Abstract
Zn2+ inhibits currents through γ-aminobutyric acid (GABA)A receptors. Its affinity depends on the subunit composition; α1β1 receptors are inhibited with high affinity (IC50 = 0.54 μm). We sought to identify the residues that form this high affinity Zn2+ binding site. β1His267 aligns with α1Ser272, a residue near the extracellular end of the M2 membrane-spanning segment that we previously demonstrated to be exposed in the channel. The Zn2+ affinity of α1β1 H267S was reduced by 300-fold (IC50 = 161 μm). Addition of a histidine at the aligned position in α1 creates a receptor, α1S272Hβ1, that should have five channel-lining histidines; the Zn2+ affinity was increased 20-fold (IC50 = 0.025 μm). Shifting the position of the histidine from the β1 subunit to the aligned position in α1 with the two mutants α1S272Hβ1H267S reduced the affinity (IC50 = 26 μm) compared with wild-type. We infer that the high affinity Zn2+ binding site involves β1His267 from at least two subunits. For two histidines to interact with a Zn2+ ion, the α carbons must be separated by <13 Å. This limits the separation of the subunits and provides a constraint on the possible quaternary structures of the channel. The ability of a divalent cation to penetrate from the extracellular end of the channel to β1His267 implies that the charge-selectivity filter, the structure that discriminates between anions and cations, is located at a more cytoplasmic position than β1His267; this is consistent with our previous work that showed that positively charged sulfhydryl-specific reagents reacted with an engineered cysteine residue as cytoplasmic as α1T261C.
The GABAA receptors are members of the ligand-gated ion channel gene superfamily and form anion-selective channels (Macdonald and Olsen, 1994; Karlin and Akabas, 1995; Sieghart, 1995). The functional receptor complex is formed as a pentamer of homologous subunits arranged pseudosymmetrically around the central channel axis (Unwin, 1993; Macdonald and Olsen, 1994; Nayeem et al., 1994). Numerous GABAA receptor subunits have been cloned, including six α, four β, three γ, one δ, one ε, and three ρ subunits (Macdonald and Olsen, 1994; Sieghart, 1995). The subunits have a similar transmembrane topology with an ∼200-amino acid extracellular amino terminus, three closely spaced membrane-spanning segments (M1, M2, M3), a cytoplasmic loop of variable length, a fourth membrane-spanning segment (M4), and an extracellular carboxyl terminus (Macdonald and Olsen, 1994). Using the substituted-cysteine-accessibility method, we have shown that the channel lining is formed, at least in part, by residues from the M2 segment (Xu and Akabas, 1996).
Functional receptors are formed by coexpression of α and β subunits, although the presence of the γ subunit is essential for benzodiazepine potentiation (Schofield et al., 1987;Pritchett et al., 1989; Gorrie et al., 1997). In heterologous expression systems, the subunit stoichiometry for receptors formed by expression of the α and β subunits is uncertain; support has been provided for two α and three β subunits (Tretter et al., 1997) and for three α and two β subunits (Im et al., 1995; Kellenberger et al., 1996), as well as other stoichiometries (Gorrie et al., 1997). When the γ subunit is included, the stoichiometry seems to be two α, two β, and one γ subunit (Chang et al., 1996;McKernan and Whiting, 1996; Tretter et al., 1997). Some β subunits also form homomeric channels, but they tend to be constitutively open (Krishek et al., 1996).
The divalent cation Zn2+ blocks GABA-induced currents with variable affinity depending on the subunit composition of the receptors (Westbrook and Mayer, 1987; Draguhn et al., 1990; Legendre and Westbrook, 1991; Smart et al., 1991;Harrison and Gibbons, 1994; Saxena and Macdonald, 1994; Smart et al., 1994; Chang et al., 1995; Wang et al., 1995). It is likely that the effects of Zn2+ are mediated by interactions with different sites in receptors with different subunit compositions (Harrison and Gibbons, 1994; Smartet al., 1994; Saxena et al., 1997). Receptors formed by coexpression of α and β subunits display the highest affinity for Zn2+ block with an IC50 of ∼1 μm (Draguhn et al., 1990; Smart et al., 1991). In these receptors, Zn2+ block is slightly voltage dependent (Draguhnet al., 1990), implying that the binding site may be in the channel. The β homomeric channels also display high affinity Zn2+ block (Draguhn et al., 1990;Krishek et al., 1996). The addition of the γ2 subunit to the functional complex markedly reduced Zn2+block so that 100 μm Zn2+ caused only 17% inhibition of the GABA-induced currents (Draguhn et al., 1990; Smart et al., 1991). Zn2+ has been proposed to inhibit GABA-induced currents by stabilizing the closed state of the receptor (Smartet al., 1994). We sought to identify the residues that form the high affinity Zn2+ binding site in the α1β1 GABAA receptor.
