Inhibitory synapse deficits caused by familial α1 GABA_A receptor mutations in epilepsy

Epilepsy is a spectrum of neurological disorders with many causal factors. The GABA type-A receptor (GABA_AR) is a major genetic target for heritable human epilepsies. Here we examine the functional effects of three epilepsy-causing mutations to the α1 subunit (α1^T10'I, α1^D192N and α1^A295D) on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic GABA_AR isoform, α1β2γ2L. We employed a neuron - HEK293 cell heterosynapse preparation to record IPSCs mediated by mutant-containing GABA_ARs in isolation from other GABA_AR isoforms. IPSCs were recorded in the presence of the anticonvulsant drugs, carbamazepine and midazolam, and at elevated temperatures (22, 37 and 40°C) to gain insight into mechanisms of febrile seizures. The mutant subunits were also transfected into cultured cortical neurons to investigate changes in synapse formation and neuronal morphology using fluorescence microscopy. We found that IPSCs mediated by α1^T10'Iβ2γ2L, α1^D192Nβ2γ2L GABA_ARs decayed faster than those mediated by α1β2γ2L receptors. IPSCs mediated by α1^D192Nβ2γ2L and α1^A295Dβ2γ2L receptors also exhibited a heightened temperature sensitivity. In addition, the α1^T10'Iβ2γ2L GABA_ARs were refractory to modulation by carbamazepine or midazolam. In agreement with previous studies, we found that α1^A295Dβ2γ2L GABA_ARs were retained intracellularly in HEK293 cells and neurons. However, pre-incubation with 100nM suberanilohydroxamic acid (SAHA) induced α1^A295Dβ2γ2L GABA_ARs to mediate IPSCs that were indistinguishable in magnitude and waveform from those mediated by α1β2γ2L receptors. Finally, mutation-specific changes to synaptic bouton size, synapse number and neurite branching were also observed. These results provide new insights into the mechanisms of epileptogenesis of α1 epilepsy mutations and suggest possible leads for improving treatments for patients harbouring these mutations.