title: Altered Channel Conductance States and Gating of GABAAReceptors by a Pore Mutation Linked to Dravet Syndrome.
abstract: We identified ade novomissense mutation, P302L, in the γ-aminobutyric acid type A (GABAA) receptor γ2 subunit geneGABRG2in 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. GABAAreceptor 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 GABAAreceptors 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 GABAAreceptor-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 GABAAreceptor 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 GABAAreceptors, which results in hyperexcitability and contributes to the pathogenesis of the genetic epilepsy Dravet syndrome.
authors.affiliation: Department of Neurology, Vanderbilt University Medical Center , Nashville, TN 37240-7915.
authors.forename: Ciria C
authors.initials: CC
authors.lastname: Hernandez
authors.affiliation: Department of Pediatrics, Peking University First Hospital , Beijing, China 100034.
authors.forename: Weijing
authors.initials: W
authors.lastname: Kong
authors.affiliation: Department of Neurology, Vanderbilt University Medical Center , Nashville, TN 37240-7915.
authors.forename: Ningning
authors.identifier: 0000-0002-4854-6219
authors.initials: N
authors.lastname: Hu
authors.affiliation: Department of Pediatrics, Peking University First Hospital , Beijing, China 100034.
authors.forename: Yujia
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Neurology, Vanderbilt University Medical Center , Nashville, TN 37240-7915.
authors.forename: Wangzhen
authors.identifier: 0000-0002-1784-511X
authors.initials: W
authors.lastname: Shen
authors.affiliation: Department of Neurology, Vanderbilt University Medical Center , Nashville, TN 37240-7915.
authors.forename: Laurel
authors.initials: L
authors.lastname: Jackson
authors.affiliation: Department of Pediatrics, Peking University First Hospital , Beijing, China 100034.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Pediatrics, Peking University First Hospital , Beijing, China 100034.
authors.forename: Yuwu
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Neurology, Vanderbilt University Medical Center , Nashville, TN 37240-7915.
authors.forename: Robert L
authors.identifier: 0000-0002-9539-5556
authors.initials: RL
authors.lastname: Macdonald
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D004831:Epilepsies, Myoclonic / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D053444:Inhibitory Postsynaptic Potentials / Q000502:physiology; D008810:Mice, Inbred C57BL; D055369:Miniature Postsynaptic Potentials / Q000502:physiology; D008958:Models, Molecular; D020125:Mutation, Missense; D009474:Neurons / Q000378:metabolism; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: C546279:GABRG2 protein, human; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D015032:Zinc
keywords: Dravet syndrome; GABRG2; conductance states; gating; pore mutation; structural modeling