Insights into the Structural Basis for Zinc Inhibition of the Glycine Receptor*

Simon T. Nevin {ddagger} §, Brett A. Cromer § ¶ ||, Justine L. Haddrill {ddagger}, Craig J. Morton ¶, Michael W. Parker ¶ || and Joseph W. Lynch {ddagger} **

From the {ddagger}Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia, and St. Vincent's Institute of Medical Research, 9 Princes St., Fitzroy, Victoria 3065, Australia

Received for publication, January 6, 2003 , and in revised form, April 25, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Histidines 107 and 109 in the glycine receptor (GlyR) {alpha}1 subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR {alpha}1 subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His107 from one subunit and His109 from an adjacent subunit. This was tested by co-expressing {alpha}1 subunits containing the H107A mutation with {alpha}1 subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type {alpha}1 homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent {alpha}1 subunits. No evidence was found for {beta} subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per {alpha}1{beta} receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of {alpha}1 homomers. The binding of zinc at the interface between adjacent {alpha}1 subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Zinc is concentrated into round, clear presynaptic vesicles in central nerve terminals and is released into the synaptic cleft by nerve stimulation (13). During synaptic stimulation, zinc is thought to reach a peak external concentration of more than 100 µM (1, 3, 4). At such concentrations, zinc is potentially able to modulate a wide variety of pre- and postsynaptic ion channels (5). Several lines of evidence suggest that the glycine receptor chloride channel (GlyR),1 which mediates inhibitory neurotransmission in the spinal cord and brainstem (6), may be a physiological target for zinc modulation. First, zinc exerts potent effects on the GlyR; low concentrations (0.01–10 µM) potentiate glycinergic currents by increasing the apparent glycine affinity, whereas higher concentrations (>10 µM) inhibit the current by reducing the apparent glycine affinity (7). Second, an ultrastructural study has found evidence for zinc and glycine co-localization in individual presynaptic terminals in the spinal cord (8). Third, at glycinergic synapses onto intact zebrafish hindbrain (Mauthner) neurons, zinc chelators decreased the amplitude, duration, and frequency of glycinergic inhibitory postsynaptic currents, whereas zinc application had the opposite effect (9).

The GlyR is a member of the ligand-gated ion channel (LGIC) receptor family, which includes the nicotinic acetylcholine receptor cation channel (nAChR), the {gamma}-aminobutyric acid type-A and type-C receptor chloride channels (GABAAR and GABACR), the serotonin receptor cation channel (5HT3R), and a recently identified zinc-gated cation channel (10) as well as invertebrate glutamate and histamine receptors (11). These receptors are composed of five structurally similar subunits arranged in a ring to form a central ion-conducting pore (12). Adult GlyRs in vivo are composed of {alpha} and {beta} subunits in a 3{alpha}/2{beta} stoichiometry (13). Although {alpha} subunits readily form homomeric GlyRs in heterologous expression systems, {beta} subunits form functional receptors only as heteromers with {alpha} subunits. The molecular mechanisms of zinc inhibition and potentiation have recently been investigated in several laboratories (7, 1416). Although the molecular basis of potentiation has so far proved elusive, a strong case has been advanced for a candidate inhibitory site. Harvey et al. (15) showed that zinc inhibition of the recombinant {alpha}1 GlyR was selectively abolished by either reducing the pH or by pretreating receptors with a histidine-specific modifying agent. Since both treatments effectively reduce the ability of zinc to bind to histidine imidazole rings, this implicated histidines in the coordination of inhibitory zinc. Mutations of either His107 or His109 were subsequently shown to abolish zinc inhibition, strongly suggesting that these residues coordinate zinc at its inhibitory site (15).

For a pair of histidine side chains to coordinate a zinc ion, the {alpha}-carbon atoms need to be within 13 Å of each other (17). Since histidines 107 and 109 are separated by only one residue, it is certainly feasible that this pair could coordinate zinc ions within individual {alpha} subunits. Indeed, histidines with this spacing occur in many structurally defined zinc-binding sites (17), including carbonic anhydrase II (Protein Data Bank code 1CA2 [PDB] ) (18), which contains the same HFH tripeptide as GlyR {alpha}1 107–109, thereby providing a plausible model for zinc binding within individual GlyR {alpha}1 subunits (15). The recently resolved crystal structure of a soluble acetylcholine-binding protein (AChBP) from the snail, Lymnaea stagnalis, has provided a structural model for the N-terminal ligand-binding domain of LGICs (19). Modeling of the GlyR N-terminal domain, based on AChBP, places His107 and His109 near the lumen of the channel vestibule close to the axis of symmetry (20). In this position, His107 and His109 from adjacent {alpha} subunits could be close enough to coordinate zinc ions across the subunit interface, as recently predicted by Laube et al. (21).

The aim of this study was to use a combination of homology modeling based on the AChBP crystal structure, coupled with functional analysis of GlyRs incorporating site-directed mutations in putative zinc-binding regions, to provide insights into the structure of the inhibitory zinc-binding site.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutagenesis and Expression of GlyR cDNAs—The human GlyR {alpha}1 and {beta} subunit cDNAs were subcloned into the pCIS2 and pIRES2-EGFP plasmid vectors (Clontech, Palo Alto, CA), respectively. Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA), and the successful incorporation of mutations was confirmed by sequencing the clones. HEK293 cells were transfected using a calcium phosphate precipitation protocol. When co-transfecting the GlyR {alpha} and {beta} subunits, their respective cDNAs were combined in a ratio of 1:10 (22). After exposure to transfection solution for 24 h, cells were washed twice using the culture medium and used for recording over the following 24–72 h.

