 1C could be due to voltage sensitivity in steps that are downstream to the activation of the mGluR3. For example, Hommers et al. (27) suggested that G subunits not only activate the GIRK channel but also, at high concentrations, directly affect the rectification properties of the channel. To check for this possibility, we compared the I-V relation of the basal GIRK current, I_K-V, to the I-V relation of mGluR3-induced GIRK current, I_{Glu(20 µM)}-V, obtained with saturating Glu concentration. At a saturating Glu concentration, any putative effect of membrane potential on the receptor affinity is abolished. Hence, if the I_{Glu(20 µM)}-V differs from I_K-V, it will support the suggestion of Hommers et al. (27).

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Voltage-dependent shift in the DR curves of mGluR3-induced GIRK currents. A, control experiments for DR curve measurements. The oocyte was voltage-clamped to –80 mV. Four concentrations of Glu (1, 2, 3, and 4, represent 30, 80, 300, and 20,000 nM, respectively) were applied sequentially. After a 10-min washout in ND96 solution, only two concentrations of Glu (30 and 20,000 nM) were applied. In both cases, the ratio between IGlu(30 n) and IGlu(20,000 n) was similar (0.21 and 0.23). B, mGluR3-induced GIRK currents. Top, the oocyte was voltage-clamped to –80 mV. Four Glu concentrations were applied sequentially (1, 2, 3, and 4 represent 25, 150, 250, and 20,000 nM, respectively). Bottom, after a 10-min washout in ND96 solution, the holding potential was switched to +40 mV, and the protocol described above was repeated. Notice changes in the calibration bars. Broken line, extrapolated IK; dotted line, IGlu. C, full DR curves of mGluR3-induced GIRK currents at –80 mV () and +40 mV ()(n = 28 oocytes taken from four donors). Empty symbols represent the results from three oocytes that did not contain voltage-dependent Na+ channels, at –80 mV () and +40 mV (). D, DR curves at –80 mV before () and after () 10 min of depolarization at +40 mV (n = 3). Data points represent mean ± S.E. Solid lines in C and D, fit with Equation 1; broken lines, fit with Equation 2 (see "Experimental Procedures").

The Binding of [³H]Glu to Oocytes Expressing mGluR3 Is Voltage-dependent—To check directly whether the voltage sensitivity seen in Fig. 1C resides in the activation of mGluR3, we measured binding of [³H]Glu to individual oocytes expressing mGluR3 at two membrane potentials. Variations in membrane potential were induced by changing the [K⁺]_out, using ND96 and high K⁺ solutions.

