Different Residues in the GABAA Receptor α1T60-α1K70 Region Mediate GABA and SR-95531 Actions
2002; Elsevier BV; Volume: 277; Issue: 21 Linguagem: Inglês
10.1074/jbc.m111778200
ISSN1083-351X
AutoresJessica H. Holden, Cynthia Czajkowski,
Tópico(s)Insect and Pesticide Research
ResumoAlthough γ-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from α1T60 to α1K70 was mutated to cysteine and expressed with wild-type β2 subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with α1T60C, α1D62C, α1F64C, α1R66C, α1S68C, and α1K70C. γ-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of α1F64C, α1R66C, and α1S68C, whereas SR-95531 slowed modification of α1D62C, α1F64C, and α1R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for α1F64C and α1R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized α1F64C and α1R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of α1D62C and α1S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating. Although γ-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from α1T60 to α1K70 was mutated to cysteine and expressed with wild-type β2 subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with α1T60C, α1D62C, α1F64C, α1R66C, α1S68C, and α1K70C. γ-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of α1F64C, α1R66C, and α1S68C, whereas SR-95531 slowed modification of α1D62C, α1F64C, and α1R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for α1F64C and α1R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized α1F64C and α1R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of α1D62C and α1S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating. Few studies of ligand-gated ion channels (LGICs) 1The abbreviations used are: LGICligand-gated ion channelGABAγ-aminobutyric acid, GABAA, γ-aminobutyric acid type ASCAMsubstituted cysteine accessibility methodMTSmethanethiosulfonateMTSEA2-aminoethyl methanethiosulfonateMTSET2-(trimethylammonium)ethyl methanethiosulfonateMTSES2-sulfonatoethyl methanethiosulfonate2-ME2-mercaptoethanolAChBPacetylcholine-binding protein have addressed the question of how the binding of compounds with divergent structure leads to dramatic functional differences. For example, agonist binding induces conformational changes that result in channel opening, whereas binding of competitive antagonists does not. Distinguishing the specific amino acid residues involved in the binding of agonists and antagonists will help to elucidate the structural rearrangements that govern the pharmacological effects of these compounds. In this paper, we examined the molecular determinants important for the binding of the agonist GABA and the competitive antagonist SR-95531 to the γ-aminobutyric acid type A (GABAA), receptor and explored the conformational changes that occur within the GABA-binding site during channel activation by a barbiturate. ligand-gated ion channel γ-aminobutyric acid, GABAA, γ-aminobutyric acid type A substituted cysteine accessibility method methanethiosulfonate 2-aminoethyl methanethiosulfonate 2-(trimethylammonium)ethyl methanethiosulfonate 2-sulfonatoethyl methanethiosulfonate 2-mercaptoethanol acetylcholine-binding protein GABAA receptors are heteropentameric chloride channels that mediate fast synaptic inhibition in the brain and are members of an evolutionarily related superfamily of LGICs that also includes nicotinic acetylcholine, glycine, and serotonin-type 3 receptors (1Ortells M.O. Lunt G.G. Trends Neurosci. 1995; 18: 121-127Abstract Full Text PDF PubMed Scopus (469) Google Scholar). To date, 16 different GABAA receptor subunit isoforms (α1–6, β1–3, γ1–3, δ, ε, π, and θ) have been cloned (2Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1793) Google Scholar, 3Davies P.A. Hanna M.C. Hales T.G. Kirkness E.F. Nature. 1997; 385: 820-823Crossref PubMed Scopus (368) Google Scholar, 4Hedblom E. Kirkness E.F. J. Biol. Chem. 1997; 272: 15346-15350Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 5Bonnert T.P. McKernan R.M. Farrar S. le Bourdelles B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J. Brown N. Wafford K.A. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9891-9896Crossref PubMed Scopus (281) Google Scholar, 6Barnard 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, 7Whiting P.J. 