Electrophysiology—The cells were observed using a fluorescent microscope, and currents were measured using the whole cell patch clamp configuration. Cells were perfused by a control solution that contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, with the pH adjusted to 7.4 with NaOH. Patch pipettes were fabricated from borosilicate hematocrit tubing (Vitrex, Modulohm, Denmark) and heat-polished. Pipettes had a tip resistance of 1.5–3 megaohms when filled with the standard pipette solution, which contained 145 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA, with the pH adjusted to 7.4 with NaOH. After establishment of the whole cell configuration, cells were voltage-clamped at –40 mV, and membrane currents were recorded using an Axopatch 1D amplifier and pclamp6 software (Axon Instruments, Foster City, CA). The cells were perfused by a parallel array of microtubular barrels through which solutions were gravity-induced.

Because the {alpha} subunit can efficiently assemble into functional GlyRs as either {alpha} homomers or {alpha}{beta} heteromers, it is necessary to confirm the incorporation of {beta} subunits into functional receptors. This was achieved in two ways. First, green fluorescent protein expression was used to identify cells expressing GlyR {beta} subunit protein. Second, picrotoxin sensitivity was used as a functional assay of the incorporation of {beta} subunits into heteromeric GlyRs (22). When co-expressed with {alpha} subunits in HEK293 cells, incorporation of {beta} subunits increases the picrotoxin IC50 from around 25 to 500 µM in the presence of an EC50 glycine concentration (23). In the present study, we measured the effect of 1 mM picrotoxin on the magnitude of currents activated by an EC20 glycine concentration, and cells were assumed to express {alpha}{beta} heteromeric GlyRs if 1 mM picrotoxin inhibited the current by less than 50%.

Data Analysis—The empirical Hill equation, fitted by a nonlinear least squares algorithm (Sigmaplot; Jandel Scientific, San Rafael, CA), was used to calculate the EC50, the IC50, and the Hill coefficients (nH) of excitatory and inhibitory dose-response curves. Statistical significance was determined by a one-way analysis of variance using the Student-Newman-Keuls post hoc test for unpaired data (Sigma Stat; Jandel Scientific), with p < 0.05 representing significance.

Molecular Modeling—Models of the extracellular region of the human GlyR {alpha}1 subunit were constructed by homology modeling, using the crystal structure of AChBP from the snail Lymnaea stagnalis as a template (Protein Data Bank entry 1I9B [PDB] ) (19). Sequence identity between LGICs and AChBP is only 15–24%, in the "twilight zone" for effective alignment and homology modeling (24). To improve the reliability of the alignment, we used ClustalW (25) to align AChBP with a large number of Cys loop LGICs, particularly those that are the most similar to AChBP, such as nAChRs, as described in Ref. 20. Secondary structure predictions using the PHD prediction algorithm (26) assisted the alignment in regions of particularly low homology, such as the N-terminal {alpha}-helix. Swiss-Model (27) was used to create independent monomer models for each alignment tested. Loop regions that correspond to gaps in the alignment were modeled by fitting structures from a loop data base and are the least reliable sections of the models. Acceptable monomer models were then assembled into pentamers using AChBP as a scaffold. The assembled pentamer was energy-minimized using INSIGHT II DISCOVER (Accelrys, San Diego, CA) to eliminate any obvious problems such as steric clashes. Ramachandran plots and Verify-3D scores (28) were used to assess the quality of each model. For regions of a model that scored poorly, other possible alignments were tested. Four different alignments were tested in the region in the putative inhibitory zinc-binding region surrounding His107 and His109 (Fig. 1A). Excluding the inhibitory zinc-binding region, there are only three residues that fall in the disallowed region of a Ramachandran plot, Leu83 and His201, which are in loops corresponding with gaps in the alignment, and Lys33, which corresponds to Arg23 in AChBP, which also falls in the disallowed region. Total Verify-3D scores were above 64 for all models, which is quite acceptable for this type of model (28). With a sliding window of 21 residues, there are no regions with a negative score, but there are three regions that score below 0.2. These are the N-terminal helix and the loop between the second and third {beta}-strands, which are both likely to interact with residues 1–10, and the signature Cys loop, which is likely to interact with the membrane domain. Since our models do not include residues 1–10 or the membrane domain, they cannot fully represent the native environment for the three regions mentioned. Finally, a conserved N-glycosylation site at Asn38 is on the surface of the model, consistent with it being glycosylated, and the unique disulfide bond of GlyRs is present in our models.



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FIG. 1.
Model of the {alpha}1 GlyR. A, alignment used for homology modeling of {alpha}1 GlyR based on AChBP structure. The boxes show identical residues (open) and residues conserved throughout LGIC superfamily (shaded). Secondary structure elements of AChBP structure are shown, with {beta}-strands in orange and the {alpha}-helix in green. The open green box shows the predicted {alpha}-helix using PHD (26). The putative inhibitory zinc-binding region is shown in blue, with His107 and His109 in pink. Alternative sequence alignments of AChBP to this region are shown as models A–D (in parentheses). B, backbone ribbon representation of model homopentameric {alpha}1 GlyR (built using alignment C above) viewed along the axis of symmetry from the synaptic cleft side. The colors indicate each subunit. Dotted lines mark the subunit interfaces, with plus and minus signs indicating the subunit faces that contribute to the interface. The putative inhibitory zinc-binding region is shown in blue, with His107 and His109 side chains shown as bonds in pink. The arrow shows the point of view used in Fig. 4.

