Investigating the Mechanism by Which Gain-of-function Mutations to the α1 Glycine Receptor Cause Hyperekplexia

Abstract

Hyperekplexia is a rare human neuromotor disorder caused by mutations that impair the efficacy of glycinergic inhibitory neurotransmission. Loss-of-function mutations in the GLRA1 or GLRB genes, which encode the α1 and β glycine receptor (GlyR) subunits, are the major cause. Paradoxically, gain-of-function GLRA1 mutations also cause hyperekplexia, although the mechanism is unknown. Here we identify two new gain-of-function mutations (I43F and W170S) and characterize these along with known gain-of-function mutations (Q226E, V280M, and R414H) to identify how they cause hyperekplexia. Using artificial synapses, we show that all mutations prolong the decay of inhibitory postsynaptic currents (IPSCs) and induce spontaneous GlyR activation. As these effects may deplete the chloride electrochemical gradient, hyperekplexia could potentially result from reduced glycinergic inhibitory efficacy. However, we consider this unlikely as the depleted chloride gradient should also lead to pain sensitization and to a hyperekplexia phenotype that correlates with mutation severity, neither of which is observed in patients with GLRA1 hyperekplexia mutations. We also rule out small increases in IPSC decay times (as caused by W170S and R414H) as a possible mechanism given that the clinically important drug, tropisetron, significantly increases glycinergic IPSC decay times without causing motor side effects. A recent study on cultured spinal neurons concluded that an elevated intracellular chloride concentration late during development ablates α1β glycinergic synapses but spares GABAergic synapses. As this mechanism satisfies all our considerations, we propose it is primarily responsible for the hyperekplexia phenotype.

Keywords: glycine receptor; glycinergic; inhibition mechanism; patch clamp; site-directed mutagenesis; startle disease; synapse; tropisetron.

FIGURE 1.

Model of the α1 GlyR viewed from within the membrane. The model is based on the cryoEM structure of zebrafish α1 GlyR in the glycine-bound conformation (PDB access code 3JAE) (39). One subunit is depicted in gold with gain-of-function hyperekplexia mutations (red) shown in stick form.

FIGURE 2.

Effect of the α1 subunit I43F mutation examined by whole cell patch clamp recording. Recordings were performed at −60 mV. A, sample whole cell recordings for heteromeric α1β and α1^I43Fβ GlyRs in the presence of the indicated glycine concentrations. B, averaged whole cell glycine dose-response curves for α1, α1β, α1^I43F, and α1^I43Fβ GlyRs. Mean parameters of best fit are provided in Table 1.

FIGURE 3.

Spontaneous activity in α1^W170Sβ and α1^I43Fβ GlyRs. Whole cell steady-state currents showing the effect of 100 μm picrotoxin (PTX) on α1^W170Sβ (A) and α1^I43Fβ (B) GlyRs. Paired single channel recordings of the α1^W170Sβ GlyR in the absence of applied glycine (C) and in the presence of 100 μm glycine (D). The patch contained an estimate of 6 channels. Paired single channel recordings of the α1^I43Fβ GlyR in the absence of applied glycine (E) and in the presence of 100 μm glycine (F). The patch contained an estimate of 4 channels.

FIGURE 4.

Properties of spontaneous IPSCs mediated by wild-type and mutant GlyRs in artificial synapses. Recordings were performed at −60 mV. A, representative recordings of glycinergic IPSCs in HEK293 cells expressing the indicated homomeric GlyRs at three different temporal resolutions. B, averaged, normalized IPSCs each averaged from >50 events from the corresponding cell in A. C, comparison of mean IPSC amplitude, decay time constant, and 10–90% rise time for the indicated GlyRs. Statistical significance was determined via one-way analysis of variance followed by Bonferroni's post hoc correction with significance represented by *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001 relative to α1β GlyRs.

FIGURE 5.

Distribution histograms for the peak amplitude and decay time constants of IPSCs recorded from wild-type and mutant GlyRs (n = 6 cells for each subtype). Curves represent single Log(Gaussian) functions of best fit to the data.

FIGURE 6.

Comparison of the IPSC decay rate with the corresponding intrinsic receptor deactivation rate. Averaged macropatch currents recorded from outside-out patches containing the indicated GlyR. The currents were activated by brief (∼1 ms) exposure to saturating (1 mm) glycine. Recordings were performed at −60 mV. Corresponding averaged IPSCs (right panel, black traces), reproduced from Fig. 3B, are included for comparison.

FIGURE 7.

A clinically relevant concentration of tropisetron prolongs the IPSC duration. A, representative IPSC recordings from an α1β GlyR-expressing cell before, during, and after the application of 1 nm tropisetron. Recordings were performed at −60 mV. B, superimposed averaged traces of IPSCs (n > 200 events) were recorded in control (black trace), 1 nm tropisetron exposure (red trace), and 5 min after the tropisetron washout (gray trace). Currents have been normalized. C, the decay time constant of IPSCs mediated by α1β GlyRs was significantly prolonged by 1 nm tropisetron, whereas the amplitude and 10–90% rise times were unchanged. *, p < 0.05 relative to drug-free control.