An example of one experiment is depicted in Fig. 2B, presenting the average total, nonspecific, and specific binding of 100 nM [³H]Glu to individual oocytes. The nonspecific binding was measured in the presence of the selective mGluR3 agonist LY-379268 (28). It is seen that the specific binding of [³H]Glu is voltage-dependent; it is significantly higher at ND96 solution than at high K⁺ (p = 0.0183, n = 6–14). In a similar experiment, but with control oocytes that do not express mGluR3, no specific binding was observed. In particular, in high K⁺, the total binding was 2573 ± 123 dpm, and the nonspecific binding was 2434 ± 227 dpm (p = 0.6, n = 6). At ND96, the total binding was 2245 ± 157 dpm, and the nonspecific binding was 2386 ± 180 dpm (p = 0.57, n = 7). These results indicate that the specific binding obtained with LY-379268 reflects binding only to the mGluR3 and not to other putative endogenous receptors. Fig. 2C depicts results of experiments similar to the experiment described in Fig. 2B, obtained with various [³H]Glu concentrations. The average specific binding of [³H]Glu was significantly higher at ND96 solution than at high K⁺ at all Glu concentrations. To examine whether it is the K⁺-induced depolarization or the actual K⁺ concentration that produces the results of Fig. 2A, we measured the binding of one concentration of [³H]Glu to perforated oocytes that are depolarized also at ND96 (see "Experimental Procedures"). The binding of 75 nM [³H]Glu to perforated oocytes was similar at ND96 and high K⁺ solution (5.4 ± 1.1 and 6.1 ± 1.4 fmol of [³H]Glu/oocyte, respectively; p = 0.7, n = 8–10) and was not significantly different from the binding to unperforated oocytes at high K⁺ (7.8 ± 0.36 fmol of [³H]Glu/oocyte; p = 0.42). This result indicates that it is indeed the voltage, and not K⁺ concentration, that affects [³H]Glu binding to mGluR3.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. The voltage sensitivity of mGluR3 does not reside downstream to its activation. A, example of I-V curves of IK (black) measured at 24 mM K+ solution and IGlu (gray) measured at 24 mM K+ solution with 20 µM Glu. Curves are normalized; for each, the current at –80 mV was referred to as –1. B, an example of [3H]Glu binding to oocytes expressing mGluR3 and GIRK channels at –82 mV (ND96, left) and at 0 mV (high K+, right). Presented are average dpm values of 100 nM [3H]Glu total binding (Total, white bars), nonspecific binding (measured in the presence of 5 µM LY-379268; n.s., gray bars), and specific binding (s.b., black bars) obtained by subtraction of the average nonspecific binding from the total binding to individual oocytes. The average total binding was significantly different from the average nonspecific binding for both ND96 and high K+ (***, p < 0.001, n = 8–16). C, specific binding of various [3H]Glu concentrations to individual oocytes at –82 mV (ND96 solution, ) and at 0 mV (high K+ solution, )(n = 8–35 taken from seven donors). D, as in B but with 10 nM [3H]LY341495 (n = 6–12). E, as in C but with [3H]LY341495 (n = 6–24 taken from five donors). Solid lines, fit with Equation 1. Data points represent mean ± S.E.

We next examined whether the binding of a selective mGluR3 antagonist, [³H]LY341495 (29), to oocytes expressing mGluR3 is voltage-dependent. Fig. 2, D and E, shows that this is not the case. Fig. 2D describes an example of one experiment, presenting the average total, nonspecific, and specific binding of 10 nM [³H]LY341495 to individual oocytes. Fig. 2E depicts results of similar experiments obtained with various [³H]LY341495 concentrations. Fitting the data of Fig. 2E with Equation 1 showed that both the affinity and the maximal binding of [³H]LY341495 are similar at ND96 and high K⁺ (K_d = 5.7 ± 1.3 and 6.2 ± 0.9 nM at ND96 and high K⁺, respectively (p = 0.76, n = 5); B_max = 24.9 ± 2.8 and 24.3 ± 1.8 at ND96 and high K⁺, respectively (p = 0.84, n = 5)).

Membrane Potential Affects the Dependence of mGluR1a-mediated Cl^– Currents on Glu Concentration—We used the mGluR1a-mediated Ca²⁺-activated Cl^– currents, I_Cl(Ca), (30) to assay the activity of mGluR1a. The Cl^– channel undergoes outward rectification at potentials more negative than –60 mV (31). Hence, in order to expose possible voltage sensitivity of the mGluR1a, we constructed, as for the mGluR3, full DR curves at the two holding potentials of –80 and +40 mV.

The mGluR1a-evoked I_Cl(Ca) undergoes rapid inactivation upon prolonged exposure to Glu or following repeated agonist applications, and full recovery requires several min (21, 32). To ensure viability of the oocytes, we applied in each oocyte only two concentrations of Glu (submaximal and saturating), with a 20-min washout between the applications, at only one holding potential (see experimental protocol in Fig. 3A). Notice that the decay of I_Cl(Ca) is faster at –80 mV than at +40 mV at both Glu concentrations, presumably as a result of the intrinsic properties of the Cl^– channel (33).

