Basis of Voltage & Chemical Gating in Connexin Channels

缝隙连接通道是连接蛋白的十二聚体，在相邻细胞的细胞质之间提供直接连接，每个细胞提供一个六角体连接蛋白或半通道。连接蛋白基因的突变与包括X连锁Charcot-Marie-Tooth病(CMTX)在内的疾病有关，CMTX是一种由连接蛋白32缺陷引起的外周神经病变。申请人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，这两种连接蛋白在门控方面具有不同的特性。实验结果表明，正庚醇、氟烷和二价阳离子对这些半通道的阻断作用与完整的细胞间通道相同，说明缝隙连接解偶联是由半通道阻断引起的。还将评估电压对二价块的影响。HCx37的单通道电流将决定半通道电流在生理条件下是否起作用。将使用本地制剂来进一步探索这一可能性。最后，将制作包含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管中进行评估，这些区域与连接蛋白的化学门控有关。电压门控特性将在半通道和由交换细胞外环域的嵌合体形成的细胞间通道中进行检查。也可以在与门控有关的结构域中进行定点突变。一旦确定了门控的分子基础，特定的残留物可能被用作治疗CMTX等疾病的基本药物设计的靶点。

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.