Molecular domains determining gap junction channel properties

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Background***Gap junction channels are specialized membrane pore structures between cells, allowing ions and small signalling/metabolic molecules in one cell to pass on to the neighbour cells. The building blocks for gap junction channels are a group of transmembrane proteins called connexins (Cxs). 20 different types of connexins are identified in the rodent genome. Six connexins oligomerize to form a cylinder-shaped half channel known as a hemichannel. Docking of two hemichannels forms a complete gap junction channel. Communication through gap junction channels is essential for synchronized, coordinated cellular activities and exchanging of small molecules between cells in tissues. The gap junction channel pore size, the switch control for opening/closing, and the modulations by chemicals are different depending on the connexin subtypes that organize the channel. The molecular domains responsible for these channel properties are largely unknown.***Objectives***This research program aims to reveal the critical domains in connexins responsible for the gap junction channel pore size and gating controls, by voltage and/or chemicals, in the gap junctions of the neuronal connexin (Cx36), the eye lens connexin (Cx50) and the inner ear connexin (Cx26).***Approaches***To evaluate the role of the critical domains, we will use molecular techniques to engineer cDNA constructs with the candidate domains of Cx36 swapping the corresponding domains of Cx50 (or Cx26). Mutagenesis will be used to generate point mutations to evaluate the roles of individual amino acid residues in the channel properties. These chimeras and mutants will be introduced into gap junction deficient neuroblastoma (N2A) cells for functional tests. Dual patch clamp will be used to study voltage- and chemical-dependent gating and single channel conductance. Fluorescent proteins and immunolabeling with antibodies will also be used to study the localization of these chimera/mutant connexins. We will also study the structure model of Cx26 and homology structure models of Cx36 and Cx50 to reveal the structural basis of the functional changes of the chimeras/mutants. We have successfully used these approaches to identify the roles of the amino terminus on single channel conductance and transjunctional voltage-dependent gating of Cx50 and Cx36.***Significance***Gap junction channels are ubiquitous in various tissues and play important roles in development, growth, differentiation, and many physiological processes, including synchronized neuronal activities. How each connexin domain contributes to the whole gap junction, and the structure-function relationships for gap junction channels are not clear for Cx36, Cx50, Cx26 and closely related connexins. Yet the knowledge is crucial in understanding physiological functions of these channels in synchronized neuronal activities and possibly other synchronized physiological processes.**