The mechanisms underlying gating of ion channels are not yet fully understood. The application of molecular genetic and biophysical techniques should lead to the description of the properties, organization and primary sequence of the protein domains that are responsible for the voltage dependence of gap junctions. The regulation of intercellular communication by voltage dependent gap junctions has been postulated to play a role in development, neural signalling and integration, and control of secretion. In vertebrates, gap junction proteins are known to be encoded by a small gene family that shares no extensive sequence homology with other ion channels. Several gap junction proteins (connexins) for which cloned DNA are available have been shown to form channels with different voltage sensitivities and kinetics, but the divergence in their primary protein sequence is sufficient to prevent the identification of regions that are required for the expression of voltage dependence. We will focus our initial investigations on two vertebrate connexins Cx26 and Cx32. Gap junctions formed from homopolymers of Cx26 differ markedly in the form and types of voltage dependence when they are compared to junctions formed by homopolymers of the closely related protein, Cx32. Heterotypic channels resulting from the union of Cx26 hemichannels with Cx32 hemichannels are unique in that they display junctional currents that rectify with a fast time course when transjunctional voltages, Vj, are applied. This fast Vj dependent rectification is similar to that described for some electronic synapses formed by gap junctions in the nervous system. We have developed a new procedure for the formation of gene chimeras that is not dependent on the existence of sequence homology between the two domains. We will use this procedure to determine the protein sequences that are responsible for the differences in voltage dependence of Cx26 and Cx32 and the fast rectification of heterotypic channels by examining the properties of channels formed by the expression these chimeras in pairs of Xenopus oocytes. The role of identified protein domains in the process voltage dependent gating can be inferred from biophysical analyses and tested lines the channel lumen and forms a gate that regulates ion flow. If this hypothesis is verified, the relationship between this domain and other regions of the molecule that function in the expression of voltage dependence will be explored. In the long term these studies should provide an account of the molecular mechanisms that underlie the process of voltage gating of gap junctions. The descriptions of such molecular mechanisms may have applicability to gating of other voltage dependent of ion channels and the should provide information concerning the relationship between protein structure and its function.
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