Activation of Go-proteins by membrane depolarization traced by in situ photoaffinity labeling of Gαo-proteins with [α32P]GTP-azidoanilide

Evidence for depolarization-induced activation of G-proteins in membranes of rat brain synaptoneurosomes has been previously reported (Cohen-Armon, RI,, and Sokolovsky, M. (1991) J, Biol, Chem, 266, 2595-2605; Cohen-Armon, RI., and Sokolovsky, M. (1993) J, Biol, Chem, 268, 9824-9838). In the present work we identify the activated G-proteins as G(o)-proteins by tracing their depolarization-induced in situ photoaffinity labeling with [alpha(32)P]GTP-azidoanilide (GTPAA), Labeled GTPAA was introduced into transiently permeabilized rat brainstem synaptoneurosomes. The resealed synaptoneurosomes, while being UV-irradiated, were depolarized. Relative to synaptoneurosomes at resting potential, the covalent binding of [alpha(32)P]GTPAA to G alpha(o1)- and G alpha(o3)-proteins, but not to G alpha(o2)- isoforms, was enhanced by 5- to 7-fold in depolarized synaptoneurosomes, thereby implying an accelerated exchange of GDP for [alpha(32)P]GT-PAA. Their depolarization-induced photoaffinity labeling was independent of stimulation of G(o)-proteincoupled receptors and could be reversed by membrane repolarization, thus excluding induction by transmitters release. It was, however, dependent on depolarization-induced activation of the voltage-gated sodium channels (VGSC), regardless of Na+ current. The alpha subunit of VGSC was cross-linked and co-immunoprecipitated with G alpha(o)-proteins in depolarized brain-stem and cortical synaptoneurosomes. VGSC alpha subunit most efficiently cross-linked with guanosine 5'-O2-thiodiphosphate-bound rather than to guanosine 5'-O-(3-thiotriphosphate)-bound G alpha(o)-proteins in isolated synaptoneurosomal membranes. These findings support a possible involvement of VGSC in depolarization-induced activation of G(o)-proteins.