Involvement of regions in domain I in the opioid receptor sensitivity of alpha 1B 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 demonstrate d previously that a region extending from the N terminus to the I/II loop o f the Ca2+ channel is involved in determining the differences between alpha 1B and alpha 1E Ca2+ channels with respect to inhibition by G proteins. He re we explore this region of the channel in greater detail in an effort to further define the regions involved in determining inhibition. Chimeric Ca2 + channels constructed from alpha 1B and alpha 1E Ca2+ channels revealed th at the N terminus, the I/II loop, and domain I all play an important role i n determining inhibition. We identified a 70-amino acid fragment from domai n I that mediates the effects of domain I, and a 50-amino acid fragment fro m the I/II loop that mediates the effects of the I/II loop. When these regi ons from alpha 1B were exchanged into alpha 1E, inhibition identical with t hat of alpha 1B was observed. The differences between alpha 1B and alpha 1E in the identified region of domain I involve residues that are predicted t o be almost exclusively extracellular. Mutations to some of the high-affini ty G protein binding regions of alpha 1B (alpha interaction domain, CC14, a nd a C-terminal G alpha binding site) caused relatively little change in in hibition, which suggests that these sites are not necessary individually fo r G protein-mediated inhibition and may help to explain the small effects o f exchanging these regions in isolation.