APPS November 2002 Meeting Abstract 2417


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

Native GABAA channels have conductances ranging from 10 to 90pS whereas the conductance of recombinant receptors ranges from 10 to 30pS1-4. GABARAP is a protein that can bind to the GABAA γ subunit 5 and when co-expressed with GABAA subunits in QT6 cells, induces receptor clustering and changes in channel kinetics 6.

Human α1, β1 and γ2s GABAA subunits were transfected in mouse fibroblast L929 cells. Single channel currents activated by GABA (1-100ÁM) were recorded in the cell-attached (c/a) and outside-out (o/o) configurations. In c/a patches the mean conductance (γm) was 22.3 ▒ 1.2pS (n=15); in o/o patches γm was 29.7 ▒ 0.3pS (n=39). Currents reversed at 0mV (ECl ~0) and showed a linear IV relationship. Cells co-transfected with α11 and γ2s subunits plus GABARAP often displayed much higher conductances: 16 of 25 c/a patches had γm above 40pS (γm = 60.7 ▒ 4.3pS), 6 of 20 o/o patches showed γm 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 GABAA channels. The effects of diazepam (1-10ÁM) and pentobarbitone (100ÁM) on γ were observed only in cells co-transfected with GABAA 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 GABAA receptors 10), nor α1 and β1 subunits plus full GABARAP.

Our results suggest coexpression of GABAA 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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