Cx43 Hemichannels: Gating, Modification and Function

Cx43 半通道：门控、修改和功能

• 批准号: 7643110
• 负责人: MICHAEL V L BENNETT
• 金额: $ 32.68万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7643110
• 关键词: Acids; Acute; Affect; Antioxidants; Astrocytes; Binding Sites; Biological Assay; Biotinylation; Brain; C-terminal; Cell Line; Cells; Clinical; Communication; Connexin 43; Connexins; Connexon; Coupled; Cultured Cells; Cysteine; Data; Dependence; Derivation procedure; Diffuse; Docking; Dyes; Excision; Exhibits; Gap Junctions; Glucose; Glutathione; Golgi Apparatus; Heart Arrest; Image; In Vitro; Ions; Ischemia; Kinetics; Luciferases; Measurement; Measures; Mediating; Membrane; Membrane Potentials; Metabolic; Modification; Mutate; Myocardium; Oxidation-Reduction; Oxygen; Permeability; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Positioning Attribute; Probability; Protein Dephosphorylation; Reaction; Reducing Agents; Relative (related person); Research Personnel; Role; Side; Site; Site-Directed Mutagenesis; Slice; Solutions; Surface; Techniques; Time; Tissues; Titrations; Western Blotting; base; cell killing; deprivation; extracellular; gap junction channel; immunocytochemistry; luciferin; mutant; neutravidin; oxidation; prevent; research study; response; tool; uptake; voltage; voltage clamp

DESCRIPTION (provided by applicant): Gap junctions are formed of two hemichannels (or connexons), one from each of the apposed cells. Hemi- channels are formed in the ER or a post ER compartment and inserted into the surface with little localization. They diffuse over the surface to dock with a partner in an apposed membrane and then open. Non-junctional surface hemichannels are for the most part closed, which is reasonable in view of their large conductance and relatively non-specific permeability. However some hemichannels open in physiological or pathological conditions. Cx43, a prevalent connexin in many tissues, has been little characterized in respect to hemi- channel opening. This application proposes to ameliorate that deficiency. Techniques include time lapse recording of dye uptake, recording of single channel activity, isolation of surface Cx43 by biotinylation/ NeutrAvidin pull down, Western blot analysis and site directed mutagenesis. Aim 1 is to analyze gating of Cx43 hemichannels as a function of voltage and reduced divalent ion concentration. Aim 2 is to identify sites of modification of Cx43 by oxidizing and reducing agents and by metabolic inhibition (Ml), treatments that affect voltage dependence and open probability. Ml and NO donors induce S-nitrosylation of Cx43, an effect blocked by reducing agents such as DTT. Truncation that removes all cytoplasmic cysteines greatly atten- uates the effect of metabolic inhibition. Now we will remove the cysteines individually and in combination. We will assay phosphorylation of surface hemichannels (isolated by biotinylation) to determine relation to effects of metabolic inhibition. Phosphorylation at several sites modulates gating but does not affect responses to Ml. Aim 3 is to localize the relative position of the gate closed by acidification with the H3O+ binding site. Preliminary data indicate that the site on the cytoplasmic side of the gate, i.e. weak, membrane permeant acids rapidly and reversibly block the hemichannels, and strong, relatively membrane impermeant acids do not block hemichannels until they open. Aim 4 is to extend these data to astrocytes, both in culture and in brain slices. Our preliminary data indicate high degree of similarity in culture. These studies should clarify controls of Cx43 hemichannel opening in physiological and pathological conditions. Cx43 is the primary connexin expressed by astrocytes; responses to metabolic challenge will relate to the clinical conditions of focal and global ischemia in the CNS, where the role of astrocytes remains largely unexplored.

描述（由申请人提供）：间隙连接由两个半通道（或连接）组成，一个是来自每个相关单元的。半通道在ER或ER后室形成，并插入表面几乎没有定位。他们在表面上扩散，以与伴侣在拟议的膜上进行对接，然后打开。非界面的表面半通道在大多数情况下是封闭的，鉴于它们的大电导率和相对非特异性的渗透性，这是合理的。但是，有些半通道在生理或病理状况下开放。 CX43是许多组织中普遍存在的连接蛋白，在半通道开放方面几乎没有表征。该申请建议可以改善这种缺陷。技术包括染料摄取的延时记录，单个通道活性记录，通过生物素化/中性素降低，蛋白质印迹分析和位点定向诱变分离表面CX43。目的1是分析CX43半通道的门控为电压的函数，并降低了二价离子浓度。目的2是通过氧化和还原剂以及代谢抑制（ML），影响电压依赖性和开放概率的治疗方法来鉴定CX43修饰的位点。 ML和NO供体诱导CX43的S-亚硝基化，这种效应被降低剂（例如DTT）所阻断。去除所有细胞质半胱氨酸的截断极大地减轻了代谢抑制的作用。现在，我们将单独删除半胱氨酸。我们将测定表面半通道（通过生物素化分离）的磷酸化，以确定与代谢抑制作用的影响。在几个部位的磷酸化调节门控，但不会影响对ML的反应。 AIM 3是通过用H3O+结合位点酸化闭合门的相对位置。初步数据表明，门的细胞质侧的位点，即弱的，膜的胶原蛋白酸会迅速和可逆地阻止半渠道，强，相对较强的，相对膜的渗透酸不会阻止半冰川，直到它们打开。 AIM 4是将这些数据扩展到培养物和大脑切片中的星形胶质细胞。我们的初步数据表明培养中的相似性高度。这些研究应阐明在生理和病理条件下对CX43半通道开放的控制。 CX43是由星形胶质细胞表达的主要连接素。对代谢挑战的反应将与中枢神经系统中局灶性和全球性缺血的临床状况有关，在那里，星形胶质细胞的作用仍然在很大程度上没有探索。