Cardiac Kir2.1 and Na1.5 Channels Traffic Together to the Sarcolemma to Control Excitability.

Rationale: In cardiomyocytes, Na(v)1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. Objective: The objective of this study was to test whether Na(v)1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. Methods and Results: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1(Delta 314-315) or Kir2.1(R44A/R46A) with wild-type (WT) Na(v)1.5(WT) in heterologous cells reduced inward sodium current compared with Na(v)1.5(WT) alone or coexpressed with Kir2.1(WT). In cell surface biotinylation experiments, expression of Kir2.1(Delta 314-315) reduced Na(v)1.5 channel surface expression. Glycosylation analysis suggested that Na(v)1.5(WT) and Kir2.1(WT) channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of Na(v)1.5(WT), and vice versa, whereas coexpression with Kir2.1(Delta 314-315) reduced mobility of both channels. Viral gene transfer of Kir2.1(Delta 314-315) in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with Na(v)1.5(WT) and Kir2.1(WT) within areas corresponding to t-tubules and intercalated discs. Like Kir2.1(WT), Na(v)1.5(WT) coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that Na(v)1.5(WT) channels interact with AP1 through the Na(v)1.5(Y1810) residue, suggesting that, like for Kir2.1(WT), AP1 can mark Na(v)1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 (sic)-adaptin subunit in human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. Conclusions: The Na(v)1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of Na(v)1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.