Vaghy P L, Striessnig J, Miwa K, Knaus H G, Itagaki K, McKenna E, Glossmann H, Schwartz A
Department of Pharmacology and Cell Biophysics, University of Cincinnati, Ohio 45267-0575.
J Biol Chem. 1987 Oct 15;262(29):14337-42.
A 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide has been identified by photoaffinity labeling of purified rabbit and guinea pig skeletal muscle calcium channel preparations. The arylazide ligands (-)-[3H]azidopine and (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile [( N-methyl-3H]LU 49888) were used to label 1,4-dihydropyridine- and phenylalkylamine-binding sites, respectively. A single, 155 to 170-kDa polypeptide was specifically labeled by both ligands in rabbit and guinea pig preparations provided that the skeletal muscle membranes used for purification were derived from fresh and not previously frozen and thawed tissue. The photoaffinity labeled polypeptide (termed here alpha 1) is different from the previously described alpha subunit in that it has the identical electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels irrespective of pretreatment either with N-ethylmaleimide or with dithiothreitol. The use of transverse tubular membranes isolated from previously frozen and thawed skeletal muscle results in a purified calcium channel preparation devoid of the alpha 1 subunit. In these preparations proteolytic degradation products of alpha 1 are labeled with both (-)-[3H]azidopine and [N-methyl-3H]LU 49888. Another large molecular weight polypeptide (termed here alpha 2) was also present in every purified calcium channel preparation studied. alpha 2 is distinct from alpha 1 in that reduction with dithiothreitol changes its apparent mass from 160-190 to 130-150 kDa. The alpha 2 subunit is not photoaffinity labeled either with (-)-[3H]azidopine or [N-methyl-3H]LU 49888. These data suggest that two distinct high molecular weight polypeptides (termed alpha 1 and alpha 2) are putative subunits of skeletal muscle calcium channels. Only the alpha 1 subunit contains both 1,4-dihydropyridine and phenylalkylamine receptors. alpha 2 is the same as the previously described alpha subunit (Curtis, B. M., and Catterall, W. A. (1984) Biochemistry 23, 2113-2118), but is neither a 1,4-dihydropyridine- nor a phenylalkylamine-binding protein.
通过对纯化的兔和豚鼠骨骼肌钙通道制剂进行光亲和标记，已鉴定出一种1,4 - 二氢吡啶和苯烷基胺结合多肽。芳基叠氮化物配体（-）-[³H]叠氮平与（-）-5 - [（3 - 叠氮苯乙基）[N - 甲基 - ³H]甲氨基]-2 - （3,4,5 - 三甲氧基苯基）-2 - 异丙基戊腈[（N - 甲基 - ³H]LU 49888）分别用于标记1,4 - 二氢吡啶和苯烷基胺结合位点。在兔和豚鼠制剂中，只要用于纯化的骨骼肌膜来自新鲜组织而非先前冻融过的组织，这两种配体均可特异性标记一条分子量为155至170 kDa的单一多肽。光亲和标记的多肽（此处称为α1）与先前描述的α亚基不同，在于其在十二烷基硫酸钠 - 聚丙烯酰胺凝胶中的电泳迁移率相同，无论是否用N - 乙基马来酰亚胺或二硫苏糖醇预处理。使用从先前冻融过的骨骼肌中分离的横管膜，会得到一种不含α1亚基的纯化钙通道制剂。在这些制剂中，α1的蛋白水解降解产物会被（-）-[³H]叠氮平和[N - 甲基 - ³H]LU 49888标记。在研究的每一种纯化钙通道制剂中还存在另一种大分子多肽（此处称为α2）。α2与α1不同，用二硫苏糖醇还原后其表观分子量从160 - 190 kDa变为130 - 150 kDa。α2亚基不会被（-）-[³H]叠氮平或[N - 甲基 - ³H]LU 49888光亲和标记。这些数据表明，两种不同的高分子量多肽（称为α1和α2）可能是骨骼肌钙通道的亚基。只有α1亚基同时含有1,4 - 二氢吡啶和苯烷基胺受体。α2与先前描述的α亚基相同（柯蒂斯，B.M.，和卡特拉尔，W.A.（1984年）《生物化学》23卷，2113 - 2118页），但既不是1,4 - 二氢吡啶结合蛋白也不是苯烷基胺结合蛋白。