Before constructing the DR curves, we should address the following point. As mentioned, the mGluR1a-induced I_Cl(Ca) undergoes rapid inactivation. It is therefore possible that the actual magnitude of the current is underestimated due to the inactivation. This is, however, not the case. Fig. 3A (insets) shows that I_Cl(Ca) currents reach a steady-state prior to inactivation. Hence, the peak current (Fig. 3A, insets, dotted lines) reliably reflects the full magnitude of I_Cl(Ca).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. The apparent affinity of mGluR1a and the binding of [3H]Glu to oocytes expressing mGluR1a are voltage-dependent. A, mGluR1a-induced Cl– current measurements. Top, the oocyte was voltage-clamped to +40 mV in ND96 solution, and a submaximal Glu concentration (10 µM) was applied. After a 20-min washout in ND96, saturating concentration of Glu (200 µM) was applied. Bottom, the same procedure was repeated in a different oocyte at –80 mV. See insets for faster time scale of the dashed rectangles, notice the different calibration bars. Dotted lines, ICl(Ca). B, full DR curves of mGluR1a-induced Cl– currents at –80 mV () and at +40 mV () (n = 72 taken from 15 donors). Empty symbols represent the results from oocytes that did not contain voltage-dependent Na+ channels, at –80 mV () and +40 mV ()(n = 37 taken from seven donors). C, an example of [3H]Glu binding to oocytes expressing mGluR1a and GIRK channels at +0 mV (high K+, left) and at –82 mV (ND96, right). Presented are average dpm values of 500 nM [3H]Glu total binding (Total, white bars), nonspecific binding (in the presence of 0.3 mM quisqualate; n.s., gray bars), and specific binding (s.b., black bars) obtained by subtraction of the average nonspecific binding from the total binding to individual oocytes. The average total binding was significantly different from the average nonspecific binding for both ND96 and high K+ (***, p < 0.0001, n = 10; *, p < 0.05, n = 16–19). The difference between the nonspecific binding at ND96 and at high K+ is not significant (p = 0.147, n = 10–16). D, specific binding of various [3H]Glu concentrations to individual oocytes at –82 mV (ND96 solution, ) and at 0 mV (high K+ solution, )(n = 6–14 taken from eight donors). Solid lines in B and D, fit with Equation 1. Data points represent mean ± S.E.

The mGluR1a is reported to undergo desensitization (34). If this desensitization is voltage-dependent, it could explain the observed voltage dependence of the DR curves. Due to the rapid inactivation of the I_Cl(Ca) (21), we could not exploit these currents to examine this possibility. However, we used mGluR1a-induced GIRK currents (activated via the chimeric G_i3/q and described in detail in Fig. 4) to check whether the desensitization of mGluR1a is voltage-dependent. We measured mGluR1a-induced GIRK currents at –80 and +40 mV, in a continuous presence of a saturating concentration of Glu (200 µM; see example in Fig. 4D). The falling phase of the current, which under these experimental conditions reflects desensitization, was fitted by a single exponent. Fig. 4E shows that the rate of desensitization, _Des, of mGluR1a is not voltage-dependent; it is similar at +40 and –80 mV (32.2 ± 3.6 and 35.3 ± 2s, respectively; p = 0.43, n = 14 and 18, respectively). These results imply that the DR curves of Fig. 3B reflect genuine voltage-dependent affinity of mGluR1a toward Glu.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. The voltage dependence of mGluR1a coupled to chimeric Gi3/q is similar to that of mGluR1a coupled to native Gq. A, amino acid sequence of native Gi3 (white background) and native Gq (gray background). Chimeric Gi3/q was constructed by replacing the four C-terminal amino acids of Gi3 with those of Gq. B, mGluR1a-Gi3/q activated GIRK current measurements. Top, the oocyte was voltage-clamped to –80 mV, and 10 µM Glu was applied. Bottom, the same procedure was repeated in a different oocyte, at +40 mV. Note the different calibration bars. C, full DR curves of mGluR1a-Gi3/q activated GIRK currents at +40 mV () and at –80 mV () (n = 145, taken from seven donors). Solid lines, fit with Equation 1. D, an example of IGlu desensitization in the continuous presence of agonist (200 µM), at a holding potential of –80 mV. Broken line, fit to a single exponent. E, Des of mGluR1a-induced IGlu (200 µM), at holding potentials of –80 mV (gray) and +40 mV (black)(n = 18 and 14, respectively). Data points represent mean ± S.E.

