The GLRA1 Missense Mutation W170S Associates Lack of Zn²⁺ Potentiation with Human Hyperekplexia

Abstract

Hyperekplexia is a neurological disorder associated primarily with mutations in the α1 subunit of glycine receptors (GlyRs) that lead to dysfunction of glycinergic inhibitory transmission. To date, most of the identified mutations result in disruption of surface expression or altered channel properties of α1-containing GlyRs. Little evidence has emerged to support an involvement of allosteric GlyR modulation in human hyperekplexia. Here, we report that recombinant human GlyRs containing α1 or α1β subunits with a missense mutation in the α1 subunit (W170S), previously identified from familial hyperekplexia, caused remarkably reduced potentiation and enhanced inhibition by Zn²⁺. Interestingly, mutant α1^W170Sβ GlyRs displayed no significant changes in potency or maximum response to glycine, taurine, or β-alanine. By temporally separating the potentiating and the inhibitory effects of Zn²⁺, we found that the enhancement of Zn²⁺ inhibition resulted from a loss of Zn²⁺-mediated potentiation. The W170S mutation on the background of H107N, which was previously reported to selectively disrupt Zn²⁺ inhibition, showed remarkable attenuation of Zn²⁺-mediated potentiation and thus indicated that W170 is an important residue for the Zn²⁺-mediated GlyR potentiation. Moreover, overexpressing the α1^W170S subunit in cultured rat neurons confirmed the results from heterologous expression. Together, our results reveal a new zinc potentiation site on α1 GlyRs and a strong link between Zn²⁺ modulation and human disease.

Introduction

Hyperekplexia is an inherited neuronal disorder characterized by hypertonia and exaggerated startle reflex to unexpected sensory stimuli (Bakker et al., 2006). Hyperekplexia is caused by dysfunction of glycinergic transmission that can result from diverse root causes that include loss of function of glycine receptors (GlyRs), reduced receptor clustering of GlyRs at synapses, or reduced glycine release from presynaptic terminals (Harvey et al., 2008). Most cases of identified familial hyperekplexia are associated with dominant or recessive missense mutations of the glra1 gene, which encodes the α1 subunit of GlyRs (Lynch, 2004). These mutations commonly lead to severe impairments of α1 GlyR-mediated synaptic inhibition either by reducing surface expression of synaptic GlyRs or by disrupting basic channel properties, such as agonist binding affinity, channel gating, and channel conductance (Chung et al., 2010). Complete loss of function mutations of α1 subunit has also been reported, causing hyperekplexia symptoms that are clinically indistinguishable from patients carrying missense glra1 mutations (Brune et al., 1996). However, in animal models, deletion of functional α1 subunits caused more severe symptoms and juvenile death in mutant mice, suggesting that deficits in human α1 subunit might be more efficiently compensated (Buckwalter et al., 1994).

GlyRs are activated by multiple endogenous agonists, including glycine, β-alanine, and taurine. In addition, GlyRs also exhibit significant allosteric modulation by various effectors, such as Zn²⁺, endocannabinoids, steroids, and alcohols (Lynch, 2004). Among these allosteric modulators, Zn²⁺ is enriched in presynaptic vesicles in many regions of the CNS and acts as a potent allosteric modulator of inhibitory GlyRs (Sensi et al., 2011). Low concentrations (<10 μm) of Zn²⁺ potentiate GlyR currents, whereas high concentrations (>10 μm) inhibit GlyR-mediated responses (Bloomenthal et al., 1994; Laube et al., 1995). Before this study, there has been no evidence from reported human mutations that hyperekplexia is caused by impaired allosteric modulation without affecting basic receptor properties of GlyRs. Although selectively removing sensitivity to Zn²⁺-mediated potentiation produced exaggerated startle reactions in knockin mice (Hirzel et al., 2006), the different phenotype severities between human and murine glra1 null mutations make it difficult to predict whether simply disrupting Zn²⁺-mediated modulation of GlyRs also causes hyperekplexia in humans.

