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An N-Terminal Histidine Is the Primary Determinant of Subunit-Dependent Cu²⁺ Sensitivity of 32L GABA_A Receptors

Department of Neurology, University of Michigan Health System, University of Michigan, Ann Arbor, Michigan (H.K.); and Departments of Neurology, Molecular Physiology and Biophysics, and Pharmacology, Vanderbilt University, Nashville, Tennessee (R.L.M.)

Received April 21, 2003; accepted August 4, 2003

	Abstract

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Copper (Cu²⁺) is a physiologically important cation and is released from nerve terminals. Cu²⁺ modulates GABA_A receptor currents in an subunit subtype-dependent manner; 132L receptors are more sensitive to Cu²⁺ than 632L receptors. We compared the effect of Cu²⁺ on 32L receptors containing each of the six subtypes and generated 1/6 chimeras and mutants to determine the functional domain(s) and specific residues responsible for subtype-dependent differences in Cu²⁺ sensitivity. Whole-cell GABA_A receptor currents were obtained from L929 fibroblasts coexpressing wild-type, chimeric and mutant subunits with 3 and 2L subunits. Maximal Cu²⁺ inhibition of 132L and 232L receptor currents was larger (52.2 ± 3.0 and 59.0 ± 2.5%, respectively) than maximal inhibition of 332L, 432L, 532L, and 632L receptor currents (22.6 ± 3.1, 19.2 ± 3.4, 20.2 ± 4.8, and 21.2 ± 3.6%, respectively). Receptors containing chimeric constructs with 1 subtype N-terminal sequence between residues 127 and 232 were inhibited by Cu²⁺ to an extent similar to those with 1 subtypes, suggesting that this N-terminal region (127-232) contains a major determinant for high Cu²⁺ sensitivity. 1 subtype residues V134, R135, and H141 in a VRAECPMH motif (VQAECPMH in the 2 subtype) conferred higher Cu²⁺ sensitivity, and the H141 residue was the major determinant in the motif. The 3 subtype M2 domain residue H267, which is a major determinant of Zn²⁺ inhibition, and 6 subtype M2-M3 loop residue H273, which is responsible for the increased Zn²⁺ sensitivity of the 6 subtype, also seemed to contribute to Cu²⁺ inhibition. These data suggest that the N-terminal VR(Q)AECPMH motif in 1 and 2 subtypes is the major determinant of increased subtype-dependent inhibition by Cu²⁺, that residue H141 is the major determinant in that motif, and that Cu²⁺ may also interact with GABA_A receptors at sites similar to or overlapping Zn²⁺ sites.

Cations have been shown to modulate voltage- and ligand-gated ion channels (Ma and Narahashi, 1993; Smart et al., 1994; Fisher and Macdonald, 1998; Nagaya and Macdonald, 2001). Cu²⁺ plays important roles physiologically and pathologically as a cofactor for many enzymes or sources of free radicals (for review, see Pena et al., 1999). It has been reported that Cu²⁺ is released from nerve terminals in some brain regions during depolarization (Hartter and Barnea, 1988; Kardos et al., 1989), and Cu²⁺ levels are higher in brain than in other organs (Hui et al., 1977). The concentration of synaptic Cu²⁺ is estimated to be in the micromolar range (Kardos et al., 1989). Because it has been shown that Zn²⁺ is released from nerve terminals and can be a potential endogenous neuronal modulator (Assaf and Chung, 1984; Smart et al., 1994), most studies of cation modulation of ligand-gated ion channel have been on Zn²⁺. However, several recent studies have shown that Cu²⁺ inhibited GABA_A receptor currents in native and transfected cells (Ma and Narahashi, 1993; Narahashi et al., 1994; Fisher and Macdonald, 1998; Sharonova et al., 1998). In addition, Cu²⁺ inhibited -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, N-methyl-D-aspartate, and glycine receptor currents (Trombley and Shepherd, 1996; Vlachova et al., 1996; Weiser and Wienrich, 1996) and blocked long-term potentiation in hippocampus (Doreulee et al., 1997). These findings imply that Cu²⁺ may play an important role in synaptic transmission as a modulator, possibly similar to the action of Zn²⁺.

