Cx43 Hemichannels: Gating, Modification and Function

描述(申请人提供)：缝隙连接由两个半通道(或连接子)组成，每个相对的细胞一个。半通道形成于内质网或内质网后室，并插入表面，几乎没有定位。它们在表面扩散，与对置的膜中的伴侣对接，然后打开。非连接表面半通道大部分是封闭的，考虑到它们的大电导和相对非比渗透性，这是合理的。然而，有些半管在生理或病理条件下开放。Cx43是一种广泛存在于多种组织中的连接蛋白，但在半通道开放方面却鲜有报道。这个应用程序建议改善这一缺陷。技术包括延时记录染料摄取，记录单通道活性，通过生物素化/中性亲和素下拉分离表面Cx43，Western印迹分析和定点突变。目标1是分析Cx43半通道的门控与电压和还原二价离子浓度的函数关系。目的2是确定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处理对Cx43的修饰位置。ML和NO供体可诱导Cx43的S亚硝化，该作用可被脱氧核糖核酸等还原剂阻断。截断除去细胞质中的所有半胱氨酸，大大减弱了代谢抑制的效果。现在我们将分别和组合去除半胱氨酸。我们将检测表面半通道的磷酸化(通过生物素化分离)，以确定与代谢抑制效应的关系。几个位点的磷酸化调节门控，但不影响对m1的反应。目标3是定位通过酸化关闭的门与H3O结合部位的相对位置。初步数据表明，门的细胞质一侧的位置，即弱的膜透性意味着酸迅速并可逆地阻断半通道，而强的相对膜的非通透性意味着酸在半通道打开之前不会阻挡半通道。目标4是将这些数据扩展到星形胶质细胞，无论是在培养中还是在脑切片中。我们的初步数据表明，在文化上有很高的相似性。这些研究应该阐明在生理和病理条件下对Cx43半通道开放的控制。Cx43是星形胶质细胞表达的主要连接蛋白；对代谢挑战的反应将与中枢神经系统局灶性和全局性缺血的临床状况有关，星形胶质细胞在其中的作用在很大程度上尚不清楚。

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.