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.
Simen
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AA
Department of Neurobiology, Pharmacology, and Physiology, and Committee on Neurobiology, The University of Chicago, IL 60637, USA.
Miller
R J
RJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D017473:Receptors, Opioid, kappa / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D015220:Calcium Channels; D017473:Receptors, Opioid, kappa; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins