Gating and Regulation of Connexin Hemichannels

心脏中的连接蛋白43(Cx43)通常在其通常形成的间盘表达 连接通道(缝隙连接)。许多心脏疾病导致Cx43表达增加或异常， 这会导致心肌细胞中蛋白质的偏侧化。我们的团队和其他人提供了证据 偏侧化的Cx43在血浆中形成不成对的高活性半通道(非连接通道) 薄膜。这些Cx43半通道的过度开放与急性期细胞死亡有关 缺血性和心肌营养不良。细胞死亡被认为是由于电化学作用的丧失所致。 梯度和小的细胞质代谢物穿过连接蛋白的大而适度的选择性水孔 半条腿。我们和其他人有间接证据表明，一氧化氮(NO)能打开Cx43 半脑沟，促进心脏和大脑的细胞功能障碍。此外，我们有证据表明，在 在导致心肌细胞偏侧化的条件下，Cx43被S亚硝化。其分子机制是 原因Cx43由NO引起的半通道开放尚未阐明，尽管这可能在 心脏和其他病理改变。我们认为，导致Undocked的异常打开的一个主要因素是 半通道为Cx43-S-亚硝化。在本提案中，我们将利用我们在连接蛋白方面的专业知识 半通道生物物理学，以确定无门Cx43半通道的机制。具体目标1将 确定NO是否通过S亚硝化直接开放Cx43半通道，并鉴定涉及的残基。 特定目标2将确定介导NO对Cx43影响的精确细胞质结构域相互作用 门控。特异靶3将确定S亚硝化的Cx43半核细胞是否促进细胞和组织 损坏。我们计划使用综合的多学科方法来实现这些具体目标 结合了电生理学、诱变、荧光和核磁共振光谱、质谱学和 心脏病模型。我们期待我们的工作将为新世纪的合理发展做出贡献。 针对参与异常半通道门控的特定连接蛋白结构域的药理学策略。 这将使我们能够达到特定的治疗结果，例如，减少多动的开放 心肌梗塞期间的半心肌梗死可预防心律失常和组织损伤。

Connexin 43 (Cx43) in the heart is normally expressed at the intercalated disk where it commonly forms junctional channels (gap junctions). Many cardiac diseases produce increased or aberrant expression of Cx43, which results in lateralization of the protein in cardiomyocytes. Our group and others have provided evidence that lateralized Cx43 forms unpaired hyperactive hemichannels (non-junctional channels) at the plasma membrane. Excessive opening of these Cx43 hemichannels is associated with cell death during acute ischemia and cardiac muscular dystrophy. Cell death is thought to result from the loss of electrochemical gradients and small cytoplasmic metabolites through the large and modest selective aqueous pore of connexin hemichannels. We and others have indirect evidence indicating that nitric oxide (NO) opens Cx43 hemichannels, promoting cellular dysfunction in the heart and brain. In addition, we have evidence that under conditions that cause lateralization in cardiomyocytes, Cx43 is S-nitrosated. The molecular mechanisms that cause Cx43 hemichannel opening by NO have not been elucidated, despite the importance this may have in cardiac and other pathologies. We propose that a major contributor to the aberrant opening of undocked hemichannels is Cx43 S-nitrosation. In this proposal, we will take advantage of our expertise in connexin hemichannel biophysics to identify the mechanisms by which NO gates Cx43 hemichannels. Specific Aim 1 will determine whether NO directly opens Cx43 hemichannels via S-nitrosation and identify residues involved. Specific Aim 2 will identify the precise cytoplasmic domain interactions that mediate the effects of NO on Cx43 gating. Specific Aim 3 will determine whether S-nitrosated Cx43 hemichannels promote cell and tissue damage. We plan to accomplish these specific aims using an integrative multidisciplinary approach that combines electrophysiology, mutagenesis, fluorescence and NMR spectroscopy, mass spectrometry, and a cardiac disease model. We expect that our work will contribute to the rational development of new pharmacological strategies to target the specific connexin domains involved in aberrant hemichannel gating. This will allow us to achieve specific therapeutic outcomes, for example, to decrease opening of hyperactive hemichannels during myocardial infarction preventing arrhythmias and tissue damage.