Identification of the Bovine γ-Aminobutyric Acid Type A Receptor α Subunit Residues Photolabeled by the Imidazobenzodiazepine [3H]Ro15-4513
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m209281200
ISSN1083-351X
AutoresGregory W. Sawyer, David C. Chiara, Richard W. Olsen, Jonathan B. Cohen,
Tópico(s)Neurotransmitter Receptor Influence on Behavior
ResumoLigands binding to the benzodiazepine-binding site in γ-aminobutyric acid type A (GABAA) receptors may allosterically modulate function. Depending upon the ligand, the coupling can either be positive (flunitrazepam), negative (Ro15-4513), or neutral (flumazenil). Specific amino acid determinants of benzodiazepine binding affinity and/or allosteric coupling have been identified within GABAA receptor α and γ subunits that localize the binding site at the subunit interface. Previous photolabeling studies with [3H]flunitrazepam identified a primary site of incorporation at α1His-102, whereas studies with [3H]Ro15-4513 suggested incorporation into the α1 subunit at unidentified amino acids C-terminal to α1His-102. To determine the site(s) of photoincorporation by Ro15-4513, we affinity-purified (∼200-fold) GABAAreceptor from detergent extracts of bovine cortex, photolabeled it with [3H]Ro15-4513, and identified 3H-labeled amino acids by N-terminal sequence analysis of subunit fragments generated by sequential digestions with a panel of proteases. The patterns of 3H release seen after each digestion of the labeled fragments determined the number of amino acids between the cleavage site and labeled residue, and the use of sequential proteolytic fragmentation identified patterns of cleavage sites unique to the different α subunits. Based upon this radiochemical sequence analysis, [3H]Ro15-4513 was found to selectively label the homologous tyrosines α1Tyr-210, α2Tyr-209, and α3Tyr-234, in GABAA receptors containing those subunits. These results are discussed in terms of a homology model of the benzodiazepine-binding site based on the molluscan acetylcholine-binding protein structure. Ligands binding to the benzodiazepine-binding site in γ-aminobutyric acid type A (GABAA) receptors may allosterically modulate function. Depending upon the ligand, the coupling can either be positive (flunitrazepam), negative (Ro15-4513), or neutral (flumazenil). Specific amino acid determinants of benzodiazepine binding affinity and/or allosteric coupling have been identified within GABAA receptor α and γ subunits that localize the binding site at the subunit interface. Previous photolabeling studies with [3H]flunitrazepam identified a primary site of incorporation at α1His-102, whereas studies with [3H]Ro15-4513 suggested incorporation into the α1 subunit at unidentified amino acids C-terminal to α1His-102. To determine the site(s) of photoincorporation by Ro15-4513, we affinity-purified (∼200-fold) GABAAreceptor from detergent extracts of bovine cortex, photolabeled it with [3H]Ro15-4513, and identified 3H-labeled amino acids by N-terminal sequence analysis of subunit fragments generated by sequential digestions with a panel of proteases. The patterns of 3H release seen after each digestion of the labeled fragments determined the number of amino acids between the cleavage site and labeled residue, and the use of sequential proteolytic fragmentation identified patterns of cleavage sites unique to the different α subunits. Based upon this radiochemical sequence analysis, [3H]Ro15-4513 was found to selectively label the homologous tyrosines α1Tyr-210, α2Tyr-209, and α3Tyr-234, in GABAA receptors containing those subunits. These results are discussed in terms of a homology model of the benzodiazepine-binding site based on the molluscan acetylcholine-binding protein structure. γ-Aminobutyric acid type A (GABAA) 1The abbreviations used are: GABAAreceptor, GABA receptor type A; GABA, γ-aminobutyric acid; ACh, acetylcholine; AChBP, molluscan acetylcholine-binding protein; V8 protease, S. aureus glutamyl endopeptidase; EndoLys-C, endoproteinase Lys-C; EndoAsp-N, endoproteinase Asp-N; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high pressure liquid chromatography1The abbreviations used are: GABAAreceptor, GABA receptor type A; GABA, γ-aminobutyric acid; ACh, acetylcholine; AChBP, molluscan