Gap junctions formed by Cx26 and Cx32 differ in molecular permeability and in sensitivity to transjunctional voltage. These differences will be exploited to define the molecular mechanisms that underlie voltage dependence and ionic fluxes of intercellular and membrane channels formed by proteins of the connexin gene family. In the long term, integration of data from biophysical, molecular genetic, NMR and computer modeling studies will lead to understanding of how conformational changes in this membrane protein and others dynamically relate to their function and how fixed charges modulate ion flux. In the proposed studies the pore-lining sequence of the Cx32 channel will be further defined and the atomic structure of the amino terminus of wild type and mutated Cx32 will be solved by high resolution NMR. The position of the voltage sensor and its relation to the transjunctional electric field will be explored. Studies are proposed to test the hypothesis that the conformational flexibility of a proline kink motif underlies the conformational changes required for voltage dependent gating. Studies are also proposed to distinguish between concerted and individual subunit gating models. Data from molecular and electrophysiological studies of ionic flux will be examined using the electrodiffusive model of Chen and Eisenberg to further define the roles of charged amino acids in determining permeation and selectivity of gap junction channels. X-linked Charcot-Marie-Tooth disease (CMTX) is caused by mutations of human Cx32, and nonsyndromic deafness by mutations of Cx26. Biophysical and molecular studies have shown that changes in permeability of Cx32 are likely to underlie the etiology of CMTX. The proposed studies will advance the basic scientific knowledge required to define the molecular basis of this and other connexin-related diseases, and the biological role of gap junctions.
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