The results so far are compatible with the notion that both mGluR3 and mGluR1a may be, by themselves, voltage-sensitive. However, although not likely, the voltage sensitivity could reside in the G-protein, since its coupling regulates the affinity of GPCRs (25). This possibility is examined below.

The Voltage Sensitivity Does Not Reside in the G-protein—Because mGluR1a couples both to G_q (mainly) and to G_i (to a lesser extent) (32), the ideal way to check whether the voltage sensitivity resides in the G-protein would be to compare the behavior of mGluR1a when coupled to G_q with its behavior when coupled to G_i. However, this is not possible, because the mGluR1a-G_i-induced GIRK currents are extremely small (32). We therefore used a chimeric G subunit, G_i3/q, which is identical to G_i3, excluding the last four C-terminal residues that were replaced by the corresponding ones of G_q (Fig. 4A). These four residues determine the specificity of the G-protein toward the receptor (36). The chimeric G_i3/q, unlike the native G_i3, couples efficiently to mGluR1a as evident from the large GIRK currents produced in oocytes expressing mGluR1a, GIRK channels, and the chimeric G_i3/q (Fig. 4B). The oocytes were also injected with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, to prevent the activation of the endogenous Cl^– channels, and with the phosphlipase C inhibitor U-73122, to prevent inhibition of the GIRK channel by endogenous G_q (17). These treatments affected the viability of the oocytes and hence limited the permissible duration of an experiment. We thus measured in each oocyte the response to only one Glu concentration at only one holding potential. An example of such experiment is seen in Fig. 4B.

Fig. 4C depicts normalized DR curves of mGluR1a-G_i3/q-activated GIRK currents. It is seen that as for mGluR1a-G_q, mGluR1a-G_i3/q complex shows a higher apparent affinity at +40 mV than at –80 mV. The apparent K_d was 6.4 ± 0.85 µM at –80 mV and decreased 1.8-fold at +40 mV (3.6 ± 0.6 µM; p = 0.029, n = 42 and 24, respectively), K_d values similar to those obtained with mGluR1a-G_q-induced Cl^– currents. Thus, the voltage sensitivity does not reside in the G-protein.

The Intracellular Loops of mGluR3 and mGluR1a Are Involved in Determining the Voltage-dependent Affinity of the Receptors—The next question is then which sites in the receptors could be involved in determining their voltage-dependent affinity toward Glu. Recalling that the affinity of GPCRs toward their agonists is regulated by their coupling to the G-protein (25) and acknowledging the results of Fig. 4C, it is possible that the intracellular loops that couple to the G-protein are involved in determining the voltage-dependent affinity. In mGluRs, it was shown that the second intracellular loop (i2) plays a key role in the coupling to the G-protein (37), but the first (i1) and the third (i3) intracellular loops are also required to render the coupling efficient (15). Examining the amino acid sequence of the intracellular loops in both receptors (Fig. 5A), we see that these intracellular loops contain clusters of charged residues that differ in charge distribution in the two receptors, which is compatible with the suggestion raised above.

We thus examined the voltage sensitivity of two mGluR1a chimeric receptors. In one chimera, R1a/3(i2,i3), i2 and i3 were replaced, whereas in the other chimera, R1a/3(i1,i2), i2 and i1 were replaced by the corresponding ones of mGluR3 (Figs. 5, B and C, respectively, right). These two chimeric mGluR1a/3 do not activate the Ca²⁺-activated Cl^– currents in oocytes (15). We therefore first checked whether they do activate GIRK currents as the mGluR3 does. Fig. 5, B and C (upper left), shows that large GIRK currents were produced by the two chimeric mGluR1a/3. We thus repeated the experiment of Fig. 1C but now with R1a/3(i1,i2) or R1a/3(i2,i3). It should be noted that the chimeric receptors, unlike the parental mGluR1a, did not exhibit desensitization. This is presumably because the substrate for protein kinase C-mediated phosphorylation that is responsible for mGluR1a desensitization, Thr⁶⁹⁵ (34), is located within mGluR1a i2 but is not present in i2 of mGluR3.

