Electrophysiological effects of non-vitamin K antagonist oral anticoagulants on atrial repolarizing potassium channels.

AIMS: Non-vitamin K antagonist oral anticoagulants (NOACs) are widely used in the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation (AF). The efficacy of NOACs has been attributed in part to pleiotropic effects that are mediated through effects on thrombin, factor Xa, and their respective receptors. Direct pharmacological effects of NOACs and cardiac ion channels have not been addressed to date. We hypothesized that the favourable clinical outcome of NOAC use may be associated with previously unrecognized effects on atrial repolarizing potassium channels. METHODS AND RESULTS: This study was designed to elucidate acute pharmacological effects of NOACs on cloned ion channels Kv11.1, Kv1.5, Kv4.3, Kir2.1, Kir2.2, and K2P2.1 contributing to IKr, IKur, Ito, IK1, and IK2P K+ currents. Human genes, KCNH2, KCNA5, KCND3, KCNJ2, KCNJ12, and KCNK2, were heterologously expressed in Xenopus laevis oocytes, and currents were recorded using voltage-clamp electrophysiology. Apixaban, dabigatran, edoxaban, and rivaroxaban applied at 1 µM did not significantly affect peak current amplitudes of Kv11.1, Kv1.5, Kv4.3, Kir2.1, Kir2.2, or K2P2.1 K+ channels. Furthermore, biophysical characterization did not reveal significant effects of NOACs on current-voltage relationships of study channels. CONCLUSION: Apixaban, dabigatran, edoxaban, and rivaroxaban did not exhibit direct functional interactions with human atrial K+ channels underlying IKr, IKur, Ito, IK1, and IK2P currents that could account for beneficial clinical outcome associated with the drugs. Indirect or chronic effects and potential underlying signalling mechanisms remain to be investigated.

N-Glycosylation of TREK-1/hK2P2.1 Two-Pore-Domain Potassium (K2P) Channels.

Mechanosensitive hTREK-1 two-pore-domain potassium (hK2P2.1) channels give rise to background currents that control cellular excitability. Recently, TREK-1 currents have been linked to the regulation of cardiac rhythm as well as to hypertrophy and fibrosis. Even though the pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, relatively little is known about their posttranslational modifications. This study aimed to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Following pharmacological inhibition of N-glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-293T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. TREK-1 channel subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrated that nonglycosylated hTREK-1 channel subunits are able to reach the cell surface in general but with seemingly reduced efficiency compared to glycosylated subunits. These findings extend our understanding of the regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how altered ion channel glycosylation may promote arrhythmogenesis.

N-glycosylation-dependent regulation of hK2P17.1 currents.

Two pore-domain potassium (K2P) channels mediate potassium background currents that stabilize the resting membrane potential and facilitate action potential repolarization. In the human heart, hK2P17.1 channels are predominantly expressed in the atria and Purkinje cells. Reduced atrial hK2P17.1 protein levels were described in patients with atrial fibrillation or heart failure. Genetic alterations in hK2P17.1 were associated with cardiac conduction disorders. Little is known about posttranslational modifications of hK2P17.1. Here, we characterized glycosylation of hK2P17.1 and investigated how glycosylation alters its surface expression and activity. Wild-type hK2P17.1 channels and channels lacking specific glycosylation sites were expressed in Xenopus laevis oocytes, HEK-293T cells, and HeLa cells. N-glycosylation was disrupted using N-glycosidase F and tunicamycin. hK2P17.1 expression and activity were assessed using immunoblot analysis and a two-electrode voltage clamp technique. Channel subunits of hK2P17.1 harbor two functional N-glycosylation sites at positions N65 and N94. In hemi-glycosylated hK2P17.1 channels, functionality and membrane trafficking remain preserved. Disruption of both N-glycosylation sites results in loss of hK2P17.1 currents, presumably caused by impaired surface expression. This study confirms diglycosylation of hK2P17.1 channel subunits and its pivotal role in cell-surface targeting. Our findings underline the functional relevance of N-glycosylation in biogenesis and membrane trafficking of ion channels.