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

We identified a de novo missense mutation, P302L, in the gamma-aminobutyric acid type A (GABAA) receptor gamma 2 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 gamma 2 subunit that faces the pore lumen. GABAA receptor alpha 1 and beta 3 subunits were coexpressed with wild-type (wt) gamma 2L or mutant gamma 2L(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 GABAA receptors using structural modeling. The gamma 2(P302L) subunit mutation produced an similar to 90% reduction of whole-cell current by increasing macroscopic desensitization and reducing GABA potency, which resulted in a profound reduction of GABAA 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 GABAA 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 gamma 2 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.