In this study, we report that the missense mutation W170S in the GlyR α1 subunit, which was recently identified from Omani families with hyperekplexia and mild mental retardation (Al-Futaisi et al., 2012), caused almost complete loss of Zn²⁺-mediated potentiation and enhancement of Zn²⁺-mediated inhibition. Unlike previously reported missense α1 mutations from human cases of hyperekplexia, α1^W170S-containing GlyRs displayed no significant alterations in agonist sensitivities, maximal current responses, or current–voltage (I-V) relations. The major alteration of α1^W170S was the ablated sensitivity to Zn²⁺-mediated potentiation, which was determined in both a recombinant expression system and in cultured neurons. Collectively, our findings demonstrate that impaired allosteric modulation of α1 GlyRs by Zn²⁺ may directly lead to human hyperekplexia.

Materials and Methods

cDNA constructs and transfection.

Wild-type (WT) human GlyR α1 (hGlra1) and the human GlyR β subunit (hGlrb) were subcloned into the pBK-CMV NB-200 expression vector (Liu et al., 2010). α1^W170S, α1^H107N, and α1^W170S/H107N plasmids were constructed by site-directed mutagenesis of hGlra1. The sequences of all the plasmids were confirmed by automated DNA sequencing. Purified plasmids encoding the WT or mutant GlyR α1 alone or with β subunits (1:10; total plasmid amount 3–4.5 μg) were transfected into HEK293T cells by electroporation (NEPA21, NEPA GENE). The α1 and α1β compositions of GlyRs were confirmed by their shifted sensitivity to picrotoxin using whole-cell patch-clamp recordings. A small amount (∼0.5 μg) of pcDNA3-GFP was cotransfected along with GlyR subunits to act as a transfection marker and facilitate the visualization of transfected cells during electrophysiological experiments. Cells were replated on glass coverslips after transfection and cultured for an additional 15–24 h before patch-clamp recordings.

Neuronal culture and overexpression.

Cultured cortical neurons were prepared from the neocortex of day 18 fetal rats (from embryos of either sex) as described previously (Liu et al., 2010) and were transiently transfected with α1 subunits (1 μg) and GFP (0.3 μg) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. Electrophysiological recordings were performed 3–7 d after plating.

Whole-cell patch-clamp recordings.

Whole-cell recordings were performed under voltage-clamp mode using an Axopatch 200B (Molecular Devices). Whole-cell currents were recorded at a holding potential of −60 mV, and signals were low-pass filtered at 2 kHz and digitized at 10 kHz (Digidata, 1440A). Recording pipettes (3–5 MΩ) were filled with intracellular solution that contained the following (in mm): 140 CsCl, 10 HEPES, 4 Mg-ATP, and 0.5 BAPTA (pH 7.20, osmolarity, 290–295 mOsm). The coverslips were continuously superfused with the extracellular solution containing the following (in mm): 140 NaCl, 5.4 KCl, 10 HEPES, 1.0 MgCl₂, 1.3 CaCl₂, and 20 glucose (pH 7.4, 305–315 mOsm). To evoke glycine currents, we used fast perfusion of glycine and other agonists with a computer-controlled multibarrel fast perfusion system (Warner Instruments). Maximum currents (I_max) were evoked by the highest concentration of the agonist as determined by the dose–response curves. Zn²⁺ was applied both in the bath and together with the agonist-containing solutions. All experiments were performed at 23–25°C.

Homology modeling.

The mature hGlyR α1 subunit was modeled on the crystal structure of the glutamate-gated chloride channel α (GluCl) (Hibbs and Gouaux, 2011) using I-TASSER server (Zhang, 2008; Roy et al., 2010, 2012). All 3D images were subsequently rendered using the UCSF Chimera package (Pettersen et al., 2004).

Data analysis.

Values are expressed as mean ± SEM. One-way ANOVA or a two-tailed Student's t test was used for statistical analysis, and p values <0.05 were considered to be statistically significant. Dose–response curves were created by fitting data to the Hill equation: I = I_max/[1 + (EC₅₀/[A])^nH], where I is the current, I_max is the maximum current, [A] is a given concentration of agonist, and n_H is the Hill coefficient.

