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

Abstract

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 Gα 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. 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 Gα 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. G-protein-coupled receptors (GPCRs) 2The 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. 2The 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. comprise the largest superfamily of proteins in the body (1Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1215) Google Scholar) 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 (2Ganitkevich V. Isenberg G. J. Physiol. 1993; 470: 35-44Crossref PubMed Scopus (130) Google Scholar), a purinergic receptor P2Y (3Martinez-Pinna J. Gurung I.S. Vial C. Leon C. Gachet C. Evans R.J. Mahaut-Smith M.P. J. Biol. Chem. 2005; 280: 1490-1498Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), or by an adrenergic receptor (4Itoh T. Seki N. Suzuki S. Ito S. Kajikuri J. Kuriyama H. J. Physiol. 1992; 451: 307-328Crossref PubMed Scopus (173) Google Scholar) 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 (5Perroy J. Richard S. Nargeot J. Bockaert J. Fagni L. J. Biol. Chem. 2002; 277: 1223-1228Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). In all studies mentioned above, as well as in other studies demonstrating a regulatory effect of membrane potential on GPCR-mediated processes (6Mason M.J. Hussain J.F. Mahaut-Smith M.P. J. Physiol. 2000; 524: 437-446Crossref PubMed Scopus (23) Google Scholar, 7Slutsky I. Parnas H. Parnas I. J. Physiol. 1999; 514: 769-782Crossref PubMed Scopus (72) Google Scholar, 8Slutsky I. Wess J. Gomeza J. Dudel J. Parnas I. Parnas H. J. Neurophysiol. 2003; 89: 1954-1967Crossref PubMed Scopus (47) Google Scholar), 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. (9Ben-Chaim Y. Tour O. Dascal N. Parnas I. Parnas H. J. Biol. Chem. 2003; 278: 22482-22491Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) 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 (10Pin J.P. Duvoisin R. Neuropharmacology. 1995; 34: 1-26Crossref PubMed Scopus (1228) Google Scholar) (i.e. control of release of various neurotransmitters (11Schoepp D.D. J. Pharmacol. Exp. Ther. 2001; 299: 12-20PubMed Google Scholar), neurotoxicity, and synaptic plasticity (12Conn P.J. Pin J.P. Annu. Rev. Pharmacol. PubMed Scopus Google We selected mGluRs that are coupled to different mGluR3 that is coupled to (12Conn P.J. Pin J.P. Annu. Rev. Pharmacol. PubMed Scopus Google Scholar) and mGluR1a that is coupled to P.J. PubMed Scopus Google Scholar). Using Xenopus oocytes, we show that both mGluR3 and mGluR1a are of and oocytes were and in of the of and N. I. in Scholar). their the oocytes were the In of from the was using N. I. in Scholar). The chimeric receptors and a from J. Pin were in by Gomeza et al. J. C. R. T. Bockaert J. Pin J.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were the J. 1992; Full Text PDF PubMed Scopus Google Scholar). The chimeric was a from N. Dascal The of were as and and mGluR1a, and and For the binding both mGluR1a and mGluR3 were the which and of the Xenopus a of expression in the oocytes J. 1992; Full Text PDF PubMed Scopus Google Scholar). The of were the as the the mGluR1a, which was currents were and were using the voltage The was two from were in to of The and the were and was used and mGluR3-induced of mGluR3-induced currents Dascal N. J. Physiol. PubMed Scopus Google Scholar), oocytes of and mGluR3 were at either or in in of was in to the that is by N. PubMed Scopus Google Scholar) and to the activation of the channels by the S. T. Dascal N. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). To K+ was replaced by K+ to but and and G. I. Dascal N. J. Physiol. PubMed Scopus Google Scholar). were in an and the mGluR3-induced currents was upon in a in the was to the potential, and the was The of the was a to the the receptor mGluR3 does not exhibit in a of Dascal N. J. Physiol. PubMed Scopus Google Scholar) it a in the of To this the was in of and were of and were The induced by and induced by was the two The of was to be at the and at the In a of the was to be of were and and were results indicate that the mGluR3 not in the of the The results further show that the the in the two does not the of the and activity of and was as the mGluR3 in oocytes of and or control to that in was the chimeric receptors, results not Cl– of mGluR1a-induced Ca2+-activated Cl– currents oocytes of mGluR1a were in and to either or a specific was and in The Cl– and of are R. Dascal N. J. Physiol. PubMed Scopus Google Scholar). We the of of the To a of was and the induced was various of the was and the of and the was We that the of is In a of the and the was not to mGluR1a-induced currents were in oocytes of mGluR1a, and chimeric the the oocytes were of and of and in Dascal N. J. Physiol. PubMed Scopus Google Scholar). was as mGluR3. of was by a Xenopus oocytes (9Ben-Chaim Y. Tour O. Dascal N. Parnas I. Parnas H. J. Biol. Chem. 2003; 278: 22482-22491Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). We binding of or to oocytes of mGluR3 or mGluR1a or at two membrane were also of and and also in