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J. Biol. Chem., Vol. 280, Issue 45, 37877-37884, November 11, 2005

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Molecular Basis for Zinc Potentiation at Strychnine-sensitive Glycine Receptors^*

From the Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom

Received for publication, July 28, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
The divalent cation Zn²⁺ is a potent potentiator at the strychnine-sensitive glycine receptor (GlyR). This occurs at nanomolar concentrations, which are the predicted endogenous levels of extracellular neuronal Zn²⁺. Using structural modeling and functional mutagenesis, we have identified the molecular basis for the elusive Zn²⁺ potentiation site on GlyRs and account for the differential sensitivity of GlyR ₁ and GlyR ₂ to Zn²⁺ potentiation. In addition, juxtaposed to this Zn²⁺ site, which is located externally on the N-terminal domain of the subunit, another residue was identified in the nearby Cys loop, a region that is critical for receptor gating in all Cys loop ligand-gated ion channels. This residue acted as a key control element in the allosteric transduction pathway for Zn²⁺ potentiation, enabling either potentiation or overt inhibition of receptor activation depending upon the moiety resident at this location. Overall, we propose that Zn²⁺ binds to a site on the extracellular outer face of the GlyR subunit and exerts its positive allosteric effect via an interaction with the Cys loop to increase the efficacy of glycine receptor gating.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
The glycine receptor is a major component of inhibitory neurotransmission in the spinal cord and brainstem (1). It forms part of the Cys loop receptor family, which includes acetylcholine, -aminobutyric acid type A (GABA_A),³ and serotonin type 3 receptors (2). Glycine receptors are pentameric assemblies of ligand binding _(1–4) subunits and the homologous structural subunit (3). Each subunit has an extracellular N-terminal domain followed by four transmembrane (TM) segments connected by two intracellular regions and an extracellular TM2-TM3 linker, which is important for receptor gating (4). The function of these receptors can be enhanced by a variety of agents, including alcohols, anesthetics, neurosteroids, and Zn²⁺ (5–8), but their exact binding sites and transduction pathways remain controversial.

However, Zn²⁺ binding sites do provide realistic targets for identification, as these are traditionally compact, consisting of only 3–4 residues, and their coordination chemistry is understood (9). With regard to GlyRs, Zn²⁺ exhibits biphasic activity, potentiating receptor activation at submicromolar Zn²⁺ concentrations and causing inhibition at concentrations >10 µM (8). A previous study demonstrates that the mutation D80A in the extracellular domain of the GlyR ₁ subunit ablated Zn²⁺ potentiation, and it is proposed that this residue participates in the direct coordination of Zn²⁺ (10, 11). However, an additional report indicates that the Zn²⁺ potentiation of responses to the partial agonist taurine, which binds to the same agonist site as glycine, is unaffected by mutating Asp-80. This suggests that either multiple Zn²⁺ binding sites exist or that this mutation induces an indirect allosteric effect on receptor function that selectively disrupts Zn²⁺ potentiation of responses to glycine rather than those to taurine (12). In accord with an indirect allosteric effect, mutations of several other residues in the TM2-TM3 linker are capable of disrupting Zn²⁺ potentiation, though none of these residues are chemically suitable for the direct coordination of Zn²⁺.

The high sensitivity of the strychnine-sensitive GlyR to Zn²⁺ potentiation makes this receptor an ideal substrate for modulation by basal levels of Zn²⁺. In a physiological context, Zn²⁺ is released following neuronal stimulation (13, 14) and can also modulate inhibitory neurotransmitter receptors at basal concentrations (15, 16). Furthermore, Zn²⁺ is concentrated into synaptic boutons that also contain either glutamate, GABA, or glycine in many areas of the brain, including the cortex, hippocampus, and spinal cord (17–19). Although the predicted concentration of Zn²⁺ resulting from presynaptic release is probably <10 µM (20), this is more than sufficient to modulate N-methyl-D-aspartate receptors, certain GABA_A receptor subtypes, and GlyRs (8, 21). Indeed, low nanomolar basal Zn²⁺ concentrations are adequate to prolong the decay phase of glycinergic inhibitory postsynaptic currents (22).

