Identification of a Tyrosine in the Agonist Binding Site of the Homomeric ρ1 γ-Aminobutyric Acid (GABA) Receptor That, When Mutated, Produces Spontaneous Opening

Mutagenesis of recombinant ρ1 γ-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the ρ1 receptor is also associated with the agonist binding site. Wild-type and mutant ρ1 receptors were expressed inXenopus laevis oocytes and examined using the two-electrode voltage clamp. When Tyr-102 was mutated to cysteine, serine, tryptophan, or glycine the EC50 increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC50 was also observed for the competitive antagonist 3-APMPA, but not for the non-competitive antagonist picrotoxin. Y102C was accessible to modification by methanethiosulfonate, and this modification was prevented by both GABA and 3-APMPA. An interesting characteristic of the Y102S mutant receptor was that, in the absence of GABA, there was an unusually high oocyte resting conductance that was blocked by both 3-APMPA and picrotoxin, indicating spontaneously opening GABA receptors. It appears that mutation of Tyr-102 perturbs the binding site and gates the pore. We conclude that Tyr-102 is a component of the GABA binding domain and speculate that Tyr-102 might be important for coupling agonist binding to channel opening. Mutagenesis of recombinant ρ1 γ-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the ρ1 receptor is also associated with the agonist binding site. Wild-type and mutant ρ1 receptors were expressed inXenopus laevis oocytes and examined using the two-electrode voltage clamp. When Tyr-102 was mutated to cysteine, serine, tryptophan, or glycine the EC50 increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC50 was also observed for the competitive antagonist 3-APMPA, but not for the non-competitive antagonist picrotoxin. Y102C was accessible to modification by methanethiosulfonate, and this modification was prevented by both GABA and 3-APMPA. An interesting characteristic of the Y102S mutant receptor was that, in the absence of GABA, there was an unusually high oocyte resting conductance that was blocked by both 3-APMPA and picrotoxin, indicating spontaneously opening GABA receptors. It appears that mutation of Tyr-102 perturbs the binding site and gates the pore. We conclude that Tyr-102 is a component of the GABA binding domain and speculate that Tyr-102 might be important for coupling agonist binding to channel opening. GABA 1The abbreviations used are: GABA, γ-aminobutyric acid; GABAA, GABAB, and GABAC, γ-aminobutyric acid, types A, B, and C, respectively; TACA, trans-4-aminocrotonic acid; I4AA, imidazole-4-acetic acid is the main inhibitory neurotransmitter in the central nervous system where its action is mediated by three classes of receptors: GABAA, GABAB, and GABAC. Both GABAAreceptors (typically comprised of α, β, and γ subunits) and GABAC receptors (presumably comprised of ρ subunits) are evolutionarily related to a ligand-gated ion channel family that includes nicotinic acetylcholine (nAChR), glycine, and serotonin type 3 (5-HT3) receptors (1Ortells M.O. Lunt G.G. Trends Neurosci. 1995; 18: 121-127Abstract Full Text PDF PubMed Scopus (471) Google Scholar). Heteromeric GABAA and homomeric GABAC receptors can be differentiated by their physiological and pharmacological properties. For example, GABAC receptors have a higher sensitivity to GABA (2Cutting G.R., Lu, L. O'Hara B.F. Kasch L.M. Montrose-Rafizadeh C. Donovan D.M. Shimada S. Antonarakis S.E. Guggino W.B. Uhl G.R. Kazazian H.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2673-2677Crossref PubMed Scopus (408) Google Scholar), they do not desensitize (3Amin J. Weiss D.S. Receptors Channels. 1994; 2: 227-236PubMed Google Scholar), they have a smaller single channel conductance (4Wotring V.E. Chang Y. Weiss D.S. J. Physiol. (Lond.). 1999; 521: 327-336Crossref Scopus (40) Google Scholar), and they are insensitive to benzodiazepines, barbiturates, and bicuculline (5Shimada S. Cutting G. Uhl G.R. Mol. Pharmacol. 1992; 41: 683-687PubMed Google Scholar, 6Kusama T. Spivak C. Whiting P. Dawson V. Schaeffer J. Uhl G. Br. J. Pharmacol. 1993; 109: 200-206Crossref PubMed Scopus (145) Google Scholar). Another important difference is their distribution in the central nervous system. GABAA receptors are broadly expressed in the central nervous system, whereas GABAC receptors are found mainly in the retina, with lower levels in the brain and spinal cord (7Wegelius K. Pasternack M. Hiltunen J.O. Rivera C. Kaila K. Saama M. Reeben M. Eur. J. Neurosci. 1998; 10: 350-357Crossref PubMed Scopus (109) Google Scholar, 8Enz R. Brandstatter J.H. Wassle H. Bormann J. J. Neurosci. 