Connexins and Heart Function

DESCRIPTION (provided by applicant): Gap junction (GJ) channels, which are multimers of connexin (Cx) proteins, mediate direct cell-cell transfer of ions and metabolites and are required for propagation of excitation in the heart. This project is based on our recent discovery of a new cardiac Cx (mCx30.2) and data showing that knockout of mCx30.2 accelerates conduction in the atrioventricular (AV) node and reduces the Wenckebach period. mCx30.2 is found to be expressed not only in the heart but also in many other organs and tissues including neurons. The experiments will combine immunohistochemical and biophysical characterization of Cx-based channels in cardiomyocytes and exogenously expressed in mammalian cell lines. Specific Aim 1 focuses on gating and permeability properties of GJ channels and unapposed hemichannels formed of mCx30.2. We will determine whether two gates, 'fast' and 'slow', are also present in mCx30.2 GJs. We will determine the extent to which mCx30.2 hemichannels influence metabolic exchange across the plasma membrane. In Specific Aim 2, we will determine gating and permeability properties of heterotypic gap junction channels formed from cardiac Cxs. To establish whether mCx30.2 exhibits a dominant negative effect on junctional communication and slows atrioventricular conduction, we will determine whether mCx30.2 together with other cardiac connexins can oligomerize into heteromeric connexons (hemichannels) and whether these connexons can form functional heteromeric hemichannels and cell-cell channels. To determine the domains underlying the distinctive biophysical properties of mCx30.2 channels, we will examine chimeras formed of mCx30.2 and Cx43. In Specific Aim 3, we will examine an expression pattern of mCx30.2 and other cardiac Cxs in the AV-nodal region and correlate these data with measurements of passive electrical properties, excitability, refractoriness and resting potential to find why deletion of mCx30.2 accelerates AV conduction and reduces the Wenckebach period. In isolated cardiomyocytes of wild type and transfected with mCx30.2, we will examine whether homotypic, heterotypic and heteromeric GJ channels containing mCx30.2 form and function. We will determine whether mCx30.2 hemichannels are functional in cardiomyocytes and whether they contribute to slow propagation of excitation in the AV node and protection of ventricles from over excitation during atrial tachyarrhythmia.