GABARAP AFFECTS CONDUCTANCE OF GABA_A RECEPTORS

Andrea B. Everitt, Tien Luu, Peter W. Gage, Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, ACT.

Native GABA_A channels have conductances ranging from 10 to 90pS whereas the conductance of recombinant receptors ranges from 10 to 30pS^1-4. GABARAP is a protein that can bind to the GABA_A γ subunit ⁵ and when co-expressed with GABA_A subunits in QT6 cells, induces receptor clustering and changes in channel kinetics ⁶.

Human α₁, β₁ and γ₂s GABA_A subunits were transfected in mouse fibroblast L929 cells. Single channel currents activated by GABA (1-100µM) were recorded in the cell-attached (c/a) and outside-out (o/o) configurations. In c/a patches the mean conductance (γ_m) was 22.3 ± 1.2pS (n=15); in o/o patches γ_m was 29.7 ± 0.3pS (n=39). Currents reversed at 0mV (E_Cl ~0) and showed a linear IV relationship. Cells co-transfected with α₁,β₁ and γ₂s subunits plus GABARAP often displayed much higher conductances: 16 of 25 c/a patches had γ_m above 40pS (γ_m = 60.7 ± 4.3pS), 6 of 20 o/o patches showed γ_m of 54.3 ± 1.8pS. Both were significantly higher than controls (p<.0001, Student's t test). Currents were blocked by bicuculline and outwardly rectified.

Diazepam and barbiturates increase channel open probability ⁷ and conductance ^8,9 of native GABA_A channels. The effects of diazepam (1-10µM) and pentobarbitone (100µM) on γ were observed only in cells co-transfected with GABA_A subunits and GABARAP. High conductance channels were not seen in cells expressing α₁, β₁ and γ₂s subunits plus truncated GABARAP (residues 36-117, unable to cluster GABA_A receptors ¹⁰), nor α₁ and β₁ subunits plus full GABARAP.

Our results suggest coexpression of GABA_A α₁, β₁ and γ₂s subunits with GABARAP can produce high conductance channels with properties and pharmacology resembling native receptors.These effects on conductance may be related to receptor clustering.

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