In this study, we accounted for the differential sensitivity to Zn²⁺ potentiation of GlyR ₁ and GlyR ₂ by identifying the location of a single conserved residue in the N-terminal domain. Subsequently, by using structural homology modeling together with the identified residue underlying Zn²⁺ sensitivity, we established the molecular determinants for the elusive Zn²⁺ potentiation binding site. In doing so, we uncovered a prospective transduction residue for this site that is located in the Cys loop-gating domain, providing a plausible molecular pathway for Zn²⁺ potentiation of glycine receptor gating.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
cDNA Constructs—Human (h) wild-type cDNA constructs were used for hGlyR _1L, hGlyR _2A, and hGlyR . Site-specific mutant cDNAs were prepared using the Stratagene Quikchange mutagenesis kit. The mutated sequences were confirmed by complete sequencing of the cDNA insert using an ABI sequencer.

Cell Culture and Transfection—Human embryonic kidney (HEK) cells (American Type Culture Collection CRL1573) were grown and transfected as previously documented (23). Plasmids of hGlyR cDNA clones were co-transfected in a ratio of 1:1 with enhanced green fluorescent protein (24). To co-express GlyR heteromers, subunit cDNA was added in excess at a ratio of 20:1 to subunit cDNA. HEK cells were plated onto poly-L-lysine-coated coverslips (100 µg/ml) sufficient to achieve 20% confluence and used for recording on the next day.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Zn2+ potentiation of glycine-activated currents for GlyR 1 and GlyR 2. Zn2+ concentration response curves were determined for the modulation of EC50 responses to glycine recorded in the presence of 10 mM tricine from HEK cells. A, typical glycine (EC50)-activated currents in the presence and absence of 30 µM Zn2+ for GlyR 1, 2, and 2E201D. Zn2+ concentration curves for homomeric GlyR 1, 2, or 2E201D (B) or heteromeric GlyR 1 or 2 (C) (n = 4–6). D, amino acid sequence alignment of GlyR 1 and GlyR 2 showing the region of the N-terminal extracellular domain that contains differences in potential Zn2+ binding residues (bold). A gray filled box highlights the residue responsible for the differential sensitivity to Zn2+-mediated potentiation between GlyR 1 and 2. All numbering is for the mature protein.

Electrophysiology—An Axopatch 200B amplifier (Axon Instruments) recorded whole-cell currents from single HEK cells using the patch clamp technique. HEK cells exhibited resting potentials between -10 and -40 mV and were voltage-clamped at a -40 mV holding potential. The cells were visualized with differential interference contrast optics using a Nikon Optiphot microscope with an epifluorescence attachment to identify green fluorescent protein-transfected cells. A Y-tube was used to rapidly apply drugs and Krebs solutions (exchange rate 50–100 ms) to the recorded cells. Patch electrodes were fabricated using a Narashige PC-10 puller with resistances, after polishing, of 4–5 megohms. All recordings were performed in constantly perfusing Krebs-Ringer solution at room temperature (20–22 °C).

Data Acquisition and Analysis—Recorded currents were filtered using a high pass Bessel filter at 3 kHz (-36 db/octave), and series resistance compensation was achieved up to 70%. Data were recorded in 20-s acquisition epochs directly to a Pentium IV, 1.8 GHz computer into Clampex software, version 8.0, via a Digidata 1322A (Axon Instruments) sampling at 200-µs intervals. Zn²⁺ was co-applied with the agonist to attenuate any delayed onset of Zn²⁺-mediated inhibition. Strychnine and picrotoxin were pre-incubated for 15 s, sufficient to attain equilibrium, and then also co-applied with the agonist. The digitized membrane current records were analyzed off-line using Axoscope, version 8.2. Biphasic (potentiation and inhibition) Zn²⁺ concentration response curves were fitted according to a modified Hill equation as previously described (26). Where a single component to the concentration response relationships was evident, it was fitted with a form of the Hill equation. For the agonist and Zn²⁺ concentration potentiation curves, I = I_min + (I_max - I_min)([1/(1 + (EC₅₀/A)^nH)]), and for the strychnine and Zn²⁺ concentration-inhibition curves, I/I_max = 1 - [1/(1 + (IC₅₀/B)^mH)].