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The neurotransmitter recognition site, where agonists such as GABA and muscimol and antagonists such as SR-95531 and bicuculline bind, is located at the interface between the α and β subunits because residues have been identified on both subunits that are important for ligand recognition. On the α1 subunit, residues identified include Phe64 (12Smith G.B. Olsen R.W. J. Biol. Chem. 1994; 269: 20380-20387Abstract Full Text PDF PubMed Google Scholar, 13Sigel E. Baur R. Kellenberger S. Malherbe P. EMBO J. 1992; 11: 2017-2023Crossref PubMed Scopus (169) Google Scholar), Arg66, Ser68 (14Boileau A.J. Evers A.R. Davis A.F. Czajkowski C. J. Neurosci. 1999; 19: 4847-4854Crossref PubMed Google Scholar), Arg119, and Ile120 (15Westh-Hansen S.E. Rasmussen P.B. Hastrup S. Nabekura J. Noguchi K. Akaike N. Witt M.R. Nielsen M. Eur. J. Pharmacol. 1997; 329: 253-257Crossref PubMed Scopus (43) Google Scholar, 16Westh-Hansen S.E. Witt M.R. Dekermendjian K. Liljefors T. Rasmussen P.B. Nielsen M. Neuroreport. 1999; 10: 2417-2421Crossref PubMed Scopus (46) Google Scholar). On the β2 subunit, residues Tyr157, Thr160 (17Amin J. Weiss D.S. Nature. 1993; 366: 565-569Crossref PubMed Scopus (374) Google Scholar), Thr202, Ser204, Tyr205, Arg207, and Ser209 (17Amin J. Weiss D.S. Nature. 1993; 366: 565-569Crossref PubMed Scopus (374) Google Scholar, 18Wagner D.A. Czajkowski C. J. Neurosci. 2001; 21: 67-74Crossref PubMed Google Scholar) have been identified. Based on work on the related nicotinic acetylcholine receptor, residues that contribute to forming the binding site are located in at least six different non-contiguous extracellular N-terminal regions of the α and β subunits. These regions have been designated loops A–F (19Corringer P.J., Le Novere N. Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar). Residues within these loops likely have different functional roles. Some residues may directly contact ligand, some may be important for maintaining the structural integrity of the binding site, and others may mediate local conformational movements within the site. In the present study, we examined the binding site region surrounding α1F64 (loop D) of the GABAA receptor. In the homologous region of the serotonin-type 3 receptor, White and colleagues (20Yan D. Schulte M.K. Bloom K.E. White M.M. J. Biol. Chem. 1999; 274: 5537-5541Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) used alanine-scanning mutagenesis and determined that different amino acid residues contribute to the binding of agonists and antagonists. We hypothesized that the region surrounding α1F64 of the GABA-binding site also contains unique residues important for agonist and antagonist binding, and we tested this hypothesis by using the substituted cysteine accessibility method (SCAM). SCAM has been used on a variety of ion channels to elucidate channel lining and binding site residues, to determine the location of channel gates and selectivity filters, and to identify regions of the protein that are involved in conformational rearrangements during state changes (21Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (544) Google Scholar). In this method, individual amino acid residues are mutated to cysteine, and the ability of sulfhydryl-specific reagents to modify covalently each introduced cysteine is assessed by observing the effect of the reagent on receptor function. We measured the rates of sulfhydryl modification of accessible introduced cysteine residues in the presence and absence of GABA and SR-95531. We identified a subsite important for agonist binding that includes α1F64, α1R66, and α1S68 and an antagonist-binding subsite that includes α1D62, α1F64, and α1R66. In addition, we used sulfhydryl-specific reagents of different charge and determined that a negative subsite exists within the binding pocket. Finally, we measured rates of sulfhydryl modification in the presence of pentobarbital (a GABAAreceptor modulator that opens the channel), and we identified conformational changes that occur within the GABA-binding site during channel activation. The α1 cysteine mutants were engineered using the Altered Sites II® in vitro Mutagenesis Systems (Promega Corp., Madison, WI) or by recombinant PCR as described previously (14Boileau A.J. Evers A.R. Davis A.F. Czajkowski C. J. Neurosci. 1999; 19: 4847-4854Crossref PubMed Google Scholar, 22Kucken A.M. Wagner D.A. Ward P.R. Boileau J.A. Czajkowski C. Mol. Pharmacol. 2000; 57: 932-939PubMed Google Scholar). Cysteine substitutions were made in the rat α1 subunit at positions Tyr59, Thr60, Ile61, Asp62, Val63, Phe64, Phe65, Arg66, Gln67, Ser68, Trp69, and Lys70, where the number reflects the position in the mature α1 subunit protein. The cysteine mutants were subcloned into pGH19 (23Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (979) Google Scholar, 24Robertson G.A. Warmke J.M. Ganetzky B. Neuropharmacology. 