 



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FIG. 4.
Close-up view of putative inhibitory zinc-binding site in models A, B, C, and D, viewed from within the vestibule lumen as indicated by the arrow in Fig. 1. Only two subunits are shown for clarity. Side chains of His107 and His109 from the plus face (right) and His107, His109, Glu110, and Thr112 from the minus face (left) are shown as bonds, with standard coloring according to atom. Pink spheres indicate possible locations for bound zinc.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Homology Modeling Indicates That His107 and His109 Face the Vestibule Lumen—Based on earlier findings (1416), we started with the premise that His107 and His109 coordinate zinc at the inhibitory site of {alpha}1 homomeric GlyRs. In an attempt to understand the structural basis for zinc inhibition, we built models of the GlyR {alpha}1 subunit extracellular region, based on homology to AChBP, as described in Cromer et al. (20), using the alignments shown in Fig. 1A. Histidines 107 and 109 of the GlyR {alpha}1 subunit fall within a region from Lys104 to Asn115, which we have termed the "inhibitory zinc-binding region" (Fig. 1A), that shares little homology with AChBP but is flanked by sequences with strong homology. These flanking sequences anchor the zinc-binding region within the model such that it faces the lumen of the extracellular vestibule, near the axis of symmetry and subunit interfaces (Fig. 1B; see also Ref. 20). Although the detailed structure of the zinc-binding region cannot be reliably predicted, its location near the axis of symmetry immediately raises the possibility that His107 and His109 from adjacent subunits could be close enough to coordinate zinc in an intersubunit site, as recently suggested by others (21). On the other hand, the possibility of an intrasubunit site with zinc coordinated by His107 and His109 from the same subunit, as proposed by Harvey et al. (15), remains also feasible. Consequently, our first aim, experimentally, was to discriminate between these alternative models for inhibitory zinc binding.

His107 and His109 from Adjacent Subunits Form the Inhibitory Zinc-binding Site—To discriminate experimentally between intrasubunit and intersubunit models for the inhibitory zinc-binding site, we examined the zinc sensitivity of GlyRs formed from co-expression of {alpha}H107A and {alpha}H109A subunits, each of which individually shows reduced zinc sensitivity. The rationale was that if the inhibitory zinc-binding site is formed within a subunit, then these mixed receptors should be zinc-insensitive, since each subunit contains only one of the two required histidine residues. Therefore, if the resultant recombinant receptors possess a high affinity zinc inhibitory site, then this site must be formed at the interface between subunits. It is important to note that this approach cannot address the question of whether zinc can also be coordinated within individual subunits. Hence, in all our experiments, it is necessary to eliminate at least one histidine (His107 or His109) per subunit to ensure that this does not confound the interpretation of our results.

The mean glycine EC50, nH, and Imax values for the WT and all mutant GlyRs employed in this study are shown in Table I. As demonstrated previously, the {alpha}WT GlyR is highly sensitive to zinc inhibition. As expected from previous studies employing mutations at these positions (7, 15, 16, 29), none of these mutations had dramatic effects on the glycine EC50, nH, or Imax values. The inhibitory zinc dose responses for the {alpha}H107A GlyR, the {alpha}H109A GlyR, the double mutant {alpha}H107A,H109A GlyR, and the co-expressed ({alpha}H107A + {alpha}H109A) GlyR were measured in the presence of an EC20 glycine concentration. Zinc inhibition was quantitated by measuring the steady-state level of the current following application of zinc and expressing this as a fraction of the peak magnitude of the zinc-potentiated current (14). Sample responses to increasing concentrations of zinc for each of these receptor constructs are shown in Fig. 2A. Averaged zinc inhibitory dose responses are plotted in Fig. 2B, and the mean zinc IC50 and nH values for these and all other recombinant constructs examined in this study are summarized in Table I. The H107A mutation reduced the inhibitory potency by a factor of 15, whereas the H109A mutation almost entirely abolished zinc inhibition (Table I), consistent with earlier results (15) and the proposed role of these residues in zinc coordination. The difference in the magnitude of the effect of the two mutations indicates that the zinc coordination is not perfectly symmetric, consistent with the probable involvement of other residues (16). As expected, the double mutant {alpha}H107A,H109A GlyR was also insensitive to zinc inhibition (Fig. 2A).


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TABLE I
Functional properties of WT and mutant GlyRs

 


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FIG. 2.
Zinc inhibition of {alpha} homomeric GlyRs. A, examples of zinc inhibitory dose responses are shown for the {alpha}H107A GlyR (top left panel), the {alpha}H109A GlyR (top right), the double mutant {alpha}H107A,H109A GlyR (bottom left), and GlyRs comprising both {alpha}H107A and {alpha}H109A subunits (bottom right). EC20 glycine applications are indicated by the thin lines, and periods of zinc application are shown by the thick lines. Zinc concentrations applied in each panel are indicated. In each case, progressively increasing the zinc concentration caused an increased degree of block. The horizontal scale bar represents 5 s and applies to all panels. The vertical scale bar represents 0.8 nA (top left panel), 1.2 nA (top right), 1.3 nA (bottom left), and 0.2 nA (bottom right). B, averaged dose responses for the {alpha}WT GlyR (filled circles), the {alpha}H107A GlyR (filled triangles), and the {alpha}H107A + {alpha}H109A GlyR (open triangles). Error bars, S.E.; curves, Hill equation fits to averaged data. Averaged parameters of best fit to individual dose responses are given in Table I.

