Molecular domains determining gap junction channel properties

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Background Gap junction (GJ) 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 GJ channels are connexins. 20 different connexin genes are identified in the rodent genome. Six identical or different connexins oligomerize to form a hemichannel. Back-to-back docking of two hemichannels forms a whole GJ channel. Intercellular communication through GJ channels is essential for synchronized, coordinated cellular activities in many tissues and organs. The GJ channel pore size, the switch control for opening/closing, and the modulations by ions and chemicals are different depending on the connexin subtypes that organize the channel. The molecular domains and residues responsible for these channel properties are largely unknown. Long-term Objectives To reveal how neuronal and lens connexin (Cx36, Cx46, and Cx50) domains/residues and their interactions with chemicals regulate GJ channel properties. Short-term Objectives In this proposal we aim to identify the roles of pore-lining domains/residues of Cx36, Cx46, and Cx50 on their GJ channel properties, including single channel conductance, transjunctional voltage (Vj) and pH dependent gating. Approaches To evaluate the role of the critical domains and individual residues, we will use molecular biology techniques to engineer cDNA constructs with a candidate domain switched among Cx36, Cx46, and Cx50. Mutagenesis will be used to generate point variants to further evaluate the roles of individual amino acid residues. These cDNA construct of chimeras and variants will be transfected into connexin deficient N2A cells for functional tests. Dual patch clamp will be used to study voltage- and chemical-dependent gating as well as single channel conductance. Immunolabelling with connexin-specific antibodies or tagging fluorescent protein (GFP or RFP) at the carboxyl terminus will be used to study the localization of these connexin chimeras/variants. We have successfully used these approaches to identify the roles of amino terminus (NT) and the first extracellular domain (E1) in single channel conductance and transjunctional voltage-dependent gating of Cx50. Significance GJ channels are ubiquitous in various tissues and play important roles in development, growth, differentiation and many physiological processes. How each connexin domain or residue controls to its GJ channel properties are not clear. Recent studies with cryo-electron microscopy (CryoEM) revealed high resolution Cx46/Cx50 GJ structures with many novel interactions among domains, subunits, and local water/lipids. Yet the functional implications are not identified. The knowledge on structure-function relationship is crucial in understanding of the physiological functions of these channels in synchronized neuronal activities and the lens physiology.