Cai Y C, Douglass J
Vollum Institute, Oregon Health Sciences University, Portland 97201.
J Biol Chem. 1993 Nov 5;268(31):23720-7.
The major species of voltage-gated potassium channel found on mammalian T lymphocytes is referred to as the type n channel. This potassium channel exhibits unique functional properties which distinguish it from other species of potassium channels, including a potential role in the onset of cellular events associated with T cell activation. As a first step in characterizing specific biochemical properties of the type n channel, we have generated polyclonal antisera against bacterial fusion proteins containing peptide regions unique to the mouse and human type n channel. From membranes of T cell lines derived from both mouse (SAK 8 cell line) and human (Jurkat cell line), the type n channel can be immunoprecipitated following either surface labeling with 125I or metabolic labeling with 32P. The apparent molecular mass of the immunoprecipitated type n channel is approximately 65 kDa, significantly greater than that of the 58-kDa in vitro translated product, and suggestive of post-translational modification events. Phosphoamino acid analysis of the metabolically labeled Jurkat type n channel reveals phosphorylation of serine residues exclusively. In vitro studies also describe the ability of both protein kinase A and protein kinase C to phosphorylate the Jurkat type n channel. The former kinase also appears to phosphorylate a 40-kDa protein which co-immunoprecipitates with the type n channel. These data suggest that direct phosphorylation of the T lymphocyte type n potassium channel or its associated 40-kDa subunit may serve as a means by which channel activity is regulated.
在哺乳动物T淋巴细胞上发现的主要电压门控钾通道类型被称为n型通道。这种钾通道具有独特的功能特性，使其有别于其他种类的钾通道，包括在与T细胞激活相关的细胞事件发生过程中可能发挥的作用。作为表征n型通道特定生化特性的第一步，我们针对包含小鼠和人类n型通道特有的肽段区域的细菌融合蛋白制备了多克隆抗血清。从小鼠（SAK 8细胞系）和人类（Jurkat细胞系）来源的T细胞系膜中，在用125I进行表面标记或用32P进行代谢标记后，n型通道都可以被免疫沉淀。免疫沉淀的n型通道的表观分子量约为65 kDa，明显大于58 kDa的体外翻译产物，这表明存在翻译后修饰事件。对代谢标记的Jurkat n型通道进行的磷酸氨基酸分析显示，仅丝氨酸残基发生了磷酸化。体外研究还描述了蛋白激酶A和蛋白激酶C对Jurkat n型通道进行磷酸化的能力。前一种激酶似乎还能使与n型通道共免疫沉淀的一种40 kDa蛋白发生磷酸化。这些数据表明，T淋巴细胞n型钾通道或其相关的40 kDa亚基的直接磷酸化可能是调节通道活性的一种方式。
