Regulation of KV1.5 Channel Density in the Rat Atria

Abstract

The density of functional Kv1.5 channels underlying IKur, the main repolarizing current in human atria, is a result of the equilibrium between exocytosis and endocytosis. We have shown that in addition to constitutive exocytosis, Kv1.5 channels can also undergo regulated exocytosis, for instance following changes in the mechanical environment or changes in the cholesterol content of the sarcolemma. Although constitutive and triggered endocytosis have been investigated for Kv1.5, its internalization pathway has not been described. Using high-resolution 3-D deconvolution microscopy, we showed that Kv1.5 channels are associated with clathrin vesicles (CVs) in atrial myocytes. Electron microscopy (EM) showed that CVs are found both at the intercalated disc and at the lateral sarcolemma, aligned along z-bands. Blockade of the clathrin pathway using hypertonic media or SiRNA increased IKur densityin atrial myocytes and led to Kv1.5 channels accumulating at the sarcolemma, as shown by biotinylation assays and fluorescence recovery after photobleaching (FRAP) experiments. These data support the hypothesis that Kv1.5 channels are internalized via the clathrin pathway.Next, we investigated Kv1.5 channel internalization in a rat model of atrial hemodynamic overload. Despite reduced Kv1.5 protein expression in dilated atria, IKur density was unchanged, suggesting increased functional Kv1.5 channels at the sarcolemma. Clathrin expression was reduced in dilated atria, and a decreased colocalization between Kv1.5 channels and CVs was observed. However, EM showed no significant difference in internalization activity between sham and dilated atria. Therefore, the reduced clathrin protein synthesis observed in dilated atria is not likely to be responsible for the accumulation of Kv1.5 channels at the sarcolemma. Other mechanisms such as increased recycling and/or membrane stabilization must be investigated to understand how IKur is maintained in dilated atria.