CELLULAR REGULATION OF POTASSIUM CHANNELS

电压依赖性钾通道在设定 自发性放电的模式和动作电位的形状 肌肉细胞和神经元。表达水平的变化和 钾通道的电特性是作为响应而发生的 荷尔蒙以及影响第二信使通路的药物 与蛋白激酶有关。这个项目将研究这些机制 它调节了两个不同的钾通道，它们的基因已经 分离出并命名为水貂基因和Kv1基因。两个基因 在心脏细胞和其他组织中都有表达。水貂RNA，当 注射到非洲爪哇卵母细胞中，产生缓慢激活的钾 其幅度因激活循环而大大增强的电流 AMP依赖的蛋白激酶。电子录音和生化 异源表达的正常和突变水貂通道的检测 然后将执行信元以确定该信道是否 直接磷酸化以及是否发生通道活动的调制 通过快速向质膜募集新的通道。 正常表达水貂RNA和蛋白质的哺乳动物细胞株将是 确定了该通道的生理作用和调节 在这样的牢房里接受调查。第二系列实验将是 与Kvl一起进行的，Kvl是Shaker家族的哺乳动物成员 蛋白质。尽管许多属于这个家族的通道基因 已经被确认，但还没有基因被证明能解释 正常细胞中钾电流的特定成分。混合逮捕 因此，将使用技术来确定Kv1蛋白是否 参与GH3细胞的电压依赖性钾电流，a 高水平表达Kv1基因的克隆性垂体细胞系。 电生理测量，结合生物化学研究 然后将使用与KV1特异的抗血清鉴定的KV1蛋白 研究KV1通道的短期和长期调节 荷尔蒙和第二信使。

Voltage-dependent potassium channels play a central role in setting the pattern of spontaneous firing and the shape of action potentials of muscle cells and neurons. Alterations in the level of expression and in the electrical characteristics of potassium channels occur in response to hormones, as well as to drugs that influence second messenger pathways linked to protein kinases. This project will investigate the mechanisms that regulated two different potassium channels whose genes have been isolated and have been termed the minK gene and the Kvl gene. Both genes are expressed in heart cells as well as in other tissues. MinK RNA, when injected into Xenopus oocytes, generates slowly activating potassium currents whose amplitude is greatly enhanced by activation of the cyclic AMP-dependent protein kinase. Electrical recordings and biochemical measurements on normal and mutant minK channels expressed in heterologous cells will then be carried out to determine whether the channel is phosphorylated directly and whether modulation of channel activity occurs through the rapid recruitment of new channels to the plasma membrane. Mammalian cells lines that normally express minK RNA and protein will be identified and the physiological role and regulation of the channel will be investigated in such cells. A second series of experiments will be carried out with Kvl, a mammalian member of the Shaker family of proteins. Although a number of channel genes belonging to this family have been identified, no gene has yet been shown to account for a specific component of potassium current in a normal cell. Hybrid arrest techniques will therefore be used to determine whether the Kvl protein contributes to the voltage-dependent potassium current in GH3 cells, a clonal pituitary cell line that expresses Kvl mRNA at high levels. Electrophysiological measurements, coupled with biochemical studies of the Kvl protein identified with Kvl-specific antisera, will then be used to study the short-term and long-term regulation of the Kvl channel by hormones and second messengers.