1996; 16: 4479-4490Crossref PubMed Google Scholar). Although both GABAA and GABAC receptors are found in bipolar cells of the retina, their different pharmacological and physiological properties suggest distinct roles in synaptic transmission. As for nAChR, the ligand binding domains of GABA receptors are most likely located at subunit-subunit interfaces. By mutational analysis, two domains have been identified in the β2 subunit of GABAA receptors important for activation of the receptor by GABA (9Amin J. Weiss D.S. Nature. 1993; 366: 565-569Crossref PubMed Scopus (376) Google Scholar). These domains correspond to loops B and C of the nAChR ligand binding sites (10Corringer P.-J., Le Novère N. Changeux J.P. Ann. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (707) Google Scholar). Important amino acids in loop B of the GABAA β2 subunit are Tyr-157 and Thr-160, and in loop C, Thr-202 and Tyr-205. Mutagenesis and photoaffinity labeling studies of the α1 subunit of the GABAA receptor have also identified a phenylalanine at position 64 (loop D; Fig.1), which appears to be involved in agonist binding (11Smith G.B. Olsen R.W. J. Biol. Chem. 1994; 269: 20380-20387Abstract Full Text PDF PubMed Google Scholar, 12Sigel E. Baur R. Kellenberger S. Malherbe P. EMBO J. 1992; 11: 2017-2023Crossref PubMed Scopus (170) Google Scholar). The residues homologous to α1F64 in the β2 (Tyr-62) and γ2 (Phe-77) subunits do not seem to play a key role in agonist binding as mutation of β2Y62 and γ2F77 resulted in a negligible shift in GABA sensitivity (12Sigel E. Baur R. Kellenberger S. Malherbe P. EMBO J. 1992; 11: 2017-2023Crossref PubMed Scopus (170) Google Scholar). However, a complete loss of high affinity binding was reported when β2Y62 was mutated to serine (13Newell J. Davies M. Bateson A. Dunn S. J. Biol. Chem. 2000; 275: 14198-14204Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). They suggested that β2Y62 might be a component of the high affinity GABA binding site, but not part of the binding site linked to channel gating. For the ρ1 GABAC receptor, five residues have been identified as part of the GABA binding site (3Amin J. Weiss D.S. Receptors Channels. 1994; 2: 227-236PubMed Google Scholar). The role of the residue in the homologous position to α1F64 and β2Y62 for homomeric ρ1 receptors (Tyr-102) has yet to be evaluated. In the present study, via site-directed mutagenesis, oocyte expression, and the two-electrode voltage clamp technique, we examined the effect of mutating Tyr-102 on the activation properties of the GABAC receptor. Our results suggest that Tyr-102 is an important component of the GABA binding domain essential for channel gating. Furthermore, serine substitution of this position creates a receptor that opens in the absence of GABA. We speculate that Tyr-102 might be important for coupling agonist binding to channel opening. The human ρ1 cDNA was cloned into the pGEMHE vector (14Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (982) Google Scholar) and site-directed mutagenesis was achieved by the polymerase chain reaction overlap extension method (15Kammann M. Laufs J. Schell J. Gronenborn B. Nucleic Acids Res. 1989; 12: 4445-4452Google Scholar). pGEMHE-ρ1 was linearized withNheI, and cRNA synthesis was carried out using standardin vitro transcription procedures as previously described (16Chang Y. Weiss D.S. Mol. Pharmacol. 1998; 53: 511-523Crossref PubMed Scopus (70) Google Scholar). Integrity, as well as yield, of the cRNA was verified on a 1% agarose gel. FemaleXenopus laevis (Xenopus I, Ann Arbor, MI) were anesthetized by 0.2% MS-222 (3-aminobenzoic acid ethyl ester, methanesulfonate salt), and oocytes were surgically removed from the frog and placed in calcium-free oocyte Ringers-2 (OR2) incubation solution consisting of 92.5 mm NaCl, 2.5 mmKCl, 5 mm HEPES, 1 mm CaCl2, 1 mm MgCl2, 1 mmNa2HPO4, 50 units/ml penicillin, and 50 μg/ml streptomycin, pH 7.5. Oocytes were dispersed in this solution plus 0.3% collagenase A (Roche Molecular Biochemicals) with constant stirring at room temperature for 1.5–2 h. The dispersed oocytes were thoroughly rinsed with the above solution plus 1 mmCaCl2. After this, stage VI oocytes were isolated and incubated at 18 °C. Micropipettes for injecting cRNA were pulled from borosilicate glass (Drummond Scientific, Broomall, PA) on a Sutter P87 horizontal puller, and the tips were cut off with microscissors to a 40-μm outer diameter. cRNAs were diluted 10- to 50-fold with diethyl pyrocarbonate-treated water. The cRNA was injected into the oocytes with a Nanoject delivery system (Drummond Scientific) at a total injection volume of 40–50 nl (25–100 ng/μl). One to 3 days after injection, oocytes expressing ρ1 wild-type or mutant receptors were placed