the of mGluR3 and in membrane potential were by the and were by channels were in to the the membrane The membrane potential of the oocytes at the different K+ was a intracellular not the at a potential of the potential of oocytes the to In K+ to K+ but and the oocytes, either or were to oocytes were the of in either or K+ was a of or K+ In the the of the and was by a a of binding was but in the of of a in the of mGluR3 and mGluR1a and be that binding oocytes from different binding was by of the binding at and membrane potential from the binding to of as above, was also in control oocytes of was also as above, in The oocytes were a and their potential was to be at and at K+ electrophysiological as well as the binding were a two binding In the of the is the of the at is the to of as is the of the and the In the of the binding is the specific is the and and are as The of mGluR3-induced currents were also a that the of two of receptors and two binding the of the affinity the of the affinity is the of the affinity and is the of the affinity The of the two is to be and are the as in For all of the the were the of two binding in in the is in the of mGluRs and the that activation binding of to both in the P.J. J. J. Pharmacol. Exp. Ther. 2005; PubMed Scopus Google Scholar, J. H. L. Pin J.P. Biol. PubMed Scopus Google Scholar). The of currents and were by a to the of the In all the was and were results are as were from the of mGluR3 and mGluR1a. an of mGluR3-induced currents in the by of and at a potential of and a of to a a of and from the of and of mGluR3-induced and a of and from the of and and of mGluR3-induced at of and a of from the of and at and of mGluR1a-induced coupled to chimeric at of and a of from the of and at and of chimeric and chimeric at of and a of from the of For and at and For and at and the of K+ on the activity of we used the mGluR3-induced currents Dascal N. J. Physiol. PubMed Scopus Google Scholar). The that at J. 1992; PubMed Scopus Google Scholar) In to whether membrane potential in to effect on the also an effect on mGluR3 we the of on DR, at two of and The the is in the to and To we that the of in G. I. Dascal N. J. Physiol. PubMed Scopus Google Scholar). the of was by of the from the The were by to the the of at the potential. is to the different at the two of and in a and to be to oocytes in their a to the of the at the that the apparent from at to at (i.e. a it was that they in two affinity toward their affinity coupled to the G-protein and affinity R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that the of also be by an the of two of Indeed, the could be that at a affinity at the affinity of the receptors, and of the receptors are at a affinity both the we at this whether the itself or the affinity The in the be by changes to the rather by a specific effect of on the mGluR3. To this a at the potential was to a of and to and a was this the established at and the were of the oocytes channels C. J. Physiol. PubMed Scopus Google Scholar) that could potentially the be the results in oocytes that not channels are from the of the of receptors or channels other is oocytes, as well as oocytes of mGluR3 but the not show in to at both of and not The in the of the in the in could be to voltage sensitivity in steps that are downstream to the activation of the mGluR3. For example, et al. M.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) that not the but at directly the of the To this we the of the to the of mGluR3-induced a effect of membrane potential on the receptor affinity is the from it the of et al. M.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The results of are in is that the two In particular, in a of the was and that the voltage sensitivity in does not reside in the step of activation of the The of to mGluR3 directly whether the voltage sensitivity in resides in the activation of we binding of to oocytes expressing mGluR3 at two membrane in membrane potential were induced by the using and K+ of is in the and specific binding of to The binding was in the of the mGluR3 M.J. J.P. R. R. J.P. D.D. J. Chem. 1999; PubMed Scopus Google Scholar). is that the specific binding of is it is at at K+ In a but control oocytes that not specific binding was In particular, in the binding was and the binding was the binding was and the binding was results indicate that the specific binding binding to the mGluR3 and not to other results of to the in various The specific binding of was at at K+ at all To examine whether it is the or the K+ that the results of we the binding of of to oocytes that are also at The binding of to oocytes was at and K+ and of and was not different from the binding to oocytes at K+ of that it is the and not K+ that binding to mGluR3. 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Dascal N. J. Physiol. PubMed Scopus Google Scholar, J. PubMed Google Scholar). To of the oocytes, we in two of and a the at potential in that the of is at at at both as a of the of the Cl– N. I. J. Physiol. Physiol. 