Cardiac electrical conduction depends on intercellular passage of ions through gap-junction channels. Alterations in gap-junction distribution and subunit protein expression have been implicated in the pathogenesis of re-entrant arrhythmias. Gap-junction channels are formed by the joining of two hexameric hemichannels (connexons) composed of subunit proteins called connexins (Cx). Individual connexins form channels with different biophysical properties. Many cardiovascular cells contain multiple connexins which frequently co-localize to identify gap-junction. The major goals of the current proposal is to define the parameters of mixing of the cardiovascular connexins and the functional consequence of that mixing. The investigators hypothesize that the most physiological consequential mixing of connexins results in the formation of mixed hexamers (heteromeric connexons). The investigators propose a three-part program which will focus particularly on the mixing of the major cardiac connexin (Cx43) with Cx37 or Cx40, since Cx37 and Cx40 are abundantly expressed in distinct expression patterns (which are largely subsets of the Cx43 distribution), but these proteins individually make channels with substantially different properties from Cx43.
The specific aims are as follows 1) To examine, biochemically, the extent to which combinations of the cardiovascular connexins form mixed connexons. Connexin pairs (Cx37 plus Cx43, Cx40 plus Cx43) will be expressed by stable transfection of immortalized cell lines. Cross-linking and co-immunoprecipitation will be used to determine the extend of mixing of connexins within these cells and within individual channels; 2) To define physiologically, how intercellular communication is modified by the formation of heteromeric channels. Physiologic properties of the juncitonal channels in co- transfected cells will be compared to ones containing only a single connexin. Unitary conductance and channel gating will be studied by double whole-cell and dual permeabilized patch methods. Channel permeability/selectivity will be assessed by intercellular transfer of micro-injected fluorescent tracers; 3) To determine the functional consequences of connexin mixing on cardiac conduction. Cx40 or Cx37 will be over-expressed in cardiac myocytes (by production of transgenic mice or adenoviral infection). Channel gating and action-potential prolongation will be determined in cell pairs. Conduction in patterned arrays in cultured neonatal myocytes will be examined by optical mapping. Electrocardiograms and epicardial mapping will be used to examine ventricular conduction.
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