In proteins of known crystal structure, bound Zn2+ ions interact directly with two to four amino acids. The amino acids found at Zn2+binding sites include histidine, cysteine, aspartate, or glutamate (Higaki et al., 1992; Regan, 1993; Berg and Shi, 1996). Because charged, water-soluble, sulfhydryl reactive reagents applied extracellularly have no effect on GABAA receptors (Xu and Akabas, 1993, 1996) it is unlikely that a cysteine residue is available to interact with extracellularly applied Zn2+. In GABA receptors formed by the ρ1 subunit, a histidine residue in the extracellular domain was shown to mediate lower affinity Zn2+ block (IC50 = 16 μm) (Wang et al., 1995); histidine, however, is not conserved at the aligned position in other subunits. In the aligned sequences of GABAA receptor subunits, we noted that histidine was conserved at the position aligned with β1His267 in all β subunits but is not found at the aligned position in other subunit subtypes. This position aligns with α1Ser272 in the M2 membrane-spanning segment; we had previously shown that this residue was exposed in the channel lining (Fig.1) (Xu and Akabas, 1996). We hypothesized that the high affinity Zn2+ binding site was formed by histidine residues from position β1 267 contributed by at least two β subunits.
Materials and Methods
Oligonucleotide-mediated mutagenesis.
The cDNAs encoding the rat α1 and γ2 subunits in the pBluescript SK(−) plasmid (Stratagene, La Jolla, CA) were obtained from Dr. P. Seeburg (Max-Planck Institute for Medical Research, Heidelberg, Germany), and the β1 subunit in the pBluescript SK vector was from Dr. A. Tobin (University of California, Los Angeles). The subunits were excised from the pBluescript clones by restriction digestion with the enzymes α1XhoI, β1 XbaI and HindIII, and γ2EcoRI. The β1 and γ2 subunits were ligated into the pGEMHE vector (Liman et al., 1992) digested with the corresponding enzymes. For the α1 subunit, the XhoI cut fragment was blunt ended and ligated into pGEMHE, which had been digested with SmaI. Subcloning and orientation were confirmed by restriction digestion and DNA sequencing. The Altered-Sites Mutagenesis procedure (Promega, Madison, WI) was used to generate mutations as described previously (Xu et al., 1995). Mutations were confirmed by DNA sequencing.
Preparation of mRNA and oocytes.
Plasmids were linearized with NheI for in vitro mRNA transcription with T7 RNA polymerase. In vitro mRNA transcription and the preparation and injection of Xenopus laevisoocytes were performed as described previously (Xu et al., 1995). Oocytes were injected with 10 ng of mRNA encoding the α1 and β1 subunits in a 1:1 ratio or with α1, β1, and γ2 subunits in a 1:1:1 ratio.
Electrophysiology.
GABA-induced currents were recorded from individual oocytes under two-electrode voltage-clamp, at a holding potential of −50 mV. Electrodes were filled with 3 m KCl and had a resistance of <2 MΩ. The ground electrode was connected to the bath via a 3 m KCl/Agar bridge. Data were acquired and analyzed on a 486/33 MHz computer using a TEV-200 amplifier (Dagan Instruments, Minneapolis, MN), a Digidata 1200 data interface (Axon Instruments), and pCLAMP 6 software (Axon Instruments, Foster City, CA). The oocyte was perfused at 5 ml/min with CFFR (115 mmNaCl, 2.5 mm KCl, 1.8 mmMgCl2, 10 mm HEPES, pH 7.5, with NaOH) at room temperature. The recording chamber had a volume of ∼0.25 ml.
Experimental protocol.
In all experiments, GABA was applied at a concentration 10 times the GABA EC50 value of the mutant or wild-type unless otherwise indicated. Applications of GABA were separated by 3–5-min washes with CFFR. In all experiments used for analysis, the magnitude of the GABA-induced current changed by <10% between two consecutive applications of GABA.
