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）形成，每个半通道（或连接子）来自每个并置细胞。半通道在ER或ER后区室中形成，并以很少的定位插入表面。它们在表面扩散，与并置膜中的伴侣对接，然后打开。非交界表面半通道大部分是封闭的，鉴于其大的传导性和相对非特定的渗透性，这是合理的。然而，一些半通道在生理或病理条件下打开。Cx43是许多组织中普遍存在的连接蛋白，其半通道开放的特征很少。本申请提出改善该缺陷。技术包括染料摄取的延时记录、单通道活性的记录、通过生物素化/ NeutrAvidin pull down分离表面Cx43、Western印迹分析和定点诱变。目的1是分析作为电压和还原二价离子浓度的函数的Cx43半通道的门控。目的2是通过氧化剂和还原剂以及通过代谢抑制（MI）、影响电压依赖性和开放概率的治疗来鉴定Cx43的修饰位点。Ml和NO供体诱导Cx43的S-亚硝基化，这种作用被还原剂如DTT阻断。去除所有细胞质半胱氨酸的截短大大减弱了代谢抑制的作用。现在我们将单独和联合去除半胱氨酸。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是定位酸化关闭的门与H3 O+结合位点的相对位置。初步数据表明，在细胞质侧的网站的门，即弱，膜渗透酸快速和可逆地阻止半通道，和强，相对膜不渗透酸不阻止半通道，直到他们打开。目的4是将这些数据扩展到培养和脑切片中的星形胶质细胞。我们的初步数据表明，文化的高度相似性。这些研究应该澄清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CNS中的局灶性和全脑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尚未探索。

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.