It has been demonstrated that Cu²⁺ modulates GABA_A receptors in a subunit-subtype dependent manner: 1 subtype-containing receptors cotransfected with 3 and 2L subunits are more sensitive to Cu²⁺ than 6 subtype-containing receptors (Fisher and Macdonald, 1998). In a previous study using 1/6 chimera and 6/1 chimeras made by replacing entire N-terminal sequence of 1 and 6 subunit with 6 and 1 sequences, respectively, from the middle of TM1 to the N terminus, we suggested that the functional domain regulating subtype-dependent Cu²⁺ sensitivity of GABA_A receptors was located in the N-terminal extracellular domain of subunits (Fisher and Macdonald, 1998). In present study, we identify further the functional domains and specific residues responsible for subtype-dependent Cu²⁺ sensitivity of 32L GABA_A receptors.

	Materials and Methods

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Construction of Chimeric and Mutant cDNAs. The chimeras were generated by interchanging restriction fragments between 1 and 6 cDNAs. Point mutations were generated using the QuikChange site-directed mutagenesis procedure and products (Stratagene, La Jolla, CA). Oligonucleotide primers were synthesized by the University of Michigan DNA synthesis core (Ann Arbor, MI). Sequences of chimeras and point mutants were verified by fluorescent DNA sequencing (University of Michigan DNA sequencing core).

Transient Transfection of L929 Cells. Full-length cDNAs for rat 1, 3, 5, 6, 3, 2L, and human 2 GABA_A receptor subtypes were subcloned into the pCMVneo vector and the rat 4 subtype cDNA was subcloned into the pRK5 expression vector. All of the cDNAs were transfected into the mouse fibroblast cell line L929 (American Type Culture Collection, Manassas, VA) using a modified calcium phosphate method (Chen and Okayama, 1987). Plasmids encoding , 3, and 2L GABA_A receptor subtype cDNAs were added to the cells in 1:1:1 ratios of 4 µg each plus 4 to 8 µg of the plasmid-encoding sFv. After 4 to 6 h of incubation at 3% CO₂, the cells were treated with a 15% glycerol solution in Bis-buffered saline for 30 s. L929 cells were maintained in Dulbecco's modified Eagle's medium plus 10% heat-inactivated horse serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. For selection of transfected cells, the plasmid pHook-1 (Invitrogen) containing cDNA that encoded the surface antibody sFv was cotransfected into the cells. Twenty to 28 h later, the cells were passaged and mixed with 5 µl of magnetic beads coated with hapten. After a 30- to 60-min incubation to allow the beads to bind to positively transfected cells, the beads and bead-coated cells were isolated using a magnetic stand. The selected cells were used for recording 18 to 28 h later.

Electrophysiological Recording Techniques and Analysis of Whole-Cell Currents. For whole-cell recording, the external solution consisted of 142 mM NaCl, 8.1 mM KCl, 6 mM MgCl₂,1mM CaCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4, and osmolarity was adjusted to 295 to 305 mOsM. Recording electrodes were filled with an internal solution of 153 mM KCl, 1 mM MgCl_2, 5m EGTA, 10 mM HEPES, and 2 mM MgATP, pH 7.3, and osmolarity was adjusted to 295 to 305 mOsM. These solutions provided a chloride equilibrium potential near 0 mV. Patch pipettes were pulled from microhematocrit tubes made of soda lime glass (World Precision Instruments, New Haven, CT) on a P 87 Flaming Brown puller (Sutter Instrument Co., San Rafael, CA). The drugs were applied to cells using a modified U-tube delivery system with a 10-to-90% rise time of 70 to 150 ms (Greenfield and Macdonald, 1996). Currents were recorded with a List EPC-7 (List Electronics, Darmstadt, Germany) patch clamp amplifier. All experiments were performed at room temperature. Whole cell currents were analyzed using the programs Axoscope (Axon Instruments) and Prism (GraphPad, San Diego, CA). All whole-cell current amplitudes were obtained by measuring the peak current evoked during the application of GABA or GABA plus Cu²⁺. All data for Cu²⁺ modulation of GABA_A receptor currents were normalized to the response to GABA alone. Normalized concentration-response data for the different isoforms were fitted with a four-parameter logistic equation (I = I_max /(1 + [10(logEC₅₀ - log[drug])^nH]), where n_H is the Hill coefficient, and I represented currents expressed as a percentage of the current elicited by GABA alone (I_max). Data were presented as mean ± S.E.M. Statistical comparisons among GABA_A receptor subunit combinations were performed with one-way analysis of variance, Newman-Keuls multiple comparison test.

