When a Gain Becomes a Loss: Gain-of-Function Glycine Receptors and Hyperekplexia

Abstract

Glycine receptors (GlyRs) mediate rapid synaptic inhibition in mammalian spinal cord and brainstem. GlyRs mutations often produce abnormalities that result in hyperekplexia. Most mutations cause a loss of GlyR function such as decreased expression, conductance or ligand sensitivity. It is easily seen how a reduction in inhibition could result in the increased excitatory symptoms of hyperekplexia. Paradoxically, the α1V280M mutation triggers hyperekplexia but exhibits gain of GlyR function. The α1V280M mutation increases GlyR sensitivity to glycine and produces spontaneous channel activity but does not alter GlyR expression. Because CSF contains resting levels of 10µM glycine and because GlyRs undergo desensitization, we sought to determine the degree of desensitization induced by resting glycine levels for wild-type (α1WT) and α1V280M GlyRs. HEK-293 cells transfected with cDNA for either human α1WT or α1V280M GlyR were prepared for outside-out patch clamp recording. Currents were activated by five, 300ms applications of 3mM glycine every 5s (–50mV). Between applications (background), patches were perfused with glycine-free solution. Subsequently, the background was switched to a solution containing between 3nM and 60µM glycine; and currents recorded. Finally, recovery currents were obtained. Desensitization was calculated as the peak current in the presence of glycine background relative to that in glycine-free background. Background glycine decreased peak currents through both α1WT and α1V280M GlyR with a sigmoidal concentration dependence. IC50s for current inhibition were 14µM for α1WT GlyR and 0.3µM for α1V280M GlyR. The 10µM glycine in CSF would desensitize about half of α1WT GlyRs. In contrast, 98% of α1V280M GlyR would be desensitized. Thus, even though the V280M mutation produces a gain-of-function at the receptor level, it results in a loss-of-function at the synaptic level. This may explain why patients with this mutation exhibit the symptoms of hyperekplexia.