Hyperekplexia, or human startle disease, is a rare neurological disorder characterized by excessive startle reflexes to unexpected auditory and tactile stimuli. It is mainly caused by hereditary mutations in the α1 subunit of the postsynaptic human glycine receptor (hGlyR) chloride channel. GlyRs belong to the family of pentameric ligand-gated ion channels (pLGICs) and mediate inhibitory neurotransmission in the spinal cord, brainstem and retina, with most synaptic GlyRs consisting of two α1 and three β subunits. Mutations in the genes that encode hGlyR subunits can cause changes in expression efficiency and function, thereby disrupting inhibitory neurotransmission, leading to hyperekplexia. Recently, the cognate presynaptic human glycine transporter type-2 (hGlyT2) was discovered as the second major gene of effect.
A large genetic screening of hyperekplexia patients by our collaborators revealed twelve novel mutations in the α1 subunit and thirteen novel mutations in the β subunit of the hGlyR, the latter establishing the GLRB gene as the third major gene of effect. We also described the first cases of digenic inheritance, two cases with mutations in the α1 and β subunit and one case with mutations in the α1 subunit and in the hGlyT2. The effects of these new mutations on hGlyR expression and function were quantified via both a fluorescence flux assay to average effects over large cell numbers and via patch-clamp electrophysiology for higher resolution analysis from individual cells. Surface expression studies and molecular modelling were also conducted. These analyses demonstrated that the newly discovered β mutations were inherited in a recessive mode and reduced cell surface expression of wild type hGlyRs, with L285R representing an exemption due to its dominant mode of inheritance and its spontaneous channel activity.
The dominant mutations in the α1 subunit, Q226E, V280M and R414H, also induced spontaneous channel activity indicating this is a recurring mechanism in hGlyR pathophysiology. We also showed that Q226E and V280M receptors caused slower synaptic events whereas the synaptic properties of R414H receptors were similar to wild type hGlyRs. Q226, located near the top of TM1, is closely apposed to R271 at the top of TM2 in the neighbouring subunit. Using mutant cycle analysis, we inferred that Q226E induces spontaneous activation via an enhanced electrostatic attraction to R271. This would tilt the top of TM2 towards TM1 and hence away from the pore axis to open the channel. We also concluded that the increased side chain volume of V280M, in the TM2-TM3 loop, exerts a steric repulsion against I225 at the top of TM1 in the neighbouring subunit. This would tilt the top of TM3 radially outwards against the stationary TM1 and thus provide space for TM2 to relax away from the pore axis to create an open channel. Because the transmembrane domain movements inferred from this functional analysis are consistent with the structural differences evident in the X-ray atomic structures of closed and open state bacterial pLGICs, we propose that this model of activation may be broadly applicable across the eukaryotic pLGIC receptor family.
Recessive mutations in the α1 hGlyR mainly precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive E375X mutation resulted in subunit truncation upstream of TM4. Surprisingly, the truncated subunit was incorporated into functional hGlyRs where it conferred reduced glycine sensitivity. To our knowledge, the efficient functioning of receptors lacking TM4 domains has not been reported for any pLGIC receptor. As the E375X truncation occurs upstream of the naturally occurring premature stop codon in the human GLRA4 gene, we speculate the corresponding α4 subunit may have residual function. However, our results indicate that the α4 hGlyR is indeed a pseudogene. Interestingly, the non-conserved residues, E59K and Y204C, preclude cell surface expression and not the premature stop codon at residue 390 as suggested previously. Receptors incorporating P230S (compound heterozygous with the recurrent mutation R65W) desensitized much faster than wild type hGlyRs representing a new TM1 domain site capable of modulating desensitization. Taken together, our analysis of hyperekplexia mutations has provided important insights into the structure and function of GlyRs and into glycinergic signalling mechanisms in health and disease.