Gating and Regulation of Connexin Hemichannels

连接蛋白半通道的门控和调节

• 批准号: 9402712
• 负责人: Jorge Enrique Contreras
• 金额: $ 39.38万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9402712
• 关键词: Acute; Arrhythmia; Binding; Biophysics; Blood Vessels; Brain; Brain Diseases; C-terminal; Cardiac; Cardiac Myocytes; Cardiac health; Cardiovascular Diseases; Cardiovascular Pathology; Cell Death; Cell membrane; Cells; Cessation of life; Complex; Connexin 43; Connexins; Cysteine; Cytoplasmic Tail; Data; Development; Disease; Disease model; Electrophysiology (science); Fluorescence Spectroscopy; Functional disorder; Gap Junctions; Goals; Heart; Heart Diseases; Hereditary Disease; Hyperactive behavior; Intercalated disc; Ions; Ischemia; Knowledge; Lateral; Lead; Mass Spectrum Analysis; Mediating; Membrane; Metabolic; Methodology; Modification; Molecular; Muscle Cells; Muscular Dystrophies; Mutagenesis; Myocardial Infarction; NMR Spectroscopy; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrosation; Organ; Pathologic; Pathology; Peptides; Permeability; Pharmacologic Substance; Pharmacology; Play; Protein Isoforms; Proteins; Regulation; Research; Role; Signal Transduction; Site; Spectrum Analysis; System; Testing; Therapeutic; Tissues; Ventricular; Work; aqueous; connexin pore; design; extracellular; genetic approach; innovation; insight; interdisciplinary approach; novel; prevent; small molecule; therapy outcome; tool

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.

心脏中的连接蛋白43（Cx43）通常在其通常形成的闰盘处表达 连接通道（缝隙连接）。许多心脏疾病引起Cx43表达增加或异常， 这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 侧化Cx43在血浆中形成不成对的过度活跃的半通道（非连接通道）， 膜的这些Cx43半通道的过度开放与急性期细胞死亡相关。 局部缺血和心肌营养不良。细胞死亡被认为是由于电化学损失造成的 梯度和小的细胞质代谢物通过连接蛋白的大的和适度的选择性水孔 半通道我们和其他人有间接证据表明，一氧化氮（NO）打开Cx43 半通道，促进心脏和大脑的细胞功能障碍。此外，我们有证据表明， 在导致心肌细胞偏侧化的条件下，Cx43被S-亚硝化。的分子机制 导致Cx43半通道开放的NO尚未阐明，尽管这可能具有重要性， 心脏病和其他疾病。我们认为，一个主要的贡献者异常开放的非对接 半通道是Cx43 S-亚硝化。在这个提议中，我们将利用我们在连接蛋白方面的专长， 半通道生物物理学以鉴定NO门控Cx43半通道的机制。具体目标1将 确定NO是否通过S-亚硝化直接打开Cx43半通道并鉴定所涉及的残基。 特异性目的2将鉴定介导NO对Cx43的作用的精确胞质结构域相互作用 门控具体目标3将确定S-亚硝化的Cx43半通道是否促进细胞和组织 损害我们计划采用综合性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NMR光谱学、质谱学，以及 心脏病模型我们期望，我们的工作将有助于合理发展新的 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。 这将使我们能够实现特定的治疗结果，例如，减少过度活跃的开放 在心肌梗死期间的半通道，防止心律失常和组织损伤。