To determine the Zn2+ IC50value, an increasing series of Zn2+concentrations were applied. Each concentration of ZnCl2 was applied according to the following protocol: ZnCl2 in CFFR, 1 min; ZnCl2 plus GABA in CFFR, 20 sec; ZnCl2 in CFFR, 30 sec; CFFR, 5 min.
The experiments to determine the effects of the sulfhydryl reagents on the β1H267C mutant were performed as described previously (Xu and Akabas, 1996). The sequence of reagents applied was GABA, 20 sec; GABA, 20 sec; sulfhydryl reagent, 1 min; GABA, 20 sec; GABA, 20 sec. For a given oocyte, the concentration of GABA used was either the GABA EC50 or 10 times the EC50value of the receptor (see Table 1 for EC50 values). The fractional effect was taken as [(IGABA, after/IGABA, before) − 1]. The sulfhydryl reagents were dissolved in CFFR immediately before application.
Curve fitting.
The concentration dependence of the inhibition of the GABA-induced currents by Zn2+was fit with the Hill equation, I/Imax = 1/[1 + (IC50/Zn)n], where IC50 is the concentration of Zn2+ that causes half-maximal inhibition, Zn is the concentration of Zn2+, and n is the Hill coefficient, using either Prism 2.0 (GraphPAD, San Diego, CA) or custom software kindly provided by Dr. Juan Pascual (Columbia University, New York, NY).
Reagents.
A 1 m stock solution of ZnCl2 was prepared by adding sufficient HCl to eliminate all visible precipitates. All working solutions of ZnCl2 were prepared daily by diluting the 1m stock solution in CFFR. The addition of ZnCl2 to CFFR did not change the pH of the solution.
The MTS derivatives MTSES−, MTSET+, and MTS ethylammonium were synthesized as described previously (Stauffer and Karlin, 1994) or obtained commercially (Toronto Research Chemicals, North York, Ontario, Canada). These reagents react with cysteine and add —SCH2CH2X, the charged portion of the molecule onto the sulfhydryl; where Xis SO3− for MTSES−, NH3+ for MTS ethylammonium, and N(CH3)3+for MTSET+. The organic mercurial pCMBS− was obtained from Sigma Chemical (St. Louis, MO). It adds HgC6H4SO3−onto the cysteine.
Results
Characterization of the mutants.
We expressed all of the mutants in X. laevis oocytes as either α1β1 or α1β1γ 2 combinations where the mutant subunit or subunits replaced the corresponding wild-type subunit or subunits. GABA-induced currents were observed in oocytes expressing each of the combinations. For all of the subunit combination, we determined the EC50 value for GABA and the Hill coefficient of the GABA dose-response relationship (Table 1). For wild-type α1β1, the GABA EC50 value was 3.4 ± 0.7 μm and the Hill coefficient was 0.97 ± 0.12. The EC50 values of the mutants ranged from 5-fold smaller than wild-type for α1β1H267S to 1.6-fold larger than wild-type for the α1S272Hβ1 mutant. The EC50 value is a function of both the intrinsic affinity of the binding sites for GABA and the isomerization rate constants for transitions between the various closed, open, and desensitized states (Akabas et al., 1992). Because channel gating involves conformational changes in the membrane-spanning segments to transduce ligand binding in the extracellular domain to opening of the gate near the cytoplasmic end of the channel (Xu and Akabas, 1996), mutations of residues in membrane-spanning segments can alter the isomerization rate constants. Thus, the observed changes in EC50 probably arise from effects of the mutations on the transduction process.
Residues forming the high affinity Zn2+ binding site.
Zn2+ blocked GABA-induced currents arising from wild-type α1β1 GABAA receptors with an IC50 value of 0.54 ± 0.02 μm (four experiments) (Fig.2) similar to the IC50 values reported by other investigators for αxβy receptors (Draguhnet al., 1990; Smart et al., 1991). β1His267 aligns with α1Ser272, a residue that we had previously shown to be a channel-lining residue (Xu and Akabas, 1996). To determine whether β1His267 was involved in forming the high affinity Zn2+ binding site, we mutated the histidine to the amino acids found at the aligned positions in the α1 and γ2 subunits, serine and isoleucine, respectively (Fig. 1). The affinity for Zn2+ block of both mutants was reduced by >300-fold (Fig. 2); for α1β1H267S, the IC50value was 161 ± 40 μm (three experiments), and for α1β1H267I, the IC50 value was 654 ± 113 μm (nine experiments) (Table2). The residual inhibition likely arises from an interaction of Zn2+ with a different site or sites on the receptor; removal of the high affinity site has unmasked this previously unrecognized low affinity site or sites. The location of this low affinity binding site is unknown.