Results

The startle disease mutation W170S did not alter heteromeric GlyR agonist sensitivities or channel permeability

Glycine receptor-mediated currents were tested by whole-cell voltage-clamp recordings in HEK293T cells expressing WT or mutant GlyRs. Because the extracellular N terminus of α1 contains the GlyR agonist binding site and previously reported missense mutations at this region typically exhibit a feature of impaired sensitivity to agonist activation (Chung et al., 2010), we first examined whether agonist binding affinities were altered in α1^W170S GlyRs. The dose–response curve of glycine-activated, homomeric α1^W170S GlyRs currents was shifted toward the right compared with homomeric α1^WT GlyRs, with the EC₅₀ increased from 62.9 ± 5.0 μm to 127.6 ± 6.9 μm (n_H = 3.38 ± 0.49 and 1.69 ± 0.14; n = 5 and n = 10, respectively; p < 0.05; Fig. 1A). The agonist sensitivity of α1^W170S was also examined with the other two endogenous GlyR partial agonists, β-alanine and taurine. Homomeric α1^W170S showed increased sensitivity to activation by β-alanine (EC₅₀ = 226.9 ± 14.3 μm and 99.4 ± 8.0 μm, n_H = 2.40 ± 0.03 and 1.56 ± 0.03, n = 9 and n = 9 in α1^WT and α1^W170S, respectively; p < 0.01; Fig. 1B) and no change in sensitivity to activation by taurine (EC₅₀ = 256.6 ± 29.9 and 213.7 ± 19.1 μm, n_H = 1.55 ± 0.05 and 1.57 ± 0.04, n = 8 and n = 8, respectively; p > 0.05; Fig. 1C). As the majority of α1-containing GlyRs in the adult CNS consist of heteromeric α1β GlyRs that may exhibit different current kinetics than homomeric receptors (Lynch, 2004), we also investigated the potential for altered agonist sensitivities in heteromeric α1^W170Sβ GlyRs. Interestingly, the α1^W170Sβ receptors did not show significant impairment of the sensitivity to glycine (EC₅₀ = 116.4 ± 5.1 and 80.0 ± 5.1 μm, n_H = 2.45 ± 0.02 and 1.96 ± 0.03, n = 5 and n = 6 in α1^WTβ and α1^W170Sβ, respectively; p > 0.05; Fig. 1D), β-alanine (EC₅₀ = 91.0 ± 7.6 and 95.5 ± 8.0 μm, n_H = 1.55 ± 0.03 and 1.83 ± 0.04, n = 9 and n = 8, respectively; p > 0.05; Fig. 1E), or taurine (EC₅₀ = 371.1 ± 25.6 and 194.4 ± 18.9 μm, n_H = 1.60 ± 0.03 and 1.45 ± 0.04, n = 8 and n = 8, respectively; p < 0.05; Fig. 1F). These data suggest that the hyperekplexia phenotype observed in humans carrying W170S alleles is not likely to be mediated by impaired agonist responsiveness of mutant GlyRs.

Figure 1.

The W170S mutation in α1β GlyRs showed no significant changes in agonist sensitivities, maximal current responses, or I-V relationship. A–C, Dose–response curves for α1^WT and α1^W170S homomeric GlyRs normalized to maximal currents induced by glycine (A), β-alanine (B), and taurine (C). D–F, Dose–response curves for α1^WTβ and α1^W170Sβ heteromeric GlyRs normalized to maximal currents induced by glycine (D), β-alanine (E), and taurine (F). G, Maximal responses (I_max) of glycine-, β-alanine-, and taurine-induced currents in α1^WTβ and α1^W170Sβ GlyRs. H, The β-alanine- and taurine-mediated I_max were normalized to glycine-mediated I_max from the same cell. I, Representative traces of 100 μm glycine-induced currents at different holding potentials from −60 mV to 60 mV. J, Peak I-V relations (normalized to the current recorded at −60 mV) of α1^WTβ and α1^W170Sβ GlyRs. n.s., Not significant.

