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.

Glycine is able to induce both a motility speed in- and decrease during zebrafish neuronal migration

“…We aimed to increase the speed reduction effect by overexpressing gain-of-function mutations in glycine receptors. The mutations are modelled on available human patient data, and correspond to zebrafish GlyRa1 I67F [18,19], GlyRa1 V304M [18,20,21], and rat GlyRa3 P185L [22]. In contrast to expectations, all of these mutations led to a moderate THN speed increase in phase 2 (approx.…”

The role of tonic glycinergic conductance in cerebellar granule cell signalling and the effect of gain‐of‐function mutation

“…To date, the only proposed explanation is that enhanced spontaneous GlyR activity due to gain‐of‐function mutation during nervous system development prevents formation of glycinergic synapses and thus induces shortage of inhibitory signalling in adult brain (Zhang et al . ). This, however, is unlikely the case for CGCs in cerebellar vermis, which are deprived of glycinergic synapses under healthy conditions.…”

“…) via the ablation of α1β glycinergic synapses (Zhang et al . ). This is, however, unlikely the case for the cerebellar effects associated with CGCs due to the absence (or extreme scarcity) of glycinergic synaptic connections at these cells.…”

Physiological properties of Glycinergic synapses with defined subunit compositions

“…Recessive hyperekplexia mutations generally result in the loss of α1 or β GlyR protein expression at the cell surface, whereas dominant mutations usually allow strong surface expression but impair channel function via reduced open probability, single channel conductance or glycine sensitivity . In addition, several GLRA1 gain-of-function hyperekplexia mutations have been detected to evoke spontaneous channel activity, which is paradoxical in that these mutations generated different levels of prolonged postsynaptic glycinergic currents but full hyperekplexia phenotype (Zhang et al, 2016). All genetic forms of hyperekplexia are successfully treated with the benzodiazepine or clonazepam Thomas et al, 2013), which acts by enhancing GABAergic synaptic transmission.…”

“…b GlyR also exhibits spontaneous activation (Zhang et al, 2016) and suggested that, like other gain-of-function startle disease mutations, W170S may cause startle disease via a developmental defect that prevents the maturation of a1b GlyR synapses (Zhang et al, 2016).…”