Interactions with residues in other subunits.
To investigate whether a histidine at the aligned position in the α1 subunit would interact with β1His267, we generated the mutant α1S272H and expressed it with the wild-type β1 subunit. With five histidines in the channel lining, the IC50 value of the α1S272Hβ1 receptor was 0.025 ± 0.007 μm (three experiments) (Fig. 3); that the affinity was significantly higher than wild-type suggests that the aligned residues in different subunits are in close proximity in the channel lumen. To ensure that the higher affinity was not solely due to placement of the histidine in the α1 subunit, we examined the effect of Zn2+ on the receptor that at this position only contained histidine in the α1 subunit, α1S272Hβ1H267S; the IC50 value was 26 ± 1 μm(eight experiments) (Fig. 3, Table 2). The affinity of α1S272Hβ1H267S for Zn2+ was lower than that in wild-type but higher than that in the α1β1H267S receptor, a receptor with no histidines at this level of the channel.
Cysteine substitution for β1His267.
It was hypothesized that Zn2+ inhibits GABA-induced currents by binding to and stabilizing the closed state of the GABAAreceptor (Smart et al., 1994). Therefore, we sought to determine whether β1His267 was accessible to interact with ions in the closed state of the channel. We used the substituted-cysteine-accessibility method (Akabas et al., 1992; Xu and Akabas, 1993) to probe the accessibility of a cysteine residue substituted at position 267 in the closed state of the channel. In this approach, a cysteine residue is engineered into the protein. The cysteine-substitution mutant is expressed, and the ability of charged, sulfhydryl-specific reagents to react with the engineered cysteine residue is tested. The magnitude of the GABA-induced current is determined; then, the sulfhydryl reagent is applied for 1 min in the absence of GABA (i.e., in the closed state of the channel) and washed out, and the magnitude of the GABA-induced current is determined again. If the magnitude of the GABA-induced currents after application of the sulfhydryl reagent is significantly different than the magnitude of the GABA-induced currents before application of the sulfhydryl reagent, we infer that the sulfhydryl reagent has covalently modified the engineered cysteine. The sulfhydryl reagents we used are pCMBS−, MTSES−, and MTSET+ (Xu and Akabas, 1996). Because these reagents react more rapidly with the thiolate anion (S−) than with the neutral thiol (SH) (Hasinoffet al., 1971; Roberts et al., 1986) and because only cysteines on the water-accessible surface of the protein are likely to ionize to a significant extent, we assume that these reagents will only react with cysteine residues on the water-accessible surface of the protein at an appreciable rate. Covalent modification of a channel-lining cysteine may alter the single-channel conductance and/or the gating kinetics (Akabas et al., 1994). To detect the effects of covalent modification, we used two different test concentrations of GABA: one at the GABA EC50value and the other at 10 times the EC50 value.Zhang and Karlin (1997) have shown that applying the agonist at the EC50 value for the test responses before and after the reagent provides a more sensitive test for detecting covalent modification because it allows one to detect reaction when the effect of modification is a change in gating alone with little or no change in single-channel conductance. In contrast, application at 10 times the EC50 value will detect changes in conduction but should be insensitive to changes in gating.
A 1-min application of 0.5 mmpCMBS−, 10 mmMTSES−, or 1 mmMTSET+ in the absence of GABA had no significant effect on wild-type α1β1 GABAA receptors whether the test concentration of GABA was at the EC50 or 10 times the EC50value (Fig. 4C, open bars). For the α1β1H267C mutant, when the test concentration of GABA was 5 μm, the EC50 value for the mutant (Table 1), a 1-min application of 0.5 mmpCMBS−, 10 mmMTSES−, or 1 mmMTSET+ in the absence of GABA potentiated the subsequent GABA-induced currents by ∼50% (Fig. 4, A and C,left). For the α1β1H267C mutant, when the test concentration of GABA was 50 μm, 10 times the EC50 value for the mutant, a 1-min application of 1 mm MTSET+ caused a 31% potentiation of the subsequent GABA-induced currents but a 1-min application of 0.5 mm pCMBS− or 10 mm MTSES− had no significant effect on the subsequent GABA-induced currents (Fig. 4, B and C,right). Thus, in the closed state of the receptor, the engineered cysteine was accessible to react with all three of the reagents. Therefore, we infer that the corresponding wild-type residue, β1His267, is on the water-accessible surface of the protein in the closed state of the channel and therefore available to interact with Zn2+.