2000; 278: PubMed Google Scholar). the we address the the mGluR1a-induced is that the of the is to the however, not the that currents a to the the of of mGluR1a-induced Cl– by the to the at the in the the that the apparent is at and at and The of could not be by because to of were to be as also mGluR1a the apparent is but in to the apparent of mGluR1a is at at Here as channels not the results and in The mGluR1a is reported to Duvoisin S. 2000; PubMed Scopus Google Scholar). this is it could the voltage of the to the of the R. Dascal N. J. Physiol. PubMed Scopus Google Scholar), we could not currents to examine this we used mGluR1a-induced currents the chimeric and in in to whether the of mGluR1a is voltage-dependent. We mGluR1a-induced currents at and in a of a of in The of the which was by a that the of of mGluR1a is not it is at and and and results that the of affinity of mGluR1a toward The of to mGluR1a the binding of to oocytes expressing mGluR1a, as mGluR3. of is in where the and specific binding of to oocytes at two membrane are The binding was in the of the mGluR1a C. 1993; PubMed Scopus Google Scholar). be that the specific binding of is it is at K+ at In a but control oocytes that not mGluR1a, specific binding was In particular, in the binding was and the binding was the binding was and the binding was results indicate that the specific binding binding to the mGluR1a and not to other results of to the in various is that as the electrophysiological the specific binding of is at K+ at the a at both membrane and at and at the binding is and of and at also mGluR1a, the of affinity receptors and not the The results are the notion that both mGluR3 and mGluR1a by themselves, not the voltage sensitivity could reside in the the affinity of GPCRs R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is The in the mGluR1a both to and to a J. PubMed Google Scholar), the to whether the voltage sensitivity resides in the G-protein would be to the of mGluR1a coupled to coupled to this is not because the currents are J. PubMed Google Scholar). We used a chimeric Gα subunit, which is to the that were replaced by the of the of the G-protein toward the receptor 1993; PubMed Scopus Google Scholar). The chimeric the to mGluR1a as from the currents in oocytes expressing mGluR1a, and the chimeric The oocytes were also to the activation of the Cl– and the to inhibition of the by Dascal N. J. Physiol. PubMed Scopus Google Scholar). affected the of the oocytes and the of an We in the to at potential. of is in of is that as a apparent affinity at at The apparent was at and at and to those Cl– the voltage sensitivity does not reside in the G-protein. The of mGluR3 and mGluR1a in the of the question is which sites in the receptors could be involved in their affinity toward that the affinity of GPCRs toward their is by their to the G-protein R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and the results of it is that the intracellular loops that couple to the G-protein are involved in the In mGluRs, it was that the intracellular a key role in the to the G-protein J. I. J. L. Pin J.P. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), but the and the intracellular loops are also to the J. C. R. T. Bockaert J. Pin J.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the intracellular loops in both receptors we that intracellular loops of that in in the two receptors, which is the We the voltage sensitivity of two mGluR1a chimeric In and were in the other and were replaced by the of mGluR3 and two chimeric not the Ca2+-activated Cl– currents in oocytes J. C. R. T. Bockaert J. Pin J.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We whether they currents as the mGluR3 and that currents were by the two chimeric We the of but or be that the chimeric receptors, the parental mGluR1a, not exhibit is because the that is mGluR1a Duvoisin S. 2000; PubMed Scopus Google Scholar), is within mGluR1a but is not in of mGluR3. The in show that the chimeric receptor in voltage sensitivity the mGluR3 and not the mGluR1a. The apparent was at and at The the of at the two that of the mGluR3 however, that the of of that of mGluR1a. be the chimeric receptors the binding of the mGluR1a. The chimeric also voltage sensitivity that in that of the mGluR3 but to a The apparent was at and at be that the apparent of and at are rather at the of is that of and results further the that the voltage sensitivity resides in the receptors themselves and that the two intracellular and and to a play a crucial role in relaying changes in membrane potential to changes in the affinity of the We that a the chimeric voltage sensitivity as the mGluR3 does and to the The of to of to the chimeric in oocytes was as mGluR3 and mGluR1a. an of in The binding was in the of the mGluR1a be that the specific binding of is it is at at K+ results of various is as the electrophysiological the voltage of mGluR3 and not that of mGluR1a. Here as mGluR3 and mGluR1a, of the binding that the was not affected by and and and the binding was upon and of and The of of the from mGluR3 and mGluR1a to examine the that the voltage of the receptor To this we whether the at and in their of or in their of or in it to that are the step in a of currents induced by it was that the be from the of M.J. L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and S. 2003; PubMed Scopus Google Scholar) We here to whether the are and