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 beta-subunit of neuronal calcium channels plays an important role in this process, because when it is absent, greater receptor-mediated inhibition is observed (). We therefore hypothesized that the calcium channel beta-subunits normally may occlude G-protein-mediated inhibition. Calcium channel beta-subunits bind to the cytoplasmic loop between transmembrane domains I and II of the alpha1-subunits (). We have examined the hypothesis that this loop is involved in G-protein-mediated inhibition by making chimeras containing the I-II loop of alpha1B or alpha1A inserted into alpha1E (alpha1EBE and alpha1EAE, respectively). This strategy was adopted because alpha1B (the molecular counterpart of N-type channels) and, to a lesser extent, alpha1A (P/Q-type) are G-protein-modulated, whereas this has not been observed to any great extent for alpha1E. Although alpha1B, coexpressed with alpha2-delta and beta1b transiently expressed in COS-7 cells, showed both kinetic slowing and steady-state inhibition when recorded with GTPgammaS in the patch pipette, both of which were reversed with a depolarizing prepulse, the chimera alpha1EBE (and, to a smaller extent, alpha1EAE) showed only kinetic slowing in the presence of GTPgammaS, 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 alpha1B may involve a separate site on this calcium channel.