acetylcholine-binding protein; V8 protease, S. aureus glutamyl endopeptidase; EndoLys-C, endoproteinase Lys-C; EndoAsp-N, endoproteinase Asp-N; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high pressure liquid chromatographyreceptors, the major inhibitory ligand-gated ion channels in the mammalian central nervous system, comprise a family in the Cys loop receptor superfamily of homologous ligand-gated ion channels that also includes glycine and invertebrate glutamateCl receptors with anion-selective channels and nicotinic acetylcholine (ACh) and serotonin 5-HT3 receptors with cation-selective channels (1Ortells M.O. Lunt G.G. Trends Neurosci. 1995; 18: 121-127Abstract Full Text PDF PubMed Scopus (467) Google Scholar, 2LeNovere N. Corringer P.J. Changeux J.P. Biophys. J. 1999; 76: 2329-2345Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Although multiple types of GABAA receptor existin vivo differing in subunit composition, distribution, and pharmacological characteristics, each receptor is composed of homologous subunits arranged in a pentamer around a central axis that is the chloride-selective channel. Whereas the most abundant GABAA receptor in mammalian brain is made up of α1, β2, and γ2 subunits in a stoichiometry of 2:2:1 (3McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1073) Google Scholar, 4Chang Y. Wang R. Barot D.S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar), other GABAA receptors containing two α subunits(1–6), two β subunits(1–4), and one additional subunit (β, γ(1–3), ρ(1–3), δ, ε, and π subunits) can form functional channels (5Barnard E.A. Skolnick P. Olsen R.W. Mohler H. Sieghart W. Biggio G. Braestrup C. Bateson A.N. Langer S.Z. Pharmacol. Rev. 1998; 50: 291-313PubMed Google Scholar). Site-directed mutagenesis studies have shown that the two agonist-binding sites in the GABAA receptor are located extracellularly at β-α subunit interfaces in a ligand-binding pocket whose residues are partially conserved between superfamily members (6Smith G.B. Olsen R.W. Trends Pharmacol. Sci. 1995; 16: 162-168Abstract Full Text PDF PubMed Scopus (451) Google Scholar, 7Cromer B. Morton C.J. Parker M.W. Trends Biochem. Sci. 2002; 27: 280-287Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar).Interactions of γ-aminobutyric acid (GABA) with GABAAreceptors are modulated by many drugs including benzodiazepines, barbiturates, anesthetics, alcohols, and neurosteroids (8Sieghart W. Pharmacol. Rev. 1995; 47: 181-234PubMed Google Scholar, 9Olsen R.W. Gordey M. Handb. Exp. Pharmacol. 2000; 147: 499-517Crossref Google Scholar). Benzodiazepines are of significant therapeutic interest and are among the most widely prescribed drugs in the world, used to treat anxiety and insomnia, to induce anesthesia, and to reduce seizure activity. The binding of many clinically useful benzodiazepines, such as flunitrazepam, is positively coupled to the agonist-binding sites and results in an allosteric potentiation of apparent GABA binding affinity as measured electrophysiologically. Other drugs, such as the imidazobenzodiazepine Ro15-1788 (flumazenil), can bind to the benzodiazepine site without energetic coupling to the GABA site (zero modulator or benzodiazepine antagonist), whereas for Ro15-4513, an azide analog of Ro15-1788 and the focus of this study, there is negative allosteric coupling with GABA binding (negative modulator or benzodiazepine inverse agonist) for some GABAA receptor subunit compositions (5Barnard E.A. Skolnick P. Olsen R.W. Mohler H. Sieghart W. Biggio G. Braestrup C. Bateson A.N. Langer S.Z. Pharmacol. Rev. 1998; 50: 291-313PubMed Google Scholar, 8Sieghart W. Pharmacol. Rev. 1995; 47: 181-234PubMed Google Scholar, 9Olsen R.W. Gordey M. Handb. Exp. Pharmacol. 2000; 147: 499-517Crossref Google Scholar, 10Sieghart W. Eichinger A. Richards J.G. Mohler H. J. Neurochem. 1987; 48: 46-52Crossref PubMed Scopus (178) Google Scholar, 11Knoflach F. Benke D. Wang Y. Scheurer L. Lüddens H. Hamilton B.J. Carter D.B. Mohler H. Benson J.A. Mol. Pharmacol. 