on a 300-μm nylon mesh suspended in a small volume chamber (< 100 μl). The oocyte, voltage-clamped at −70 mV, was continuously perfused at a rate of 150–200 μl/s with a solution consisting of (in mm): NaCl, 92.5; KCl, 2.5; HEPES, 5; CaCl2, 1; MgCl2, 1; pH 7.5, and briefly switched to the test solution that consisted of this same perfusion solution plus drug (e.g. GABA). Recording microelectrodes filled with 3m KCl had resistances of 1–3 MΩ. Standard two-electrode voltage clamp techniques were used to record currents in response to application of agonist. Dose-response and dose-inhibition relations were fit with the following forms of the Hill equation using a nonlinear least-squares method, ActivationI=Imax1+(EC50/[A])n(Eq. 1) InhibitionI=Imax1+([A]/IC50)n(Eq. 2) where I is the current amplitude,I max is the maximum current amplitude for that particular agonist or antagonist ([A]), EC50is the agonist concentration that induces a 50% maximal response, IC50 is the antagonist concentration that produces half-maximal inhibition, and n is the Hill coefficient. Stock solutions of 100 mm 2-aminoethyl methanethiosulfonate (MTSEA; Toronto were in and on solutions were in for GABA oocytes expressing ρ1 wild-type and Y102C receptors were for in 2.5 mm for 5 with and to the For we used the concentration of to maximal oocytes expressing Y102C were in the chamber for with 1 in the absence or of GABA, or and for 5 with Wild-type receptors with 2.5 mm in the EC50 and Hill there was a of the maximum current We do not the of this increase in the maximum current by We observed a from Y102C receptors not A has been previously described for modification of the acetylcholine receptor M. A. 1992; PubMed Scopus Google Scholar). the role of Tyr-102 in receptor this residue was mutated to five different amino cysteine, serine, tryptophan, and Wild-type and mutant receptors were expressed in and currents were examined with the two-electrode voltage clamp. A the current from and the expressed receptors with current The concentration of GABA to half-maximal activation was increased for and by 31-, 214-, 664-, and 8752-fold, The phenylalanine not a in the this position has a for substitution as of the GABA and Hill for Tyr-102 of are in a are It has previously been that ρ1 homomeric GABAC receptors can be by GABA a and I4AA, which in their and T. Spivak C. Whiting P. Dawson V. Schaeffer J. Uhl G. Br. J. Pharmacol. 1993; 109: 200-206Crossref PubMed Scopus (145) Google Scholar, R. L. R. Mol. Pharmacol. 1992; Google Scholar, Y. Weiss D.S. Mol. Pharmacol. 2000; PubMed Scopus Google Scholar). A relations in oocytes expressing and Y102S ρ1 receptors. The EC50 for Y102C and Y102S were increased and respectively. 3 relations for The EC50 for increased and in the Y102C and Y102S respectively. the EC50 and Hill for GABA, TACA, and Hill and for activation by different for ρ1 and Y102C shift max for are in a are It has been described that GABA and have in the activation of ρ1 the of is lower Y. Weiss D.S. Mol. Pharmacol. 2000; PubMed Scopus Google Scholar). the maximum for for ρ1 Y102S and Y102C receptors. was from the maximum current to max for GABA. not be for the and the shift in EC50 the of a complete the for to GABA was increased in the Y102S and Y102C receptors to and with for wild-type receptors. The of this in be the were to agonist binding (e.g. of the might the EC50 for the different to be in a mutant J. Physiol. (Lond.). 1994; Scopus Google Scholar). As in 3 and the were not at the that the receptor affinity has been By a competitive antagonist to the same site as the agonist. a antagonist is to the receptor by at a site distinct from the binding a mutation in the agonist binding site might be to the EC50 for as well as the IC50 for competitive but not the IC50 for We the effect of the ρ1 competitive antagonist 3-APMPA and the antagonist on currents in oocytes expressing Y102S and wild-type receptors. and B the effect of of 3-APMPA on the current by a GABA concentration to the and for wild-type and respectively. The Y102S mutation increased the IC50 for 3-APMPA from to a in that in oocytes injected with application of 3-APMPA resulted in a As we in a this is to of the spontaneously opening Y102S receptors. C the effect of of on currents for wild-type and Y102S receptors. The application of in a the The ρ1 wild-type IC50 was with for As to 3-APMPA, the sensitivity to was not The in evidence that Tyr-102 is associated with the agonist binding site. The method has been to domains of receptors A. 1998; PubMed Scopus Google Scholar). of mutating the amino acid of to and the of this with 5 A currents in a oocyte expressing the Y102C receptor and after application of 5 B the GABA The