The fundamental property of connexin channels is that they are the pathways for direct movement of cytoplasmic molecules between cells. In spite of considerable knowledge about connexin channels, the character and mechanism of their most salient property - selective permeability among signaling molecules - remains largely unknown. Which chemical signals pass though connexin channel, and how selectivity among them is achieved, are fundamental issues with far-reaching impact. We seek to define these processes. The channels formed by each of the approximately 20 varieties of connexin differ from one another with regard to molecular permeability. The importance of this specificity is demonstrated by the fact that every functional deletion of a connexin type produces a distinct pathology, such as neuronal demyelination, deafness, cardiac defects, cataracts or infertility. The pathologies that arise from altered connexin channel function must arise from abnormal molecular movement through connexin pores, whether in magnitude, regulation or molecular specificity. The proposed studies seek to define mechanism(s) by which connexin channels select among cytoplasmic signaling molecules. We also seek to further develop, characterize and utilize a class of open pore blockers of connexin channels that we have identified. The experiments address the following specific questions: Do connexin channels select among signaling molecules on the basis of specific molecular affinities and how is this achieved? What is the mechanism of pore block by cyclodextrins (CDs) and how can CDs be used to investigate pore structure? For both issues (selective permeability and block) we seek to identify the sites in the pore that are involved. Identification of such sites would be of key importance and will provide new information on the location of pore-lining parts of the protein. The work on the blockers will lead to their utilization as molecular tools to investigate connexin pore structure and function. The projects utilize a well-characterized reconstitution system to study native and heterologously-expressed connexin hemichannels to obtain information about connexin permeation selectivity and its structural basis that has been long desired, and unavailable by other means. The information obtained will inform cellular and physiological studies of intercellular communication in disease and development.
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