The DR curves in Fig. 5B show that the chimeric receptor R1a/3(i2,i3) resembles in its voltage sensitivity the mGluR3 and not the mGluR1a. The apparent K_d was 0.62 ± 0.01 µM at –80 mV and increased 3.7-fold at +40 mV (2.3 ± 0.06 µM). The ratio between the K_d values of R1a/3(i2,i3) at the two holding potentials resembles that of the native mGluR3 (3.5). Notice, however, that the actual range of K_d values of R1a/3(i2,i3) resembles that of mGluR1a. This may be expected, since the chimeric receptors possess the agonist binding site of the native mGluR1a. The second chimeric receptor, R1a/3(i1,i2), also exhibits voltage sensitivity that resembles in its direction that of the native mGluR3 (Fig. 5C), but to a lesser extent. The apparent K_d was 0.53 ± 0.02 µM at –80 mV and increased only 2.4-fold at +40 mV (1.26 ± 0.06 µM). It should be noted that the apparent K_d values of R1a/3(i2,i3) and R1a/3(i1,i2) at –80 mV are rather similar, whereas at +40 mV, the K_d of R1a/3(i1,i2) is significantly lower than that of R1a/3(i2,i3) (p < 0.0001, n = 15 and 23, respectively). These results further strengthen the conclusion that the voltage sensitivity resides in the receptors themselves and suggest that the two intracellular loops, i2 and i3, and to a lesser extent i1 play a crucial role in relaying changes in membrane potential to changes in the affinity of the receptor. We argue that i1 plays a minor role, since the chimeric R1a/3(i2,i3) exhibits similar voltage sensitivity as the mGluR3 does and to the same extent.

The Binding of [³H]Glu to Oocytes Expressing R1a/3(i1,i2) Is Voltage-dependent—Binding of [³H]Glu to the chimeric R1a/3(i2,i3) expressed in oocytes was measured as described for mGluR3 and mGluR1a. Fig. 5D shows an example of one experiment in 600 nM [³H]Glu. The nonspecific binding was measured in the presence of the selective mGluR1a agonist quisqualate. It can be seen that the specific binding of [³H]Glu is voltage-dependent; it is significantly higher at ND96 than at high K⁺ (p = 0.006, n = 10–12). Fig. 5E depicts results of similar experiments obtained with various [³H]Glu concentrations. It is seen that, as for the electrophysiological results, R1a/3(i2,i3) retained the voltage dependence of mGluR3 and not that of mGluR1a. Here too, as for mGluR3 and mGluR1a, analysis of the binding curve revealed that the K_d was not affected by depolarization (K_d = 0.34 ± 0.03 and 0.34 ± 0.06 µM for ND96 and high K⁺, respectively; p = 0.9812), and the maximal binding was significantly reduced 1.6-fold upon depolarization (B_max = 172 ± 11 and 94 ± 14 fmol of [³H]Glu/oocyte for ND96 and high K⁺, respectively; p = 0.0007).

