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.
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