HUMAN CARDIAC NA+ CHANNELS--EFFECTS OF PHOSPHORYLATION

虽然诱发心律失常的因素主要是 未知，毫无疑问，急性自主神经刺激是 会引起过敏。电压门控钠通道是一个关键的 心脏中正常和异常传导的决定因素，以及 具有细微功能障碍的突变通道可导致危及生命的 心律不齐目前，肾上腺素能刺激的作用， 激活蛋白激酶A和C，对心脏钠电流 仍然存在争议。本提案的目的是测试 蛋白激酶激活调节细胞功能假说 人心脏钠通道的磷酸化 α亚基电生理学研究将表征 蛋白激酶A和C刺激对主要细胞的功能影响 人心脏电压门控钠通道 在两种不同的异源系统中表达（非洲爪蟾 卵母细胞和哺乳动物细胞系）。大量初步数据 这表明两种激酶的激活都会引起显著的影响， HH 1电流将进行更多的研究， 激酶对hHI影响的生化基础。免疫沉淀 技术将用于确定通道是否直接 在体外和体内条件下通过激酶磷酸化。到 阐明假定的磷酸化位点的功能作用， hHI序列，实验将使用两种嵌合的 人心脏-骨骼肌通道和定点突变， 来精确定位通道序列的区域， 氨基酸的功能和生物化学的重要性。最后 蛋白激酶C同工型存在于细胞表达 将识别所使用的系统和人类心肌。的影响 hHI细胞系统中缺乏的人类同种型 然后将进行测试，以更全面地了解hHI的相关性 对人类心脏的调节。从这些中获得的知识 研究将提高我们对自然和分子的理解 人心脏钠通道调节的基础和条件 这可能会促进或抑制心律失常， 渠道功能的变化。

While the factors which precipitate cardiac arrhythmias are largely unknown, there is no doubt that acute autonomic stimulation is arrhythmogenic. Voltage-gated sodium channels are a critical determinant of normal and abnormal conduction in the heart, and mutant channels with subtle dysfunction can cause life-threatening arrhythmias. At present, the effects of adrenergic stimuli, which activate protein kinase A and C, on the cardiac sodium current remain controversial. The goal of this proposal is to test the hypothesis that protein kinase activatiOn modulates function of human cardiac sodium channels by phosphorylation of the channel alpha-subunit. Electrophysiologic studies will characterize the functional effects of protein kinase A and C stimulation on the major voltage-gated sodium channel in human heart, hHI, with the channel expressed in two different heterologous systems (Xenopus Laevis oocytes and a mammalian cell line). Substantial preliminary data demonstrate that activation of both kinases causes significant effects on hH1 current. Additional studies will be undertaken to define the biochemical basis for kinase effects on hHI. Immunoprecipitation techniques will be used to determine if the channel is directly phosphorylated by kinase under in Vitro and in vivo conditions. To elucidate the functional role of putative phosphorylation sites in the hHI sequence, experiments will be performed using both chimeric human heart-skeletal muscle channels and site-directed mutagenesis, to pinpoint regions of channel sequence and, ultimately, individual amino acids of functional and biochemical importance. Finally, the protein kinase C isoforms present in both the cellular expression systems used and human myocardium will be identified. The effects of human isoforms which are lacking in the cellular systems on hHI will then be tested to understand more fully the relevance of hHI modulation for human heart. The knowledge gained from these studies will improve our understanding of the nature and molecular basis of sodium channel modulation in human heart, and conditions which could conceivably promote or suppress arrhythmias due to changes in channel function.