not to the of the We whether two of mGluR3 that in their different of the mGluR3-induced currents by of and of the affinity M.J. J.P. R. R. J.P. D.D. J. Chem. 1999; PubMed Scopus Google Scholar), were that the of the two currents are the of is that of The of of is that of and and and results indicate that also mGluR3-induced currents in oocytes, the is to is by the of the The of the of the currents was not different and and and not We examine whether the We of mGluR3-induced at the two of and that the is at in that at and and In other the of from mGluR3 is at at We the of currents but induced by mGluR1a coupled to the chimeric mGluR1a was reported to Duvoisin S. 2000; PubMed Scopus Google Scholar). we showed that this is not voltage-dependent. to of in the of we the of currents at (i.e. Indeed, that at a of mGluR1a that in this in to the results in and at and and results were Glu, where In was at at not The that the of currents voltage activated by mGluR1a that activated by that it is the of from mGluR3 that is depolarization. results also that mGluR1a, in to the of the is depolarization. the two chimeric receptors that both as the mGluR3 and not as the parental mGluR1a. was at at and and and and and results are the notion that two GPCRs belonging to the mGluRs mGluR3 and mGluR1a, are the that the effect at the of the is the that the binding of is both mGluR3 and mGluR1a. The other is the that the intracellular loops of mGluR1a those of mGluR3 results where in the receptors the voltage we show that the intracellular loops that couple to the in and play a crucial role in changes in membrane potential to changes in the binding affinity of the on cumulative results R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Pharmacol. 1999; PubMed Scopus Google Scholar), we the to the effect of membrane potential on the binding affinity of GPCRs. The results of the the chimeric receptors indicate that also mGluR3 and mGluR1a, as other GPCRs R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Pharmacol. 1999; PubMed Scopus Google Scholar), the to the G-protein is involved in the affinity of the receptors toward their other V. G. S. J. V. J. 2000; PubMed Scopus Google Scholar, C. C. G. S. H. J. T. V. Neuropharmacology. 2000; PubMed Scopus Google we that a that the intracellular loops that couple to the the of the receptors to couple to their G-protein. binding that (mGluR3) or increases (mGluR1a) the of the affinity that GPCRs are in a affinity coupled to the G-protein and in a affinity Pharmacol. 1999; PubMed Scopus Google Scholar), the binding results be to that (mGluR3) or increases (mGluR1a) the of receptors coupled to the G-protein. in turn the affinity of the receptors toward their In particular, the receptor is coupled to the it is by a and affinity toward the from the is and it affinity toward the The that the binding of the to mGluR3 was further the notion that the of receptors coupled to the G-protein. is studies that in to agonists, a affinity toward GPCRs whether coupled or to their G-protein R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.J. Pharmacol. Google Scholar). that it is the affinity of the two GPCRs that is is because the step in GPCR-mediated signal transduction is binding and a activation of the The affinity of the binding being the regulatory role of GPCRs in to be We the of the affinity of binding by two the mGluR1a, it was to play a role in in different Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The is the of the Annu. Rev. PubMed Scopus Google Scholar). of this the and the which are the two to the are of mGluR1a that is at the of this was to play a key role in of R. T. S. S. T. Full Text PDF PubMed Scopus Google Scholar). is that the affinity of mGluR1a toward a role in the and processes. this be to be the the mGluR1a be example, in to the that mGluR3 is to be involved in inhibition of release M.J. H. P.J. J. 2000; PubMed Google Scholar, V. Neuropharmacology. 2001; PubMed Scopus Google Scholar, J. Pharmacol. PubMed Scopus Google Scholar). GPCR that it was I. G. Parnas H. Parnas I. J. 2002; PubMed Google Scholar) that inhibition of ACh release is by two in the of a of ACh and at all membrane depolarization. The other was to be and to at a of ACh the of but not the is at The I. G. Parnas H. Parnas I. J. 2002; PubMed Google Scholar) that the affinity which is mediated by a is to the potential and of of the affinity is to regulate the ACh mGluR3 is a to regulate both and is because it was here to be in a affinity at and in a affinity at potential. Furthermore, mGluR3 was to be in the Y. S. N. R. 2001; PubMed Scopus Google Scholar), and as mentioned above, it inhibition of to be in further whether also mGluR3 the two of inhibition in the of the The mGluRs and the muscarinic ACh receptors, the other where voltage sensitivity was directly (9Ben-Chaim Y. Tour O. Dascal N. Parnas I. Parnas H. J. Biol. Chem. 2003; 278: 22482-22491Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), to different of GPCRs and (12Conn P.J. Pin J.P. Annu. Rev. Pharmacol. PubMed Scopus Google Scholar). that voltage sensitivity is to be a of GPCRs. We are to J. the of to J. the of the chimeric to N. the of the chimeric and the and to the