Two Different Mechanisms of Disinhibition Produced by GABAAReceptor Mutations Linked to Epilepsy in Humans

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Abstract
The first mutations of the GABAA receptor channel linked to familial epilepsy in humans were reported recently (Baulac et al., 2001; Wallace et al., 2001). Preliminary functional analysis of α1β2γ2 GABAA receptors expressed inXenopus oocytes suggested that the γ2 subunit R43Q mutation abolished current enhancement by the benzodiazepine, diazepam, and that the γ2 subunit K289M mutation decreased current amplitudes. We used single-channel recording and concentration jump techniques applied to outside out patches to evaluate the impact of these mutations on GABAA receptor channel function of the highly conserved rat ortholog subunits expressed in human embryonic kidney cells. When coexpressed with α1 and β3 subunits, no differences were observed between wild-type and mutant GABAA receptor current activation rates or rates or extent of desensitization during prolonged (400 msec) GABA application (1 mm). Although deactivation after brief (5 msec) or prolonged (400 msec) GABA application was unaltered by the R43Q mutation, deactivation (a correlate of IPSC duration) was accelerated for the K289M mutation. Faster deactivation was likely a consequence of altered gating, because single-channel openings had shorter mean duration. Interestingly, the R43Q mutation did not alter diazepam potentiation. It did, however, substantially decrease current amplitude, which was not caused by decreased single-channel conductance or open time, suggesting reduced surface expression of functional receptors. The two γ2 subunit mutations likely produce disinhibition and familial epilepsy by distinct mechanisms, suggesting that maintenance of neuronal inhibition depends not only on the peak amplitude of IPSCs, but also on their time course.