The EC₅₀ represents either the concentration of agonist-inducing or Zn²⁺-potentiating (A) 50% of the maximal current (I_max) evoked or potentiated by a saturating concentration of agonist or Zn²⁺, and n is the Hill coefficient. For Zn²⁺ potentiation, I_min represents the control glycine current in the absence of Zn²⁺ and was set to 100%. For inhibition, the IC₅₀ defines the antagonist concentration (B) producing a 50% inhibition of the current, and m_H represents the Hill coefficient. When Zn²⁺ induced a biphasic inhibition, the concentration response data were fitted with a two-component inhibitory curve using I/I_max = 1 - (aB^mH/(B^mH + IC₅₀^mH)) + (bB^mH/(B^mH + IC₅₀^mH)), where a and b represent the relative proportions of each inhibitory component. All statistical comparisons used an unpaired t test.

Structural Homology Modeling—The mature N-terminal extracellular domain of the hGlyR₁ subunit was modeled on the crystal structure of the acetylcholine-binding protein (27) using SwissProt DeepView, version 3.7, in accordance with a ClustalW protein alignment. All three-dimensional images were subsequently rendered using the freeware program POV-Ray.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
GlyR ₁ and ₂ Exhibit Distinct Sensitivities to Zn²⁺ Potentiation— The sensitivity of the GlyR ₁ and ₂ subtypes to Zn²⁺ potentiation was assessed from whole-cell recordings of half-maximal (EC₅₀) responses to glycine obtained from transfected HEK cells maintained at -40 mV holding potential (Fig. 1A). To accurately compare the modulation of GlyR ₁ and GlyR ₂ at submicromolar concentrations of Zn²⁺, the buffer tricine (25) was used to remove Zn²⁺ contamination in the external Krebs solution (22). The ₁ subtype, considered to be an adult form of the GlyR and highly expressed throughout the spinal cord and brainstem (1), exhibited a very high sensitivity to Zn²⁺ potentiation with an EC₅₀ of only 37 ± 10 nM (n = 5) (Fig. 1B). This sensitivity is comparable with the high affinity inhibitory Zn²⁺ site found on the N-methyl-D-aspartate receptor NR2A subunit (25). By comparison, the principal embryonic subtype GlyR 2 (1) also displayed a high nanomolar sensitivity to Zn²⁺ potentiation with an EC₅₀ of 540 ± 180 nM (n = 5) but was nevertheless 15-fold less sensitive than the GlyR ₁ isoform (p < 0.05). This difference in sensitivity was unaffected by co-expression with the GlyR ancillary subunit (p < 0.05) (Fig. 1C), which assemble to form heteromeric receptors, as confirmed by an approximate 20-fold shift in the sensitivity to picrotoxin (data not shown) (28). The Hill slopes for Zn²⁺-mediated potentiation varied between 1.2 and 1.5, suggesting more than one Zn²⁺ ion was probably coordinated by each receptor. To identify the structural determinant(s) responsible for differential Zn²⁺ sensitivity, the extracellular domains were scanned for the classical Zn²⁺ binding residues, Cys, Asp, Glu, and His (9), focusing particularly on differences between GlyR ₁ and ₂. On this basis, four residues were prioritized (Fig. 1D). Upon individual substitution of the GlyR ₁ variants into GlyR ₂, no influence on Zn²⁺ sensitivity was observed for S179E, D180Q, or E187D mutated receptors (data not shown). However, a single conservative E201D substitution was found to be sufficient and necessary to enable GlyR ₂ to exhibit a similar sensitivity to GlyR ₁ toward potentiation by Zn²⁺ (Fig. 1, B and D).

