Molecular Modulation of Cardiac Na+ Channel Function

细胞信号系统的激活不仅被认为是质膜蛋白功能的潜在调节器，而且还被认为是细胞膜内外运输的潜在调节器。在心脏中，钠离子通道对正常电生理和危及生命的心律失常的发生都起着关键作用。虽然心肌钠电流是由蛋白激酶(PKA)调节的，但这种调节的性质仍有争议，其机制尚不清楚。基于我们已经产生的数据，本研究的目的是验证PKA激活调节心肌Na+通道转运的假设，并探讨其分子机制。在表达人心肌Na+通道HHL的细胞和大鼠心肌细胞中，将使用电生理和生化方法、免疫定位和荧光通道融合蛋白来显示活细胞内的运输，以确定PKA激活对Na+电流、质膜通道密度和细胞分布的影响。我们的初步数据表明，I-II域间连接子是促成这一效应的一个区域。为了研究基础状态和刺激状态下通道循环所需的结构和分子组成，将探索该区域以及与其他蛋白质(例如，具有PDZ结构域的蛋白质)相互作用的羧基末端的结构-功能假说。通过使用参与PKA效应的通道区域(S)的过度表达和结合研究，将寻找相互作用或适配蛋白的证据。为了探索调节Na+通道转运的细胞机制，我们将研究特定分子成分在胞吐和胞内途径中的作用。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，并可能确定调节心肌Na+电流的新策略。

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.