De Jongh K S, Merrick D K, Catterall W A
Department of Pharmacology, University of Washington, Seattle 98195.
Proc Natl Acad Sci U S A. 1989 Nov;86(21):8585-9. doi: 10.1073/pnas.86.21.8585.
Antibodies prepared against peptides CP2, CP4, and CP5, which occur within the first 1522 amino acid residues of the alpha 1 subunit of dihydropyridine-sensitive skeletal muscle calcium channels, specifically recognized a 175-kDa form of the alpha 1 subunit in immunoblots and immunoprecipitation experiments. In contrast, antibodies prepared against peptide CP1, which represents the C-terminal 18 amino acid residues predicted by cloning and sequence analysis of the alpha 1 subunit, recognized a minor, previously undescribed 212-kDa protein, which is the size predicted for the full length of the alpha 1 subunit from cDNA cloning [Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1987) Nature (London) 328, 313-318]. Both the 175-kDa and 212-kDa forms were phosphorylated by cAMP-dependent protein kinase and both were present in isolated transverse tubule membranes. The 175-kDa form may arise from posttranslational proteolytic cleavage of the C terminus of the 212-kDa form of the alpha 1 subunit predicted by cDNA cloning and sequence analysis. Partial amino acid sequencing of the 54-kDa beta subunit of the calcium channel indicated this protein was not derived from the proteolytically cleaved C terminus of the alpha 1 subunit. This analysis identified a threonine residue in the sequence (Lys/Arg)-Arg-Pro-Thr-Pro of the beta subunit that was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of this residue in the beta subunit may play a role in modulation of calcium channel function. Separate functional roles of the 175-kDa form of the alpha 1 subunit in excitation-contraction coupling and of the 212-kDa form in ion conductance are proposed.
针对位于二氢吡啶敏感性骨骼肌钙通道α1亚基前1522个氨基酸残基内的肽段CP2、CP4和CP5制备的抗体，在免疫印迹和免疫沉淀实验中特异性识别出α1亚基的一种175 kDa形式。相比之下，针对肽段CP1制备的抗体识别出一种较小的、先前未描述的212 kDa蛋白，CP1代表通过α1亚基的克隆和序列分析预测的C末端18个氨基酸残基，212 kDa是根据cDNA克隆预测的α1亚基全长大小[田边，T.，竹岛，H.，三上，A.，弗洛克齐，V.，高桥，H.，川合，K.，小岛，M.，松尾，H.，广濑，T.和沼田，S.（1987年）《自然》（伦敦）328，313 - 318]。175 kDa和212 kDa形式均被cAMP依赖性蛋白激酶磷酸化，且二者均存在于分离的横管膜中。175 kDa形式可能源于通过cDNA克隆和序列分析预测的α1亚基212 kDa形式C末端的翻译后蛋白水解切割。钙通道54 kDaβ亚基的部分氨基酸测序表明该蛋白并非来自α1亚基经蛋白水解切割的C末端。该分析确定了β亚基序列（Lys/Arg）-Arg-Pro-Thr-Pro中的一个苏氨酸残基，它被cAMP依赖性蛋白激酶磷酸化。β亚基中该残基的磷酸化可能在钙通道功能调节中起作用。有人提出α1亚基175 kDa形式在兴奋 - 收缩偶联中具有独立的功能作用，而212 kDa形式在离子传导中具有独立的功能作用。
