Abstract
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 α1E Ca2+ 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 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 α1B (α interaction domain, CC14, 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.
Footnotes
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Send reprint requests to: Richard J. Miller, Ph.D., Dept. of Neurobiology, Pharmacology, and Physiology, University of Chicago, 947 East 58th Street, Chicago IL 60637. E-mail:rjmx{at}midway.uchicago.edu
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This work was supported by National Institute of Health Grants DA02121, MH40165, NS33826, DK44840, and NS21442. A.A.S. was supported by Grants HD07009 and DA02575.
- Abbreviations:
- DHP
- dihydropyridine
- norBNI
- nor-binaltorphimine
- AID
- α interaction domain
- ND1
- N-terminal domain I region
- GID
- G protein interaction domain
- κOR
- κ-opioid receptor
- Received September 29, 1999.
- Accepted February 3, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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