Molecular domains determining gap junction channel properties

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Molecular domains determining gap junction channel properties. 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 identical or different connexins oligomerize to form a cylinder-shaped half channel known as a hemichannel. Back-to-back docking of two hemichannels forms a complete gap junction channel. Communication through gap junction channels is essential for synchronized, coordinated cellular activities and exchanging 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 proposal aims to reveal the critical domains in connexin molecule responsible for the gap junction channel pore size and the gating controls by voltage and/or chemicals in the gap junctions of the neuronal connexin (Cx36) and the eye lens connexin (Cx50). 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. Mutagenesis will be used to generate point mutations to further evaluate the roles of individual amino acid residues in the channel properties. These chimeras and mutants will be transfected into gap junction deficient neuroblastoma (N2A) cells for functional tests. Functional investigation with dual patch clamp will be used to study voltage- and chemical-dependent gating and single channel conductance of the gap junction channels. Fluorescent proteins (GFP and RFP) and immunolabelling with antibodies will also be used to study the localization of these chimera/mutant connexins. We have successfully used these approaches to identify the roles of amino terminus in single channel conductance and transjunctional voltage-dependent gating of these two connexins (Xin et al, 2010; 2012). Significance Gap junction channels are ubiquitous in various tissues and play important roles in development, growth, differentiation and many physiological processes, including synchronized heart beat and 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 and many other connexins. Yet the knowledge is crucial in understanding of the physiological functions of these channels in synchronized neuronal activities and possibly other synchronized physiological processes.