The intracellular loop between domains I and II of the B-type calcium channel confers aspects of G-protein sensitivity to the E-type calcium channel

Abstract

Neuronal voltage-dependent calcium channels undergo inhibitory modulation by G-protein activation, generally involving both kinetic slowing and steady-state inhibition. We have shown previously that the β-subunit of neuronal calcium channels plays an important role in this process, because when it is absent, greater receptor-mediated inhibition is observed (Campbell et al., 1995b). We therefore hypothesized that the calcium channel β- subunits normally may occlude G-protein-mediated inhibition. Calcium channel β-subunits bind to the cytoplasmic loop between transmembrane domains I and II of the α1-subunits (Pragnell et al., 1994). We have examined the hypothesis that this loop is involved in G-protein-mediated inhibition by making chimeras containing the I-II loop of α1B or α1A inserted into α1E (α1EBE and α1EAE, respectively). This strategy was adopted because α1B (the molecular counterpart of N-type channels) and, to a lesser extent, α1A (P/Q-type) are G-protein-modulated, whereas this has not been observed to any great extent for α1E. Although α1B, coexpressed with α2-δ and β1b transiently expressed in COS-7 cells, showed both kinetic slowing and steady- state inhibition when recorded with GTPγS in the patch pipette, both of which were reversed with a depolarizing prepulse, the chimera α1EBE (and, to a smaller extent, α1EAE) showed only kinetic slowing in the presence of GTPγS, and this also was reversed by a depolarizing prepulse. These results indicate that the I-II loop may be the molecular substrate of kinetic slowing but that the steady-state inhibition shown by α1B may involve a separate site on this calcium channel.