 

When the two single mutant {alpha}H107A + {alpha}H109A subunits were co-expressed, high sensitivity zinc inhibition was observed (Fig. 2, A and B). The zinc sensitivity (21 ± 5 µM; Table I) was not significantly different from that of the {alpha}WT GlyR (16 ± 8 µM), consistent with it being due to the same zinc-binding site. This inhibition could not have been mediated by zinc ions binding within individual subunits, since each subunit contained only one of the two required histidine residues. Therefore, it must have been due to intersubunit coordination of zinc ions by His107 and His109 from different subunits, supporting the hypothesis of an intersubunit zinc site and the positioning of the inhibitory zinc-binding region in our initial model (Fig. 1). The two coordinating histidines almost certainly come from adjacent subunits, since the constraints imposed on the model by the conserved anchor points make it virtually impossible for His107 and His109 from nonadjacent subunits to get close enough to coordinate zinc across the vestibule lumen. The fact that the inhibition seen with the mixed {alpha}H107A + {alpha}H109A GlyRs was not seen in either of the single mutants alone indicates that His107 and His109 each occupy a specific subunit "face" (either + or –; see Fig. 1) and are not flexible to coordinate zinc at either face. Therefore, an intersubunit zinc site can only occur at one of the two possible +/–interfaces, either H107A/H109A or H109A/H107A. This limits the number of inhibitory zinc sites in a pentamer of mixed {alpha}H107A + {alpha}H109A subunits to a maximum of two (Table II), demonstrating that two such sites are sufficient for inhibition by zinc. The relatively low nH for zinc inhibition in WT GlyRs and our finding that it is unchanged in the mixed {alpha}H107A + {alpha}H109A GlyRs support the view that one or two bound zinc molecules may be sufficient for maximal zinc inhibition in the WT GlyR (Table II).


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TABLE II
Possible subunit arrangements resulting from the recombinant expression of {alpha}H107A and {alpha}H109A subunits

The probability of each combination, together with the number of intersubunit zinc-binding sites, is also shown. Net probability of 0 zinc sites: 6.2%, 1 zinc site: 62.4%, 2 zinc sites: 31.2%.

 

Zinc inhibition of the mixed {alpha}H107A + {alpha}H109A GlyRs was incomplete (Fig. 1, A and B), however, inhibiting a maximum of 55 ± 5% (n = 6) of the glycine-activated current. In contrast, a saturating (1 mM) zinc concentration inhibits the {alpha}WT GlyR current by 100% (Fig. 1B; see also Refs. 7 and 14). The reduced extent of zinc inhibition is not unexpected, given that each receptor in the mixed population of mutant receptors contains zero, one, or two zinc-binding sites, relative to five sites in WT receptors. Assuming the {alpha}H109A and {alpha}H107A subunits recombine in a random binomial manner to form pentameric GlyRs, 6.2% of recombinant GlyRs should have no zinc-binding sites, 62.4% should have one inhibitory zinc-binding site, and 31.2% should contain two inhibitory zinc-binding sites per receptor (Table II). The 55% overall inhibition could be explained by GlyRs with two zinc sites being inhibited completely and those with one site being partially inhibited. Although this is a reasonable explanation, random recombination cannot be assumed, and other explanations are possible. In summary, the data above show that an intersubunit zinc-binding site can account for the zinc inhibition observed in WT {alpha}1 homomeric GlyRs but do not rule out an additional role for intrasubunit binding.

Possible Involvement of Thr112 in Zinc Coordination—Laube et al. (16) showed that a T112A mutation abolished inhibition by zinc and proposed that Thr112 is also involved in the coordination of zinc at its inhibitory site. If Thr112 directly coordinates zinc at an intersubunit inhibitory site, then co-expression of either the {alpha}H107A or {alpha}H109A subunits with the {alpha}T112A subunit should be able to restore zinc sensitivity, as observed for the two histidines above. To test this concept, we made the more conservative T112V mutation, which simply replaces the hydroxyl group of the threonine with a methyl group. As with the T112A mutation (16), the T112V mutation had little effect on glycine sensitivity but completely eliminated sensitivity to zinc inhibition (Table I). As expected, the double mutant {alpha}H107A,T112V and {alpha}H109A,T112V GlyRs were also insensitive to zinc inhibition (Table I). However, these experiments provide no information as to whether T112V caused a nonspecific structural disruption to the zinc coordination scaffold or whether it directly participated in zinc coordination. To test the possibility of direct coordination, we co-expressed {alpha}H107A + {alpha}T112V, {alpha}H109A + {alpha}T112V, or {alpha}H107A + {alpha}H109A,T112V subunits. As summarized in Table I, none of these mixed receptors was significantly inhibited by 1 mM zinc. The fact that co-expression did not restore zinc sensitivity provides no evidence to support a direct role for Thr112 in coordinating zinc. Although T112V mutant subunits contain both His107 and His109, the T112V mutation eliminates functional inhibitory zinc-binding sites at both faces of the subunit, by either 1) disrupting the structure of the zinc-binding region, either directly or allosterically, or 2) precluding the formation of GlyRs containing both mutant subunits. These data do not, however, rule out a direct role for Thr112 in zinc coordination, since it could have both a coordinating and a more structural role.

