Structural Analysis of the Drosophila Innexin ShakB: Role of the N-Terminus in Rectifying Electrical Synapses

OF THESIS Structural Analysis of the Drosophila Innexin ShakB: Role of the N-Terminus in Rectifying Electrical Synapses Gap junction channels mediate direct intercellular communication in all multicellular animals. They are comprised of the connexin family of proteins in vertebrates and the innexin family in prechordates. Connexins and innexins share many functional and structural similarities as orthologous proteins. Both types are capable of forming electrical synapses. Rectifying junctions are specialized electrical synapses found in neural systems that control escape responses. It has been shown that heterotypic gap junction channels mediate asymmetric properties of rectifying junctions. Shaking B N+16 and Shaking B lethal are variants of the ShakB locus in Drosophila and the organization of these innexins into heterotypic junctions underlies electrical rectification in the Giant Fiber System. The goal of this study was to further explore molecular mechanisms of rectification by establishing the role of the N-terminus. After creating a series of deletions and chimeric proteins in which the N-terminus of the innexin Shaking B Lethal was modified, proteins were characterized in paired Xenopus oocytes and analyzed electrophysiologically. Deletion of the N-terminus of Shaking B lethal resulted in loss of function. Replacing the N-terminus with that of Shaking B N+16 produced a chimeric protein that formed rectifying junctions when paired with wildtype, thus demonstrating that the Nterminus of innexins is crucial for channel function and plays a key role in rectification. The chimera gated symmetrically with characteristics similar to those of ShakB L, when paired homotypically, providing insight into the mechanism of voltage gating. State University of New York College at Buffalo Department of Biology Structural Analysis of the Drosophila Innexin ShakB: Role of the Amino-Terminus in Rectifying Electrical Synapses A Thesis in Biology by William D. Marks Submitted in Partial Fulfillment Of the Requirements For the Degree of Master of Arts May 2012