Abstract 14862: Clathrin-dependent Internalization of KV1.5 Channels is a Major Regulatory Pathway of Potassium Current in Atrial Myocytes
Introduction: KV1.5 channels carry the ultra-rapid delayed rectifier current IKur that controls action potential duration in the atria. Whilst the cellular machinery controlling Kv1.5 trafficking has started to be elucidated, the mechanisms regulating its internalization remain fragmentary.
Hypothesis: The aim of the present study was to identify the internalization pathway of KV1.5 channels and the role of the cytoskeleton in native adult atrial cardiomyocytes (CM).
Methods: We used an interdisciplinary approach based on adenoviral transfer technology to overexpress human KV1.5 channel in fusion with the GFP protein (h-Kv1.5-GFP) in adult rat cardiac myocytes, high resolution 3-dimensional deconvolution, Total Internal Reflection Fluorescence (TIRF) an electron microscopy (EM), biochemistry, and patch clamp electrophysiology.
Results: In CM, KV1.5 channels localize with clathrin vesicles but not with caveolae both at the intercalated disc (ID) and at the lateral membrane (LM). Quantification of the number of clathrin buds and clathrin coated vesicles at the ID and LM from EM images showed that the LM is an active endocytosis zone. Clathrin blockade increased both KV1.5 surface expression and density of the related current, IKur. TIRF microscopy showed that KV1.5 channel displayed average run length of 5.90 μm, average speed of 0.05 μm/s and intermediate straightness movements in the x/y axis suggestive of structured displacement in control conditions. Clathrin blockade was characterized by an increase in GFP florescence intensity, a reduction in KV1.5 vesicle track length and velocity and by the formation of large clusters that were preferentially associated with stable microtubules. As for clathrin blockade, microtubule disruption, and not actin cytoskeleton disruption, reduced KV1.5 dynamics and redistributed KV1.5 channels into large clusters.
Conclusion: The present study uncovers new mechanisms regulating the trafficking of KV1.5 channels in native atrial myocytes. The clathrin pathway is a major regulator of the functional expression of KV1.5 channels in atrial myocytes. The high mobility of KV1.5 channels in the plasma membrane depends on integrity of the microtubule network and not on the actin cytoskeleton.
Author Disclosures: E. Balse: None. C. Barbier: None. C. Eichel: None. C. Rucker-Martin: None. A. Coulombe: None. S. Hatem: None.
- © 2016 by American Heart Association, Inc.