	Results

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Cu²⁺ Inhibited 132L to a Greater Extent Than 632L Receptor Currents. Cu²⁺ inhibited 1-receptor currents (we use the short-hand notation of "n-receptors" to signify "n32L receptors") to a greater extent than 6-receptor currents. Cu²⁺ IC₅₀ values for both receptors were similar, but the maximal Cu²⁺ inhibition of 1-receptor currents (52.2%) was greater than maximal inhibition of 6-receptors (21.2%) (p < 0.001) (Fig. 1 and Table 1). In a previous study, we reported that the Cu²⁺ concentration-response relationship curve for inhibition of GABA_A receptor currents had a two population inhibition pattern for both 1- and 6-receptor currents with both "high-" and "low-affinity" block (Fisher and Macdonald, 1998). In the present study, we focused on the high affinity Cu²⁺ inhibition of GABA_A receptor currents.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Cu2+ sensitivity of wild-type subunit subtype-containing receptors. A, representative whole-cell currents from L929 fibroblasts coexpressing 1 and 6 subtype-containing receptors with 3 and 2L subunits. GABA or GABA plus Cu2+ was applied for 8 to 12 s (as indicated by horizontal bar) and voltage clamped at -50 mV. The GABA concentration used was near the EC50 value for each isoform, and the Cu2+ concentration used was 10 µM. B, Cu2+ concentration-response relationship for 1 and 6 subtype-containing receptors were obtained by normalizing peak response to GABA (near EC50 value for each isoform) plus each Cu2+ concentration as a percentage of maximum current response to GABA alone for each cells. Symbols and vertical bars represent means and S.E.M. Data were fitted with four-parameter logic equation. The average IC50 values for Cu2+ are presented in Table 1.

Cu²⁺ Modulated GABA_A Receptor Currents in an Subtype-Dependent Manner. We examined the effect of Cu²⁺ on currents from GABA_A receptors containing the other subtypes (2, 3, 4, and 5). Each of the subtype-containing receptors produced functional GABA_A receptors when cotransfected with 3 and 2L subunits in L292 fibroblasts. Cu²⁺ modulated GABA_A receptor currents in an subtype-dependent manner (Fig. 2A). Cu²⁺ inhibited 2-receptor currents (59.0%) to an extent that was similar to its effect on 1-receptor currents (52.2%) but to a greater extent than 3-, 4-, or 5-receptor currents (19.2 to 22.6%) (Fig. 2B and Table 1). The maximal Cu²⁺ inhibition of 3-, 4-, and 5-receptor currents was similar to the 21% inhibition of 6-receptor currents but was significantly less than that of 1-receptor currents (Fig. 2B and Table 1, p < 0.001). However, the Cu²⁺ IC₅₀ values for all six wild-type -subunit receptors (2.4 to 3.2 µM) were not different (Table 1). These results indicated that the maximal extent of Cu²⁺ inhibition varied with subtypes rather than the IC₅₀.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Differential subunit subtype dependent Cu2[supi]+ modulation of GABAA receptor currents. A, the Cu2+ concentration-response relationship for 1, 2, 3, 4, 5, and 6 subtype-containing receptors were obtained by normalizing peak response to GABA (near EC50 value for each isoform) plus each Cu2+concentration as a percentage of maximum current response to GABA alone. Symbols and vertical bars represent means and S.E.M. Data were fitted with a four-parameter logic equation. The average IC50 values for Cu2+ are presented in Table 1. B, comparison of maximal Cu2+ inhibition of six subtype-containing receptors. Each bars represent mean ± S.E.M.

