title: Endocytosis of HERG is clathrin-independent and involves arf6.
abstract: The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Rucha
authors.initials: R
authors.lastname: Karnik
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom ; Faculty of Biological Sciences, Multidisciplinary Cardiovascular Centre, University of Leeds, Leeds, United Kingdom.
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Ludlow
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Nada
authors.initials: N
authors.lastname: Abuarab
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Smith
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Matthew E L
authors.initials: ME
authors.lastname: Hardy
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: David J S
authors.initials: DJ
authors.lastname: Elliott
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom ; Faculty of Biological Sciences, Multidisciplinary Cardiovascular Centre, University of Leeds, Leeds, United Kingdom.
authors.forename: Asipu
authors.initials: A
authors.lastname: Sivaprasadarao
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D000072237:ERG1 Potassium Channel; D004705:Endocytosis; D051638:Ether-A-Go-Go Potassium Channels / Q000378:metabolism*; D006367:HeLa Cells; D006352:Heart Ventricles / Q000166:cytology; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000089982:ADP-Ribosylation Factor 6; D002966:Clathrin; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D002784:Cholesterol; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715349:Arf6 protein, rat; D034281:Dynamins