1996; 50: 1253-1261PubMed Google Scholar).Benzodiazepines bind to a distinct binding site on the GABAA receptor located between an α and the γ subunits. The benzodiazepine-binding site is homologous to the GABA-binding sites located at the β-α interfaces, with many binding site residues conserved (12Galzi J.L. Changeux J.P. Curr. Opin. Struct. Biol. 1994; 4: 554-565Crossref Scopus (198) Google Scholar, 13Sigel E. Buhr A. Trends Pharmacol. Sci. 1997; 18: 425-429Abstract Full Text PDF PubMed Scopus (345) Google Scholar). Mutational analyses have identified amino acids in three discrete regions of the primary structure of an α subunit (14Buhr A. Schaerer M.T. Baur R. Sigel E. Mol. Pharmacol. 1997; 52: 676-682Crossref PubMed Scopus (87) Google Scholar) and three regions of a γ subunit (15Buhr A. Baur R. Sigel E. J. Biol. Chem. 1997; 272: 11799-11804Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 16Kucken A.M. Wagner D.A. Ward P.R. Teissere J.A. Boileau A.J. Czajkowski C. Mol. Pharmacol. 2000; 57: 932-939PubMed Google Scholar, 17Sigel E. Schaerer M.T. Buhr A. Baur R. Mol. Pharmacol. 1998; 54: 1097-1105Crossref PubMed Scopus (55) Google Scholar) that function as determinants of benzodiazepine binding affinity and/or allosteric coupling to the agonist-binding site. Mutational analyses allow the identification of amino acids that are important determinants of the energetics of ligand binding, but these amino acids need not be direct contributors to the ligand-binding site.Photoaffinity labeling provides an alternative experimental approach to identify amino acids in proximity to a bound ligand (reviewed in Refs.18Brunner J. Annu. Rev. Biochem. 1993; 62: 483-514Crossref PubMed Scopus (438) Google Scholar and 19Kotzyba-Hibert F. Kapfer I. Goeldner M. Angew. Chem. Int. Ed. Engl. 1995; 34: 1296-1312Crossref Scopus (383) Google Scholar). For the nicotinic ACh receptor, photoreactive agonists and antagonists have provided extensive identification of amino acids contributing to the agonist-binding sites and to the ion channel (see Refs. 20Wang D. Chiara D.C. Xie Y. Cohen J.B. J. Biol. Chem. 2000; 275: 28666-28674Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar and 21Pratt M.B. Pedersen S.E. Cohen J.B. Biochemistry. 2000; 39: 11452-11462Crossref PubMed Scopus (26) Google Scholar and references therein). For the GABAAreceptor, irradiation at 254 nm results in the covalent incorporation of the agonist [3H]muscimol, with Phe-65 in the bovine α1 subunit identified as a labeled amino acid (22Smith G.B. Olsen R.W. J. Biol. Chem. 1994; 269: 20380-20387Abstract Full Text PDF PubMed Google Scholar). The benzodiazepine [3H]flunitrazepam photoincorporates into several α subunit isoforms (23Mohler H. Battersby M.K. Richards J.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1666-1670Crossref PubMed Scopus (217) Google Scholar, 24Fuchs K. Mohler H. Sieghart W. Neurosci. Lett. 1988; 90: 314-319Crossref PubMed Scopus (50) Google Scholar, 25Stephenson F.A. Duggan M.J. Pollard S. J. Biol. Chem. 1990; 265: 21160-21165Abstract Full Text PDF PubMed Google Scholar), and α1His-102 has been identified as the primary site of photoincorporation within the α1 subunit (26Duncalfe L.L. Carpenter M.R. Smillie L.B. Martin I.L. Dunn S.M.J. J. Biol. Chem. 1996; 271: 9209-9214Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 27Smith G.B. Olsen R.W. Neuropharmacology. 2000; 39: 55-64Crossref PubMed Scopus (33) Google Scholar). [3H]Ro15-4513 can also be photoincorporated into α subunits of the GABAAreceptor (10Sieghart W. Eichinger A. Richards J.G. Mohler H. J. Neurochem. 1987; 48: 46-52Crossref PubMed Scopus (178) Google Scholar), and its site(s) of incorporation was shown to be C-terminal to α1His-102 (28Duncalfe L.L. Dunn S.M.J. Eur. J. Pharmacol. 1996; 298: 313-319Crossref PubMed Scopus (18) Google Scholar) and possibly within the transmembrane domains of the receptor (29Davies M. Dunn S.M.J. Biochem. Biophys. Res. Commun. 