Structural Characterization of the Dihydropyridine Receptor-Linked Calcium Channel from Porcine Heart

• Published in: Journal of biochemistry (Tokyo) 1992-08, Vol.112 (2), p.235-242
• Main Authors: Kuniyasu, Akihiko, Oka, Eozo, Ide-Yamada, Tomoko, Hatanaka, Yasumaru, Abe, Teruo, Nakayama, Hitoshi, Kanaoka, Yuichi
• Format: Article
• Language: English
• Subjects: Animals; Antibodies - immunology; Biological and medical sciences; Calcium Channels - chemistry; Calcium Channels - immunology; Calcium Channels - metabolism; Cell receptors; Cell structures and functions; Dihydropyridines - metabolism; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Immunoblotting; Miscellaneous; Molecular and cellular biology; Myocardium - chemistry; Phosphorylation; Precipitin Tests; Protein Kinases - metabolism; Swine
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a123883

Ca2+-cbannel was purified 230-fold from digitonin extracts of the porcine cardiac sarcolemmal membranes by means of a four-step procedure. Two antibodies, a site-directed antibody against the sequence 1691–1707 of the rabbit cardiac α1 subunit (anti-CCP5) and a monoclonal antibody directed to rabbit skeletal muscle α2δ subunit-complex (MCC-1), effectively immunoprecipitated the 125I-labeled cardiac Ca2+-channel complex in 0.2% digitonin. SDS-PAGE analysis of the immunoprecipitates under reducing conditions revealed that the cardiac channel is mainly composed of two large polypeptides of 190 and 150 kDa, and five smaller polypeptides of 60, 56, 35, 30, and 25 kDa. An additional polypeptide of either 79 or 55 kDa is crosslinked with the 190 kDa component to form 250–270 kDa (˜270kDa) to the extent of 15-20% through disulfide bond(s). The 190 kDa component (α1) is responsible for photoafflnity labeling with [3H]diazipine, since minor photolabeled ˜270kDa was converged to the major labeled 190 kDa component when electrophoresed under reducing conditions. The 150 kDa component (α2) was derived by reduction of disulfide bonds from another 190 kDa component of glycopolypeptide which was separated from the channel complex in 1% Triton X-100 and capable of binding to WGA-Sepharose. The four smaller components of 60, 35, 30, and 25 kDa were not covalently associated with the large components through disulfide bonds, whereas the 55 kDa polypeptide was suggested to be a mixture of two kinds of peptides with respect to the disulfide bond: one was crosslinked with al through disulfide linkage and the other was not covalently associated with any other component. None of the polypeptides in the purified preparation or the sarcolemmal membranes which were phosphorylated by the cAMP-dependent protein kinase, was immunoprecipitated by either of the antibodies.