Identification and Subcellular Localization of the Subunits of L-type Calcium Channels and Adenylyl Cyclase in Cardiac Myocytes*
- Tianyan Gao‡§,
- Tipu S. Puriद,
- Brian L. Gerhardstein‡‖,
- Andy J. Chien‡**,
- Richard D. Green‡ and
- M. Marlene Hoseyत
- From the ‡Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611 and the ‡Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60612
Abstract
The properties of cardiac L-type channels have been well characterized electrophysiologically, and many such studies have demonstrated that the channels are regulated by a cAMP-dependent pathway. However, the subunit composition of native cardiac L-type calcium channels has not been completely defined. Furthermore, a very important question exists regarding the status of the C-terminal domain of the pore-forming α1subunit, as this domain has the potential to be the target of protein kinases but may be truncated as a result of post-translational processing. In the present studies, the α1C and β2 subunits were identified by subunit-specific antibodies after partial purification from heart membranes, or immunoprecipitation from cardiac myocytes. Both the β2and the full-length α1C subunits were found to be expressed and co-localized in intact cardiac myocytes along T-tubule membranes. Using a quantitative antibody binding analysis, we demonstrated that the majority of the α1C subunits in intact cardiac myocytes appear to be full-length. In addition, we observed that adenylyl cyclase is localized in a pattern similar to the channel subunits in cardiac myocytes. Taken together, our results provide new insights into the structural basis for understanding the regulation of L-type calcium channels by a cAMP-mediated signaling pathway.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant HL23306 (to M. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Contributed equally to the results in this article.
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↵¶ Supported by a predoctoral fellowship from the March of Dimes Birth Defects Foundation and by a Howard Hughes Research Training Fellowship for Medical Students.
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↵‖ Supported by a National Research Service Award Training Grant T32 DK07169.
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↵** Supported by individual National Research Service Award F30 MH10770.
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↵§§ To whom correspondence should be addressed: Dept. of Molecular Pharmacology & Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave., S215, Chicago, IL 60611. Tel.: 312-503-3692; Fax: 312-503-5349; E-mail: mhosey{at}nwu.edu.
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↵1 The abbreviations used are: PKA, protein kinase A; HEK, human embryonic kidney; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; PAGE, polyacrylamide gel electrophoresis; AC, adenylyl cyclase; PBS, phosphate-buffered saline; WGA, wheat germ agglutinin.
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↵2 Puri, T. S., Gerhardstein, B. L., Zhao, X.-L., Ladner, M. B., and Hosey, M. M. (1997) Biochemistry, in press.
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↵3 J. Krupinski, personal communication.
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- Received April 17, 1997.
- Revision received May 29, 1997.