The Rate Constant of Dissociation of the Agonist from Both mGluR3 and mGluR1a Is Voltage-dependent—We next attempted to examine the biophysical mechanism that underlies the voltage dependence of the receptor affinity. To this end, we tested whether the K_d values obtained at –80 and +40 mV differ in their rate constant of agonist association (on-rate) or in their rate constant of agonist dissociation (off-rate) or in both. At first, it may seem impossible to detect rate constants that are associated with the first step in a long cascade of reactions. However, concerning GIRK currents induced by GPCRs, it was shown that the off-rate can be estimated from the decay of 2-adrenergic (38) and adenosine A1 receptor-induced (39) GIRK currents.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. The intracellular loops of mGluR3 and mGluR1a are involved in determining the voltage-dependent affinity of the receptors. A, amino acid sequence alignment of the intracellular loops of mGluR3 and mGluR1a. i1, i2, and i3 correspond to the first, second, and third intracellular loops, respectively. Positively charged residues (+) and negatively charged residues (–) are indicated. B and C (right side), schematic presentation of chimeric R1a/3(i2,i3) and R1a/3(i1,i2), respectively. The schemes present parental mGluR1a with the specific intracellular loops from mGluR3 (in light gray). B, DR curves of R1a/3(i2,i3)-induced GIRK currents. Top, experimental protocol at a holding potential of –80 mV. Four Glu concentrations were applied sequentially (1, 2, 3, and 4 represent 0.75, 2.5, 5, and 100 µM Glu, respectively). Broken line, extrapolated IK; dotted line, calculated IGlu. Bottom, full DR curves at –80 mV () and +40 mV ()(n = 28 taken from four donors). C, DR curves of R1a/3(i1,i2)-induced GIRK currents. Top, experimental protocol as in B, with Glu concentrations of 0.5, 0.9, 1.4, and 100 µM. Bottom, full DR curves at –80 mV () and +40 mV ()(n = 26 taken from three donors). Data points represent mean ± S.E. D, an example of [3H]Glu binding to oocytes expressing R1a/3(i2,i3) and GIRK channels at +0 mV (high K+, left) and at –82 mV (ND96, right). Presented are average dpm values of 600 nM [3H]Glu total binding (Total, white bars), nonspecific binding (measured in the presence of 0.3 mM quisqualate; n.s., gray bars), and specific binding (s.b., black bars) obtained by subtraction of the average nonspecific binding from the total binding to individual oocytes. The average total binding was significantly different from the average nonspecific binding for both ND96 and high K+ (**, p < 0.01, n = 6–12). E, specific binding of various [3H]Glu concentrations to individual oocytes at –82 mV (ND96 solution, ) and at 0 mV (high K+ solution, )(n = 6–18 taken from eight donors). Solid lines in B, C, and E, fit with Equation 1. Data points represent mean ± S.E.

We thus determined _D of mGluR3-induced I_Glu at the two holding potentials of –80 and +40 mV. Fig. 6C shows that the _D is significantly smaller at +40 mV in comparison with that at –80 mV (17 ± 1 and 29 ± 1.7 s, respectively; p < 0.0001; n = 19 and 39, respectively). In other words, the dissociation of Glu from mGluR3 is faster at +40 than at –80 mV.