Zinc Coordination by {alpha}{beta} Heteromeric GlyRs—To further characterize the inhibitory zinc-binding site, we examined its properties in the {alpha}1{beta} heteromeric GlyR. As stated under "Experimental Procedures," the incorporation of {beta} subunits into {alpha}{beta} heteromers was confirmed for each cell by measuring the percentage inhibition of the EC20 glycine current that was inhibited by 1 mM picrotoxin. If the picrotoxin inhibited this current by no more than 50% (c.f. Refs. 22, 23, and 30), it was assumed that all recombinant receptors in that cell contained 3{alpha} and 2{beta} subunits (13). Since the arrangement of the {alpha} and {beta} subunits around the pentamer has not been determined, we considered the possibility that the {beta} subunits may be located either side-by-side or separated by an {alpha} subunit (Table III).


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TABLE III
Possible arrangements of intersubunit zinc binding sites resulting from the recombinant expression of WT and mutant {alpha} and {beta} subunits

 

Examples of the effects of zinc on heteromeric {alpha}WT{beta}WT GlyRs are shown in Fig. 3A, and the averaged zinc dose response is plotted in Fig. 3B. The mean zinc IC50 and nH values are summarized in Table I. As previously demonstrated (7), the zinc inhibitory potency of these receptors is similar to that of the homomeric {alpha}WT GlyR (Table I). The {beta} subunit contains a histidine at position 132, which is homologous with His109 in the {alpha} subunit. However, it contains an asparagine (Asn130) at the position homologous with His107. Since asparagine residues are not known to coordinate zinc ions (17), it is feasible that only the {beta} subunit His132, located at the plus side of the {beta}-{alpha} subunit interface, may coordinate zinc ions. Given this possibly, heteromeric {alpha}WT{beta}WT GlyRs may contain a maximum of three intersubunit zinc coordination sites as shown in Table III (top row).



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FIG. 3.
Effects of zinc on {alpha}{beta} heteromeric GlyRs. A, examples of zinc dose responses are shown for the {alpha}WT{beta}WT GlyR (top left), the {alpha}WT{beta}H132A GlyR (top right), the {alpha}H109A{beta}WT GlyR (bottom left), and the {alpha}H107A,H109A{beta}N130H GlyR (bottom right). Zinc concentrations applied in each panel are indicated. In each case, progressively increasing the zinc concentration caused an increased degree of block. The horizontal scale bar indicates 5 s and applies to all panels. The vertical scale bar represents 0.5 nA (top left panel), 0.4 nA (top right), 1.1 nA (bottom left), and 1 nA (bottom right). B, averaged dose responses for the {alpha}WT{beta}WT GlyR (filled circles) and the {alpha}WT{beta}H132A GlyR (filled triangles). Error bars, S.E.; curves, Hill equation fits to averaged data. Averaged parameters of best fit to individual dose responses are given in Table I.

 

Several approaches were employed to determine whether {beta}-{alpha} subunit interfaces could coordinate zinc ions. First, we incorporated the {beta} subunit H132A mutation to determine its effect on zinc inhibitory potency. The resultant {alpha}WT{beta}H132A GlyRs contain either one or two {alpha}-{alpha} subunit interfaces, depending on the subunit arrangement (Table III, row 2). As shown in Fig. 3, A and B, and summarized in Table I, these receptors were highly sensitive to zinc inhibition, with a mean IC50 of 11 ± 4 µM (n = 3). Thus, heteromeric GlyRs containing a maximum of two zinc inhibitory sites can be maximally inhibited by zinc. These results provide no evidence for a contribution of putative {beta}-{alpha} interface zinc sites to zinc-induced inhibition of glycine currents.

The second approach involved removing all {alpha}-{alpha} subunit zinc sites via the H109A mutation. The resultant heteromeric GlyRs would then have contained either one or two {beta}-{alpha} interface sites, depending on the subunit arrangement (Table III, row 3). Since the resultant {alpha}H109A{beta}WT GlyRs receptors were completely insensitive to zinc inhibition (Fig. 3, A and B, and Table I), whereas heteromers containing a similar number of sites at {alpha}-{alpha} subunit interfaces were highly zinc-sensitive, it is concluded that inhibitory zinc-binding sites are not formed at {beta}-{alpha} interfaces or at {beta}-{beta} interfaces. In a final experiment, we investigated the zinc sensitivity of the {alpha}H107A,H109A{beta}N130H GlyR. Although experiments described above suggest that this construct would not be zinc-sensitive, it was considered worth trying, since a high zinc sensitivity would have been evidence for a side-by-side arrangement of {beta} subunits (Table III, row 4). As anticipated, however, the construct was completely insensitive to zinc inhibition (Fig. 3, A and B, and Table I). In summary, we found no evidence for a direct contribution of the {beta} subunit to inhibitory zinc-binding sites.

