Probing the cardiac Kir2.1-Nav1.5 channelosome using trafficking and gating-defective arrhythmia-associated KCNJ2 variants

Kir2.1 potassium channels (encoded by KCNJ2) contribute to the inward rectifier K+ current that provides ventricular myocytes a stable resting potential and participates in terminal cardiac action potential repolarization. Loss of function Kir2.1 variants underlie Anderson-Tawil syndrome (ATS). Previously, it has been suggested that Kir2.1 form functional complexes with another cardiac channel, Nav1.5. This existence of a so-called “channelosome” has profound implications in cardiac physiology and, indeed, pathology. In this study, we utilized both trafficking and gating-defective ATS-associated KCNJ2 variants to interrogate possible interactions between Kir2.1 and Nav1.5. Introduction of ATS-associated KCNJ2 variants abolished whole-cell IKir2.1, in transfected HEK-293 cells. Co-expression of variant and wild-type (WT) channels partially recovered current, without changes in conductance or activation kinetics. Under ventricular action potential clamp, the Ba2+-sensitive current (300 μM) was significantly reduced, consistent with impaired repolarizing capacity. A western In-Cell/On-Cell LI-COR® assay was used to distinguish Kir2.1 variants that resulted from perturbed membrane localization (Δ314-315) from gating/conduction (E138V). Co-expression of these variants with WT-Nav1.5 did not significantly change the membrane expression of trafficking competent or incompetent Kir2.1, nor was the total channel synthesis altered. Notably, trafficking-defective Kir2.1 (Δ314-315) significantly reduced whole-cell INav1.5, whereas gating-defective Kir2.1 (E138V) had no effect; WT-Nav1.5 did not recover IKir2.1 for either trafficking or gating-defective channels. To determine whether there was any direct Nav1.5-dependent modulation of Kir2.1, as has previously been suggested, cell-attached single-channel recording was used. This revealed no change in Kir2.1 unitary conductance or opening probability when alone or co-expressed with WT-Nav1.5. Taken together, our data support a role for trafficking defective Kir2.1 in modulating Nav1.5. This has significant implications for the characterization and classification of future arrhythmia-associated KCNJ2 variants.