1998; 246: 650-653Crossref PubMed Scopus (10) Google Scholar). Ro15-4513 contains a photoreactive azide that upon UV excitation forms a reactive nitrene. Therefore [3H]Ro15-4513 should photolabel amino acids in close proximity to the azide, and identification of those residues will provide a first definition of the orientation of Ro15-4513 within the benzodiazepine-binding site.In this report we identify the amino acids photolabeled by [3H]Ro15-4513 in GABAA receptors purified by affinity chromatography from detergent extracts of bovine cerebral cortex. Labeled amino acids in the α1, α2, and α3 subunits were determined by use of a protein sequencing strategy that depended upon the pattern of 3H release rather than the direct identification of GABAAreceptor subunit amino acids.DISCUSSIONThe goal of this study was to use photoaffinity labeling to identify the amino acid(s) in the benzodiazepine-binding site that is photolabeled by [3H]Ro15-4513, an imidazobenzodiazepine that contains an azide substituent that upon UV illumination will produce a reactive nitrene. Because of the defined structure of the photoreactive intermediate, identification of the amino acids labeled by [3H]Ro15-4513 will define the orientation of the ligand within the benzodiazepine-binding site. Previous studies have shown that for receptors containing the α1, α2, α3, or α5 subunits, the binding of Ro15-4513 was negatively coupled to the binding of GABA, in contrast to the positive allosteric coupling seen for flunitrazepam at those receptor subtypes or for Ro15-4513 binding to receptors containing α4 or α6 subunits (11Knoflach F. Benke D. Wang Y. Scheurer L. Lüddens H. Hamilton B.J. Carter D.B. Mohler H. Benson J.A. Mol. Pharmacol. 1996; 50: 1253-1261PubMed Google Scholar).[3H]Flunitrazepam has been shown to photoincorporate into α1His-102 (26Duncalfe L.L. Carpenter M.R. Smillie L.B. Martin I.L. Dunn S.M.J. J. Biol. Chem. 1996; 271: 9209-9214Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 27Smith G.B. Olsen R.W. Neuropharmacology. 2000; 39: 55-64Crossref PubMed Scopus (33) Google Scholar), which had been identified by mutational analyses as a major affinity determinant of benzodiazepine binding (39Wieland H.A. Luddens H. Seeburg P.H. J. Biol. Chem. 1992; 267: 1426-1429Abstract Full Text PDF PubMed Google Scholar). α1His-102 is also an important determinant of Ro15-4513 efficacy, as substitutions at that position regulate equilibrium binding affinity (40Wingrove P.B. Safo P. Wheat L. Tompson S.A. Wafford K.A. Whiting P.J. Eur. J. Pharmacol. 2002; 437: 31-39Crossref PubMed Scopus (32) Google Scholar) and determine whether it acts as a positive or negative modulator of GABA responses (41Dunn S.M.J. Davies M. Muntoni A.L. Lambert J.J. Mol. Pharmacol. 1999; 56: 768-774PubMed Google Scholar). However, the extent of overlap between the binding site for flunitrazepam and Ro15-4513 is uncertain. Photolabeling of recombinant α1β3γ2 GABAAreceptors by Ro15-4513 resulted in a 90% decrease of high affinity [3H]flunitrazepam or [3H]Ro15-4513-binding sites, but after photolabeling by flunitrazepam there was a 90% reduction of flunitrazepam sites, whereas the number of [3H]Ro15-4513 sites were reduced by less than 10% (42McKernan R.M. Farrar S. Collins I. Emms F. Asuni A. Quirk K. Broughton H. Mol. Pharmacol. 1998; 54: 33-43Crossref PubMed Scopus (39) Google Scholar). Although [3H]flunitrazepam is photoincorporated into α1His-102, the amino acid(s) photolabeled by [3H]Ro15-4513 are contained within a subunit fragment extending from residue 104 to the C terminus of the α1subunit (43Davies M. Martin I.L. Bateson A.N. Hadingham K.L. Whiting P.J. Dunn S.M. Neuropharmacology. 1996; 35: 1199-1208Crossref PubMed Scopus (19) Google Scholar), possibly within amino acids 247–289 that span the end of the first transmembrane segment to the beginning of the third transmembrane segment (29Davies M. Dunn S.M.J. Biochem. Biophys. Res. Commun. 1998; 246: 650-653Crossref PubMed Scopus (10) Google Scholar).In this report we have used a radiochemical sequencing strategy to identify the amino acids labeled by [3H]Ro15-4513 in a heterogeneous preparation of GABAA receptors purified from bovine cortex on a benzodiazepine affinity column. The purification procedure resulted in an ∼200-fold purification of a population of receptors containing, based upon immunoblot analysis, α1, α2, or α3 subunits. Based upon the level of specific [3H]muscimol binding, active GABA receptors comprise ∼1% of the protein in the preparation, and, not surprisingly, GABAA receptor subunits or subunit fragments could not be directly detected by Edman degradation when labeled subunits were isolated by SDS-PAGE or when subunit fragments were further purified by SDS-PAGE and reversed-phase HPLC. However, by use of N-terminal sequence