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.
Highlights
The hERG potassium channel (Kv11.1), encoded by the KCNH2 gene, underlies the rapidly activating delayed rectifier K+ current (IKr)
Antibody recognition requires only one of the four subunits of a tetrameric hERG channel to contain the haemagglutinin A (HA) sequence, cells were transfected with a 1:1 ratio of HA-hERG:hERG cDNA as this provided the best balance between signal strength within the assays and HA-hERG subunit inclusion
All findings made here were equivalent for HA-hERG and HA-hERGhERG channels, suggesting that any disruption caused by the presence of four HA epitopes within the channel is only minor with respect to endocytic trafficking
Summary
The hERG (human ether-a-go-go related gene) potassium channel (Kv11.1), encoded by the KCNH2 gene, underlies the rapidly activating delayed rectifier K+ current (IKr) This forms a crucial component of the repolarisation phase of the cardiac action potential and a reduction in its activity is associated with prolongation of the QT interval in the electrocardiogram (long QT syndrome 2; LQT2), which increases the risk of ventricular fibrillations and sudden death [1,2]. Endocytic trafficking of hERG can be disrupted, altering channel removal from the surface, recycling back to the cell membrane and/or targeting for endosomal degradation This mechanism is less established, but has been implicated in the impact of certain drugs [8,9] and pathophysiological conditions such as hypokalaemia [10,11] and hyperglycaemia [12,13]. It is important that we understand the fate of hERG after it is inserted in the plasma membrane, something that has so far received little attention
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