Possible Conformations of the Inhibitory Zinc-binding Region—In our initial model of the GlyR extracellular domain, the inhibitory zinc-binding region is anchored by the flanking sequences, which have strong homology to AChBP, but the zinc-binding region itself is not reliably modeled because of weak homology and the inclusion of 3 extra residues relative to AChBP. It is now worth reconsidering models of possible conformations of this region in terms of how well they fit our experimental evidence for intersubunit inhibitory zinc-binding sites. Since {beta} subunits do not appear to contribute directly to these sites, we have restricted the modeling to {alpha}1 homopentamers. To sample possible conformations of this region, we built a series of models (A–D), each based on a different alignment in the 104–115 region as shown in Fig. 1A. To maintain as much of the AChBP secondary structure as possible, the gaps in the alignment with the zinc-binding region were placed at either end of the short {beta}-strand in AChBP. Each model was built independently from scratch, as described under "Experimental Procedures." Models were then assessed in terms of how well they fit the experimental data and their quality as protein structures.

We have shown here that intersubunit coordination of zinc by His107 and His109 can account for essentially all the properties of zinc inhibition. Therefore, we examined each of the models to see whether they were consistent with such coordination. The constraints of zinc coordination by histidine imidazole nitrogens (17) place the zinc less than 6 Å from the histidine {beta}-carbon atom. Consequently, the {beta}-carbon atoms of two histidines coordinating the same zinc must be within 12 Å of each other. For tetrahedral geometry, the most common coordination geometry for zinc, the {beta}-carbons must be within 10 Å of each other. The intersubunit distance between {beta}-carbons of His107 and His109 is <10 Å for model C (making tetrahedral coordination possible), <12 Å for models A and D (suggesting that coordination is possible), but >15 Å for model B (essentially ruling this model out as a viable model for an intersubunit zinc site). Given appropriate distance constraints, side chain orientation and surrounding structure also determine the feasibility of zinc coordination. To examine the feasibility of zinc binding in each model, we tested whether a reasonable zinc site could be produced by manually adjusting the side chain position of the two histidines within allowed rotamers while keeping the backbone fixed. Model C was able to most closely approach ideal tetrahedral geometry, but models A and D could also approach a reasonable zinc-binding site. The best positions achieved are shown in Fig. 4, together with a possible location for bound zinc in models A, C, and D. Interestingly, for models C and D, coordination by His109 and His107 was from the minus and plus faces, respectively, whereas this was reversed in model A.

We have also shown that Thr112 appears to have a structural role in the zinc-biding site but may have an additional direct role in zinc coordination. None of our models are consistent with Thr112 directly coordinating zinc, together with His107 and His109. Models C and D do, however, indicate that the side chain hydroxyl, which is removed in both the T112V (this study) and T112A (16) mutants that abolish zinc inhibition, could form hydrogen bonds with other parts of the structure. These potential hydrogen bonds could be determinants of a structural role for Thr112. Although we have not tested its role here, an E110A mutation has been shown to reduce sensitivity to zinc inhibition by 16-fold (16), indicating that it could be a third ligand for coordinating zinc. Of the models that could coordinate zinc by the two histidines, only in models C and D is Glu110 close enough to play a role in coordinating zinc, either directly or through a water molecule. Ramachandran plots and Verify-3D scoring both measure how well a model structure fits the characteristics of real protein structures, independent of the modeling process. Ramachandran plots for our models reveal that the inhibitory zinc-binding region of model C has no residues in the disallowed regions, whereas the other models have one or two residues each in the disallowed regions. Model C also gave a markedly higher Verify-3D score than the other three models, indicating that it can be objectively considered the most reasonable protein structure of the four models. In summary, whereas models A and D are possible models of an intersubunit inhibitory zinc-binding site, model C is our preferred working model.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanism of Zinc Inhibition—This study shows that inhibitory zinc-binding sites are formed at GlyR {alpha}1 subunit interfaces, where zinc is coordinated by His107 and His109 residues from adjacent subunits. We cannot rule out the possibility that His107 and His109 are also able to coordinate zinc within the same subunit, but an intersubunit site is able to account for essentially all of the inhibitory properties of zinc in WT receptors. Although zinc bound within proteins is normally coordinated by at least three side chains, coordination by only these two histidines may be sufficient to explain the relatively low binding affinity of the inhibitory zinc-binding site. Similar low micromolar affinity is seen in a mutant carbonic anhydrase that retains two histidine ligands and probably a water (18). Thr112 appears to have a significant structural role in this region, but we cannot rule out the possibility that it may also contribute to the coordination of zinc. Glu110 has also been shown to be important for zinc inhibition (16) and could be involved in coordinating zinc, either directly or through a water molecule, but this was not tested here. The present study also shows that functional zinc inhibitory sites are not formed at the interfaces between {beta} and {alpha} subunits or between {beta} subunits. Furthermore, since inhibitory zinc sites are formed only at the interface between {alpha} subunits, a maximum of two occupied sites is sufficient to completely inhibit the {alpha}{beta} heteromeric GlyR. A maximum of two occupied sites is also sufficient to give significant, possibly complete, inhibition of the {alpha} homomeric GlyR.

