Gap junctions provide for the homeostasis of nearly every tissue by metabolically, chemically, and electrically coupling the cells into a functional syncytium. The heart is the only organ that requires gap junctions for rapid electrical transmission of the heartbeat that is essential to function for every second of life. Of the over twenty identified mammalian connexin genes, only four connexins, connexin40 (Cx40), connexin43 (Cx43), connexin45 (Cx45), and connexin 31.9 (Cx31.9), are expressed in the heart. It is not fully known how the differential functional properties of Cx40, Cx43, Cx45, and Cx31.9 contribute to the normal function of the heart. This project seeks to understand how the connexins form an intercellular ion channel and the molecular basis for the selective ionic conductance and permeability differences between Cx40 and Cx43. Utilizing a structure-function mutagenesis approach, we will provide new detailed information about the contribution of the cardiac connexin cytoplasmic amino terminal (NT) domain residues make to the formation of the cytoplasmic pore and its blockade by endogenous intracellular polyamines. Many of these loci have been associated with disease-causing connexin mutations such as oculodigitodental dysplasia (ODDD) and ascertaining the function of these specific amino acid loci sights will provide further insights into the possible disease-causing consequences of these connexin mutations. We have also produced novel connexin-mimetic NT peptides that exhibit specific inhibitory properties of heteromeric Cx43-Cx40 channel function or the blockade of Cx40 gap junctions by spermine. In a new final aim of this proposal, we demonstrate the functional significance of recently predicted Cx43 calmodulin (CaM) binding domain to the calcium regulation of myocardial gap junctions formed predominantly by Cx43 and Cx40. We further propose to examine the effect that ODDD mutations occurring on the second half of the connexin cytoplasmic loop (CL) domain might have on the newly identified intracellular Ca2?dependent gating mechanism of Cx43. Together, these connexin structural and functional approaches will provide new mechanistic insights into the contributions that the NT and CL domains make to the channel conductance, selective ion permeability, selective polyamine blockade, calcium open-closed gating, and functional consequences of genetically-linked human connexin disease mutations such as ODDD. These multiple mechanistic approaches to Cx40 and Cx43 gap junction channel function will identify new sub domains on the connexin molecules as possible selective therapeutic targets for future drug development. PUBLIC HEALTH RELEVANCE: The synchronized contraction of the heartbeat depends on the rapid electrical activation of the cardiac muscle carried by gap junctions. Connexin40 (Cx40) and connexin43 (Cx43) form the vast majority of these atrial and ventricular gap junctions. This project will determine the how these two proteins conduct electrical current, the molecular basis for their different conductance properties, and their differential block by intracellular polyamines, how these cell-to-cell channels are regulated open or closed by intracellular calcium and calmodulin, and effect that disease-causing mutations have on these vital functional properties. These combined experimental approaches will identify new molecular targets on the connexin proteins for the possible pharmacological modulation of cardiac electrical communication by new therapeutic agents.