STRUCTURE, FUNCTION, AND REGULATION OF CARDIAC GAP JUNCTION PROTEINS

氢离子被认为是生物化学的主要调节剂之一， 反应. 因此，活细胞中的功能过程可以是 细胞内pH值的变化而急剧改变。 心脏，细胞内酸化是已知的改变功能， 各种通道膜蛋白，包括缝隙连接通道。 在 事实上，有人提出，酸化诱导的解偶联在 心脏可能在恶性肿瘤的发展中起重要作用。 心肌梗死后室性心律失常。 我们建议 表征参与心脏pH门控的分子机制 缝隙连接蛋白43（Cx43）。我们实验室的最新结果 结合分子生物学和电生理学 技术已经表明，Cx43的pH敏感性可以改变， 通过在氨基酸257处截短羧基尾，或通过取代 组氨酸95具有不同电荷和极性氨基酸。 我们的数据 因此，我已经表明，Cx43蛋白至少有两个区域 与pH门控有关。 数据进一步表明， 这两个区域的唯一负责通道关闭，因为 一个突变足以部分阻止pH诱导的解偶联 尽管另一个存在。 我们将结合使用光学， 电生理学和分子生物学技术， 表达的Cx43通道pH门控的结构和功能研究 非洲爪蟾卵母细胞。 具体目标是：1）本地化， 确定Cx43羧基尾部区域的精确限值 参与连接电导的pH门控，并确定 该区域中的特定残基是否在酸化中发挥作用- 诱导解偶联; 2）表征组氨酸95在 Cx43的pH门控确定了Cx43的其他残基 细胞质环可能在酸化诱导的解偶联中发挥作用; 3） 确定负责pH门控的羧基尾区是否 可以作为独立于连接蛋白的单独结构域发挥功能;并且 4)以表征pH门控反应的动力学，并确定 钙作为发展中的限速步骤的可能作用 酸化诱导的Cx43通道解偶联。 总体而言，这些 实验应该提供更好的理解分子 介导酸化诱导的Cx43通道关闭的机制。 最终，我们的结果应该有助于阐明 心脏中细胞间通讯的pH调节。

Hydrogen ions are recognized as one of the major regulators of biochemical reactions. As such, functional processes in living cells can be drastically altered by changes in intracellular pH. In the case of the heart, intracellular acidification is known to alter the function of various channel membrane proteins, including gap junctional channels. In fact, it has been proposed that acidification-induced uncoupling in the heart may play an important role in the development of malignant ventricular arrhythmias following myocardial infarction. We propose to characterize the molecular mechanisms involved in pH gating of the cardiac gap junction protein, connexin43 (Cx43). Recent results from our laboratory using a combination of molecular biological and electrophysiological techniques have shown the that pH sensitivity of Cx43 can be altered either by truncation of the carboxyl tail at amino acid 257, or by replacing histidine 95 with amino acids of different charge and polarity. Our data have therefore shown that there are at least two regions of th Cx43 protein that are involved in pH gating. The data further suggest that neither one of these two regions is solely responsible for channel closure, since mutation of one is enough to partially prevent pH-induced uncoupling despite the presence of the other. We will use a combination of optical, electrophysiological and molecular biological techniques to carry out structure function studies of pH gating of Cx43 channels expressed in Xenopus laevis oocytes. The specific aims are; 1) To localize and establish the precise limits of the region of the carboxyl tail of Cx43 that is involved in pH gating of junctional conductance, and determine whether specific residues in that region play any ole in acidification- induced uncoupling; 2) to characterize the role played by histidine 95 in the pH gating of Cx43, nd identify the role that other residues of the cytoplasmic loop may play in acidification-induced uncoupling; 3) to determine whether or not the carboxyl tail region responsible for pH gating can function as a separate domain, independent of the connexin protein; and 4) to characterize the kinetic of the ph gating reaction, and identify the possible role of calcium as the rate-limiting step in the development of acidification-induced uncoupling of Cx43 channels. Overall, these experiments should provide a better understanding of the molecular mechanisms mediating acidification-induced closure of Cx43 channels. Ultimately, our results should help to elucidate the molecular bases for the pH regulation of intercellular communication in the heart.