βγ dimers derived from G0 and G1 proteins contribute different components of adrenergic inhibition of Ca2+ channels in rat sympathetic neurones

Abstract

1. Using perforated-patch recordings, we have examined the part played by endogenous G-protein subunits in the α2-adrenoceptor-mediated inhibition of N-type Ca2+ currents in sympathetic neurones. 2. Two components of I(Ca) inhibition by noradrenaline were recorded: a prominent, high affinity and voltage-dependent pertussis toxin (PTX)-sensitive pathway and a minor, low affinity and mostly voltage-insensitive PTX-resistant pathway. 3. PTX-sensitive inhibition was reduced by microinjection of antibodies against either Gα(oA,B) or Gα(i1,2). The voltage-dependent fraction of inhibition was reduced by anti-Gα(o) but not by anti-Gα(i) antibody. 4. Antisense depletion of Gα(oA) led to a marked reduction of noradrenaline-induced inhibition and voltage dependence. By contrast, Gα(i) depletion attenuated noradrenergic modulation without affecting the voltage dependence. 5. Expression of the βγ-binding agents β-adrenergic receptor kinase 1 (C-terminus, βARK1(C-ter) or Gα(i1) with a Cys3 to Ser mutation partially prevented noradrenergic inhibition while α-transducin abolished it. Residual inhibition was mostly voltage independent in cells expressing βARK1(C-ter) but was strongly reversed by depolarization in Gα(i1) Cys3Ser-expressing cells. 6. Expression of the PTX-resistant Gα(i1) Cys351Ile mutant in cells treated with PTX restored α2-adrenoceptor inhibition. This restored inhibition was weakly reversed by depolarization. Both the degree and voltage dependence of inhibition were correlated with the level of expression of the Gα(i1) Cys351Ile subunit. 7. Our findings identify βγ dimers associated with Gα(oA) and Gα(i) as mediators of the PTX-sensitive α2-adrenoceptor-mediated inhibition of N-type Ca2+ channels. Different βγ combinations may account for the differential voltage-dependent effects of G(o) and G(i) on I(Ca).