Lipid raft and Arf6-GTPase dependent endocytosis of the hERG potassium channel

Karnik, R. , Smith, A. J., Elliott, D. J., Taneja, T. K., Aviss, K. and Sivaprasadarao, A. (2010) Lipid raft and Arf6-GTPase dependent endocytosis of the hERG potassium channel. Biophysical Journal, 98(3 S1), 376a. (doi: 10.1016/j.bpj.2009.12.2028)

Abstract

The voltage-gated hERG potassium channel is critical for the repolarisation phase of the cardiac action potential. Genetic mutations leading to a decrease in the number of hERG channels at the cell surface lead to potentially fatal Long QT syndrome which is reflected by an elongated QT interval in the ECG. It is therefore important to understand the trafficking mechanisms that regulate the surface density of hERG. Here we have investigated the endocytic pathway of an epitope (hemagglutinin A) tagged hERG, expressed in HeLa cells, using techniques in molecular cell biology, electrophysiology and biochemistry. Our results demonstrate that the majority of the hERG channel is rapidly internalized from the plasma membrane in a dynamin and clathrin independent manner. Endocytosis of hERG is dependent on cholesterol rich lipid rafts and ADP-ribosylation factor 6 (Arf6). Depletion of cholesterol from cell membrane by treatment with methyl-β-cyclodextrin and disruption of Arf6 activity by over-expression of inactive Arf6 mutants or aluminum fluoride resulted in an inhibition of hERG endocytosis, leading to an increase in hERG currents. Majority of the internalized hERG channel was found in lipid rafts isolated by density gradient centrifugation, identified by the presence of raft marker proteins including flotillin and the co-expressed GFP-tagged GPI anchor protein. Raft associated caveolin, Rac-1 and RhoA-GTPase do not appear to be required for hERG endocytosis. A small fraction of hERG, however, appears to undergo endocytosis via clathrin mediated endocytosis. Following internalization the channel enters Rme1 positive recycling endosomes and also Lamp1-positive late endosome/lysosomal compartments. The significance of lipid-raft and Arf6 dependent endocytosis of hERG in cardiac physiology has yet to be understood. This work was funded by the British Heart Foundation.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Karnik, Dr Rucha
• Authors: Karnik, R., Smith, A. J., Elliott, D. J., Taneja, T. K., Aviss, K., and Sivaprasadarao, A.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Biophysical Journal
• Publisher: Elsevier (Cell Press)
• ISSN: 0006-3495
• ISSN (Online): 1542-0086
• Published Online: 26 January 2010