Based on the differences between the effects of modification as probed with GABA test concentrations at the EC50 and 10 times EC50 value, we infer that the cationic reagent MTSET+ alters conduction and probably also gating, but the major effect of modification by the anionic reagents pCMBS− and MTSES− is on gating. It was surprising that modification by all three reagents resulted in potentiation of the subsequent GABA-induced currents. Presumably, the presence of a charged residue, that is, the covalently modified cysteine, at this position stabilizes the open state; the mechanism, however, is unknown.
Cysteine residues are frequently involved in the formation of Zn2+ binding sites (Higaki et al., 1992; Regan, 1993; Berg and Shi, 1996); therefore, we tested the effect of Zn2+ on α1β1H267C. Zn2+ inhibited the GABA-induced currents; the IC50 value was 23 ± 3 μm(five experiments) (Table 2). Although the affinity of α1β1H267C for Zn2+ was 40-fold lower than wild-type, it was 7- and 28-fold higher than the corresponding serine and isoleucine mutants at this position. Thus, we believe that Zn2+ is binding to the engineered cysteine or cysteines. Furthermore, covalent modification of the engineered cysteine by either 10 mm MTSES− or 5 mm MTSET+ reduced the ability of Zn2+ to inhibit the α1β1H267C. Before modification, Zn2+ inhibited α1β1H267C with an IC50 of 23 ± 3 μm (five experiments) (Table 2); after modification of α1β1H267C by either MTSES− or MTSET+, the IC50 value for Zn2+inhibition of GABA-induced currents was >1 mm (three experiments) (Fig. 5). Thus, covalent modification of the cysteine prevents it from interacting with Zn2+.
Effect of the γ2 subunit on Zn2+ affinity.
It has previously been shown that the IC50 value for Zn2+ inhibition of GABAAreceptors containing the γ2 subunit is much higher than that for receptors formed from only the α and β subunits (Draguhn et al., 1990; Smart et al., 1991). Consistent with this, we found that the Zn2+ IC50value for wild-type α1β1γ2 was >1 mm (three experiments). In the γ2 subunit, the residue Ile282 aligns with β1His267 (Fig. 1). We mutated this residue to histidine, γ2I282H, and expressed the mutant with wild-type α1 and β1 subunits, but the Zn2+ IC50 value also was >1 mm (three experiments) (Table 2). Expression of the other combinations of histidines at this position α1S272Hβ1H267Sγ2I282H, and α1S272Hβ1γ2I282H, which should contain five histidines, also gave IC50 values of >1 mm (Table 2). Thus, inclusion of the γ2 subunit prevents the ability of Zn2+ to inhibit GABA-induced currents regardless of the number of histidines present at this level.
Discussion
Zn2+ is a high affinity inhibitor of α1β1 GABAA receptors (Draguhn et al., 1990; Smart et al., 1991). We have shown that the Zn2+ affinity of α1β1 GABAA receptors is reduced by >300-fold by mutation of β1His267 in the M2 membrane-spanning segment to either serine or isoleucine, the amino acids at the aligned positions in the α1 and γ2 subunits (Fig. 1). Furthermore, we have shown that β1His267 is exposed in the channel lining (Fig. 4) as we had previously shown for the aligned residue in the α1 subunit, α1Ser272 (Xu and Akabas, 1996). Histidine is conserved at the position aligned with β1His267 in all GABAAreceptor β subunits but is not found at the aligned position in other subunit subtypes. Histidine residues frequently form part of metal ion binding sites (Higaki et al., 1992; Regan, 1993; Berg and Shi, 1996). Thus, we infer that β1His267 participates in the formation of the high affinity Zn2+ site in α1β1 GABAA receptors. The position of this residue near the extracellular end of the M2 channel-lining segment is consistent with the slight voltage dependence reported for Zn2+ block of α1β1 GABAA receptors (Draguhn et al., 1990). A different histidine in the extracellular amino-terminal domain of the GABA ρ1 subunit was implicated in lower affinity Zn2+ inhibition of the GABAC receptor (Wang et al., 1995).