We next measured the decay of GIRK currents but now induced by mGluR1a coupled to the chimeric G_i3/q. mGluR1a was reported to undergo agonist-dependent desensitization (34). However, we showed (Fig. 4E) that this desensitization is not voltage-dependent. Nevertheless, to ensure minimal interference of desensitization in the estimation of _D, we measured the decay of mGluR1a-Gi_3/q-induced currents at lower than saturating Glu concentration (i.e. 10 µM). Indeed, Fig. 4B shows that at a Glu concentration of 10 µM, mGluR1a exhibits only mild desensitization. Fig. 6D shows that in this case, in contrast to the results in Fig. 6C, depolarization increased _D (6.9 ± 0.3 and 10.3 ± 0.6 s at –80 and +40 mV, respectively; p < 0.0001, n = 20 and 17, respectively). Similar results were obtained with 5 µM Glu, where desensitization hardly exists. In particular,_D was 1.75-fold longer at +40 than at –80 mV (results not shown). The observation that the decay of GIRK currents exhibited opposite voltage dependence when activated by mGluR1a (in comparison with that seen when activated by mGluR3) implies that it is indeed the dissociation of Glu from mGluR3 (off rate) that is higher under depolarization. These results also suggest that for mGluR1a, in contrast to the case of mGluR3, the off-rate is lower under depolarization. Concerning the two chimeric receptors mGluR1a/3, Fig. 6E shows that both behave as the native mGluR3 and not as the parental mGluR1a. Specifically, _D was larger at –80 than at +40 mV for R1a/3(i2,i3) (22.1 ± 0.8 and 11.7 ± 0.5 s, respectively; p < 0.0001; n = 33 and 19, respectively) and for R1a/3(i1,i2) (22.7 ± 0.9 and 12.4 ± 1.2 s, respectively; p < 0.0001; n = 8 and 6, respectively).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Our results are compatible with the notion that two GPCRs belonging to the mGluRs family, mGluR3 and mGluR1a, are voltage-sensitive. Two main findings justify the conclusion that the voltage-dependent effect occurs at the level of the receptors. One is the demonstration that the binding of [³H]Glu is voltage-sensitive for both mGluR3 and mGluR1a. The other is the observation that replacing the intracellular loops of mGluR1a with those of mGluR3 reversed its voltage-dependent affinity. Whereas our results cannot discern where in the receptors the voltage sensor resides, we do show that the intracellular loops that couple to the G-protein, in particular i2 and i3, play a crucial role in linking changes in membrane potential to changes in the binding affinity of the receptors.

Based on our cumulative results together with previous ones (25, 40), we propose the following mechanism to account for the effect of membrane potential on the agonist binding affinity of GPCRs. The results of the experiments conducted with the chimeric receptors indicate that also for mGluR3 and mGluR1a, as for other GPCRs (25, 40), the coupling to the G-protein is involved in determining the affinity of the receptors toward their agonist (but see other reports (41, 42)). Therefore, we suggest that depolarization causes a conformational change that affects the intracellular loops that couple to the G-protein, thereby regulating the likelihood of the receptors to couple to their corresponding G-protein. Our binding experiments demonstrate that depolarization reduces (mGluR3) or increases (mGluR1a) the fraction of the high affinity receptor. Recalling that GPCRs are in a high affinity state when coupled to the G-protein and in a low affinity state when free (40), the binding results may be interpreted to mean that depolarization reduces (mGluR3) or increases (mGluR1a) the fraction of receptors coupled to the G-protein. This in turn determines the affinity of the receptors toward their agonist. In particular, when the receptor is coupled to the G-protein, it is characterized by a low off-rate and hence exhibits high affinity toward the agonist, whereas when free from the G-protein, its off-rate is high and hence it exhibits low affinity toward the agonist. The observation that the binding of the antagonist [³H]LY341495 to mGluR3 was voltage-independent (Fig. 2E) further supports the notion that depolarization affects the fraction of receptors coupled to the G-protein. This result is consistent with previous studies showing that in contrast to agonists, antagonists exert a similar affinity toward GPCRs whether coupled or uncoupled to their G-protein (25, 43).

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Depolarization affects the off-rates of mGluR3 and mGluR1a. A, an example of mGluR3-induced GIRK currents evoked in the same oocyte by saturating concentrations of Glu (20 µM) and LY-379268 (1.5 µM) at a holding potential of –80 mV and a washout of 10 min between applications. Broken line, fit to a single exponent, with a delay of 30 and 80 s from the beginning of agonist washout, for Glu and LY-379268, respectively. B, D of mGluR3-induced IGlu (20 µM, gray) and ILY, (1.5 µM, black), measured with a delay of 30 and 80 s from the beginning of agonist washout, respectively (n = 39 and 5 for IGlu and ILY, respectively). C, D of mGluR3-induced IGlu (20 µM) at holding potentials of –80 mV (gray) and +40 mV (black), measured with a delay of 30 s from the beginning of agonist washout. n = 39 and 19 at –80 and +40 mV, respectively. D, D of mGluR1a-induced IGlu (10 µM, coupled to chimeric Gi3/q) at holding potentials of –80 mV (gray) and +40 mV (black), measured with a delay of 10 s from the beginning of agonist washout (n = 20 and 17 at –80 and +40 mV, respectively). E, D of chimeric R1a/3(i2,i3) and chimeric R1a/3(i1,i2)-induced IGlu (100 µM) at holding potentials of –80 mV (gray) and +40 mV (black), measured with a delay of 25 s from the beginning of agonist washout. For R1a/3(i2,i3), n = 33 and 19 at –80 and +40 mV, respectively. For R1a/3(i1,i2), n = 6 and 8 at –80 and +40 mV, respectively. Bars, mean ± S.E. ***, p < 0.001.

