Activation of Go-proteins by Membrane Depolarization Traced by in Situ Photoaffinity Labeling of Gαo-proteins with [α32P]GTP-azidoanilide

Abstract

GTP-binding trimeric proteins have been implicated in signal transduction from receptors in the cell membrane to intracellular effectors and ion channels in a variety of cells (1-5). The mechanism involves signal-induced G-protein activation initiated by an exchange of GDP for GTP on the ␣ subunit of the protein (5-7). Subsequent GTPase activity of the G␣ subunit converts the activated G-proteins into their inactive, GDPbound state (7). Activation of G-proteins has been induced experimentally by stimulation of G-protein-coupled receptors in the cell membrane (2,3,8,9). Evidence indicating activation of G-proteins in response to membrane depolarization were previously observed in brain stem synaptoneurosomes (10 -12).G␣ o -proteins are widely distributed in the central nervous system (15-17). Three subtypes showing marked homology but exhibiting functional differences have been identified (13,14,18). The G␣ o1 subtype appears to be involved in the coupling of muscarinic receptors to Ca ϩ2 channels, and the G␣ o2 subtype mediates inhibition of Ca ϩ2 current activated by somatostatin receptors (19). The function of the G␣ o3 subtype is not clear (14). Phospholipase C activation mediated by activation of G oproteins has been demonstrated in the cell-free system (20), and G o -protein-mediated activation of protein kinase C has been observed in Chinese hamster ovary (CHO) cells (21).In the present study, in situ photoaffinity labeling with [␣ 32 P]GTPAA 1 (22, 23) indicated a depolarization-induced accelerated exchange of GDP for [␣ 32 P]GTPAA in G␣ o1 -and G␣ o3 -proteins, implying a depolarization-induced activation of these G o -proteins. [␣ 32 P]-GTPAA was introduced into transiently permeabilized synaptoneurosomes as described before (10). Unlike the endogenously bound guanine nucleotides, [␣ 32 P]GT-PAA, covalently bound to G␣-proteins by photoaffinity labeling, was not displaced during SDS-polyacrylamide gel electrophoresis, providing a possible tool for identification of in situ activated G-proteins.In view of findings indicating a reciprocal influence of depolarization-induced activation of VGSC and uncoupling of Gproteins from muscarinic receptors (12, 24, 25), we examined the possibility that VGSC can be involved in depolarizationinduced activation of G␣ o -proteins. Our results indicated that depolarization-induced activation of G o -proteins can be prevented by preventing VGSC activation. In addition, in depolarized brain-stem and cortical synaptoneurosomes, the ␣ subunit of VGSC cross-linked most efficiently with G␣ o -proteins. In isolated synaptoneurosomal membranes, VGSC-␣ subunit cross-linked most efficiently with GDP␤S-bound rather than GTP␥S-bound G␣ o -proteins. These findings suggest repeated

Permissive Effect of Voltage on mGlu 7 Receptor Subtype Signaling in Neurons

“…Our results were consistent with previous biochemical studies showing a voltage-dependent interaction of VGSC with G o protein in rat brain synaptoneurosomes (15). Indeed, R-DPI 201-106, a drug that blocks depolarization-induced activation of G o protein in this preparation (15), also blocked the depolarization-induced inhibition of Ca 2ϩ channels in cerebellar granule cells.…”

“…Indeed, R-DPI 201-106, a drug that blocks depolarization-induced activation of G o protein in this preparation (15), also blocked the depolarization-induced inhibition of Ca 2ϩ channels in cerebellar granule cells. Although it is difficult to determine from the present study whether or not G o protein activation occurred during activation or inactivation of VGSCs, studies in synaptoneurosomes suggested that this may happen as long as depolarization lasts (15). We show here that voltage-dependent activation of G o protein through VGSCs resulted in IP 3 formation and intracellular Ca 2ϩ release.…”

“…Indeed, cultured cerebellar granule neurons do not express functional native mGlu5 receptors (17)(18)(19). Patch pipettes had resistances of 3-5 M⍀ when filled with the following internal solution (mM): Cs-acetate (100), CsCl (20), MgCl 2 (2), HEPES (10), glucose (15), EGTA (20 mM), Na 2 ATP (2 mM), and cAMP (1 mM), adjusted to pH 7.2 with CsOH and 300 mOsm with Cs-acetate. In some experiments, intracellular EGTA was replaced by BAPTA.…”

“…Moreover, a voltage-dependent activation of a G o protein has recently been shown in rat brain synaptoneurosomes (15). In light of these results, and because the mGlu7 receptor is coupled to a G o protein, this receptor is a potential candidate for mobilization of both voltage-and G o protein-dependent events in neurons.…”

“…4A). Interestingly, this drug has been shown to block depolarization induced activation of G o protein in synaptoneurosomes (15). The potent and selective VGSC activator, veratridine, shifted the voltageinduced inhibition of IBa toward more negative potentials (Fig.…”