Because the GABAA receptor is formed as a pentamer of subunits arranged pseudosymmetrically around the central channel axis (Unwin, 1993; Nayeem et al., 1994), one would hypothesize that the aligned channel-lining residues from each subunit should be at approximately the same distance into the channel, thereby forming a ring of residues at a given level of the channel. Insertion of histidine into the aligned position in the α1 subunit and expression with wild-type β1, which should give five histidines at this level, increases the affinity for Zn2+ by 20-fold. This suggests that the aligned residues in different subunits are in close proximity in the channel lumen and that the presence of more histidines at this level allows for the formation of a higher affinity binding site.
To form a high affinity binding site, Zn2+ must interact with more than one residue. The affinity of a site will depend on the number of chelating residues, the relative position of the residues, and the local electrostatic environment (Higaki et al., 1992; Regan, 1993; Berg and Shi, 1996). It is likely that at least two histidines form the high affinity Zn2+binding site in the α1β1 GABAA receptor. The subunit stoichiometry of the α1β1 GABAA receptor is uncertain, and evidence has been reported for both two α and three β subunits (Tretter et al., 1997) and three α and two β subunits (Im et al., 1995; Kellenberger et al., 1996; Gorrie et al., 1997). We are uncertain why the affinity for Zn2+ is 50-fold higher when the histidine is in the β subunit in wild-type receptor compared with when the histidine is in the α subunit in the α1S272Hβ1H267S mutant (Table 2). There are several potential explanations for this difference in affinity. If the subunit stoichiometry is 2α:3β, then the high affinity site could be formed between histidines in the adjacent β subunits that would be present (Fig. 6A); alternatively, all three histidines might interact with the Zn2+ (Fig. 6A). The lower affinity observed when the histidine was in the α subunit would arise because there would be two histidines in nonadjacent subunits that might be less favorable for Zn2+ binding (Fig. 6B). Alternatively, neighboring channel-lining residues, such as β1Glu270, also might influence the interaction between the histidines and Zn2+, thereby resulting in a higher affinity interaction when the histidine is in the β subunit compared with the α subunit, where the adjacent channel-lining residue is α1Asn275. Finally, the position of the α and β subunits relative to the channel may not be symmetrical, and the difference in the affinity is due to the asymmetry and not to the relative number of subunits.
In X-ray crystal structures of proteins containing Zn2+ bound through histidine residues, the separation between the Zn2+ and the ε-amino group is ∼2 Å (Higaki et al., 1992). This distance and the size of histidine constrain the maximum separation of the α carbons of two histidine residues bound to a Zn2+ion to <13 Å (Higaki et al., 1992). Thus, in the Zn2+ bound state of the GABAA receptor, at the level of β1His267, the α carbons of the aligned residues in both adjacent and nonadjacent subunits must be <13 Å apart (Fig. 7B,distances A–B and A–C).
Our previous work demonstrated that picrotoxin, a rigid, roughly spherical molecule 9 Å in diameter, binds near the cytoplasmic end of the channel in the region of α1Val257 (Fig. 7, distance D–E) (Xu et al., 1995). Because it reaches that site from the extracellular end of the channel, we inferred that the channel lumen must be ≥9 Å in diameter to the level of α1Val257 (Fig. 7) (Xu et al., 1995). Our current results constrain the maximum separation of the α carbon atoms on nonadjacent subunits at the level of β1His267 to <13 Å. It should be noted, however, that these distances may be measured in different states of the receptor. Picrotoxin binds in the open state of the channel (Newland and Cull-Candy, 1992), and Zn2+ may bind in the closed state of the channel (Smart et al., 1994). We do know, however, that charged sulfhydryl reactive reagents can react with engineered cysteine residues in the α1 M2 segment in the closed state of the channel (Xu and Akabas, 1996). These reagents would fit into a right cylinder 6 Å in diameter and 10 Å in length. Thus, in the closed state of the channel, the lumen must be ≥6 Å in diameter to allow these reagents to reach the engineered cysteine residues at positions more cytoplasmic than β1His267 (Xu and Akabas, 1996), but the α carbons of the residues aligned with β1His267 must be closer than 13 Å. The narrowest region in the channel was inferred, based on the size of the largest permeant anion, to be ∼5.6 Å (Bormannet al., 1987), but this must be at a position that is more cytoplasmic than the picrotoxin binding site (Fig. 7B, distance F–G).