A Cluster of GlyR ₁ Residues Are Essential for Zn²⁺ Potentiation— The discovery that GlyR ₁ Asp-194 (which corresponds to GlyR ₂ Glu-201) is capable of influencing the sensitivity to potentiating Zn²⁺, suggested that this moiety might be a direct contributor to Zn²⁺ binding, forming part of the potentiation site. This hypothesis was examined by mutating ₁ Asp-194 to alanine, a residue incapable of coordinating Zn²⁺. However, because of the biphasic nature of Zn²⁺ action at the GlyR, where Zn²⁺ potentiates at nanomolar to low micromolar concentrations and inhibits at doses >10 µM (8), any attempt to measure a reduced sensitivity to Zn²⁺ potentiation might be occluded by the onset of Zn²⁺ mediated inhibition. To obviate this problem, all experiments to identify the Zn²⁺ potentiation binding site were performed using an H107N "background" mutation (hereafter referred to as "reduced inhibition" (RI)). This mutation dramatically attenuated the GlyR sensitivity to Zn²⁺ inhibition, increasing the Zn²⁺ IC₅₀ from 15 µM to >3 mM without affecting other macroscopic properties of the receptor, particularly the sensitivity of the receptor to Zn²⁺ potentiation (see below next paragraph and TABLE ONE).

To assess the significance of prospective binding site residues for Zn²⁺ potentiation, the consequences of their replacement were compared for two GlyR agonists, glycine and taurine. This is necessary, as previously identified residues, which have been postulated to be part of a potentiating Zn²⁺ binding site, ablated enhancement of responses to one agonist but not the other presumably due to indirect affects on downstream transduction mechanisms (12). This emphasized the potential importance of Asp-194, because the Zn²⁺ potentiation of both glycine- and taurine-activated (EC₅₀) responses was ablated in the RI₁D194A receptor (Fig. 2, A and B). As expected, removing Zn²⁺ potentiation led to an apparent increase in sensitivity to Zn²⁺ inhibition, with the IC₅₀ shifting from >3000 to 270 ± 50 µM (n = 3). This is probably a consequence of Zn²⁺ potentiation and inhibition having overlapping concentration ranges; therefore, by removing potentiation, the inhibitory component appears to have a lower threshold. We cannot discount the possibility that there is some allosteric interaction between the two Zn²⁺ sites, but this would seem unlikely, as disruption of the inhibitory site does not affect Zn²⁺ potentiation (29). Furthermore, although not a guarantee of independence, Asp-194 of the putative potentiation site is predicted to reside on the external face of the GlyR N terminus (Fig. 2C), far away from the inhibitory Zn²⁺ site located on the opposite face of the subunit (30).

View larger version (49K): [in this window] [in a new window]   FIGURE 2. GlyR 1 subunit residues that affect Zn2+ potentiation. Zn2+ concentration response curves for the modulation of EC50 responses to glycine (A) and taurine (B) constructed for mutant homomeric GlyRs RI1, RI1E192A, RI1D194A, and RI1H215A and one heteromeric GlyR, RI1E192A. All experiments were performed on a background mutant receptor, GlyR 1H107N, that exhibited a reduced inhibition (RI)toZn2+. Note the absence of any Zn2+ potentiation for most of the mutant receptors, apart from RI1 and RI1H215A. The insets show typical glycine and taurine (EC50)-activated currents in the absence (continuous line) and presence of 10 µMZn2+ (dotted line). C, color-coded amino acid motifs identified in the N-terminal domain of the GlyR 1 subunit and homologous GlyR and subunits that reside in close proximity to the previously identified 1D194A. The single extracellular domain in the GlyR structural model illustrates the side chains of three putative Zn2+ binding residues. The -helical section at the start of the mature protein is shown in pink. The inset is a plan view of the GlyR pentamer (red circles represent the extracellular subunit N-terminal domains), and the arrow denotes the viewing angle. The protein alignments show potential Zn2+ binding residues in bold in each motif, whereas important residues for Zn2+ potentiation are on color-coded backgrounds. A divergent residue in the Cys loop is also highlighted in gray.

