Gap junctions mediate the direct intercellular passage of nutrients and cellular metabolites and also underlie the spread of action potentials essential for synchronized muscle contraction in the heart. Gap junctions in the heart and endothelium are composed of the connexin family of proteins (Cx's) and there are at least twenty different connexin proteins encoded in the human genome. Cx40, Cx43 and Cx45 are expressed in the heart. Channel properties such as permeability and gating are determined by the sequence and structure of specific domains within each connexin. This project aims to provide new information about the membrane-spanning domains. These domains anchor the protein in the membrane, line part of the gap junction pore, and play critical roles in voltage gating. Currently, the role of transmembrane domain interactions in gap junctions are poorly understood. In other proteins such interactions play essential roles in the assembly of protein complexes and establishment of stable associations between protein domains.As the first critical step to understanding the role of these interactions in gap junctions, this project aims to identify regions of protein interaction within the membrane. Tryptophan will be substituted for one amino acid at a time under the premise that the large aromatic side-chain of tryptophan will be tolerated in regions facing the lipid environment but not regions of protein interaction. The functional effect of each amino acid substitution will be determined electrophysiologically using the Xenopus oocyte expression system. A complete scan of all four transmembrane domains will be carried out for Cx32 and Cx43. This detailed set of data will be used to assign specific domains to transmembrane helices shown in published structures and models of gap junction channels for the first time. In addition, to determine whether transmembrane domain architecture is conserved amongst connexins, and between connexins from different subgroups the tryptophan scanning approach will be applied to two other beta group connexins (Cx26, Cx31) and two alpha connexins (Cx40, Cx50). A more detailed analysis of voltage-gating parameters for each tryptophan mutant will follow and is expected to reveal segments that undergo conformational changes in response to transjunctional voltage. Overall the data is expected to provide a framework for future studies of the role of transmembrane domain interactions in connexin assembly and function.
|Program type||Scientist Development Grant|
|Effective start/end date||01/01/2008 → 12/31/2011|