analysis to determine the patterns of3H release as labeled subunit fragments were digested sequentially with a panel of four proteases, we established that [3H]Ro15-4513 is photoincorporated into homologous positions in the three subunits: α1Tyr-210, α2Tyr-209, and α3Tyr-234. Our results are most compelling for the α3 subunit, for which positive identification of the 3H release profile was made after digestion with 4 proteases, whereas the identification of the labeled amino acids in the α1 and α2 subunits utilized only 3 and 1 proteases, respectively. Although the subunit heterogeneity of the brain GABAA receptor preparation initially complicated the identification of the sites by making it appear that multiple sites of labeling might exist within a single subunit, the fact that our data are consistent with photoincorporation into homologous Tyr in all three subunits actually strengthens the conclusion about each individual identification.The position in the rat GABAA receptor (α1Tyr-209) occupied by the tyrosine residues photolabeled by [3H]Ro15-4513, as well as α1Thr-206, have been shown by site-directed mutagenesis to affect the affinity of ligands whose binding is coupled either positively or negatively to the GABA site (14Buhr A. Schaerer M.T. Baur R. Sigel E. Mol. Pharmacol. 1997; 52: 676-682Crossref PubMed Scopus (87) Google Scholar, 44Amin J. Brooks-Kayal A. Weiss D. Mol. Pharmacol. 1997; 51: 833-841Crossref PubMed Scopus (106) Google Scholar). In addition, α1Val-211 and the corresponding α5Ile-215 have been identified as imidazobenzodiazepine affinity determinants (45Strakhova M.I. Harvey S.C. Cook C.M. Cook J.M. Skolnick P. Mol. Pharmacol. 2000; 58: 1434-1440Crossref PubMed Scopus (14) Google Scholar). In a pharmacophore model of the benzodiazepine-binding site developed based upon the results of mutational analyses (46He X. Zhang C. Cook J.M. Med. Chem. Res. 2001; 10: 269-308Google Scholar), α1Tyr-209 was proposed to interact with all ligands. However, in the model it was not positioned in proximity to the azide of Ro15-4513 (or to α1His-101).The radiosequence strategy that we have used permits a positive identification of [3H]Ro15-4513-labeled amino acids in the α1, α2, and α3 subunits, but in the absence of direct identification of subunit masses by Edman degradation, our results provide no information about the relative efficiency of incorporation of [3H]Ro15-4513 into the different subunits or about the relative abundance of each subunit in our receptor preparation. It is most likely that the labeled α1, α2, and α3 subunits are each contained within distinct populations of monomeric GABAA receptors. However, further immunoaffinity purification by use of subunit-specific antibodies would allow a direct determination of whether labeled α1 and α3subunits, for example, coassemble in a single receptor. For rat cortical membranes, both α1 and α3 subunits were photolabeled with [3H]Ro15-4513, and based upon immunoaffinity purification with subunit specific antisera, a minor proportion of labeled α1 subunits copurifies with α3 subunits (47Araujo F. Tan S. Ruano D. Schoemaker H. Benavides J. Vitorica J. J. Biol. Chem. 1996; 271: 27902-27911Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar).No Evidence of [3H]Ro15-4513 Labeling of α1His-102 or Amino Acids within α197–117Previous photolabeling and mutational studies of the GABAA receptor indicate that at least 2 additional regions of an α subunit primary structure may come in contact with Ro15-4513 at the benzodiazepine-binding site, specifically residues near α1His-102 and α1Tyr-160 (7Cromer B. Morton C.J. Parker M.W. Trends Biochem. Sci. 2002; 27: 280-287Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 13Sigel E. Buhr A. Trends Pharmacol. Sci. 1997; 18: 425-429Abstract Full Text PDF PubMed Scopus (345) Google Scholar). An examination of an alignment of the bovine α1–3 sequences for these regions (Fig. 9) indicated that if they were labeled, we would have seen patterns of 3H release after proteolytic fragmentation significantly different from those that we observed in our experiments. For the regions preceding α1His-102 and α1Tyr-160, all three subunits would produce the same patterns of protease cleavages, EX 19KX 4D and EX 4D X 