Gap junctions formed by Cx26 and Cx32, although closely related in sequence, display significant differences in sensitivity to transjunctional voltage (vi) and in single channel conductance. These differences provide a means to define the molecular mechanisms that underlie voltage dependence and ion permeation of intercellular channels formed by members of the connexin gene family. The X-linked form of Charcot-Marie-Tooth disease (CMT-X) appears to result from """"""""loss of function"""""""" mutations in human Cx32. The structure-function studies proposed will provide insight into the molecular basis of this disease and will provide information that is required to define the biological roles of gap junctions. In the long-term, the integration of the results of the proposed biophysical, molecular genetic and computer modelling studies will permit the construction of atomic resolution models of junctions and will further our understanding of the relationship between the structure of transmembrane proteins and their functional properties. The proposed studies of slow V-j-dependent gating should identify other components of the gap junction voltage sensor. It has been proposed that the inherent structural flexibility of proline kinks commonly found in transmembrane domains of receptors plays an important role in the mechanism of signal transduction. Studies are proposed to examine if a similar mechanism underlies the reported ability of a conserved proline residue to function as a """"""""transduction element"""""""" in voltage gating of gap junctions. Studies are proposed to refine structural models of the N-terminus of Cx32 and other Group I gap junctions. These should explain how CMT-X mutations that map to this domain have altered intercellular communication. Studies described in this proposal indicate that the fast electrical rectification of Cx32/Cx26 junctions, which resembles the properties of rectifying electrical synapses found in the central nervous system of vertebrates, results from differences in ion permeation of the two connexins. A permeation barrier model is presented that accounts for the observed rectification. Single channel studies are proposed that will refine this permeation barrier model and establish the role of specific amino acids to the formation of barriers and """"""""selectivity filters"""""""". Gene chimeras of Cx26 and Cx32 are identified that should lead to the description of the protein domains that form the ion conduction path. A new CMT-X mutation, humCx32S26L is described that forms functional channels characterized by significant reductions in unitary conductance. Studies are proposed to further examine changes in permeation caused by this and other CMT-X mutations that may form functional gap junction channels. These studies should define the molecular basis of CMT-X disease.
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