CaMKII Modulates the Cardiac Transient Outward K⁺ Current through its Association with Kv4 Channels in Non-Caveolar Membrane Rafts.

BACKGROUND/AIMS:To test whether the physiological regulation of the cardiac Kv4 channels by the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is restricted to lipid rafts and whether the interactions observed in rat cardiomyocytes also occur in the human ventricle. METHODS:Ventricular myocytes were freshly isolated from Sprague-Dawley rats. I_to was recorded by the whole-cell Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the proteins of interest was analysed by western blot. Immunogold staining and electron microscopy of heart vibrosections was performed to localize Kv4.2/Kv4.3 and CaMKII proteins. Protein-protein interactions were determined by co-immunoprecipitation experiments in rat and human ventricular mycoytes. RESULTS:Patch-Clamp recordings in control conditions and after lipid raft or caveolae disruption show that the CaMKII-Kv4 channel complex must associate in non-caveolar lipid rafts to be functional. Separation in density gradients, co-immunoprecipitation and electron microscopy show that there are two Kv4 channel populations: one located in caveolae, that is CaMKII independent, and another one located in planar membrane rafts, which is bound to CaMKII. CONCLUSION:CaMKII regulates only the Kv4 channel population located in non-caveolar lipid rafts. Thus, the regulation of cardiac Kv4 channels in rat and human ventricle depends on their subcellular localization.

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