Asymmetry of Function at the Putative Zn²⁺ Potentiation Binding Site— Demonstrating that this discrete domain is accessible to water is a vital requirement for any dynamic Zn²⁺ binding site and would strengthen the conclusion that the identified residues may act as direct coordinators of Zn²⁺. We determined this by individual cysteine substitutions of GlyR ₁ Glu-192, Asp-194, and His-215, which were then exposed to the cysteine-modifying reagent MTSEA. If MTSEA covalently binds to the potentiation site, it will replace a Zn²⁺-coordinating Cys moiety with a positively charged amine group, which should attenuate the Zn²⁺ potentiation of glycine-activated currents. Pre-application of 3 mM MTSEA for 1 min to the control GlyR RI₁ did not affect Zn²⁺ potentiation (Fig. 3A) or the glycine EC₅₀ and glycine maximal responses (data not shown). Upon individual replacement of Glu-192, Asp-194, and His-215 with cysteine, GlyRs were generated that retained high sensitivities to Zn²⁺ potentiation, with EC₅₀ values for Zn²⁺ within 10-fold of the wild-type receptor (n = 4) (Fig. 3, B–D). This was not surprising, as cysteine is quite capable of coordinating Zn²⁺. However, following exposure to MTSEA, RI₁D194C and RI₁H215C were rendered unresponsive to Zn²⁺ potentiation, suggesting that the side chains of both of these residues are surface-exposed and important for Zn²⁺ potentiation. The sensitivity of RI₁E192C, however, was largely unaffected (n = 4) (Fig. 3, B–D). The binding of MTSEA alone was insufficient to induce potentiation of glycine-activated responses at RI₁D194C or RI₁H215C (Fig. 3, B and C). Surprisingly, MTSEA did increase glycine potency in the absence of Zn²⁺ for RI₁E192C, potentiating the glycine response by 61 ± 16% (n = 4, p < 0.05) (Fig. 3E). Thus, Glu-192 appears to be accessible to MTSEA, although this covalent modification did not affect the ability of Zn²⁺ to bind to the receptor. If the carboxyl side chain of Glu-192 was not directly coordinating Zn²⁺, then perhaps substitution of this residue with alanine would perturb the -strand backbone, which then would either indirectly disrupt the Zn²⁺ potentiation site or disrupt a possible contribution of the polar peptide backbone at this locus for Zn²⁺ coordination. To determine the relevance of the backbone at Glu-192, this residue was mutated to proline (RI₁E192P), an amino acid associated with placing conformational restraints upon peptide backbones (31). Even though proline cannot coordinate Zn²⁺, potentiation in this mutated receptor was retained, although at a 5-fold reduced sensitivity (0.8 ± 0.2 µM for RI₁ and 4.2 ± 0.8 µM for RI₁E192P (n = 4, p < 0.05) (Fig. 3F). This suggested that, to retain Zn²⁺-mediated potentiation, this region must retain a specific conformation of the backbone at Glu-192, which can be accommodated by the introduction of a proline but not by insertion of an alanine. Of course, without precise structural data, it is not possible to infer any specific details about the structural organization at this locus other than that the backbone is particularly sensitive to perturbation in a fashion that affects Zn²⁺ potentiation. As a control for this strategy, the mutant GlyR RI₁D194P ablated Zn²⁺-mediated potentiation, an outcome that is expected if, indeed, the side chain moiety is contributing to Zn²⁺ coordination at this particular position (data not shown).

