MODULATION OF CONNEXIN CHANNEL ACTIVITY

描述(摘自申请人的摘要)： 通过缝隙连接的细胞间信号传递的生理学仍然是一个 神秘感。尽管在连接蛋白生物化学方面取得了相当大的进展，而且 遗传学，直接控制通道是开放还是开放的配体 关闭的都是未知的。互作细胞质因子的鉴定 直接与连接蛋白通道，以及它们如何调节通道活动，是 影响深远的根本性悬而未决的问题。 缝隙连接通道(由连接蛋白组成)是 离子和小分子的细胞间运动。它们的位置受到限制 他们的研究在原位进行；毛孔的两端是细胞内的，无法接触到 多数用于探索经络功能。由于对该通道的访问是通过 细胞质，很难确定直接作用于细胞质的因子 渠道，而不是通过蜂窝组件。 长期目标是理解这一途径的分子操作。 细胞间信号传递。该方法是研究细胞间隙连接蛋白通道。 可以很容易地探索它们的调制的重构系统。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， 表达载体将在一个特征良好的系统中进行研究，该系统将产生 细胞研究不能提供的信息。这些实验建立在初步的基础上 首次发现相互作用的化合物的研究 直接和非共价性地与连接蛋白通道一起调节其活性。 拟议的研究解决了这样的问题：什么是分子基础 质子化氨基磺酸对缝隙连接蛋白通道的作用 活动?高亲和力抑制作用的分子基础是什么？ CAMP对连接蛋白通道的高亲和力抑制作用 那cGMP呢？连接蛋白分子的哪些部分与这些化合物相互作用？为什么 这两种连接蛋白的反应是否不同？关于连接蛋白，我们可以学到什么 当这些化合物的衍生物被用作亲和力时的结构功能 试剂？ 通过对这个实验可达系统中连接蛋白通道的研究，一 希望了解细胞间信号传递的基本特性。差距 连接蛋白是如此广泛，以至于对连接蛋白通道活性的阐明 调制将在整个蜂窝和 发育生物学。已知的连接蛋白有18种以上。在人类中，基因 连接蛋白32的缺陷会导致周围神经病变，连接蛋白26的缺陷会导致 大部分为非综合征性耳聋。毫无疑问，许多其他综合征都在消失。 部分或全部源于连接蛋白通道功能的缺陷。这样的缺陷将 导致异常(即，更多或更少的)细胞间信号分子。 拟议的研究解决了这种情况可能发生的原因。

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.