Abstract
Trafficking of ion channels in cardiomyocytes is a highly dynamic process which is determinant for shaping action potential. In this study, we focused on the dynamic of Kv1.5 channel which carry the atria-specific potassium outward current implicated in the repolarisation of the action potential. We first investigated the endocytosis pathway for Kv1.5 channels in the atria. High resolution 3-D microscopy revealed that Kv1.5 channels are associated with clathrin vesicles in atrial myocytes but not with caveolin. Electron microscopy showed that vesicles are found both at the lateral sarcolemma and at the intercalated disc. Blockade of the clathrin pathway using hypertonic media or SiRNA induced an increase in IKur recorded by whole-cell patch-clamp and an accumulation of Kv1.5 channels at the sarcolemma as shown by biotinylation assay. Clathrin blockade also increased fluorescence recovery after photobleaching of Kv1.5 channels. Altogether, these data show that Kv1.5 channels are internalized through the clathrin pathway. Our objective now is to investigate the kinetics of Kv1.5 channel internalization in living atrial myocytes. Total Internal Reflection Fluorescence microscopy approach will be used to follow eGFPKv1.5-Kv1.5 channels in living cells and to establish their dynamic in control and clathrin-blocked myocytes. To deplete sub-membrane stores of Kv1.5 channels, atrial myocytes will be exposed to shear-stress and endocytosis will be followed for 30 min. The fate of internalized channels will be investigated at different time points by co-immunostainings with antibodies directed against the different endosomes. In conclusion, we have identified the clathrin pathway as the internalization route for Kv1.5 channel, in atrial myocytes. Future work will be conducted to further investigate dynamic and fate of this atria-specific channel. This study should help understanding the constitution of the sub-membrane reservoir of repolarization that we previously described.
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