1996; 35: 841-850Crossref PubMed Scopus (95) Google Scholar) for expression in Xenopus laevis oocytes. The presence of the mutations was verified by restriction endonuclease digestion and double-strand cDNA sequencing. The mutants have been named, using the single letter amino acid code, as wild-type residue, residue number, and mutated residue. X. laevis oocytes were prepared as described previously (25Boileau A.J. Kucken A.M. Evers A.R. Czajkowski C. Mol. Pharmacol. 1998; 53: 295-303Crossref PubMed Scopus (70) Google Scholar). cRNA transcripts were generated using the mMessage T7 kit (Ambion, Austin, TX). GABAA receptor rat α1 or α1mutants were expressed with wild-type rat β2 subunits by injection of cRNA into oocytes (0.3 ng of cRNA/subunit/oocyte, except for α1F64Cβ2 and α1R66Cβ2 that were injected at 7 ng of cRNA/subunit to ensure high levels of receptor expression). Mean maximal responses to GABA ranged from 1 to 10 μA. The oocytes were stored in ND96 medium (in mm: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.4) supplemented with 100 μg/ml gentamicin and 100 μg/ml bovine serum albumin for 2–14 days and used for electrophysiological recordings. Oocytes under two-electrode voltage clamp (Vhold of −80 mV) were continuously perfused with ND96 at a rate of 5 ml/min. The bath volume was 200 μl. GABA, SR-95531 (Sigma), pentobarbital (Research Biochemicals, Natick, MA), and methanethiosulfonate (MTS) reagents (Toronto Research Chemicals, Toronto, Ontario, Canada) were dissolved in ND96. Standard two-electrode voltage clamp recording was carried out using a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA) interfaced to a computer with a Digidata 1200 (Axon Instruments). Electrodes were filled with 3 m KCl and had resistances of 0.5–2.0 megaohms in ND96. Data acquisition and analysis were performed using pClamp 6 (Axon Instruments). The sulfhydryl-specific reagents used were derivatives of MTS obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). The reagents used were 2-aminoethyl methanethiosulfonate (MTSEA), 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), and 2-sulfonatoethyl methanethiosulfonate (MTSES). All oocytes were stabilized before addition of MTS reagent by application of GABA (5 s) at 10-min intervals until the GABA-activated peak currents (IGABA) varied by <10%. GABA concentrations used were EC40–EC60 for each mutant. After the GABA response stabilized, freshly diluted MTS reagent was applied for 2 min; the cell was washed for 5 min, and then GABA was applied at the same concentration used before the MTS treatment. MTSEA (2 mm), MTSET (2 mm), or MTSES (5 mm) were used. The effect of the MTS reagent was calculated as (IGABA-post/IGABA-pre) − 1, where IGABA-post is the current elicited by GABA after MTS application, and IGABA-pre is the current elicited by GABA before MTS application. The rate of MTS reagent covalent modification of introduced cysteines was determined by measuring the outcome of sequential applications of MTS reagents on IGABA. The protocol was as follows: EC20–EC60 GABA was applied for 5 s; the cell was washed for 30 s; MTS reagent was applied for 5–20 s; the cell was washed for 2.5 min; and the procedure was repeated until IGABA no longer changed indicating that the reaction was complete. Before the rate of MTS modification was measured, GABA was applied every 3 min until IGABA stabilized to within 3% demonstrating that the observed changes in IGABA after application of MTS reagent were due to the effects of the MTS reagent. Concentration of MTS reagent and time of application varied as follows: α1D62C: MTSEA, 1 mm, 20 s; α1F64C: MTSES, 10 μm, 5 s; MTSET, 10 nm, 5 s; MTSEA, 100 nm, 5 s; α1R66C: MTSES, 500 μm, 20 s; MTSET, 1 μm, 5 s; MTSEA, 10 μm, 10 s; α1S68C: MTSES: 150 μm, 10 s; MTSET: 100 μm, 5 s; MTSEA: 100 μm, 5 s. The effects of agonists and antagonists on the rate of MTS modification were tested by co-applying GABA (EC85–EC95), SR-95531 (IC90–IC95), or pentobarbital (500 μm) with MTSES for all mutants except α1D62C, in which case they were co-applied with MTSEA. For these studies, IGABA was stabilized before the rate of MTS reaction was measured as follows: apply GABA (EC20–60) for 5 s, wash for 30 s, apply GABA, SR-95531, or pentobarbital at high concentration for 5–20 s, wash