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

We investigated which subtypes of G-protein β 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 β subunits. Gβ₁ and Gβ₂ strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gβ₅ subunit produced much weaker effects than Gβ₁ and Gβ₂, whereas Gβ₃ and Gβ₄ 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, Gβ₁ or Gβ₂ coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a α₁ subunit of the class B calcium channel fused to the GAL4 DNA- binding domain. In this assay, the Gβ₅ subunit interacted weakly, and Gβ₃ and Gβ₄ failed to interact. Together, these results suggest that Gβ₁ and/or Gβ₂ 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 α₁ subunit is a principal receptor for this inhibition.