Cardiac Kir2.1 and NaV1.5 Channels Traffic Together to the Sarcolemma to Control Excitability
Rationale: In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes.
Objective: To test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains.
Methods and Results: In patch-clamp experiments, co-expression of trafficking deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wildtype (WT) NaV1.5WT in heterologous cells reduced INa, compared to NaV1.5WT alone or co-expressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that co-expression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas co-expression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1∆314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) reduced <IK1 and INa, maximum diastolic potential (MDP) and action potential depolarization rate, and increased action potential duration (APD). Upon immunostaining, the adaptor protein complex 1 (AP1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT co-immunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 ϒ-adaptin subunit in hiPSC-CMs reduced IK1, INa and MPD, and impaired rate dependent APD adaptation.
Conclusions: The NaV1.5-Kir2.1 macromolecular complex preassembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.
- Received August 9, 2017.
- Revision received February 27, 2018.
- Accepted March 5, 2018.