Cardiac electrical conduction depends on intercellular passage of ions through gap junction channels. Altered gap junction distribution and subunit protein (connexin, Cx) expression have been implicated in the pathogenesis of reentrant arrhythmias. Connexins are also crucial for normal development of the heart and for the function of non-excitable tissues, implying a role in the passage of larger solutes (like signaling molecules). Individual connexins form channels with different properties (including conductance, permeability, and gating). The major connexin in working myocardium is Cx43, but Cx37, Cx40, and Cx45 are also found in various cardiovascular cells. Multiple connexins frequently co-localize to identical gap junction plaques, and they can form hetero-oligomeric channels. Co-expression and formation of heteromeric channels between Cx43 and other connexins may be a major mechanism for modulating and regulating cardiovascular intercellular communication. 1. Biochemical studies will examine the molecular determinants of interactions between subunit connexin proteins. We hypothesize that specific sequences (likely the transmembrane domains) allow interactions between connexin subunits to form hemi-channels. We will use immunofluorescence, affinity chromatography with Ni2+NTA resins, and velocity sedimentation to examine oligomerization of different chimeric mutants to determine sequences involved in subunit interactions. A bacterial expression system (TOXCAT) will be used to define critical residues in the interacting transmembrane domains. 2. Physiological studies will examine the functional consequences of co-expression in stably transfected cell lines in which we can control the relative expression of Cx40 or Cx45 with Cx43. We hypothesize that co-expression of different relative amounts of these connexins will affect electrical conductance, permeability/selectivity, and gating by protein kinase activation. We will use the double whole-cell patch-clamp technique and fluorescence microscopy to quantitate conductance and dye flux simultaneously. 3. Cellular and physiologic studies will examine truncations and point mutants of the proximal region of the carboxyl tail of Cx43 to examine its role in the assembly and function of gap junctions. ? ?
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