HUMAN CARDIAC NA+ CHANNELS--EFFECTS OF PHOSPHORYLATION

而引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 会导致心律失常。电压门控钠通道是一个关键 心脏正常和异常传导的决定因素，以及 具有微小功能障碍的突变通道可能会导致生命危险 心律不齐。目前，肾上腺素能刺激的作用 激活心肌钠电流上的蛋白激酶A和C 仍然存在争议。这项提案的目标是测试 蛋白激酶激活调节血管紧张素转换酶功能的假设 人心脏钠通道的磷酸化 阿尔法亚单位。电生理学研究将确定 刺激蛋白激酶A和C对大鼠心脏功能的影响 人心脏电压门控性钠通道，HHI与通道 在两个不同的异源系统(非洲爪哇)中表达 卵母细胞和哺乳动物细胞系)。大量初步数据 证明这两种酶的激活都会导致显著的影响 在HH1电流上。将进行更多研究，以确定 激酶对HHI影响的生化基础。免疫沉淀 将使用技术来确定通道是否直接 在体外和体内条件下被激酶磷酸化。至 阐明可能的磷酸化位点在细胞周期中的功能作用 HHI序列，实验将使用两个嵌合体 人类心脏-骨骼肌通道和定点突变， 以精确定位通道序列的区域，并最终确定个体 具有重要功能和生物化学意义的氨基酸。最后， 蛋白激酶C亚型存在于两种细胞表达中 所使用的系统和人体心肌将被识别。其影响 在HHI上的细胞系统中缺乏的人类亚型 然后将进行测试，以更全面地了解HHI的相关性 对人类心脏的调制。从这些活动中获得的知识 研究将提高我们对自然和分子的理解 人体心脏钠通道调制的基础和条件 可以想象的是，它可以促进或抑制由于 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.