Basis of Voltage & Chemical Gating in Connexin Channels

间隙连接通道，连接蛋白蛋白的十二聚体，提供相邻细胞的细胞质之间的直接偶联，每个细胞贡献六聚体连接外显子或半通道。连接蛋白基因的突变与包括X连锁腓骨肌萎缩症（CMTX）在内的疾病相关，这是一种由连接蛋白缺陷引起的周围神经病32。申请方旨在表征由人连接蛋白37（hCx37）和牛连接蛋白44（Cx44）形成的传导半通道的药理学，其具有不同的门控特性。实验提出测试的假设，这些hemichannels块庚醇，氟烷，和二价阳离子应该是相同的hemichannels在完整的细胞间通道，表明间隙连接解偶联是由于hemichannel块。还将评估电压对二价嵌段的影响。hCx37的单通道电流将决定半通道电流是否在生理条件下起作用。将使用天然制剂来进一步探索这种可能性。最后，嵌合通道，含有hCx37和U44的结构域，将确定门控的分子基础。将在嵌合半通道中评估化学门控特性，所述嵌合半通道交换细胞质环和羧基末端结构域，所述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结构域的嵌合体形成的半通道和细胞间通道中进行检查。也可以在设门的结构域中产生定点突变体。一旦门控的分子基础被确定，特定的残基可以被用作治疗CMTX等疾病的合理药物设计的靶点。

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.