Molecular Modulation of Cardiac Na+ Channel Function

细胞信号系统的激活越来越多地被认为不仅是质膜蛋白功能的潜在调节剂，而且是它们进出细胞膜的运输的潜在调节剂。 在心脏中，Na+通道对于正常电生理学和危及生命的心律失常的发生都是关键的。 虽然心脏Na+电流是由蛋白激酶（pKA）调节的，但这种调节的性质是有争议的，其机制尚不清楚。基于我们已经获得的数据，本研究的目的是验证PKA激活调节心脏Na+通道运输的假设，并探讨其分子机制。 在表达人心脏Na+通道hH 1的细胞和大鼠心室肌细胞中，pKA激活对Na+电流、质膜通道密度和Na+通道的细胞分布的影响将使用电生理学和生物化学方法、免疫定位和荧光通道融合蛋白来确定，以可视化活细胞中的运输。我们的初步数据表明I-II结构域间连接子是促成这种效应的一个区域。 为了研究在基础和刺激状态下通道再循环所需的结构和分子组分，将探索该区域以及羧基末端的结构-功能假设，羧基末端与其他蛋白质相互作用（例如，具有PDZ域的那些）。 相互作用或衔接蛋白的证据将通过使用PKA效应中涉及的通道区域的过表达和结合研究来寻找。 为了探索介导Na+通道调控运输的细胞机制，将研究特定分子组分在胞吐和胞吞途径中的作用。 从这些研究中获得的知识将提高我们对心肌细胞调节Na+通道活性的分子机制的理解，并可以确定新的策略来调节心脏Na+电流。

Activation of cellular signaling systems is increasingly recognized as a potential modulator of not only the function of plasma membrane proteins, but also their trafficking into and out of the cell membrane. In the heart, Na+ channels are pivotal to both normal electrophysiology and the genesis of life-threatening cardiac arrhythmias. While cardiac Na+ current is modulated by protein kinase(pKA), the nature of this regulation is controversial and the mechanisms unknown. Based on data we have generated, the goal of this research is to test the hypothesis that PKA activation regulates trafficking of cardiac Na+ channels and to investigate the molecular mechanisms. In cells expressing the human cardiac Na+ channel hHl and in rat ventricular myocytes, the effects of pKA activation on N a+ current, plasma membrane channel density, and the cellular distribution of Na+ channels will be determined using electrophysiologic and biochemical methods, immunolocalization, and a fluorescent channel fusion protein to visualize trafficking in living cells. Our preliminary data have implicated the I-II interdomain linker as a region contributing to this effect. To investigate the structural and molecular components required for channel recycling under basal and stimulated states, structure- function hypotheses will be explored for this region as well as the carboxy terminus, which interacts with other proteins (e.g., those with PDZ-domains). Evidence for interacting or adaptor proteins will be sought by both overexpression and binding studies using the channel region(s) involved in the PKA effect. To explore the cellular mechanisms that mediate regulated trafficking of Na+ channels, the role of specific molecular components in both exocytotic and endocytotic pathways will be investigated. The knowledge gained from these studies will improve our understanding of the molecular mechanisms whereby cardiac cells regulate Na+ channel activity and could identify novel strategies to modulate cardiac Na+ currents.