MODULATION OF CONNEXIN CHANNEL ACTIVITY

描述（来自申请人摘要）： 通过缝隙连接的细胞间信号转导的生理学仍然是一个复杂的问题。 谜尽管在连接蛋白生物化学方面取得了相当大的进展， 遗传学，直接控制通道是否开放或 关闭是未知的。相互作用的细胞质因子的鉴定 直接与连接蛋白通道，以及它们如何调节通道活性， 影响深远的根本性未决问题。 间隙连接通道（由连接蛋白组成）是细胞间相互作用的调节途径。 离子和小分子的细胞间运动。他们的位置限制了 它们的原位研究;孔的两端都是细胞内的， 大多数用于探索通道功能。由于通道的访问是通过 细胞质，很难确定直接作用于细胞质的因子。 通道，而不是通过蜂窝组件。 长期目标是了解这一途径的分子运作 细胞间信号该方法是研究连接蛋白通道， 重组系统，其中它们的调制可以很容易地探索。渠道 由天然组织免疫纯化的连接蛋白32和连接蛋白26形成， 表达载体将在一个充分表征的系统中进行研究， 细胞研究无法提供的信息。实验建立在初步的 这些研究首次发现， 直接和非共价地与连接蛋白通道连接以调节它们的活性。 拟议的研究解决了以下问题： 质子化氨基磺酸盐如牛磺酸对连接蛋白通道的作用 活动？高亲和力抑制的分子基础是什么？ 通过cAMP高亲和力抑制连接蛋白通道 cGMP？连接蛋白分子的哪些部分与这些化合物相互作用？为什么 两种连接蛋白的反应不同吗关于连接蛋白可以学到什么 当这些化合物的衍生物用作亲和剂时， 试剂？ 通过研究连接蛋白通道在这个实验上可访问的系统， 希望了解细胞间信号传导的基本特性。间隙 连接是如此的普遍， 调制将对整个细胞产生深远影响， 发育生物学已知有18种以上的连接蛋白。在人类中，基因 连接蛋白32的缺陷导致周围神经病变，连接蛋白26的缺陷导致周围神经病变。 大部分非综合征性耳聋。毫无疑问，许多其他综合征茴香在 全部或部分来自连接蛋白通道功能的缺陷。这些缺陷将 导致异常（即，或多或少）细胞间信号分子。 拟议的研究讨论了这种情况是如何发生的。

DESCRIPTION (from applicant's abstract): The physiology of intercellular Signaling through gap junctions is still a mystery. In spite of considerable progress in connexin biochemistry, and genetics, the ligands that directly control whether the channels are open or closed are unknown. Identification of fhe cytoplasmic factors that interact directly with connexin channels, and how they modulate channel activity, are fundamental unsolved issues with far-reaching impact. Gap junction channels (composed of connexin) are regulated pathways for intercellular movement of ionsn and small molecules. Their location constrains their study in situ; both ends of the pore are intracellular, inaccessible to most used to explore channel function. Since access to the channel is via cytoplasm, it is difficult to identify factors that act directly on the channel, rather than via cellular components. The long-term goal is to understand the molecular operation of this pathway of intercellular signaling. The approach is to study connexin channels in a reconstituted system where their modulation can be readily explored. Channels formed by connexin32 and connexin26 immunopurified from native tissues and expression vectors will be studied in a well-characterized system that yields information that cellular studies cannot. The experiments build on preliminary studies that have identified, for the first time, compounds that interact directly and noncovalently with connexin channels to modulate their activity. The proposed studies address the questions: What is the molecular basis for the action of protonmated aminosulfonates such as taurine on connexin chanel activity? What is the molecular basis of the high-affinity inhibition of connexin channels by the high-affinity inhibition of connexin channels by cAMP and cGMP? What parts of connexin molecules interact with these compounds? Why do the two connexins respond differently? What can be learned about connexin structure-function when derivatives of these compounds are' used as affinity reagents? By study of connexin channels in this experimentally accessible system, one hopes to understand the fudamental properties of intercellular signaling. Gap junctions are so widespread that elucidation of connexin channel activity modulation will have profound consequences throughout cellular and developmental biology. There are over 18 known connexins. In humans, genetic defects in connexin32 cause a peripheral neuropathy, and in connexin26 cause a large fraction of nonsyndromic deafness. No doubt many other syndromes anise in toto or in part from defects in connexin channel function. Such defects will result in abnormal (i.e., greater or lesser) intercellular signaling molecules. The proposed studies address how this may occur.