EC50 modification was After we observed a to the of two Hill EC50 of and for this particular The after modification were and with of the total amplitude in the sensitivity component that the of the current agonist was also after The likely to the and the likely to the We the in 5 B as two of and These two be to an modification by the from modification or a of the the main is that this residue is accessible to and this modification increased the EC50 an above the mutation Tyr-102 is part of the agonist binding site, the of Y102C with be prevented by with agonist (GABA) or competitive antagonist but not by a non-competitive antagonist Oocytes expressing Y102C were for with 1 in the absence or in the of mm 3-APMPA, or picrotoxin. These of GABA, 3-APMPA, and are the EC50 and IC50 for the agonist or The current amplitude in response to GABA, which the was and after the current to that the After with plus GABA, plus 3-APMPA, or plus the currents were and of the both agonist and competitive but not non-competitive Y102C from modification the that Tyr-102 is part of the agonist binding site. The current after in was that in 5 The in the from modification in this the was after We have also examined the mutation and modification of the residues to and The EC50 for the mutation were and and after For the EC50 were and and after respectively. The the agonist of the and and the wild-type receptor that residues do not play a key role in ligand binding. the mutation not receptor we out the that the modification but not receptor a important role for Tyr-102 in In oocytes expressing we observed an high resting conductance that was of the maximum We that this resting conductance might be to spontaneously opening GABA receptors and this by the of 3-APMPA and in the absence of GABA. of 3-APMPA the resting conductance in a with an n was to the IC50 of for 3-APMPA in the of GABA for the same mutant of to oocytes expressing Y102S also a in the resting conductance in a with an IC50 of n 5 which was not different from that for the wild-type receptor in the of GABA n The of and 3-APMPA to the resting conductance the that the Y102S mutation spontaneously opening GABA receptors. We also examined the mutation of ligand binding residues described previously in the ρ1 receptor (3Amin J. Weiss D.S. Receptors Channels. 1994; 2: 227-236PubMed Google Scholar) opening when expressed in The current was by application of to oocytes expressing the different not The current by this concentration of was the The of current (in the absence of to the maximum currents for and receptors were and respectively. Although current was with mm GABA for and receptors (3Amin J. Weiss D.S. Receptors Channels. 1994; 2: 227-236PubMed Google Scholar), we for opening with for which there was Although a small current was observed for this was with that of the Y102S receptor. We have identified a residue of the homomeric ρ1 GABAC receptor that appears to be part of the site on mutation of Tyr-102 increased the EC50 for activation by GABA, I4AA, and An increase in EC50 has to be with a in affinity or in can agonist sensitivity (9Amin J. Weiss D.S. Nature. 1993; 366: 565-569Crossref PubMed Scopus (376) Google Scholar). An increase in the EC50 as a of an in be by a in the maximum for a receptor, as is the for ρ1 Y. Weiss D.S. Neurosci. 1999; 2: PubMed Scopus Google Scholar). A of the receptors and is there be in the cRNA the mutation However, we can the maximum current for Y102C both and after Although modification an in receptor the of the current after modification to that observed was These suggest an of with a in In the Y102C mutation increased the for to that of GABA. However, the coupling the ligand binding site and the of the receptor that ligand binding into channel opening a mutational effect on binding. As we in a this in is with of this residue a role in coupling ligand binding to channel opening. A in of Tyr-102 associated with the agonist binding site is that the sensitivity of the receptor for GABA, TACA, and were by different the mutation to agonist the mutation have a of an effect on the sensitivity to different which was not the The of Tyr-102 in agonist binding is by the that at Tyr-102 the sensitivity of the receptor for the competitive antagonist 3-APMPA. a in sensitivity for the competitive bicuculline and was observed for the homologous residue in the α1 subunit of GABAAreceptors (12Sigel E. Baur R. Kellenberger S. Malherbe P. EMBO J. 1992; 11: 2017-2023Crossref PubMed Scopus (170) Google Scholar). In at the ligand binding site not the sensitivity of the receptor for a non-competitive antagonist as picrotoxin, which was the as its site of action has been located in the of the ρ1 receptor M. S. Trends Pharmacol. