Cx43 Hemichannels: Gating, Modification and Function

描述（由申请人提供）：间隙连接由两个半通道（或连接子）组成，每个半通道来自相对的细胞。半通道在内质网或内质网后腔室中形成，并以很少的定位插入表面。它们扩散到表面，在一个相对的膜上与伴侣对接，然后打开。非结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特异性的磁导率，这是合理的。然而，一些半通道在生理或病理条件下开放。Cx43是许多组织中普遍存在的一种连接蛋白，在半通道开放方面的特征很少。本应用程序旨在改善这一缺陷。技术包括染料摄取的延时记录，单通道活性记录，生物素化/ NeutrAvidin拉下分离表面Cx43， Western blot分析和定点诱变。目的1是分析Cx43半通道的门控作为电压和还原二价离子浓度的函数。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处理来确定Cx43的修饰位点。Ml和NO供体诱导Cx43的s -亚硝基化，这一作用被DTT等还原剂阻断。切除所有的细胞质半胱氨酸大大降低了代谢抑制的效果。现在我们将把半胱氨酸单独或联合去除。我们将检测表面半通道的磷酸化（通过生物素化分离），以确定与代谢抑制作用的关系。几个位点的磷酸化调节门控，但不影响对Ml的反应。目的3是定位由h30 +结合位点酸化关闭的门的相对位置。初步数据表明，位于通道细胞质一侧的位置，即弱的膜渗透酸快速且可逆地阻断半通道，而强的相对膜不渗透酸在半通道打开之前不会阻断半通道。目的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高度相似。这些研究将阐明在生理和病理条件下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，其中星形胶质细胞的作用在很大程度上仍未被探索。

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.