Activation of G(o)-proteins by membrane depolarization traced by in situ photoaffinity labeling of G(alpha o)-proteins with [alpha P-32]GTP-azidoanilide

Evidence for depolarization-induced activation of G-proteins in membranes o f 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 thei r depolarization-induced in situ photoaffinity labeling with [alpha(32)P]GT P-azidoanilide (GTPAA), Labeled GTPAA was introduced into transiently perme abilized rat brainstem synaptoneurosomes. The resealed synaptoneurosomes, w hile being UV-irradiated, were depolarized. Relative to synaptoneurosomes a t resting potential, the covalent binding of [alpha(32)P]GTPAA to G alpha(o 1)- and G alpha(o3)-proteins, but not to G alpha(o2)- isoforms, was enhance d by 5- to 7-fold in depolarized synaptoneurosomes, thereby implying an acc elerated exchange of GDP for [alpha(32)P]GT-PAA. Their depolarization-induc ed photoaffinity labeling was independent of stimulation of G(o)-proteincou pled receptors and could be reversed by membrane repolarization, thus exclu ding induction by transmitters release. It was, however, dependent on depol arization-induced activation of the voltage-gated sodium channels (VGSC), r egardless 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-linke d with guanosine 5'-O2-thiodiphosphate-bound rather than to guanosine 5'-O- (3-thiotriphosphate)-bound G alpha(o)-proteins in isolated synaptoneurosoma l membranes. These findings support a possible involvement of VGSC in depol arization-induced activation of G(o)-proteins.