The Proximal Subtype N-Termini Contained the Domain That Determined the Maximal Extent of Cu²⁺ Inhibition. To determine which domain conferred the subtype-dependent differences in Cu²⁺ sensitivity, we previously studied 1/6 and 6/1 chimeras, made by exchanging amino acid sequence from the N terminus to the middle of TM1 of the subunits (1 at residue 232 and 6 at residue 231) (Fig. 3A), coexpressed with 3 and 2L subtypes [1/6(232/231)- and 6/1(231/232)-receptors] (Fisher et al., 1997). 1/6(232/231)-receptors were strongly inhibited by Cu²⁺ with maximal Cu²⁺ inhibition and IC₅₀ values similar to those of 1-receptors, whereas 6/1(231/232)-receptors were less inhibited by Cu²⁺ with maximal Cu²⁺ inhibition and IC₅₀ similar to those of 6-receptors (Fig. 3B, Table 1). The results from these chimeric receptors suggested that the N termini were involved in the subtype-dependent differences in maximal Cu²⁺inhibition of GABA_A receptor currents.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Chimeric structures of 1 and 6 subunits of the GABAA receptor. A, the open regions represent 1 subtype sequence and the gray regions represent 6 subtype sequence. The four boxes represent putative transmembrane domains (TM1-TM4), and the numbers in parenthesis after the subunit represent N-terminal positions of amino acid residues at the splice site of each subunit. B, the Cu2+ concentration-response relationship for 1/6 chimeric subunit-containing receptors, 1/6(126/125), 6/1(125/126), 1/6(232/231), 6/1(231/232), 1/6/1(126/125;231/232), and 6/1/6(125/126;232/231), and were obtained by normalizing peak response to GABA (near EC50 value for each isoform) plus each Cu2+ concentration as a percentage of maximum current response to GABA alone for each isoform. Symbols and vertical error bars represent mean and S.E.M., respectively. Data were fitted with a four-parameter logistic equation. The average IC50 values for Cu2+ are presented in Table 1.

To further localize the functional domain within the N-terminal domains, we created additional 1/6 chimeras (Fig. 3A). When cotransfected with 3 and 2L subunits in L292 fibroblasts, all chimeric subtypes produced functional GABA_A receptors. First, we exchanged the N-terminal amino acid sequences from the N terminus to before the cysteine loop (1 at residue 126 and 6 at 125) to create 6/1(125/126) and 1/6(126/125) chimeric subunits. Maximal inhibition of 6/1(125/126)-receptors by Cu²⁺ (44.4%) was similar to that of 1-receptors, and the magnitude of inhibition was significantly different from that of 6-receptors (p < 0.001) (Fig. 3B, Table 1). Maximal inhibition of 1/6(126/125)-receptors (27.8%) was not significantly different from that of 6-receptors but was significantly smaller than that of 1-receptors (p < 0.001) (Fig. 3B and Table 1). These results suggested that the relevant functional domains were between 1 subtype residues 127 to 232 and 6 subtype residues 126 to 231.

Second, we exchanged the N-terminal amino acid sequences between 1 subtype residues 127 to 232 and 6 subtype residues 126 to 231 to create 6/1/6 (125/126;232/231) and 1/6/1(126/125;231/232) chimeric subunits. These chimeras exchanged the regions in the proximal N terminus that contains the functional domains responsible for subtype-dependent Cu²⁺ inhibition. Maximal inhibition of 6/1/6 (125/126;232/231)-receptors by Cu²⁺ (51.4%) was similar to that of 1-receptors, and the magnitude of inhibition was significantly different from that of 6-receptors (p < 0.001) (Fig. 3B, Table 1). Maximal inhibition of 1/6/1(126/125;231/232)-receptors (28%) was not significantly different from that of 6-receptors (Fig. 3B and Table 1) but was significantly smaller than that of 1-receptors (p < 0.001). These results were consistent with the suggestion that the critical determinants for Cu²⁺ inhibition were located between the cysteine loop and the proximal N terminus, between 1 subtype residues 127 and 232 and 6 subtype residues 126 and 231.