6K, respectively. Although we have no direct proof that cleavages at these sites occurred, the efficiency of cleavages that occurred before α3Tyr-234 and the corresponding regions of the α1 and α2 subunits makes it likely that these cleavages also occur. The patterns of 3H release we observed lead us to conclude that there is no significant [3H]Ro15-4513 photoincorporation in α1His-102, in the corresponding positions in the α2 or α3 subunits, or in nearby amino acids. The 3H release patterns seen during sequence analysis of the major peak of 3H in the HPLC fractionation of EndoLys-C digests of GABAA receptor subunits (Figs. 5and 6) are also not consistent with labeling at or near α1Tyr-160, but the 3H release in the sixth cycle of Edman degradation of fractions 20 and 21 of the EndoLys-C digest (Fig. 7), which we attribute to labeling of α2Tyr-209, is also consistent with labeling of α1Tyr-162 or the corresponding position in the other subunits. However, in other experiments when the materials eluted from gel Bands 6–8 were digested with EndoAsp-N and then sequenced,3H release was limited to cycles 12 and 19 with no release in cycle 14 as would be expected for labeling of α1Tyr-162 (data not shown).A Model of the Benzodiazepine-binding SiteTo gain further appreciation of the selective labeling of α1Tyr-210 by [3H]Ro15-4513, we constructed an homology model of an extracellular domain of GABAA receptor containing 2 α1, 2 β1, and a γ2 subunits, based upon the crystal structure of the snail AChBP, a soluble, secreted protein homologous to the extracellular domain of the nicotinic ACh receptor (36Brejc K. van Dijk W.J. Klaassen R. Schuurmans M. van der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1571) Google Scholar, 48Smit A.B. Syed N.I. Schaap D. van Minnen J. Klumperman J. Kits K.S. Lodder H. van der Schors R.C. van Elk R. Sorgedrager B. Brejc K. Sixma T.K. Geraerts W.P.M. Nature. 2001; 411: 261-268Crossref PubMed Scopus (460) Google Scholar). Shown in Fig.10 is a stereo representation of the benzodiazepine-binding site within our model with Ro15-4513 docked in an energetically favored orientation. In this orientation the photoreactive nitrogen of the azide is ∼5 Å from α1Tyr-210 and 4 Å from α1His-102, the residue that is labeled by [3H] flunitrazepam, and it is also within 4–6 Å of α1Tyr-160, α1Ser-205, and γ2Phe-77. In a docking simulation with flunitrazepam (not shown), we found that it was oriented with its nitro group within 4 Å of α1His-102, consistent with one proposed mechanism of photoincorporation via a nucleophilic attack on a benzene carbon ortho to the nitrogen (49Givens R.S. Gingrich J. Mecklenburg S. Int. J. Pharm. (Amst.). 1986; 29: 67-72Crossref Scopus (11) Google Scholar), and within 5 Å of α1Tyr-210.In terms of the model, the labeling of α1Tyr-210 by [3H]Ro15-4513 and the lack of labeling of α1His-102 is very striking. This might result from the preferential reaction of the photoreactive intermediate with Tyr rather than His, as photolabeling of tyrosines by azide photoaffinity labeling has been frequently observed (19Kotzyba-Hibert F. Kapfer I. Goeldner M. Angew. Chem. Int. Ed. Engl. 1995; 34: 1296-1312Crossref Scopus (383) Google Scholar), whereas labeling of His has not been reported to our knowledge. However, the labeling of α1Tyr-210 also occurs in the absence of labeling of α1Tyr-160. Alternatively, the lack of labeling of α1His-102 or α1Tyr-160 may, in fact, occur because the structure of the benzodiazepine-binding site occupied by Ro15-4513 may differ significantly from that in the homology model. Because the binding of flunitrazepam is coupled positively to the binding of GABA, whereas the binding of Ro15-4513 is coupled negatively, flunitrazepam will stabilize the same conformation of the GABAA receptor that is favored by the binding of GABA, whereas Ro15-4513 will stabilize a conformation with low affinity for GABA, presumably the resting state of the receptor. Changes of the accessibility of substituted cysteines for chemical modification have provided evidence that the binding of GABA results in a change in the structure of the benzodiazepine-binding site (50Teissere J.A. Czajkowski C. J. Neurosci. 