Missense mutations in other regions of α1 have been previously characterized that result in only moderate changes in GlyR agonist affinity yet also a remarkable reduction of maximal whole-cell currents (Saul et al., 1999). We found that, in α1^W170Sβ receptors, the maximal currents (I_max) evoked by saturating agonist concentrations, including 3 mm glycine (α1^WTβ, I_max = 9.4 ± 1.2 nA, n = 7; α1^W170Sβ, I_max = 7.5 ± 0.9 nA, n = 11; p > 0.05), β-alanine (α1^WTβ, I_max = 8.4 ± 1.2 nA, n = 7; α1^W170Sβ, I_max = 7.3 ± 1.0 nA, n = 11; p > 0.05), or 5 mm taurine (α1^WTβ, I_max = 7.3 ± 1.4 nA, n = 7; α1^W170Sβ, I_max = 6.6 ± 0.9 nA, n = 11; p > 0.05) were not significantly changed compared with the α1^WTβ receptors (Fig. 1G). The I_max induced by β-alanine (α1^WTβ, I_ala/I_gly = 90.7 ± 8.6%, n = 7; α1^W170Sβ, I_ala/I_gly = 95.7 ± 4.5%, n = 11; p > 0.05) or taurine (α1^WTβ, I_tau/I_gly = 90.8 ± 17.5%, n = 7; α1^W170Sβ, I_tau/I_gly = 85.8 ± 5.2%, n = 11; p > 0.05) as a percentage of the glycine-induced I_max from the same cell was also indistinguishable between α1^WTβ and α1^W170Sβ GlyRs (Fig. 1H). In addition, the I-V relationship of 100 μm glycine-induced responses in α1^W170Sβ receptors showed no difference compared with α1^WTβ receptors, indicating that there were no changes in Cl⁻ ion permeability (Fig. 1I,J). Together, these results suggest that the basic channel properties of recombinant α1^W170Sβ GlyRs were not significantly different from that of α1^WTβ GlyRs.

Impaired Zn²⁺-mediated modulation in α1^W170S-containing GlyRs

To investigate how the W170S mutation affects glycine α1 receptors, we generated a homology model of α1 GlyR based upon the template of the crystal structure of glutamate-gated chloride channels, which share 45% identical and 62% positive sequences with the GlyR α1 subunit (Hibbs and Gouaux, 2011). The homology model showed that W170 was located at loop F in the outer face of the N terminus domain. It is structurally close to the previously reported Zn²⁺ potentiation site (Miller et al., 2005b), with particular proximity (<4 Å) to D194, a key residue for Zn²⁺ binding (Fig. 2A). Therefore, we investigated the possibility that the W170S mutation might affect the sensitivity of GlyRs to Zn²⁺ modulation. Low concentrations (0.1–1 μm) of Zn²⁺ remarkably enhanced homomeric α1^WT currents evoked by 30 μm glycine (EC₁₀), whereas high concentrations (>10 μm) of Zn²⁺ inhibited responses of α1^WT GlyRs (Fig. 2B top, C). Interestingly, Zn²⁺-mediated potentiation of GlyRs was substantially attenuated in α1^W170S GlyRs (Fig. 2B, bottom; C, top). Coexpression of the β subunit did not rescue the deficit of Zn²⁺ potentiation in α1^W170S, suggesting that the ablated Zn²⁺ potentiation was preserved in α1^W170Sβ GlyRs (Fig. 2C, bottom).

Figure 2.

W170S mutation impaired Zn²⁺-mediated potentiation and enhanced Zn²⁺-mediated inhibition of GlyR currents activated by different agonists. A, Left, The homology model of the GlyR α1 subunit based on the GluCl viewed from the outer face. Inset, Plan view of the GlyR pentamer. The arrow indicates the viewing angle. Right, The expanded illustration of the amino acid residues that affect Zn²⁺ potentiation. B, Representative traces represent biphasic modulation of Zn²⁺ on homomeric α1^WT (top) or α1^W170S (bottom) GlyR currents activated by glycine (EC₁₀). C, Averaged Zn²⁺ concentration–response curves for the modulation of EC₁₀ responses to glycine-activated currents in homomeric (top; α1^WT, n = 7; α1^W170S, n = 10) and heteromeric α1^W170Sβ GlyRs (bottom; α1^WT, n = 7; α1^W170S, n = 10). D, Averaged Zn²⁺ concentration–response curves for the modulation of EC₁₀ responses to β-alanine-activated currents in homomeric (top; α1^WT, n = 5; α1^W170S, n = 5) and heteromeric α1^W170Sβ GlyRs (bottom; α1^WT, n = 6; α1^W170S, n = 5). E, Averaged Zn²⁺ concentration–response curves for the modulation of EC₁₀ responses to taurine-activated currents in homomeric (top; α1^WT, n = 5; α1^W170S, n = 8) and heteromeric α1^W170Sβ GlyRs (bottom; α1^WT, n = 7; α1^W170S, n = 5).

