Sun W, Barchi R L, Cohen S A
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
J Biol Chem. 1995 Sep 22;270(38):22271-6. doi: 10.1074/jbc.270.38.22271.
Epitopes for monoclonal antibodies directed against the purified adult rat skeletal muscle sodium channel (rSkM1) were localized using channel proteolysis and fusion proteins. The interactions between these and other monoclonal antibodies with site-specific polyclonal antibodies were used to investigate the spatial relationships among rSkM1 cytoplasmic segments. Competition. between antibodies for binding was performed using a solution-phase assay in which solubilized channel protein retains many of the biophysical characteristics of the rSkM1 protein in vivo. Our results support a model in which: 1) the amino terminus assumes a rigid structure having a fixed orientation with respect to other intracellular segments; 2) the interdomain 2-3 region is centrally located on the cytoplasmic surface of the channel, extends farther into the cytoplasm, and has an intermediate degree of flexibility; 3) the beginning of the amino terminus and end of the carboxyl terminus specifically interact with each other; and 4) domains 1 and 4 are adjacent. The sequences responsible for the interaction of the amino and carboxyl termini were identified by demonstrating the specific binding of a synthetic peptide encompassing the first 30 residues of the rSkM1 amino terminus to a fusion protein containing the rSkM1 carboxyl terminus.
利用通道蛋白水解和融合蛋白对针对纯化的成年大鼠骨骼肌钠通道（rSkM1）的单克隆抗体的表位进行了定位。这些单克隆抗体与其他单克隆抗体和位点特异性多克隆抗体之间的相互作用被用于研究rSkM1细胞质区段之间的空间关系。抗体之间的结合竞争通过一种溶液相分析来进行，在该分析中，溶解的通道蛋白保留了rSkM1蛋白在体内的许多生物物理特性。我们的结果支持以下模型：1）氨基末端呈现出一种刚性结构，相对于其他细胞内区段具有固定的方向；2）结构域间2 - 3区域位于通道细胞质表面的中央，向细胞质延伸得更远，并且具有中等程度的灵活性；3）氨基末端的起始部分和羧基末端的末端相互特异性作用；4）结构域1和4相邻。通过证明包含rSkM1氨基末端前30个残基的合成肽与含有rSkM1羧基末端的融合蛋白的特异性结合，确定了负责氨基末端和羧基末端相互作用的序列。