Specific Subtype N-Terminal Residues Determine the Extent of Cu²⁺ Inhibition. The chimera studies revealed that the regions between a site slightly N-terminal to the cysteine loop (127) and the middle of TM1 (232) of the 1 subtype contained the primary functional domain responsible for high Cu²⁺ sensitivity of GABA_A receptor currents. Comparison of the sequences of 1-6 subtypes between 1 positions 127 and 232 revealed a single set of residues close to the cysteine loop that were similar in 1 and 2 subtypes but different in 3-6 subtypes. Residue 134 is a Val in the 1 subtype and equivalent residue 133 is also a Val in the 2 subtype. The equivalent residue is an Ile in the 3, 4, 5, and 6 subtypes (Fig. 4A). Residue 135 is an Arg in the 1 subtype, (Gln in residue 134 in the 2 subtype) and the equivalent residues in 3, 4, 5, and 6 subtypes are His (3), Ser (4 and 5), or Asn (6) (Fig. 4A). There was another interesting residue within the cysteine loop. The 1 subtype has a His at residue 141 and a His residue also occurred in the equivalent position of the 2 and 3 subtypes. The equivalent residue was Arg for the 4 and 6 subtypes and Gln for the 5 subtype (Fig. 4A). Thus, the 1 subtype had an eight-amino acid VRAECPMH motif, whereas the 6 subtype had a modified eight-amino acid INADCPMR motif.

View larger version (34K): [in this window] [in a new window]   Fig. 4. The effect of residues in the N-terminal region (127-231) of subunits on Cu2+ inhibition. A, sequence alignment of N-terminal regions between the front of the cysteine loop and TM1 of six wild-type subtypes Arrows indicate splice sites for 1/6 chimeras at positions 126/125 and 232/231 of each 1 and 6 subtype. Dashes represent conserved amino acid residues as occurred in 1 subtype. The boxed residues represent amino acids mutated in wild-type and chimeric subunits. B, the Cu2+ concentration-response relationship for 1/6 chimeric mutant subunit-containing 6/1/6(125/126;232/231) (H141R) and 1/6/1(126/125;231/232) (R140H) receptors were obtained by normalizing peak response to GABA (near EC50 value for each isoform) plus each Cu2+ concentration as a percentage of maximum current response to GABA alone for each isoform. Symbols and vertical error bars represent mean and S.E.M., respectively. Data were fitted with a four-parameter logistic equation. The average IC50 values for Cu2+ are presented in Table 2. C, comparison of maximal Cu2+ inhibition of 1/6 chimeric mutant subunit-containing receptors. Each bar represents mean ± S.E.M.

View this table: [in this window] [in a new window]   TABLE 2 Cu2+ sensitivity of subtype-mutated chimeric GABAA receptors

Replacement of 1 subtype H141 with 6 subtype R140 [6/1/6(125/126;232/231) H141R] resulted in a reduction in maximal Cu²⁺ inhibition of 6/1/6(125/126;232/231) receptors from 51.4 to 23.3% (Fig. 4B, Tables 1, 2), a level of inhibition similar to that of 6-receptors (21.2%) and 1/6/1(126/125;231/232)-receptors (28.0%). The IC₅₀ for Cu²⁺ was not significantly different for the mutant receptor compared with that of the 6/1/6(125/126;232/231)-receptor (Table 2). In contrast, the reverse mutation, R140H, [1/6/1(126/125;231/232) R140H] did not change maximal Cu²⁺ inhibition or IC₅₀ of 1/6/1(126/125;231/232)-receptors (29.0%).

Combination of the V134I and R135N mutations [6/1/6(125/126;232/231) V134I/R135N] caused a small reduction in maximal Cu²⁺ inhibition of 6/1/6(125/126;232/231)-receptors from 51.4 to 42% (Table 1). Combination of all three mutations (V134I, R135N, and H141R) further decreased the maximal Cu²⁺ inhibition of 6/1/6(125/126;232/231)-receptors to 17.8%, a value similar to that of 6-receptors (21.2%) (Table 1). These data suggested that H141 is the major determinant and V134 and R135 are minor determinants of high Cu²⁺ sensitivity of 1-receptors.