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). of Y102C by that this residue is in an and also that Tyr-102 is part of the GABA binding site modification of this increased the GABA this modification was by an agonist and a competitive antagonist but not by a non-competitive We amino acid at Tyr-102 with the of on the for the residue at this with amino acid properties as and are single substitution at that in amino acid at Tyr-102 increased the there was the of the residue and the EC50 the of the amino acid at this position We that as the chain at position 102 was smaller not there was a with the and in the smaller in indicating of a for The phenylalanine substitution at position 102 not receptor properties. However, of a serine which the but the increased the GABA with is that the homologous position in GABAA subunits is a tyrosine or a phenylalanine It is well that residues are part of the binding sites of this ligand-gated ion channel family Changeux Biol. 1994; Scopus Google Scholar). In to amino acids have been described at the agonist binding site of homomeric ρ1 receptors (3Amin J. Weiss D.S. Receptors Channels. 1994; 2: 227-236PubMed Google Scholar), GABAA receptors (9Amin J. Weiss D.S. Nature. 1993; 366: 565-569Crossref PubMed Scopus (376) Google Scholar), glycine receptors G. V. P. T. T. B. H. EMBO J. 9: PubMed Scopus Google Scholar), serotonin type 3 (5-HT3) receptors Ann. Rev. Physiol. 1995; PubMed Scopus Google Scholar), and receptors M. C. Changeux J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. M. C. Chang J. C. M. Changeux J. PubMed Scopus Google Scholar, 1991; PubMed Scopus Google Scholar). It has been suggested that of the are important in the the ligand and its binding site PubMed Scopus Google Scholar). However, we the of as or An interesting was that the Y102S mutation can channel opening in the absence of GABA. have been described in the of GABA receptors that channel opening both for GABA receptors (16Chang Y. Weiss D.S. Mol. Pharmacol. 1998; 53: 511-523Crossref PubMed Scopus (70) Google Scholar, A. C. K. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. M. P. Whiting P. K. Br. J. Pharmacol. 1999; PubMed Scopus Google Scholar) and K. M. J. C. S. A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, C. M. H. L. P. Nature. 1995; PubMed Scopus Google Scholar). this is the using site-directed mutagenesis at a binding site of a ligand-gated ion channel that creates opening a nAChR receptor has been using the method J. L. H. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). In that an amino acid with a ion to a chain was at the binding site residue and was that the substitution the They also observed that a competitive antagonist the spontaneously opening channel the antagonist not the binding of agonist but a on its and the receptor in a In of this, of the receptor in the of the competitive the binding in an N. E. Neuron. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). this we the serine substitution at and not the opening. We have to that the serine as a agonist. It likely the of in the binding site which on a of of the residue as and we have that Tyr-102 is associated with the ligand binding site of the ρ1 GABAC receptor, there are that Tyr-102 from the ligand binding residues that have been identified in the GABAC receptor. is the of a opening with a serine mutation at this The is the effect of the substitution at this position on the sensitivity to we the phenylalanine for the previously to play a role in agonist binding and a and in GABA sensitivity was (3Amin J. Weiss D.S. Receptors Channels. 1994; 2: 227-236PubMed Google Scholar), as with in sensitivity for we the serine at a and in GABA sensitivity was observed with for Although the are for we that this is a residue in the agonist binding site on the previously as well as the that Tyr-102 with binding site residues of the receptor as well as a ligand binding residue in the acetylcholine binding from the K. M. J. Nature. PubMed Scopus Google Scholar). In that Tyr-102 with of and in loop in the of the binding The with residues in loops B and C that the of the binding and are by the subunit that loop In we speculate that Tyr-102 play a role in the ligand binding to the of that to channel opening. In this agonist in the and is mainly via with key ligand binding the agonist is the of the binding site is of the ligand with residues (e.g. the of that to channel opening C. M. A. Nature. 2000; PubMed Scopus Google Scholar). is with the opening a in this to agonist in the wild-type receptor, the of in an pore. this a the and well as and of the GABA ligand binding domain have been

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