Involvement of regions in domain I in the opioid receptor sensitivity of α1B Ca²⁺ channels

The structural basis of Ca²⁺ channel inhibition by G proteins has received considerable attention recently, and multiple regions on Ca²⁺ 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 Ca²⁺ channel is involved in determining the differences between α1B and α1ECa²⁺ 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. Chimetic Ca²⁺ channels constructed from α1B and α1E Ca²⁺ 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 α1B were exchanged into α1E, inhibition identical with that of α1B was observed. The differences between α1B and α1E 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 α1 B (α interaction domain, 0014, and a C- terminal Gα 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.