We examined the Cu²⁺ sensitivity of the "reverse" triple mutation I133V/N134R/R140H in 1/6/1(126/125;231/ 232)-receptors with a single concentration 10 µM Cu²⁺. The amount of Cu²⁺ inhibition increased from 28.0 to 41.4% (p < 0.05), a level of inhibition approaching that of 6/1/6(125/126;232/231)-receptors (51.4%) or 1-receptors (52.2%). These results are also consistent with the suggestion that the V(R,Q)AECPMH motif may constitute a major structure for conferring stronger Cu²⁺ sensitivity to 1- and 2-subtype containing GABA_A receptors.

The TM2 Zn²⁺ "Domain" Is Involved in Cu²⁺ Inhibition. Although the His in the V(R,Q)AECPMH motif seemed to be the primary determinant of the increased sensitivity of 1-receptor currents, 6-receptor currents were still sensitive to Cu²⁺ inhibition. Thus, we hypothesized that there may exist functional domains in or other subunits responsible for Cu²⁺ sensitivity of GABA_A receptor currents. It has been shown that the H267 residue in TM2 of subtypes (Fig. 5A) is important for high-affinity Zn²⁺ inhibition of 11-receptors (Horenstein and Akabas, 1998). H272 in the TM2-TM3 loop of the 6 subtype, close to the H267 residue of subtypes, has also been shown to be a determinant of high Zn²⁺ sensitivity to 6-subtype receptors (Fisher and Macdonald, 1998). Recently, Sharonova et al. (2000) proposed that Cu²⁺ shares a binding site with Zn²⁺. Therefore, we examined the effect of the His residues in TM2 of subtypes and in the TM2-TM3 extracellular loop of 6 subtypes on Cu²⁺ inhibition to determine whether these residues are also involved in Cu²⁺ inhibition. These mutant subtypes produced functional GABA_A receptors when coexpressed as (1,6)32L receptors.

View larger version (21K): [in this window] [in a new window]   Fig. 5. The role of TM2 and the TM2-TM3 loop in Cu2+ inhibition of GABAA receptors. A, comparison of amino acid sequences in TM2-TM3 loop of , , and subunits. Dashes indicate conserved amino acids as occurred in the 1 subtype. The boxed His residues represent amino acids that are known to be involved in Zn2+ inhibition (Fisher and Macdonald, 1998; Horenstein and Akabas, 1998). B, Cu2+ concentration-response relationships from L929 fibroblast expressing 13(H267S)2L, 63(H267S)2L, and 6(H273N)3(H267S)2L mutant-containing receptors were obtained by normalizing peak response to GABA plus each Cu2+ concentration as a percentage of maximum current response to GABA alone. Symbols and vertical error bars represent mean and S.E.M., respectively. Data were fitted with a four-parameter logistic equation. The average IC50 values for Cu2+ were presented in Table 3.

View this table: [in this window] [in a new window]   TABLE 3 Cu2+ sensitivity of subtype-mutated GABAA receptors

	Discussion

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An N-Terminal His in the Cys-Loop Is the Primary Determinant of a Subtype-Dependent Sensitivity to Cu²⁺ Inhibition. In this study, we have demonstrated that Cu²⁺ produces subtype-dependent block of GABA_A receptor currents and that the subtype-dependent Cu²⁺ antagonism is caused by regulation of maximal inhibition rather than IC₅₀.Cu²⁺ produces a greater maximal inhibition of 1- and 2-receptors and lesser maximal inhibition of 3-, 4-, 5-, and 6-receptors. We have identified the structural bases for subtype-dependent Cu²⁺ inhibition of GABA_A receptors. The chimera and mutagenesis analysis suggested that an 1 subtype N-terminal functional domain (127-232 region) was important for greater Cu²⁺ inhibition of 1 subtype than of 6 subtype currents. We also demonstrated that 1 subtype residues Val134, Arg135, and His141 in a VRAECPMH motif confer higher Cu²⁺ sensitivity to 132L receptors and that His141 is the major determinant in the motif. The INADCPMR sequence confers the lower Cu²⁺ sensitivity of 632L receptors. Interestingly, Hosie et al. (2003) have shown recently that Glu137 and His141 residues in the N terminus of the 1 subtype are part of critical Zn²⁺-coordinating domains. Interestingly, these two residues occurred in the VRAECPMH motif, which is responsible for increased Cu²⁺ inhibition. 232L receptors have a Cu²⁺ sensitivity that is similar to that of 132L receptors, and the 2 subtype has a sequence (VQAECPMH) similar to that of the 1 subtype, with only a single exchange of a Gln for an Arg residue in the 2' position. The 332L, 432L, and 532L receptors have lower Cu²⁺ sensitivities, similar to that of 632L receptors, and 3, 4, and 5 subtypes have sequences that are similar to that of the 6 subtype (Fig. 4A). In the 2' position the 3, 4, 5, and 6 subtypes have His, Asn, or Ser residues rather than the 1 subtype Arg residue. In the 8' position, the 5 subtype has an exchange of a Gln for an Arg residue. The 3 subtype, however, has a His in the 8' position, as does the 1 subtype. A comparison of the 1 subtype sequence (VRAECPMH) with the 3 subtype sequence (IHAECPMH) is consistent with the observation that, although the 8' His is the major determinant of Cu²⁺ sensitivity, the Val-Arg residues play an important permissive role that the Ile-His residues cannot and is consistent with the conclusion that the entire 8-amino acid motif is important, not simply a single residue.