Zn²⁺ potentiation of GlyR current responses evoked from partial agonists, such as taurine, might behave differently from the potentiation effect of glycine-evoked responses (Lynch et al., 1998). Therefore, we tested the effects of Zn²⁺ on α1^W170S and α1^W170Sβ GlyRs-mediated currents induced by β-alanine or taurine at EC₁₀, respectively. We found that W170S similarly abolished Zn²⁺-mediated potentiation of responses induced by β-alanine or taurine in both homomeric and heteromeric receptors (Fig. 2D,E). The Zn²⁺ inhibition was also increased under these conditions, suggesting that W170S mutations cause either attenuated potentiation or enhanced inhibition of Zn²⁺ in α1-containing GlyRs.

Altered Zn²⁺ modulation in W170S mutation was mainly the result of abolished potentiation

At Zn²⁺ concentrations ranging from 0.1 to 1000 μm, the biphasic Zn²⁺-mediated modulation of α1^WT likely results from the superimposition of both positive and negative modulatory effects. Prior studies have shown that, if the GlyR has already been activated by the agonist, the positive and negative modulations of Zn²⁺ can be temporally separated by showing an initial potentiation followed by prolonged inhibition of GlyR currents (Lynch et al., 1998). By using this strategy, we further elucidated whether the altered responses of α1^W170S to Zn²⁺ were the result of an ablation of Zn²⁺ potentiation or an increase of Zn²⁺ inhibition. In homomeric α1^WT receptors, after 30 μm glycine was applied to induce a GlyR-mediated current, subsequent Zn²⁺ application caused an initial potentiation at concentrations of 0.1 to 50 μm (Fig. 3A). The Zn²⁺-mediated inhibition became more apparent when Zn²⁺ was washed out but glycine remained present (Lynch et al., 1998). In α1^W170S GlyRs, however, the initial potentiation was completely absent (Fig. 3B,C), and the inhibition by Zn²⁺ was consequently enhanced compared with α1^WT GlyRs (Fig. 3D). These data demonstrated that Zn²⁺-mediated positive allosteric modulation was ablated in the α1^W170S.

Figure 3.

Separation of Zn²⁺-mediated potentiation and inhibition indicated that the W170S mutation mainly impaired Zn²⁺-mediated potentiation of GlyRs. A, Examples of α1^WT-mediated currents showing that, after GlyR activation by glycine (EC₁₀), subsequent Zn²⁺ application (5 s) at higher concentrations (30 and 50 μm) evoked an initial potentiation followed by inhibition. B, In α1^W170Sβ GlyRs, the same application of Zn²⁺ only evoked inhibitory effects. Zn²⁺-mediated early effect was measured as the maximal potentiation of glycine-activated currents during the first 300 ms of Zn²⁺ perfusion (C). Zn²⁺-mediated late effect was measured as the maximal inhibition of glycine-activated currents during the last 300 ms before Zn²⁺ was washed out (D). C, D, Averaged dose–responses for Zn²⁺-mediated early and late effects on glycine-activated currents in homomeric α1^WT (n = 6) and α1^W170S receptors (n = 4). E, Top, Using the low Zn²⁺ potentiation background H107N, Zn²⁺ application induced only potentiation but no significant inhibition of glycine-activated currents in the homomeric α1^H170N receptors. Bottom, Zn²⁺-mediated potentiation was ablated in the α1^H170N/W170S double-mutant receptors. F, Averaged dose–responses for Zn²⁺-mediated modulations in α1^H170N (n = 5) and α1^H170N/W170S receptors (n = 16).