The potency of zinc inhibition is increased as the glycine concentration is reduced (7). In addition, zinc inhibition reaches a steady-state level much faster in the absence of glycine (14). A single channel study found that high (50 µM) zinc concentrations reduced {alpha}1 GlyR open probability by reducing mean channel open time and the relative abundance of long channel bursts (16). It concluded that zinc increased the rate at which the channel exits from the open state. Together, these results suggest that glycine-induced activation is accompanied by a structural change at the zinc inhibitory site and that zinc acts to prevent this by stabilizing the closed conformation. Based on our models, the inhibitory zinc-binding site is located in close proximity to the agonist-binding site, and allosteric interaction between the two could occur via relatively minor local movements. Alternatively, since both these sites are at subunit interfaces, they could interact via global movements of subunits relative to one another. Consistent with this idea of intersubunit movements, our models place Asp80, which has been implicated in the potentiating zinc-binding site, at the subunit interface on the outside of the pentamer. Although this position is quite distant from the inhibitory zinc-binding site, mutations of His109 affect zinc potentiation as well as inhibition (15).

A model of receptor activation involving global intersubunit movements has been proposed to account for a large body of functional data obtained mainly from the nAChR (reviewed in Refs. 31 and 32). This idea is also supported by recent structural evidence (33). An essential feature of this model is that the largest displacements occur at the subunit interfaces. The GlyR inhibitory zinc-binding site is located in an ideal position to hinder these movements by locking adjacent subunits into a fixed, closed position.

Zinc Sites in Other LGICs—Although zinc affects most LGIC members (10, 3437), to date putative zinc-binding sites have been identified only in the GABAAR and in the GABACR. In the GABAAR {alpha}5 subunit, zinc sensitivity is reduced by a H195D mutation in the extracellular domain, immediately preceding {beta}-strand 9, close to the membrane domain (38). In the GABAAR {beta} subunits, zinc sensitivity is reduced by mutating a histidine in the second transmembrane domain (39, 40) or at the position homologous to His109 in the GlyR {alpha}1 subunit (41). This latter observation is the only evidence so far that the GlyR zinc inhibitory site characterized in the present study may also be functional in other LGIC members. However, it is of interest to note that the recently identified zinc-activated cation channel (10) contains aspartic acid residues at positions homologous to 107 and 110 in the GlyR {alpha}1 subunit, which could contribute to a channel-activating zinc site. In GABACR {rho}1 homomers, zinc inhibition is abolished by mutating a histidine residue at the subunit interface, close to the ligand-binding site (42). A recent paper has identified an inhibitory zinc-binding site at the interface between {alpha}1 and {beta}3 subunits of the GABAAR, on the outside of the extracellular domain close to the membrane (43), a site that is quite distinct from the GlyR site characterized here. Finally, there is strong structural and functional evidence for a potentiating calcium binding site at the interface between adjacent nAChR {alpha}7 subunits (44). In contrast with the present study, the high affinity site is proposed to be associated with the channel open state. Intersubunit binding sites appear to be emerging as a common means for modulation of LGICs by divalent cations, although the location of the site may vary. There is some sense to this, given the intersubunit location of agonist binding sites and the evidence for intersubunit movement upon agonist binding (33).


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

|| Supported by the National Health and Medical Research Council of Australia. Back

** Research in the laboratory of this author is funded by the Australian Research Council. To whom correspondence should be addressed. Tel.: 617-3365-3157; Fax: 617-3365-1766; E-mail: j.lynch{at}mailbox.uq.edu.au.