Cations Have Multiple Interactions with GABA_A Receptors. GABA_A receptor currents are modified by several cations. It has been suggested that functional domains for cation modulation are partially shared among them (Celentano et al., 1991; Ma and Narahashi, 1993; Fisher and Macdonald, 1998). Ma and Narahashi (1993) suggested that Cu²⁺ and Zn²⁺ may share a common binding site on the GABA_A receptor. The 6 subtype H273 residue in the TM2-TM3 extracellular loop has been shown to play a role in conferring high Zn²⁺ sensitivity to 6 subtype-containing receptors (Fisher and Macdonald, 1998). This His residue in the TM2-TM3 loop was reported to regulate subunit dependent Ni²⁺-, Cd²⁺-, and La³⁺-inhibition but not Cu²⁺-inhibition of GABA_A receptor currents (Fisher and Macdonald, 1998; H. Kim and R. L. Macdonald, unpublished observations). The functional domain for Cd²⁺ inhibition has been suggested also to be localized to the N-terminal region of 1 subtypes (Fisher and Macdonald, 1998). Although we did not explore further structural determinants of Cd²⁺ inhibition in this study, it is possible that Cd²⁺ may also interact with the 1 subtype N-terminal functional domain (127-232) that determines Cu²⁺ sensitivity.

We also provide evidence that the H267 residue in the 3 subunit TM2, which is known to be associated with Zn²⁺ modulation (Fisher and Macdonald, 1998; Horenstein and Akabas, 1998), is involved in Cu²⁺ inhibition. The 3 subtype H267S mutation alone minimally affected maximal Cu²⁺ inhibition in 1-receptors and 6-receptors. However, combination 3 H267S with 6 H273N (i.e., removal of two His residues from 6-receptors) virtually abolished Cu²⁺ inhibition but not in 13(H267S)-receptors, which also lack these two His residues. These results suggest that Cu²⁺ shares a portion of the functional domain with Zn²⁺ and confirms that N-terminal determinants of 1 subtypes are important for Cu²⁺ inhibition. Taken together, these data indicate multiple cation sites in GABA_A receptor complex, and the VRAECPMH motif provides a novel cation regulatory site for polyvalent cations.