We further examined the effects of W170S on Zn²⁺-mediated potentiation by diminishing the influence of the inhibitory effects of Zn²⁺. The α1 mutation, H107N, has been reported to be at least 150-fold less sensitive than the α1^WT to Zn²⁺-mediated inhibition without significantly affecting Zn²⁺-mediated potentiation (Miller et al., 2005a, b). Therefore, we generated the double-mutation α1^H107N/W170S on the background of α1^H107N (α1^H107N, EC₅₀ = 41.6 ± 3.1 μm, n = 2; α1^H107N/W170S, EC₅₀ = 136.9 ± 6.5 μm, n = 7). In α1^H107N GlyRs, Zn²⁺ produced significant potentiation of glycine-induced responses without showing obvious inhibition at concentrations up to 100 μm (Fig. 3E,F). In contrast, Zn²⁺-mediated potentiation was abolished in α1^H107N/W170S receptors. At Zn²⁺ concentrations of 50–100 μm, the W170S mutation even partially recovered the sensitivity of α1^H107N to Zn²⁺-mediated inhibition (Fig. 3F). Together, these data revealed that W170 residue is likely a required site for Zn²⁺-mediated potentiation in α1 GlyRs.

Neuronal expressed α1^W170S GlyRs were insensitive to Zn²⁺-mediated potentiation

The above results have shown that α1^W170S-containing GlyRs lacked sensitivity to Zn²⁺-mediated potentiation in a recombinant expression system. However, the differential protein expression profiles between HEK293T cells and neurons might result in altered receptor functional characteristics in these two systems. To confirm that α1^W170S-containing GlyRs are also insensitive to Zn²⁺-mediated potentiation when expressed in nerve cells, we overexpressed α1^WT or α1^W170S subunits in cultured cortical neurons as an endogenous expression system. Young cortical neurons mainly express α2 subunits, which exhibit substantially lower sensitivity to Zn²⁺ potentiation than that of α1 (Lynch, 2004; Miller et al., 2005b). In addition, α1- and α2-containing GlyRs have very distinct activation kinetics that can facilitate their biophysical isolation and thus also allow unambiguous confirmation of successful α1^WT or α1^W170S overexpression in neurons (Mangin et al., 2003; Mohammadi et al., 2003). To establish the accuracy of this strategy, we first examined the activation kinetics of α1^WT and α1^W170S and compared them with the kinetics of α2-containing GlyRs expressed in HEK293T cells. α1^WT and α1^W170S GlyRs exhibit similar 10–50% rise time (α1^WT, 86.7 ± 6.9 ms, n = 8; α1^W170S; 83.6 ± 7.1 ms, n = 8; p > 0.05) after application of 3 s pulses of 30 μm glycine, whereas α2 receptors expressed in HEK293T cells exhibited substantially slower activation kinetics (10–50% rise time: 243.9 ± 17.3 ms, n = 8, p < 0.01 compared with α1^WT or α1^W170S; Fig. 4A–C). In nontransfected cortical neurons, the same glycine application induced currents with activation kinetics similar to that of α2 receptors expressed in HEK293T cells (206.7 ± 21.6 ms, n = 7, p > 0.05 compared with α2 in HEK293T). Neurons overexpressing α1^WT or α1^W170S receptors showed GlyR activation kinetics with rise times ranging from 50 to 290 ms, indicating variable GlyR expression profiles. Therefore, only neurons that exhibited glycine currents with rise times <100 ms were included in our analysis and were considered to predominantly express α1^WT or α1^W170S GlyRs.

Figure 4.

Neuronal expression of α1^W170S ablated Zn²⁺-mediated potentiation of glycine-activated currents. A, B, Representative traces showing different activation kinetics of recombinant α1 and α2 receptors expressed in HEK293T cells. A, Arrowheads indicate regions of the traces that are expanded in B. B, Gray dots indicate application of glycine. C, Averaged 10–50 rise time of α1^WT (n = 8), α1^W170S (n = 8), and α2 receptors (n = 8) expressed in HEK293T cells. D, Top, Whole-cell recordings from a cortical neuron overexpressing α1^WT that exhibited sensitivities to Zn²⁺-mediated biphasic modulation. Bottom, Whole-cell recordings from a cortical neuron overexpressing α1^W170S and not exhibiting sensitivity to Zn²⁺ potentiation. E, Averaged dose–responses for Zn²⁺-mediated modulation of glycine (50 μm)-activated currents in α1^WT (n = 4) and α1^W170S (n = 5) overexpressed neurons.

