Involvement of regions in domain I in the opioid receptor sensitivity of alpha1B Ca(2+) channels.

The structural basis of Ca(2+) channel inhibition by G proteins has received considerable attention recently, and multiple regions on Ca(2+) channels that interact with G protein subunits have been identified. We have demonstrated previously that a region extending from the N terminus to the I/II loop of the Ca(2+) channel is involved in determining the differences between alpha1B and alpha1E Ca(2+) channels with respect to inhibition by G proteins. Here we explore this region of the channel in greater detail in an effort to further define the regions involved in determining inhibition. Chimeric Ca(2+) channels constructed from alpha1B and alpha1E Ca(2+) channels revealed that the N terminus, the I/II loop, and domain I all play an important role in determining inhibition. We identified a 70-amino acid fragment from domain I that mediates the effects of domain I, and a 50-amino acid fragment from the I/II loop that mediates the effects of the I/II loop. When these regions from alpha1B were exchanged into alpha1E, inhibition identical with that of alpha1B was observed. The differences between alpha1B and alpha1E in the identified region of domain I involve residues that are predicted to be almost exclusively extracellular. Mutations to some of the high-affinity G protein binding regions of alpha1B (alpha interaction domain, CC14, and a C-terminal Galpha binding site) caused relatively little change in inhibition, which suggests that these sites are not necessary individually for G protein-mediated inhibition and may help to explain the small effects of exchanging these regions in isolation.