2001; 21: 4977-4986Crossref PubMed Google Scholar).The homology model is based upon the structure of the AChBP in a single conformational state (36Brejc K. van Dijk W.J. Klaassen R. Schuurmans M. van der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1571) Google Scholar), one that binds ACh with high affinity and is presumed to be closest in structure to a nicotinic ACh receptor conformation that binds ACh with high affinity, i.e. either an open channel or desensitized state. A recent comparison of the structure of the AChBP with that of the extracellular domain of theTorpedo nicotinic ACh receptor in the resting state identifies significant differences in structure within the agonist-binding site (51Unwin N. Miyazawa A. Li J. Fujiyoshi Y. J. Mol. Biol. 2002; 319: 1165-1176Crossref PubMed Scopus (225) Google Scholar). In future studies it will be important to determine whether [3H]Ro15-4513 photolabels amino acids other than α1Tyr-210 when photolabeling is carried out in the presence of GABA or another agonist. γ-Aminobutyric acid type A (GABAA) 1The abbreviations used are: GABAAreceptor, GABA receptor type A; GABA, γ-aminobutyric acid; ACh, acetylcholine; AChBP, molluscan acetylcholine-binding protein; V8 protease, S. aureus glutamyl endopeptidase; EndoLys-C, endoproteinase Lys-C; EndoAsp-N, endoproteinase Asp-N; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high pressure liquid chromatography1The abbreviations used are: GABAAreceptor, GABA receptor type A; GABA, γ-aminobutyric acid; ACh, acetylcholine; AChBP, molluscan acetylcholine-binding protein; V8 protease, S. aureus glutamyl endopeptidase; EndoLys-C, endoproteinase Lys-C; EndoAsp-N, endoproteinase Asp-N; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high pressure liquid chromatographyreceptors, the major inhibitory ligand-gated ion channels in the mammalian central nervous system, comprise a family in the Cys loop receptor superfamily of homologous ligand-gated ion channels that also includes glycine and invertebrate glutamateCl receptors with anion-selective channels and nicotinic acetylcholine (ACh) and serotonin 5-HT3 receptors with cation-selective channels (1Ortells M.O. Lunt G.G. Trends Neurosci. 1995; 18: 121-127Abstract Full Text PDF PubMed Scopus (467) Google Scholar, 2LeNovere N. Corringer P.J. Changeux J.P. Biophys. J. 1999; 76: 2329-2345Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Although multiple types of GABAA receptor existin vivo differing in subunit composition, distribution, and pharmacological characteristics, each receptor is composed of homologous subunits arranged in a pentamer around a central axis that is the chloride-selective channel. Whereas the most abundant GABAA receptor in mammalian brain is made up of α1, β2, and γ2 subunits in a stoichiometry of 2:2:1 (3McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1073) Google Scholar, 4Chang Y. Wang R. Barot D.S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar), other GABAA receptors containing two α subunits(1–6), two β subunits(1–4), and one additional subunit (β, γ(1–3), ρ(1–3), δ, ε, and π subunits) can form functional channels (5Barnard E.A. Skolnick P. Olsen R.W. Mohler H. Sieghart W. Biggio G. Braestrup C. Bateson A.N. Langer S.Z. Pharmacol. Rev. 1998; 50: 291-313PubMed Google Scholar). Site-directed mutagenesis studies have shown that the two agonist-binding sites in the GABAA receptor are located extracellularly at β-α subunit interfaces in a ligand-binding pocket whose residues are partially conserved between superfamily members (6Smith G.B. Olsen R.W. Trends Pharmacol. Sci. 1995; 16: 162-168Abstract Full Text PDF PubMed Scopus (451) Google Scholar, 7Cromer B. Morton C.J. Parker M.W. Trends Biochem. Sci. 2002; 27: 280-287Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Interactions of γ-aminobutyric acid (GABA) with GABAAreceptors are modulated by many drugs including benzodiazepines, barbiturates, anesthetics, alcohols, and neurosteroids (8Sieghart W. Pharmacol. Rev. 1995; 47: 181-234PubMed Google Scholar, 9Olsen R.W. Gordey M. Handb. Exp. Pharmacol. 2000; 147: 499-517Crossref Google Scholar). Benzodiazepines are of significant therapeutic interest and are among the most widely prescribed drugs in the world, used to treat anxiety and insomnia, to induce anesthesia, and to reduce seizure activit
Referência(s)