Neurons overexpressed with α1^WT showed remarkable increase in glycine-mediated currents by 0.1–10 μm Zn²⁺ (Fig. 4D,E; mean 10–50% rise time: 66.4 ± 8.9 ms, n = 4). In contrast, Zn²⁺ did not enhance glycine-mediated currents in neurons overexpressed with α1^W170S (Fig. 4D,E; mean 10–50% rise time: 62.9 ± 11.6 ms, n = 5). These results confirm our findings, using a recombinant expression system, that the W170S mutation also abolished sensitivity of α1 GlyRs to Zn²⁺ potentiation in neurons, and this may be a major causal factor of the symptoms of hyperekplexia in humans carrying this mutant allele.

Discussion

In the present study, we found that W170 was a novel Zn²⁺ potentiation site at the N-terminal domain of the GlyR α1 subunit. This site was identified from patients with an autosomal recessive form of hyperekplexia exhibiting homozygote W170S missense mutation (Al-Futaisi et al., 2012). We found the W170S mutation to cause complete ablation of sensitivity of α1 GlyRs to Zn²⁺ potentiation. In contrast to previously identified missense glra1 mutations, it did not affect other basic electrophysiological properties of α1 GlyRs. After recombinant expression, the W170S mutation removed Zn²⁺ potentiation and increased the sensitivity to Zn²⁺ inhibition of current responses activated by glycine, β-alanine, or taurine. This substantial reduction of Zn²⁺-mediated potentiation was further revealed by temporally separating the positive and negative modulatory phases and by examining the W170S mutation using the H107N mutation background, which exhibits low sensitivity to Zn²⁺ inhibition. Furthermore, abolishment of Zn²⁺ potentiation was also observed in α1^W170S GlyRs expressed in neuronal cultures, indicating that the alteration of Zn²⁺ modulation can also be observed in a native neuronal environment and might be a major causal factor of hyperekplexia in humans carrying W170S alleles.

Typically, missense mutations of glra1 identified from hyperekplexia might reduce agonist binding sensitivity, affect channel conductance, or disrupt receptor surface expression (Saul et al., 1999; Harvey et al., 2008; Chung et al., 2010). We found that homomeric α1^W170S receptors had a twofold decrease in sensitivity to glycine compared with the WT receptors. However, in α1^W170Sβ GlyRs, the W170S mutation exhibited no reduction in sensitivity to all tested agonists, no changes in the maximal current responses, and no change in I-V relationships, suggesting that there were no defects in the intrinsic channel properties of the GlyR and that abnormalities in these properties were unlikely to be the major cause of pathogenesis in hyperekplexia patients carrying the α1^170S mutation. Instead, our results demonstrated that the major alteration of α1^W170 was the ablated sensitivity to Zn²⁺-mediated potentiation and the consequently increased sensitivity to Zn²⁺ inhibition. To our knowledge, the present study is the first demonstration of disrupted allosteric modulation of GlyRs by Zn²⁺ as an important factor in human hyperekplexia symptoms.

Endogenous free Zn²⁺ is concentrated in synaptic vesicles at certain synapses and is speculated to regulate synaptic transmission (Sensi et al., 2011). The physiological role of Zn²⁺ in synaptic transmission has been demonstrated by studies using Zn²⁺ chelators or exogenous application of Zn²⁺ to affect the duration and amplitude of glycinergic IPSCs (Suwa et al., 2001; Eto et al., 2007). Selectively ablating sensitivity of α1 to Zn²⁺ potentiation led to hyperekplexia-like phenotypes in transgenic mice carrying α1^D80A (Hirzel et al., 2006). In line with these studies, our findings suggest that synaptic Zn²⁺ plays a crucial role in glycinergic synaptic transmission and efficacy in human CNS.