Robert Macdonald
Last active: 4/6/2017

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

Hernandez CC, Kong W, Hu N, Zhang Y, Shen W, Jackson L, Liu X, Jiang Y, Macdonald RL
eNeuro. 2017 4 (1)

PMID: 28197552 · PMCID: PMC5301078 · DOI:10.1523/ENEURO.0251-16.2017

We identified a missense mutation, P302L, in the γ-aminobutyric acid type A (GABA) receptor γ2 subunit gene 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 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 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 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 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 receptors, which results in hyperexcitability and contributes to the pathogenesis of the genetic epilepsy Dravet syndrome.

MeSH Terms (15)

Animals Cell Membrane Cerebral Cortex Epilepsies, Myoclonic HEK293 Cells Humans Inhibitory Postsynaptic Potentials Mice, Inbred C57BL Miniature Postsynaptic Potentials Models, Molecular Mutation, Missense Neurons Receptors, GABA-A Recombinant Proteins Zinc

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