Dataset Information

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

ABSTRACT: We identified a de novo missense mutation, P302L, in the ?-aminobutyric acid type A (GABAA) receptor ?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 ?2 subunit that faces the pore lumen. GABAA receptor ?1 and ?3 subunits were coexpressed with wild-type (wt) ?2L or mutant ?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 ?2(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 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 ?2 subunit missense mutation (P302L) that has a novel pathogenic mechanism to cause defects in the conductance and gating of GABAA receptors, which results in hyperexcitability and contributes to the pathogenesis of the genetic epilepsy Dravet syndrome.

SUBMITTER: Hernandez CC

PROVIDER: S-EPMC5301078 | biostudies-literature | 2017 Jan-Feb

REPOSITORIES: biostudies-literature

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

Hernandez Ciria C CC Kong Weijing W Hu Ningning N Zhang Yujia Y Shen Wangzhen W Jackson Laurel L Liu Xiaoyan X Jiang Yuwu Y Macdonald Robert L RL

eNeuro 20170101 1

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 coexpressed with wild-type (wt) γ2L or mutant γ2L(P302L) subunits in HEK 293T cells and cultured mouse ...[more]

PMID: 28197552

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Project description:The family of gamma-aminobutyric acid type A receptors (GABA(A)Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABA(A)Rs that are mainly comprised of alpha(1), beta(2), and gamma(2) subunits, whereas tonic inhibition is mediated by extrasynaptic GABA(A)Rs comprised of alpha(4/6), beta(2), and delta subunits. We investigated the activation properties of recombinant alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at alpha(4)beta(2)delta GABA(A)Rs (beta approximately 1600 s(-1)) than at alpha(1)beta(2)gamma(2S) GABA(A)Rs (beta approximately 460 s(-1)), whereas GABA activated alpha(1)beta(2)gamma(2S) GABA(A)Rs more rapidly (beta approximately 1800 s(-1)) than alpha(4)beta(2)delta GABA(A)Rs (beta < 440 s(-1)). Single channel recordings of alpha(1)beta(2)gamma(2S) and alpha(4)beta(2)delta GABA(A)Rs showed that both channels open to a main conductance state of approximately 25 pS at -70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited approximately 36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (P(O) approximately 0.3) at alpha(4)beta(2)delta GABA(A)Rs and at least two "modes" of single channel bursting activity, lasting approximately 100 ms at alpha(1)beta(2)gamma(2S) GABA(A)Rs. The most prevalent bursting mode had a P(O) of approximately 0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" P(O) mode ( approximately 0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs occurred as a single population of bursts (P(O) approximately 0.4-0.5) of moderate duration (approximately 33 ms) that could be described by schemes containing two shut and two open states for both GABA(A)Rs. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance.

Dataset Information

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

Publications

Altered Channel Conductance States and Gating of GABA<sub>A</sub> Receptors by a Pore Mutation Linked to Dravet Syndrome.

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