If the channel were lined by five α helical M2 segments arranged perpendicular to the membrane, the separation between the aligned position on the surface of adjacent α helices would be ∼4 Å and that between nonadjacent α-helices would be ∼7 Å: The channel, however, is unlikely to be lined by five helices perpendicular to the membrane. In the 9 Å resolution structure of the ACh receptor reported by Unwin (1993), the putative M2 segments are not perpendicular to the membrane but rather angle out from the central channel axis toward the extracellular end of the channel (as illustrated in Fig. 7B). In the ACh receptor, we showed that residues near the extracellular end of the M1 membrane-spanning segment also were exposed in the channel lining, and we suggested that in the closed state, the M1 segments may intercalate between the M2 segments at the extracellular end of the channel (Akabas and Karlin, 1995). Thus, the 13 Å constraint on the separation of the α carbons seems to be reasonable given our current structural picture of the channel.
The GABAA receptor forms a nearly ideally anion-selective channel (Bormann et al., 1987). The ability of a divalent cation, Zn2+, to penetrate from the extracellular end of the channel to the level of β1His267 in the M2 membrane-spanning segment indicates that the charge-selectivity filter that discriminates between anions and cations must be located at a more cytoplasmic position than β1His267. This is consistent with our previous results (Xu and Akabas, 1996) that showed that cationic sulfhydryl reagents could react with cysteines substituted for residues as cytoplasmic as α1Thr261, which aligns with β1Thr256 (Fig. 7A). We inferred that the charge-selectivity filter is located at a more cytoplasmic position than these residues, perhaps near the cytoplasmic end of the channel where the channel seems to narrow and form a picrotoxin binding site (Xu et al., 1995).
As other investigators had shown (Draguhn et al., 1990;Smart et al., 1991), the presence of the γ2 subunit markedly reduces the affinity for Zn2+. The mechanism of this inhibition is not solely due to the lack of a histidine at the aligned position in the channel because substitution of a histidine at that position did not increase the affinity for Zn2+ (Table 2). Thus, some other aspect of the γ2 subunit prevents Zn2+ inhibition of GABA-induced currents. Potential explanations include that (1) the γ2 subunit may sterically restrict the conformations of the other subunits, and particularly β1His267, from adopting the conformation to which Zn2+ binds; (2) the putative adjacent channel-lining residue γ2Lys285 may electrostatically interfere with Zn2+ binding; and (3) Zn2+might still bind to the receptor complex containing the γ2 subunit, but the presence of the γ2 subunit prevents the conformational change induced by Zn2+ in the α1β1 receptor.
Acknowledgments
We thank Drs. Peter Seeburg and Allan Tobin for the gifts of the GABAA receptor subunit cDNAs, David Liu and Dr. Steven Siegelbaum for the gift of the pGEMHE plasmid, Gilda Salazar-Jimenez for preparing X. laevis oocytes, and Drs. Jonathan Javitch, Arthur Karlin, Juan Pascual, Geoff Smith, and Gary Wilson for helpful discussions and comments on this manuscript.
Footnotes
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Send reprint requests to: Dr. Myles Akabas, Center for Molecular Recognition, Columbia University, 630 West 168th Street, Box 7, New York, NY 10032. E-mail: ma14{at}columbia.edu
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This work was supported in part by National Institutes of Health Grants NS30808 and DK51794. M.H.A. is the recipient of an Established Scientist Award from the American Heart Association, New York City Affiliate.
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A preliminary report of this work has appeared in abstract form [Horenstein J and Akabas MH (1997) Soc Neurosci Abstr23:112]. While preparing the manuscript, we were informed of a preliminary report describing similar results of mutation of the aligned residue in the β3 subunit, β3His292A [Wooltorton JRA, McDonald BJ, Moss SJ, and Smart TG (1997) Br J Pharmacol122(suppl):38P]. A full version of this work was subsequently published in J Physiol (Lond)505:633–640 (1997).
- Abbreviations:
- GABAA
- γ-aminobutyric acid type A receptor
- ACh
- acetylcholine
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- CFFR
- Ca2+-free frog Ringer’s solution
- GABA
- γ-aminobutyric acid
- MTS
- methanethiosulfonate
- MTSES−
- methanethiosulfonate ethylsulfonate
- MTSET+
- methanethiosulfonate ethyltrimethylammonium
- pCMBS
- p-chloromercuribenzenesulfonate
- Received November 10, 1997.
- Accepted January 16, 1998.
- The American Society for Pharmacology and Experimental Therapeutics