for 2.5 min, and repeat the procedure. This procedure was repeated until the peak of the GABA (EC20–60) current was within 3% of the previous GABA (EC20–60) current peak. For all rate experiments, the decrease in current was plotted versus cumulative time of MTS exposure. We assume that the concentration of MTS reagent does not change significantly during the reaction, and thus, we can determine a pseudo first–order rate constant from the rate of decrease in IGABA. Peak current at each time point was normalized to the initial peak current, and a pseudo first-order rate constant (k1) was determined by fitting the data with a single exponential decay equation: y = span·e−kt + plateau. Because the data are normalized to IGABA at time 0, span = 1 − plateau. The second-order rate constant (k2) for MTS reaction was determined by dividing the calculated pseudo first-order rate constant by the concentration of MTS reagent used (26Pascual J.M. Karlin A. J. Gen. Physiol. 1998; 111: 717-739Crossref PubMed Scopus (105) Google Scholar). To verify the accuracy of this protocol, second-order rate constants were determined using at least two different concentrations of MTS reagents for several mutants. Concentration-response experiments were performed as described previously (14Boileau A.J. Evers A.R. Davis A.F. Czajkowski C. J. Neurosci. 1999; 19: 4847-4854Crossref PubMed Google Scholar). In brief, these trials used a low concentration of GABA (EC2–EC7) immediately before the test concentration of agonist to correct for any slow drift in GABA responses that may occur during the experiment. Currents elicited by each test concentration were normalized to the corresponding low concentration current before curve fitting. Concentration-response data were fit to the following equation: I =Imax/(1 + (EC50/[A])n), where I is the peak response to a given concentration of GABA;Imax is the maximum amplitude of current; EC50 is the concentration of GABA that produces a half-maximal response; [A] is the concentration of GABA; and n is the Hill coefficient. IC50 values were measured as described previously (18Wagner D.A. Czajkowski C. J. Neurosci. 2001; 21: 67-74Crossref PubMed Google Scholar). SR-95531 IC50 values were measured by applying a fixed concentration of GABA (EC20–EC60) immediately followed by co-application of the same concentration of GABA and a test concentration of SR-95531. Inhibition was calculated as IGABA + SR-95531/IGABA. Data were fit to the following equation: inhibition = 1 − 1/(1 + (IC50/[Ant])n), where IC50 is the concentration of antagonist that blocks half of IGABA; [Ant] is the concentration of antagonist, and n is the Hill coefficient.KI values were calculated using the Cheng-Prusoff/Chou equation (27Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12288) Google Scholar, 28Chou T. Mol. Pharmacol. 1974; 10: 235-247PubMed Google Scholar): KI = IC50/(1 + [A]/EC50), where [A] is the concentration of GABA used, and EC50 is the concentration of GABA that elicits a half-maximal response. Data analysis was carried out using nonlinear regression analysis included in the GraphPad Prism software package (San Diego, CA; www.graphpad.com). Statistical analysis was conducted using a one-way analysis of variance, followed by a post hoc Dunnett's test. All compounds were measured after energy minimization (<0.5 kcal/Å; Chemsketch, ADC, Toronto, Ontario, Canada). All MTS regents were measured from the sulfur to the center of the base of the tetrahedron formed by the terminal tertiary group. GABA was measured from the nitrogen to the base of the tetrahedron formed by the carboxyl group. SR-95531 was measured from the carbon of the methyl group to the center of the base of tetrahedron formed by the carboxyl group. The mature protein sequences of the rat α1 and β2 subunits were homology modeled with a subunit of the acetylcholine-binding protein (AChBP) (29Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1579) Google Scholar). The crystal structure of the AChBP was downloaded from the RCSB Protein Data Bank (code 1I9B) and loaded into Swiss Protein Data bank Viewer (SPDBV, ca.expasy.org/spdbv). The α1 protein sequence from Thr12–Ile227 and the β2protein sequence from Ser10–Leu218 were aligned with the AChBP sequence using the alignment function of SPDBV. The aligned sequences of the α1 and β2subunits were threaded onto an AChBP subunit using the "Interactive Magic Fit" function of SPDBV. The threaded subunits were imported into SYBYL (Tripos, Inc., St. Louis, MO) where energy minimization was carried out with the first 100 iterations carried out using Simplex minimization followed by 10,000 iterations using the Powell method. We reported previously that when mutated to cysteine, alternating residues from α1T60 to α1S68 are accessible to covalent modification by MTSEA-biotin, suggesting that this region of the GABA-binding site forms a β-strand (14Boileau A.J. Evers A.R. Davis A.F. Czajkowski C. J. Neurosci. 