Graduation Date

Fall 12-14-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Paul L. Sorgen

Abstract

As critical mediators of cell-to-cell communication, gap junctions (GJs) are comprised of membrane channels that directly link the cytoplasm of adjacent coupled cells thereby allowing for the passage of ions, small metabolites, and secondary messengers. Each channel is formed by the apposition of two connexons from adjacent cells, each composed of six connexin (Cx) proteins. Each GJ channel functions to promote signal propagation and synchronization of cells and tissues in organs. Furthermore, GJs are essential for proper propagation of cardiac action potentials from one cell to the next, leading to the coordinated contraction and relaxation of heart muscle powering circulation. In diseased human hearts, the organization and expression of Cxs in the working myocardium is remodeled leading to impaired impulse propagation and risk of lethal arrhythmias. Diverse post-translational modifications (e.g., phosphorylation) and protein partner interactions, the majority of which involve the carboxyl-terminal (CT) domain, serve to regulate Cx function.

The CxCT domain serves a critical role in the regulation of all aspects of the Cx life cycle including trafficking, assembly, channel open states, as well as internalization and degradation. Importantly, the CT domain is the site at which the greatest degree of sequence divergence is observed between family members. A number of studies have highlighted that little conservation of regulatory mechanisms contained within the CT domain exists between family members. Furthermore, a clear understanding of the molecular determinants that influence different Cx channel properties between family members is lacking. Thus, investigation into the specific mechanisms that regulate each family member is essential to develop a complete picture of Cx biology and to develop novel therapeutics with which to modulate Cx function.

Previous work from our laboratory identified a novel feature of the Cx45CT domain, high-affinity (KD ~ 100 nM) dimerization. Here using a combination of biophysical, biochemical, and cell/molecular biology approaches we demonstrate that dimerization is essential for proper Cx45 turnover, localization, phosphorylation, and function. Additionally, we demonstrate that phosphorylation by three kinases that are dysregulated in heart disease alter the function of Cx45 to promote cell coupling and consequently may contribute to the pathogenic phenotype.

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