CELLULAR REGULATION OF POTASSIUM CHANNELS

电压依赖性钾离子通道在调节细胞膜电位中起着重要作用。 自发放电的模式和动作电位的形状 肌肉细胞和神经元。 表达水平的改变以及 钾离子通道的电特性发生在响应 激素以及影响第二信使途径的药物 与蛋白激酶有关。 本项目将研究 调节两种不同的钾离子通道， 分离并被称为minK基因和Kvl基因。 两个基因 在心脏细胞和其他组织中都有表达。 Mink RNA，当 注射到非洲爪蟾卵母细胞中， 电流的幅度大大增强激活循环 AMP依赖性蛋白激酶。 电子记录和生化 在异源细胞中表达的正常和突变的minK通道的测量 然后将执行单元以确定通道是否 直接磷酸化以及是否发生通道活性调节 通过快速募集新的通道到质膜。 正常表达minK RNA和蛋白质的哺乳动物细胞系将被 确定和生理作用和通道的调节将 在这样的细胞中进行研究。 第二个系列的实验将是 用Kvl进行，Kvl是Shaker家族的哺乳动物成员， proteins. 虽然属于这个家族的一些通道基因 已经被确定，还没有基因被证明可以解释一个 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Kvl蛋白是否 有助于GH3细胞中的电压依赖性钾电流， 高水平表达Kv1 mRNA的克隆垂体细胞系。 电生理测量，加上生物化学研究， 然后使用用Kv1特异性抗血清鉴定的Kv1蛋白 研究Kvl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.