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Clustering of recombinant GABA_A receptors alters channel properties

`Native' GABA_A receptors display distinct electrophysiological properties not always seen in recombinant receptors irrespective of subunit composition. Native channels can have conductances over 40pS (Gray & Johnson, 1985; Smith et al., 1989; Curmi et al., 1993). Moreover, the conductance of some channels can be increased by modulating drugs such as diazepam, pentobarbitone and propofol (Eghbali et al., 1997; Guyon et al., 1999; Eghbali et al., 2003). By contrast, conductances of recombinant channels have never exceeded about 30pS and, although their open probability can be increased by modulating drugs, conductance is not enhanced by drugs.

It has been suggested that high channel conductances may represent cooperative openings of clustered channels resulting in an apparent high single channel conductance. We tested this hypothesis in an expression system by co-expressing two proteins known to cluster GABA_A receptors. Rapsyn is a membrane associated protein that plays a crucial role in clustering ACh receptors at the neuromuscular junction, but has also been shown to cluster expressed GABA_A receptors. GABARAP interacts with the GABA_A γ subunit and promotes receptor clustering (Wang et al., 1999).

We co-transfected (lipofectin) GABA_A α5 and β1 subunit cDNAs with or without rapsyn into mouse fibroblast L929 cells. We measured single channel conductance in the cell-attached (c/a) or inside-out (i/o) configurations 24-72 hours later. In the control groups (i.e. GABA_A subunits alone), single channel conductances were within the range 10-35pS. When rapsyn was co-expressed with GABA_A subunits, 4 out of 8 patches showed single channel conductances greater than 40pS. Control patches expressing GABA_A α1, β1 and γ2s subunits alone had a mean conductance of 22.3 ± 1.2pS (n=15). In 16 out of 25 patches recorded from cells co-transfected with GABA_A α1,β1 and γ2s subunits and GABARAP, single channel conductances were above 40pS (γ=60.7 ± 4.3pS, n=16). These `high' conductance channels were never seen in control patches. High and low conductance channel activity was blocked by 100μM bicuculline. The current-voltage relationship of high conducting channels showed outward rectification of the current, similar to that seen in native receptors.

Diazepam can increase both open probability and conductance of GABA_A channels containing the γ subunit. In 5 patches from cells co-transfected with GABA_A α1,β1 and γ2s subunits and GABARAP, both of these effects were seen irrespective of initial channel conductance. In control patches where GABARAP was not expressed, diazepam did not increase channel conductance.

Immunofluorescent studies revealed that coexpression of rapsyn or GABARAP with GABA_A subunits, showed a punctate pattern of staining of surface receptors compared to a diffuse pattern in control cells.

Our results show that co-expression with “clustering” proteins can change the properties of recombinant GABA_A channels. It is possible that clustered receptors may be able to couple and open cooperatively by virtue of their close physical proximity.

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