beta gamma dimers derived from G(o) and G(i) proteins contribute differentcomponents of adrenergic inhibition of Ca2+ channels in rat sympathetic neurones

1. Using perforated-patch recordings, we have examined the part played by e ndogenous G-protein subunits: in the alpha(2)-adrenoceptor-mediated inhibit ion of N-type Ca2+ currents in sympathetic neurones. 2. Two components of I-Ca inhibition by noradrenaline were recorded: a prom inent, high affinity and voltage-dependent pertussis toxin (PTX)-sensitive pathway and a minor, low affinity and mostly voltage-insensitive PTS-resist ant pathway. 3. PTS-sensitive inhibition was reduced by microinjection of antibodies aga inst either G alpha(oA,B) or G alpha(i1,2). The voltage-dependent fraction of inhibition was reduced by anti-G alpha(o) but not by anti-G alpha(1) ant ibody. 4. Antisense depletion of G alpha(oA) led to a marked reduction of noradren aline-induced inhibition and voltage dependence. By contrast, G alpha(i) de pletion attenuated noradrenergic modulation without affecting the voltage d ependence. 5. Expression of the beta gamma-binding agents beta-adrenergic receptor kin ase 1 (C-terminus, beta ARK1(C-ter)) or G alpha(i1) with a Cys3 to Ser muta tion partially prevented noradrenergic inhibition while alpha-transducin ab olished it. Residual inhibition was mostly voltage independent in cells exp ressing beta ARKI(C-ter) but was strongly reversed by depolarization in G a lpha(i1) Cys3Ser-expressing cells. 6. Expression of the PTX-resistant Ga-i1 Cys351Ile mutant in cells treated with PTX restored alpha(2)-adrenoceptor inhibition. This restored inhibitio n was weakly reversed by depolarization. Both the degree and voltage depend ence of inhibition were correlated with the level of expression of the G al pha(il) Cys351Ile subunit. 7. Our findings identify beta gamma dimers associated with G alpha(oA) and G alpha(i) as mediators of the PTX-sensitive alpha(2)-adrenoceptor-mediated inhibition of S-type Ca2+ channels. Different beta gamma combinations may account for the differential voltage-dependent effects of G(o) and G(i) on I-Ca.