Concerning the mGluR1a, it was implicated to play a critical role in long term depression in many different brain areas (44, 45). The best characterized example is the long term depression of the parallel fibers-purkinje cell synapse (46). Long term depression of this synapse occurs only when the climbing and the parallel fibers, which are the two main excitatory inputs to the cerebellum, are co-activated. Activation of mGluR1a that is located at the postsynaptic site of this synapse was shown to play a key role in induction of cerebellar long term depression (47). It is quite possible that the high affinity of mGluR1a toward Glu under depolarization plays a role in coupling the pre- and postsynaptic processes. If this will be shown to be the case, the mGluR1a will be another example, in addition to the N-methyl-D-aspartate receptor, that accommodates Hebbian behavior.

mGluR3 is known to be involved in feedback inhibition of Glu release (48–50). Concerning another GPCR that mediates feedback inhibition, it was recently shown (51) that M2-muscarinic receptor-mediated feedback inhibition of ACh release is exerted by two mechanisms. One, fast, occurs only in the presence of a high concentration of ACh and takes place at all membrane potentials, including high depolarization. The other process was shown to be slow and to occur already at a very low concentration of ACh (in the range of about 20 nM). This slow feedback inhibition, but not the fast one, is abolished at high depolarizations. The authors (51) suggested that the slow high affinity process, which is mediated by a second messenger, is probably important to maintain the rest (resting potential and resting concentration of ACh) level of release, whereas the low affinity fast process is likely to regulate the action potential-evoked ACh release.

mGluR3 is a suitable candidate to regulate both rest and action potential-induced Glu release. This is because it was shown here to be in a low affinity state at depolarization and in a high affinity state at resting potential. Furthermore, mGluR3 was found to be localized in the presynapse (52), and as mentioned above, it mediates feedback inhibition of Glu release. It remains to be seen, in further studies, whether also mGluR3 exhibits the two types of feedback inhibition demonstrated in the case of the M2-muscarinic receptor.

The mGluRs and the muscarinic ACh receptors, the other group where voltage sensitivity was directly demonstrated (9), belong to different subfamilies of GPCRs (C and A, respectively) (12). This may imply that voltage sensitivity is likely to be a general characteristic feature of many GPCRs.

	FOOTNOTES

 
^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 391 (to Profs. J. Dudel, I. P., and H. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom all correspondence should be addressed: Dept. of Neurobiology, The Hebrew University, Jerusalem 91904, Israel. Tel.: 972-2-6585900; Fax: 972-2-6584174; E-mail: hanna{at}vms.huji.ac.il.

² The abbreviations used are: GPCR, G-protein coupled receptor; mGluR, metabotropic glutamate receptor; ACh, acetylcholine; GIRK, G-protein inwardly rectifying K⁺ channel; Glu, glutamate; DR, dose response; I-V, current-voltage.

	ACKNOWLEDGMENTS

 
We are grateful to P. J. Conn who kindly provided the cDNA clone of mGluR3; to J. P. Pin, who kindly provided the cDNA clones of the chimeric mGluR1a/3 receptors; to N. Dascal, who kindly provided the cDNA clone of the chimeric G_i3/q and the mGluR1a; and to Lilly Research Laboratories, who kindly provided the LY-379268.

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