1 The abbreviations used are: GlyR, glycine receptor; LGIC, ligand-gated ion channel; AChBP, acetylcholine-binding protein; nAChR, nicotinic acetylcholine receptor; 5HT3R, serotonin type 3 receptor; GABAAR, GABA type A receptor; GABACR, GABA type C receptor; Imax, maximum (saturating) current magnitude; WT, wild type. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Assaf, S. Y., and Chung, S. H. (1984) Nature 308, 734–736[Medline] [Order article via Infotrieve]
  2. Howell, G. A., Welch, M. G., and Frederickson, C. J. (1984) Nature 308, 736–738[Medline] [Order article via Infotrieve]
  3. Frederickson, C. J. (1989) Int. Rev. Neurobiol. 31, 145–238[Medline] [Order article via Infotrieve]
  4. Vogt, K., Mellor, J., Tong, G., and Nicoll, R. (2000) Neuron 26, 187–196[Medline] [Order article via Infotrieve]
  5. Smart, T. G., Xie, X., and Krishek, B. J. (1994) Prog. Neurobiol. 42, 393–441[CrossRef][Medline] [Order article via Infotrieve]
  6. Legendre, P. (2001) Cell. Mol. Life Sci. 58, 760–793[Medline] [Order article via Infotrieve]
  7. Laube, B., Kuhse, J., Rundstrom, N., Kirsch, J., Schmieden, V., and Betz, H. (1995) J. Physiol. (Lond.) 483, 613–619[Abstract]
  8. Birinyi, A., Parker, D., Antal, M., and Shupliakov, O. (2001) J. Comp. Neurol. 433, 208–221[CrossRef][Medline] [Order article via Infotrieve]
  9. Suwa, H., Saint-Amant, L., Triller, A., Drapeau, P., and Legendre, P. (2001) J. Neurophysiol. 85, 912–925[Abstract/Free Full Text]
  10. Davies, P. A., Wang, W., Hales, T. G., and Kirkness, E. F. (2003) J. Biol. Chem. 278, 712–717[Abstract/Free Full Text]
  11. Gisselmann, G., Pusch, H., Hovemann, B. T., and Hatt, H. (2002) Nat. Neurosci. 5, 11–12[CrossRef][Medline] [Order article via Infotrieve]
  12. Karlin, A. (2002) Nat. Rev. Neurosci. 3, 102–114[CrossRef][Medline] [Order article via Infotrieve]
  13. Langosch, D., Thomas, L., and Betz, H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7394–7398[Abstract]
  14. Lynch, J. W., Jacques, P., Pierce, K. D., and Schofield, P. R. (1998) J. Neurochem. 71, 2159–2168[Medline] [Order article via Infotrieve]
  15. Harvey, R. J., Thomas, P., James, C. H., Wilderspin, A., and Smart, T. G. (1999) J. Physiol. (Lond.) 520, 53–64[Abstract/Free Full Text]
  16. Laube, B., Kuhse, J., and Betz, H. (2000) J. Physiol. (Lond.) 522, 215–230[Abstract/Free Full Text]
  17. Auld, D. S. (2001) Biometals 14, 271–313[CrossRef][Medline] [Order article via Infotrieve]
  18. Lesburg, C. A., Huang, C., Christianson, D. W., and Fierke, C. A. (1997) Biochemistry 36, 15780–15791[CrossRef][Medline] [Order article via Infotrieve]
  19. Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K. (2001) Nature 411, 269–276[CrossRef][Medline] [Order article via Infotrieve]
  20. Cromer, B. A., Morton, C. J., and Parker, M. W. (2002) Trends Biochem. Sci. 27, 280–287[CrossRef][Medline] [Order article via Infotrieve]
  21. Laube, B., Maksay, G., Schemm, R., and Betz, H. (2002) Trends Pharmacol. Sci. 23, 519–527[CrossRef][Medline] [Order article via Infotrieve]
  22. Pribilla, I., Takagi, T., Langosch, D., Bormann, J., and Betz, H. (1992) EMBO J. 11, 4305–4311[Abstract]
  23. Handford, C. A., Lynch, J. W., Baker, E., Webb, G. C., Ford, J. H., Sutherland, G. R., and Schofield, P. R. (1996) Brain Res. Mol. Brain Res. 35, 211–219[CrossRef][Medline] [Order article via Infotrieve]
  24. Chothia, C., and Lesk, A. M. (1986) EMBO J. 5, 823–826[Abstract]
  25. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673–4680[Abstract]
  26. Rost, B., and Sander, C. (1993) J. Mol. Biol. 232, 584–599[CrossRef][Medline] [Order article via Infotrieve]
  27. Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714–2723[Medline] [Order article via Infotrieve]
  28. Luthy, R., Bowie, J. U., and Eisenberg, D. (1992) Nature 356, 83–85[CrossRef][Medline] [Order article via Infotrieve]
  29. Han, N. L., Haddrill, J. L., and Lynch, J. W. (2001) J. Neurochem. 79, 636–647[CrossRef][Medline] [Order article via Infotrieve]
  30. Shan, Q., Haddrill, J. L., and Lynch, J. W. (2001) J. Neurochem. 76, 1109–1120[CrossRef][Medline] [Order article via Infotrieve]
  31. Corringer, P. J., Le Novere, N., and Changeux, J. P. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 431–458[CrossRef][Medline] [Order article via Infotrieve]
  32. Grutter, T., and Changeux, J. P. (2001) Trends Biochem. Sci. 26, 459–463[CrossRef][Medline] [Order article via Infotrieve]
  33. Unwin, N., Miyazawa, A., and Fujiyoshi, Y. (2002) J. Mol. Biol. 319, 1165–1176[CrossRef][Medline] [Order article via Infotrieve]
  34. Draguhn, A., Verdorn, T. A., Ewert, M., Seeburg, P. H., and Sakmann, B. (1990) Neuron 5, 781–788[Medline] [Order article via Infotrieve]
  35. Calvo, D. J., Vazquez, A. E., and Miledi, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12725–12729[Abstract/Free Full Text]
  36. Gill, C. H., Peters, J. A., and Lambert, J. J. (1995) Br. J. Pharmacol. 114, 1211–1221[Abstract]
  37. Palma, E., Maggi, L., Miledi, R., and Eusebi, F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10246–10250[Abstract/Free Full Text]
  38. Fisher, J. L. (2002) Neuropharmacology 42, 922–928[CrossRef][Medline] [Order article via Infotrieve]
  39. Wooltorton, J. R., McDonald, B. J., Moss, S. J., and Smart, T. G. (1997) J. Physiol. (Lond.) 505, 633–640[Abstract]
  40. Horenstein, J., and Akabas, M. H. (1998) Mol. Pharmacol. 53, 870–877[Abstract/Free Full Text]
  41. Dunne, E. L., Hosie, A. M., Wooltorton, J. R., Duguid, I. C., Harvey, K., Moss, S. J., Harvey, R. J., and Smart, T. G. (2002) Br. J. Pharmacol. 137, 29–38[Abstract/Free Full Text]
  42. Wang, T. L., Hackam, A., Guggino, W. B., and Cutting, G. R. (1995) J. Neurosci. 15, 7684–7691[Abstract]
  43. Hosie, A. M., Dunne, E. L., Harvey, R. J., and Smart, T. G., (2003) Nat. Neurosci. 6, 362–369[CrossRef][Medline] [Order article via Infotrieve]
  44. Le Novere, N., Grutter, T., and Changeux, J. P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3210–3215[Abstract/Free Full Text]