View larger version (38K): [in this window] [in a new window]   FIGURE 3. Covalent modification by MTSEA affects Zn2+ potentiation of Gly 1 receptors. Shown are Zn2+ concentration response curves for the modulation of EC50 glycine responses measured before and after exposure to 3 mM MTSEA for 1 min for the receptors RI1 (A) and for those receptors mutated to incorporate cysteines at putative Zn2+ binding residues RI1D194C (B), RI1H215C (C), and RI1E192C (D)(n = 4). The insets present example glycine (EC50)-activated currents in the absence (solid line) and presence (broken line)of10 µM Zn2+ before and after 3 mM MTSEA. E, glycine (EC50) current amplitudes for RI1 and RI1E192C before and after 3 mM MTSEA. The inset depicts typical glycine-activated currents for the same concentration of glycine before and after exposure to MTSEA. F, Zn2+ concentration response curves for the modulation of EC50 glycine responses on RI1 and RI1E192P to explore whether inducing specific conformational restraints on the peptide backbone at this location affects Zn2+ potentiation (n = 4). * denotes significance at p < 0.05

View larger version (10K): [in this window] [in a new window]   FIGURE 4. Ablation of Zn2+ inhibition and potentiation in GlyR 1. Zn2+ concentration response curves for the modulation of EC50 glycine responses on the wild-type GlyR 1 and the triple mutant receptor RI1H109F,D194K. This mutant lacks two vital elements of the GlyR inhibitory Zn2+ site (His-107 replaced with Asn, denoted as reduced inhibition (RI), and His-109). Note that the mutation of Asp-194 is sufficient to remove enhancement up to 100 µM Zn2+, demonstrating its crucial requirement for Zn2+ potentiation.

View this table: [in this window] [in a new window]   TABLE TWO Dose response data for glycine and taurine activation and Zn2+ modulation on Thr151 mutated 1 GlyRs Data represent the mean ± S.E. for n number of experiments. ND, not determined.

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Role of Thr-151 in signal transduction from the Zn2+ potentiation site. Zn2+ concentration response curves for the modulation of glycine (A)- and taurine (B)-activated currents (at their EC50 values) for mutant GlyR 1 homomers. Note that potentiation is abolished by mutation of Thr-151 and replaced with a novel biphasic inhibition. The sensitivity of the high potency component is dependent on Glu-192, a residue originally involved in Zn2+ potentiation, whereas the low potency component is dependent on His-107, a residue involved in Zn2+ binding to the discrete inhibitory Zn2+ site. The insets show typical EC50 glycine and taurine currents in the absence (continuous line) and presence (dotted line) of 10 µM Zn2+. C, sequential mutation of Thr-151 affects the contribution of the high potency component to the Zn2+ concentration inhibition curve, denoted by the broken lines (n = 4–6). D, a single GlyR 1 subunit N-terminal extracellular domain, highlighting Thr-151 (yellow) within the putative Cys loop-gating domain (gray) (other colored motifs and residues are the same as in Fig. 2), and its proximity to another important determinant in gating, the TM2-TM3 linker region (maroon), which rests above the transmembrane helices that form the receptor ion channel (shaded blue). The inset depicts the viewing angle (blue arrow) of the GlyR pentamer.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Substitution at T151 disrupts agonist sensitivity of the GlyR. Glycine (A), and taurine (B) concentration response curves for a series of 1 Thr-151 mutants (n = 4–11). The glycine and taurine curves are normalized in each cell to the maximum response to glycine (2 mM). Note the decrease in the relative efficacy for taurine evident from the reduced maximal responses. C, strychnine concentration inhibition curves were determined for the antagonism of EC50 glycine-activated currents for the same series of receptors. The lack of deviation between the curves suggests the agonist binding region is not distorted by the mutations, supporting the hypothesis that disruption impinges on receptor gating not ligand binding. D and E, display of representative maximal glycine- and taurine-activated currents, respectively, recorded from HEK cells expressing the Thr-151 mutants.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

