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We identified a de novo missense mutation, P302L, in the γ-aminobutyric acid type A (GABA A ) receptor γ2 subunit gene GABRG2 in a patient with Dravet syndrome using targeted next-generation sequencing. The mutation was in the cytoplasmic portion of the transmembrane segment M2 of the γ2 subunit that faces the pore lumen. GABA A receptor α1 and β3 subunits were coexpressed with wild-type (wt) γ2L or mutant γ2L(P302L) subunits in HEK 293T cells and cultured mouse cortical neurons. We measured currents using whole-cell and single-channel patch clamp techniques, surface and total expression levels using surface biotinylation and Western blotting, and potential structural perturbations in mutant GABA A receptors using structural modeling. The γ2(P302L) subunit mutation produced an ∼90% reduction of whole-cell current by increasing macroscopic desensitization and reducing GABA potency, which resulted in a profound reduction of GABA A receptor-mediated miniature IPSCs (mIPSCs). The conductance of the receptor channel was reduced to 24% of control conductance by shifting the relative contribution of the conductance states from high- to low-conductance levels with only slight changes in receptor surface expression. Structural modeling of the GABA A receptor in the closed, open, and desensitized states showed that the mutation was positioned to slow activation, enhance desensitization, and shift channels to a low-conductance state by reshaping the hour-glass-like pore cavity during transitions between closed, open, and desensitized states. Our study revealed a novel γ2 subunit missense mutation (P302L) that has a novel pathogenic mechanism to cause defects in the conductance and gating of GABA A receptors, which results in hyperexcitability and contributes to the pathogenesis of the genetic epilepsy Dravet syndrome.

Altered Channel Conductance States and Gating of GABAAReceptors by a Pore Mutation Linked to Dravet Syndrome

Altered Channel Conductance States and Gating of GABA_AReceptors by a Pore Mutation Linked to Dravet Syndrome