PT - JOURNAL ARTICLE AU - Ciria C. Hernandez AU - Weijing Kong AU - Ningning Hu AU - Yujia Zhang AU - Wangzhen Shen AU - Laurel Jackson AU - Xiaoyan Liu AU - Yuwu Jiang AU - Robert L. Macdonald TI - Altered Channel Conductance States and Gating of GABA<sub>A</sub> Receptors by a Pore Mutation linked to Dravet Syndrome AID - 10.1523/ENEURO.0251-16.2017 DP - 2017 Jan 31 TA - eneuro PG - ENEURO.0251-16.2017 4099 - http://www.eneuro.org/content/early/2017/01/31/ENEURO.0251-16.2017.short 4100 - http://www.eneuro.org/content/early/2017/01/31/ENEURO.0251-16.2017.full AB - We identified a de novo missense mutation, P302L, in the γ-aminobutyric acid type A (GABAA) 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. GABAA receptor α1 and β3 subunits were co-expressed with wild type 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 GABAA 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 GABAA receptor-mediated 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 γ2 subunit missense mutation (P302L) that has a novel pathogenic mechanism to cause defects in the conductance and gating of GABAA receptors that results in hyperexcitability and contributes to the pathogenesis of the genetic epilepsy Dravet syndrome.Significance Statement Dravet syndrome is a catastrophic epileptic encephalopathy characterized by multiple types of seizures, impaired intellectual development and cognitive decline. In general, only truncation mutations of γ-aminobutyric acid type A (GABAA) receptors have been reported in Dravet syndrome. We identified a single de novo mutation, P302L, that faces the pore lumen in the γ2 subunit, the most abundant GABAA receptor subunit in the brain. GABAA receptors mediate the majority of fast inhibitory neurotransmission and control network excitability in the brain. The present study showed that the γ2(P302L) subunit mutation caused ∼90% loss of function by altering the conduction pathway of the receptor during gating transitions among closed, open and desensitized states, which ultimately enhances neuronal excitability.