In addition to those residues thought to line the Zn²⁺ binding site, Thr-151 was also identified as an important transduction component for this site because of its nearby location. The exact nature of the residue introduced at position 151 did not alter the potency of Zn²⁺, precluding an involvement in binding, but it was able to determine the "direction of output" from the Zn²⁺ site to be either potentiating (for threonine) or inhibitory (for alanine, arginine, and asparagine). Furthermore, the type of residue at position 151 also controlled the efficacy of inhibition from the Zn²⁺ site. As this site is quite distinct in its structure and location, Thr-151 is most unlikely to be associated with the previously reported Zn²⁺ inhibitory site on GlyR ₁, which resides on the other side of the subunit (10, 26, 30). In accordance with the location of Thr-151 being in the critical Cys loop-gating domain (34–36), this residue was also shown to be an important determinant of agonist potency for receptor activation. Thus, from a molecular perspective, it provides a potential connection between the Zn²⁺ potentiation binding site and the Cys loop-gating domain to possibly increase the efficacy of agonist-induced channel opening. In accordance with Zn²⁺ potentiation being mediated via an increase in agonist efficacy, the partial agonist taurine is converted to a full agonist by Zn²⁺ concentrations that cause potentiation at GlyR ₁ expressed in oocytes (10). Furthermore, residues located in the TM2-TM3 linker, which are predicted to be closely apposed to, and capable of interacting with, the Cys loop-gating domain (27, 37), also affect Zn²⁺-mediated potentiation (12), as would be expected if there is direct communication between these two domains.

The reversal of signal output, exemplified here by changing Zn²⁺ potentiation to inhibition following the mutation of Thr-151, is not a unique observation for ligand-gated ion channels. Mutation of isoleucine 307 in the TM2 domain of the GABA_C receptor to glutamine reverses the inhibition caused by the neurosteroid 5-pregnane-3-ol-20-one on the wild-type receptor to potentiation (38). This suggests that whether a modulator exerts a positive or negative effect on receptor activation may be highly dependent on the nature of the allosteric transduction pathway leading away from the binding site.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
We report here the identification of the Zn²⁺ potentiation binding site on the GlyR and a possible molecular route of action for Zn²⁺ potentiation via the Cys loop channel-gating domain. The Zn²⁺ potentiation site is a novel site on the GlyR clearly distinct from the previously identified interfacial inhibitory Zn²⁺ binding sites for GlyRs (30) and GABA_A receptors (39), which reside on the inside faces of these pentamers. The potentiation site has a high sensitivity to Zn²⁺, with the EC₅₀ estimated at <100 nM, well within the range of current estimates for physiological levels of basal and released Zn²⁺ (40). The location of this site away from the intrasubunit interfaces of the GlyR pentamer may have implications for the manner in which Zn²⁺ achieves its effect, because when Zn²⁺ is bound at the potentiation site, it is predicted to interact with the gating apparatus of the receptor. In contrast, when Zn²⁺ is bound at the interfacial inhibitory site, it acts in an apparent competitive manner, stabilizing the closed agonist-unbound state of the receptor (1).

	FOOTNOTES

 
^* This work was supported by the Medical Research Council and the Wellcome Trust. 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.

¹ A Wellcome Trust four year Ph.D. postgraduate student.

² To whom correspondence should be addressed: Dept. of Pharmacology, University College London, Gower St., London, WC1E 6BT, United Kingdom. Tel.: 0207-679-2013; Fax: 0207-679-7298; E-mail: t.smart{at}ucl.ac.uk.

³ The abbreviations used are: GABA_A, -aminobutyric acid type A; GABA_A/CR, -aminobutyric acid receptor type A/C; GlyR, glycine receptor; HEK, human embryonic kidney; MTSEA, 2-aminoethyl-methanesulphonate; RI, reduced inhibition; TM, transmembrane.

	ACKNOWLEDGMENTS

 
We thank Drs. Alastair Hosie and Philip Thomas for their helpful advice and comments on the manuscript and Dr. Robert Harvey for the wild-type cDNA construct.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 

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