CELLULAR REGULATION OF POTASSIUM CHANNELS

钾通道的细胞调节

• 批准号: 2186359
• 负责人: LEONARD K KACZMAREK
• 金额: $ 22.24万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186359
• 关键词: alternatives to animals in research; antisense nucleic acid; electrophysiology; gene expression; glucocorticoids; growth factor; messenger RNA; phosphorylation; potassium channel; protein kinase; protein kinase A; structural genes; tissue /cell culture; toad; voltage gated channel

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.

电压依赖性钾通道在设置 自发射击的模式和动作电位的形状 肌肉细胞和神经元。 表达水平和在 钾通道的电气特征发生 到激素以及影响第二通路途径的药物 与蛋白激酶有关。 该项目将调查机制 调节两个不同基因的钾通道 分离并被称为Mink基因和KVL基因。 这两个基因 在心脏细胞以及其他组织中表达。 Mink RNA，何时 注入爪蟾卵母细胞，生成缓慢激活钾 通过循环激活大大增强振幅的电流 AMP依赖性蛋白激酶。 电记录和生化 在异源表达的正常和突变膜通道上的测量 然后将执行单元格以确定通道是否为 直接磷酸化以及通道活性的调节是否发生 通过快速募集新通道到质膜。 通常表达Mink RNA和蛋白质的哺乳动物细胞系将是 确定的以及通道的生理作用和调节 在此类细胞中进行研究。 第二个实验将是 与KVL一起进行，他是摇摇酒家族的哺乳动物成员 蛋白质。 虽然许多属于这个家族的渠道基因 已经确定了，尚未证明任何基因可以解释 正常细胞中钾电流的特定成分。 混合逮捕 因此，技术将用于确定KVL蛋白是否 在GH3细胞中有助于电压依赖性钾电流 克隆垂体细胞系，表达高水平的KVL mRNA。 电生理测量，再加上生化研究 然后使用KVL特异性抗血清鉴定的KVL蛋白 研究KVL通道的短期和长期调节 激素和第二使者。