G-protein β-subunit specificity in the fast membrane-delimited inhibition of Ca2+ channels

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. G beta(1) and G beta(2) strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The G beta(5) subunit produced much weaker effects than G beta(1) and G beta(2), whereas G beta(3) and G beta(4) were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, G beta(1) or G beta(2) coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a alpha(1) subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the G beta(5) subunit interacted weakly, and G beta(3) and G beta(4) failed to interact. Together, these results suggest that GP, and/or G beta(2) subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel alpha(1) subunit is a principal receptor for this inhibition.