Multiple - and -Subtype His Residues Contribute to Cu²⁺ Inhibition of GABA_A Receptor Currents. The relevant and subtype residues that regulate Cu²⁺ sensitivity are quite close in the distal regions of the GABA_A receptor channel, being located in the distal TM2 (His267) of the subunit and in the TM2-TM3 loop (His273) of the subunit. The residues that control the -subtype dependent Cu²⁺ sensitivity of the GABA_A receptor channel (the VRAECPMH motif) are remote from this location in the N terminus and flank the N-terminal cysteine of the cysteine pair that forms the signature cys loop (loop 7) of all GABA_A, glycine, nicotinic cholinergic, and serotonin 5HT3 receptor channels. The basis for this remote interaction may be inferred from the crystal structure of the ACh binding protein (Brejc et al., 2001). The structure of this protein revealed that the cys loop in the ACh binding protein is hydrophilic and projects toward the side of the protein, which would interface with the membrane in ACh receptors. Furthermore, Kash et al. (2003) provided evidence that a GABA_A receptor 1 subtype cys loop residue Asp149 interacts with Lys279 in the middle portion of the TM2-TM3 loop of GABA_A receptors. Both of these residues are conserved in all GABA_A receptor subunits. The interaction has been shown to be electrostatic because charge reversal of the cys loop Asp and TM2-TM3 Lys residues maintained receptor function (Kash et al., 2003). The TM2-TM3 Lys residue is quite near the subunit TM2 His and the 6 subtype TM2-TM3 His, and the cys loop Asp is near the 1 subtype cys loop His is in the VRAECPMH motif. In addition, the subunit E182 has been proposed to interact with the 1 E137 and H141 residues in the VRAECPMH motif to contribute to the Zn²⁺ binding site (Hosie et al., 2003), supporting the proposal that this motif could be a critical structural determinant for Cu²⁺ inhibition. Therefore, there is a cluster of His residues in the subunit TM2, the 6 subtype TM2-TM3 loop, and the cys loop, and it is likely that Cu²⁺ interacts with these and subunit His residues to inhibit GABA_A receptor channels.

Physiological Significance of Cation Modulation of GABA_A Receptors. In dorsal root ganglion neurons, Cu²⁺ blocked 100% of GABA-induced currents with about a 16 µM IC₅₀ with a single population block pattern (Ma and Narahashi, 1993), which is different from our finding in recombinant receptors (x32L) that currents were not completely blocked (maximally about 50%) by Cu²⁺ and that the Cu²⁺ inhibition curves had two populations of block. Cu²⁺ inhibition of olfactory bulb neurons was similar to that of dorsal root ganglion neurons (Trombley and Shepherd, 1996). However, in cerebellar Purkinje cell GABA_A receptor currents, maximal Cu²⁺ block was only 60% and the IC₅₀ was lower (35 nM) (Sharonova et al., 1998) than in dorsal root ganglion neurons. Variations in Cu²⁺ pharmacology may come from different region-specific GABA_A receptor subunit subtype combinations. It has been shown that 2, 3, 3, 1, 2, and 3 transcripts were expressed in dorsal root ganglia and 1, 2, 3, and 2 mRNAs were detected in cerebella Purkinje cells (Laurie et al., 1992b). In olfactory bulb, although expression pattern and intensity were different depending on the region, except for the 6 subtype, most GABA_A receptor subunits were detected (Laurie et al., 1992a).

Although Zn²⁺ showed an inhibition pattern that was similar to that of Cu²⁺ in dorsal root ganglion and olfactory bulb neurons with similar IC₅₀ values (10 µM) and almost 100% maximal inhibition (Ma and Narahashi, 1993; Trombley and Shepherd, 1996), Zn²⁺ inhibited GABA currents with lower IC₅₀ values (35 µM) and similar maximal inhibition (66%) compared with Cu²⁺ in cerebellar Purkinje cells. Although Cu²⁺ and Zn²⁺ are released from nerve terminals during synaptic activity (Assaf and Chung, 1984; Kardos et al., 1989), the fact Cu and Zn²⁺ are coreleased at synaptic cleft is unclear. However, the data from the present and other studies (Sharonova et al., 2000) study strongly suggest that they might interact with each other with GABA_A receptor complex and participate in modulation of synaptic transmission. Further mechanistic study will be needed to understand the interaction between Cu²⁺ and Zn²⁺ in neurotransmitter receptors.

	Acknowledgements

 
We thank Helen Zhang for her help with construction of the chimeras and for making receptor subunit mutations.

	Footnotes

 
This study was supported by National Institutes of Health grant NS33300 (to R.L.M.).

ABBREVIATIONS: ACh, acetylcholine.

Address correspondence to: Dr. Robert L. Macdonald, Department of Neurology, Vanderbilt University, 2100 Pierce Ave., Nashville, TN 37212 (E-mail: robert.macdonald{at}vanderbilt.edu).

	References

Top Abstract Materials and Methods Results Discussion References

 
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