Kraner S, Yang J, Barchi R
Mahoney Institute of Neurological Sciences, University of Pennsylvania School of Medicine, Philadelphia 19104.
J Biol Chem. 1989 Aug 5;264(22):13273-80.
The alpha subunit (Mr approximately 260,000) of the rat skeletal muscle sodium channel is sensitive to cleavage by endogenous proteases during the isolation of muscle surface membrane. Antisera against synthetic oligopeptides were used to map the resultant fragments in order to identify protease-sensitive regions of the channel's structure in its native membrane environment. Antibodies to the amino terminus labeled major fragments of Mr approximately 130,000 and 90,000 and lesser amounts of other peptides as small as Mr approximately 12,000. Antisera to epitopes within the carboxyl-terminal half of the primary sequence recognized two fragments of Mr approximately 110,000 and 78,000. Individual antisera also selectively labeled smaller polypeptides in the most extensively cleaved preparations. The immunoreactivity patterns of monoclonal antibodies previously raised against the purified channel were then surveyed. The binding sites for one group of monoclonals, including several that recognize subtype-specific epitopes in the channel structure, were localized within a 12-kDa fragment near the amino terminus. The distribution of carbohydrate along the primary structure of the channel was also assessed by quantitating 125I-wheat germ agglutinin and 125I-concanavalin A binding to the proteolytic peptides. Most of the carbohydrate detected by these lectins was located between 22 and 90 kDa from the amino terminus of the protein. No lectin binding was detected to fragments arising from carboxyl-terminal half of the protein. These results were analyzed in terms of current models of sodium channel tertiary structure. In its normal membrane environment, the skeletal muscle sodium channel appears sensitive to cleavage by endogenous proteases in regions predicted to link the four repeat domains on the cytoplasmic side of the membrane while the repeat domains themselves are resistant to proteolysis.
大鼠骨骼肌钠通道的α亚基（分子量约为260,000）在分离肌膜表面时对内源蛋白酶的切割敏感。利用针对合成寡肽的抗血清来定位产生的片段，以确定该通道结构在其天然膜环境中的蛋白酶敏感区域。针对氨基末端的抗体标记了分子量约为130,000和90,000的主要片段以及少量低至分子量约为12,000的其他肽段。针对一级序列羧基末端一半内表位的抗血清识别出分子量约为110,000和78,000的两个片段。在切割最广泛的制剂中，个别抗血清还选择性地标记了较小的多肽。随后检测了先前针对纯化通道产生的单克隆抗体的免疫反应模式。一组单克隆抗体（包括几个识别通道结构中亚型特异性表位的抗体）的结合位点位于氨基末端附近的一个12-kDa片段内。还通过定量125I-麦胚凝集素和125I-伴刀豆球蛋白A与蛋白水解肽的结合来评估碳水化合物沿通道一级结构的分布。这些凝集素检测到的大部分碳水化合物位于距蛋白质氨基末端22至90 kDa之间。未检测到凝集素与蛋白质羧基末端一半产生的片段结合。根据目前的钠通道三级结构模型对这些结果进行了分析。在其正常膜环境中，骨骼肌钠通道在预计连接膜细胞质侧四个重复结构域的区域对内源蛋白酶的切割敏感，而重复结构域本身对蛋白水解具有抗性。
