N-glycosylation-dependent regulation of hK_2P17.1 currents

Two pore-domain potassium (K_2P) channels mediate potassium background currents that stabilize the resting membrane potential and facilitate action potential repolarization. In the human heart, hK_2P17.1 channels are predominantly expressed in the atria and Purkinje cells. Reduced atrial hK_2P17.1 protein levels were described in patients with atrial fibrillation or heart failure. Genetic alterations in hK_2P17.1 were associated with cardiac conduction disorders. Little is known about posttranslational modifications of hK_2P17.1. Here, we characterized glycosylation of hK_2P17.1 and investigated how glycosylation alters its surface expression and activity. Wild-type hK_2P17.1 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. hK_2P17.1 expression and activity were assessed using immunoblot analysis and a two-electrode voltage clamp technique. Channel subunits of hK_2P17.1 harbor two functional N-glycosylation sites at positions N65 and N94. In hemi-glycosylated hK_2P17.1 channels, functionality and membrane trafficking remain preserved. Disruption of both N-glycosylation sites results in loss of hK_2P17.1 currents, presumably caused by impaired surface expression. This study confirms diglycosylation of hK_2P17.1 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.