Basis of Voltage & Chemical Gating in Connexin Channels

间隙连接通道，连接蛋白的十二聚体，提供相邻细胞细胞质之间的直接偶联，每个细胞提供一个六聚体连接子，或半通道。连接蛋白基因突变与疾病相关，包括x -连锁腓骨肌萎缩症（CMTX），一种由连接蛋白缺陷引起的周围神经病变。申请人旨在描述由人连接蛋白37 （hCx37）和牛连接蛋白44 （Cx44）形成的传导半通道的药理学特征，它们在门控方面具有不同的特性。我们提出了实验来验证这一假设，即庚醇、氟烷和二价阳离子对这些半通道的阻断应该与完整的细胞间通道相同，这表明间隙连接解耦是由于半通道阻断。电压对二价块的影响也将被评估。hCx37的单通道电流将决定半通道电流在生理条件下是否有作用。我们将使用本地制剂来进一步探索这种可能性。最后，将制作含有hCx37和U44结构域的嵌合通道，以确定门控的分子基础。化学门控特性将在交换细胞质环和羧基末端结构域的嵌合半通道中进行评估，这些区域涉及连接蛋白的化学门控。电压门控特性将在交换细胞外环域的嵌合体形成的半通道和细胞间通道中进行研究。位点导向突变体也可以在与门控有关的区域中产生。一旦门控的分子基础被确定，特定残基可能被用作基本药物设计的靶标，以治疗CMTX等疾病。

Gap junction channels, dodecamers of connexin proteins, provide direct coupling between the cytoplasm of adjacent cells, each cell contributing a hexameric connexon, or hemichannel. Mutations in connexin genes are associated with diseases including X-linked Charcot-Marie-Tooth disease (CMTX), a peripheral neuropathy caused by defects in connexin32. The applicant aims to characterize the pharmacology of conducting hemichannels formed by human connexin37 (hCx37) and bovine connexin44 (Cx44), which have diverse properties with respect to gating. Experiments are proposed to test the hypothesis that block of these hemichannels by heptanol, halothane, and divalent cations should be the same in hemichannels as in intact intercellular channels, indicating that gap junctional uncoupling is due to hemichannel block. Voltage effects on divalent block will also be assessed. Single channel currents for hCx37 will determine whether the hemichannel currents have a role under physiological conditions. Native preparations will be used to further explore this possibility. Finally, chimeric channels, containing domains of the hCx37 and U44, will be made to determine the molecular basis of gating. Chemical gating properties will be assessed in chimeric hemichannels that exchange the cytoplasmic loop and carboxy-terminal domains, regions implicated in chemical gating of connexins. Voltage gating properties will be examined in hemichannels and intercellular channels formed by chimeras that exchange the extracellular loop domains. Site-directed mutants can also be made in the domains involved in gating. Once the molecular basis of gating is identified, specific residues may be used as targets for rationale drug design to treat illnesses like CMTX.