Recently, we have identified a novel Ca2+-activated K+ current (Ik,ca) in human and mouse atrial myocytes that contributes markedly towards the late phase of the atrial repolarization. The importance of the currents is underpinned by the fact that the late phase of the cardiac action potential is susceptible to aberrant excitation e.g. early after depolarization and arrhythmias. The channel is differentially expressed in atrial vs. ventricular myocytes. Finally, we have preliminary data, which suggest that the channel may co-localize with specific isoform of the voltage-gated Ca 2+ channels (VGCC). We hypothesize that the regional specific expression of the Ca 2+-activated K + channels (Kca) coordinates distinct functional roles in atria vs. ventricles. Specifically, we hypothesize that the channels are important in the repolarization process in atria, and alteration of the current can precipitate profound changes in the shape and duration of cardiac action potentials and possibly atrial arrhythmias. The following Specific Aims are proposed: 1. Functional identification of Ik, ca, in atrial and ventricular myocytes. We will determine the detail biophysical and pharmacologic properties of Ik,ca as well as quantifying the whole-cell current density in human and mouse myocytes to define the specific isoforms of Ik,ca, expressed in atria and ventricles. To conclusively establish that the activity of Ik,ca contributes towards the repolarization process in the heart, we will employ in-vitro adenoviral-mediated gene transfer to single isolated human and mouse cardiac myocytes using dominant-negative (DN) functional knockout strategies. 2. Biochemical identification of the specific isoform encoding the cardiac Ik,ca, The functional study will be corroborated using Western blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence techniques and in-situ hybridization. 3. Co-localization of cardiac Kca, channel with specific isoform of VGCC. To test the hypothesis that the channel colocalizes with specific isoform of VGCC, immunofluorescence techniques using double labeling will be used. In addition, we will use a gene targeted eqD Ca2+ channel null mutant mice as a tool to specifically examine the co-localization of the Kca channel with the different isoforms of VGCC. 4. Determination of the functional roles of Ik,ca in whole animals. In-vivo adenoviral-mediated gene transfer to overexpress the functional Kca channel or engineered DN constructs to mice and in vivo electrophysiologic studies will be used to assess the functional roles of the channel in whole animals. Complimentary experiments using transgenic mice of specific SK channel will be performed. Transgenic mice with conditional expression for each of the three SK channel genes have already been constructed. These mouse models will be used to study the functional roles of the Kca channel in the heart. ECG, telemetric ECG recordings and electrophysiologic studies will be performed. Single isolated myocytes will be studied using patch-clamp techniques as in Aim 1.
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