1999; 19: 4847-4854Crossref PubMed Google Scholar). We also determined that the presence of GABA inhibits the reaction of MTS compounds at α1F64C, α1R66C, and α1S68C, indicating that these residues may face into the agonist-binding pocket. In the present study, we used MTS reagents of different size and charge to explore the physicochemical environment of this region of the GABA-binding site. The MTS reagents used were MTSEA (3.7 Å long), which covalently adds a positively charged ethyl-ammonium group, MTSET (4.5 Å), which adds a positively charged ethyl-trimethylammonium group, and MTSES (4.8 Å), which adds a negatively charged ethyl-sulfonate group (Fig. 1). In order to determine the ability of the MTS reagents to react with each introduced cysteine, mutant α1 and wild-type β2 subunits were co-expressed in Xenopus oocytes, and IGABA (EC40–60) was measured before and after a 2-min MTS application. Because the MTS reagents did not affect the amplitude of IGABAat wild-type α1β2 receptors, we assumed that current changes observed in mutant receptors were due to covalent modification of the introduced cysteine residues (Fig. 1). In general, the residues that were reported previously (14Boileau A.J. Evers A.R. Davis A.F. Czajkowski C. J. Neurosci. 1999; 19: 4847-4854Crossref PubMed Google Scholar) to be modified by MTSEA-biotin (α1T60C, α1D62C, α1F64C, α1R66C, and α1S68C) were also accessible to modification by MTSEA, MTSET, and MTSES (Fig. 1). Reaction with MTS reagents reduced GABA current by 14 (α1K70C, MTSET) to 96% (α1F64C, MTSEA). In contrast to all other mutants, covalent modification of α1T60C caused an increase in IGABAsuggesting that the GABA EC50 value for this mutant receptor decreases following covalent modification. At any given position, the magnitude of the MTS effect on IGABA was dependent on the specific MTS reagent used (Fig. 1). The observed differences in MTS effects may be due to the charge and/or size of the functional group tethered within the binding site. However, it is also possible that the MTS reactions did not go to completion due to their varied intrinsic reactivities (21Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (544) Google Scholar). To test this possibility, we measured the rate at which each MTS reagent modified α1D62C, α1F64C, α1R66C, and α1S68C, making sure that each reaction was followed to completion. The rates of covalent modification of an introduced cysteine were obtained by measuring the effect of successive subsaturating applications of each MTS reagent on IGABA (Fig. 2A). The decrease in IGABA was plotted versus cumulative duration of MTS exposure and fit with a one-phase exponential decay curve, which yields a pseudo first-order rate constant (k1). To correct for the concentration dependence of the rate, a second-order rate constant (k2, Table I) was calculated by dividing k1 by the concentration of MTS used ("Experimental Procedures"). In general, the maximal effects of the MTS reagents observed in the rate experiments were consistent with those measured in the 2-min pulse protocol (mutant: MTSEA maximal inhibition/MTSEA 2-min inhibition; α1D62C: 64/54%; α1F64C: 92/96%; α1R66C: 32/33%; α1S68C: 34/32%). Because the reactions went to completion, these data indicate that tethering groups of different size and charge to the mutant receptors differentially affects IGABA.Table IRates of reaction of MTSES, MTSET, and MTSEA at α1D62Cβ2, α1F64Cβ2, α1R66Cβ2, and α1S68Cβ2receptorsReceptorMTSESMTSETMTSEAk2nk2nk2nm−1s−1m−1s−1m−1 s−1α1D62Cβ2NR1-aNR is no reaction.1NR116 ± 14α1F64Cβ223400 ± 600045,500,000 ± 2,800,00042,475,000 ± 235,0004α1R66Cβ250 ± 115116,000 ± 13,000413,000 ± 17004α1S68Cβ2270 ± 8072800 ± 140062000 ± 3003free sol.17,000212,00076,000Rates of covalent modification of cysteine-containing receptors were measured as described under "Experimental Procedures."k2 values represent mean second-order rate constants ± S.D. of at least three experiments. The free solution (free sol.) rates were reported by Karlin and Akabas (21Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (544) Google Scholar) and reflect the rate at which each MTS compound reacts with 2-mercaptoethanol, in solution.1-a NR is no reaction. Open table in a new tab Rates of covalent modification of cysteine-containing receptors were measured as described under "Experimental Procedures."k2 values represent mean second-order rate constants ± S.D. of at least three experiments. The free solution (free sol.) rates were reported by Karlin and Akabas (21Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (544) Google Scholar) and reflect the rate at which each MTS compound reacts with 2-mercaptoethanol, in solution. The rate of reaction of MTS modification of a binding site engineered cysteine depends on several factors as follows: 1) the movement of the MTS reagent from bulk solution to the substituted cysteine in the binding pocket (permeability of the pathway); 2) the intrinsic electrostatic potential within the pocket and along the pathway; 3) the ionization (acid dissociation) of the substituted cysteine's sulfhydryl group; and 4) steric restrictions in forming an activated complex between the thiolate of the substituted cysteine and the MTS reagent. MTS reagents react preferentially with the ionized thiolate (RS−) form of cysteine (30Roberts D.D. Lewis S.D. Ballou D.P. Olson S.T. Shafer J.A. Biochemistry. 1986; 25: 5595-5601Crossref PubMed Scopus (200) Google Scholar, 31Stauffer D.A. Karlin A. Biochemistry. 1994; 33: 6840-6849Crossref PubMed Scopus (257) Google Scholar). Of the residues tested, covalent modification of α1F64C was the fastest, indicating that this is the most accessible residue in loop D. For example, MTSET modified α1F64C with a k2 of ∼5,500,000 m−1 s−1, which was about 340-fold faster than reaction at α1R66C. Reaction at α1R66C, in turn, was about 40-fold faster than modification at α1S68C (Table I). At α1D62C, MTSEA was the only reagent tested that significantly altered IGABA. There are two possible explanations for this result. Either MTSET and MTSES do not react with α1D62C or covalent modification by these reagents does not change IGABA, implying that any apparent modification is functionally silent. To test these possibilities, we measured the ability of MTSEA to modify covalently α1D62C after application of MTSET or MTSES. If MTSET or MTSES modified α1D62C, then reaction with MTSEA should not occur, and no change in IGABA should be observed. Application of MTSES or MTSET prior to MTSEA had no effect on the ability of MTSEA to inhibit IGABA (data not shown), indicating MTSET and MTSES do not react with α1D62C. It should be noted that the reaction rate of MTSEA with α1D62C was very slow (k2 = 16 m−1s−1) indicating that α1D62C has limited accessibility. In free solution, the rates of MTSEA, MTSET, and MTSES with 2-mercaptoethanol (2-ME) are 76,000, 212,000, and 17,000m−1 s−1, respectively (21Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (544) Google Scholar) (Table I). The rate constants depend on the charges of the reactants. Because the net charge of 2-ME is −1, positively charged MTS reagents react faster than negatively charged MTS reagents with this compound (31Stauffer D.A. Karlin A. Biochemistry. 1994; 33: 6840-6849Crossref PubMed Scopus (257) Google Scholar). Interestingly, the reaction rate constants of MTSET and MTSEA with α1F64C were ∼30-fold faster than the their rates of reaction with 2-ME in free solution (Table I). The rate of reaction of the MTS reagents with α1F64C is influenced by the intrinsic electrostatic potential of the GABA-binding site, which arises from fixed charges and dipoles in the protein. The faster rates of MTSET and MTSEA modification of α1F64C compared with the rates of modification of a simple thiol in solution are likely due to these intrinsic properties of the protein and suggest that the short range interactions of MTSET and MTSEA with the GABA-binding site are stronger than those with a simple thiol. Similar fast rates were measured for MTSET and MTSEA reaction with the acetylcholine-binding site cysteines, αC192/193, in reduced, wild-type Torpedo nicotinic acetylcholine receptors, k2 ∼3 × 106m−1 s−1(31Stauffer D.A. Karlin A. Biochemistry. 1994; 33: 6840-6849Crossref PubMed Scopus (257) Google Scholar). The intrinsic electrostatic potential at a substituted cysteine can be examined by determining the rate of reactions of MTS reagents that differ in charge (26Pas
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