Abstract 19332: Cardiac Kir2.1 and NaV1.5 Channels Associate in a Macromolecular Complex Early in Their Forward Trafficking Pathway
Introduction: The factors that control the expression of Kir2.1 and NaV1.5 channels and the mechanisms that target such channels to subcellular and membrane macrodomains are incompletely understood.
Hypothesis: Expression of the trafficking defective Andersen-Tawil Syndrome mutant Kir2.1(Δ314-315) disturbs trafficking and functional expression of the associated NaV1.5 channels in the macromolecular complex, consequently altering excitability.
Methods: Currents were recorded by patch clamp in HEK cells and rat ventricular myocytes.
Results: In HEK293 cells, co-expression of Kir2.1(Δ314-315) with wildtype (WT) NaV1.5 significantly reduced INa, compared to INa generated by WT NaV1.5 alone or co-expressed with WT Kir2.1 (peak currents at -25 mV, 256±41, -634±54 and -828±63 pA/pF, respectively). We observed no differences in voltage dependence of activation, inactivation or recovery from inactivation (n=15, n=18 and n=16, respectively). In cell-surface biotinylation experiments the presence of Kir2.1(Δ314-315) reduced membrane NaV1.5 protein expression by 30% (n=6). Additionally, through its NH2 and COOH domains, Nav1.5 protein co-immunoprecipitated the AP-1 clathrin adaptor protein that drives Golgi export of Kir2.1 channel. Moreover, in rat ventricular myocytes, viral transfer of Kir2.1(Δ314-315) reduced both inward (-3±0.4 vs -10±8 pA/pF at -120 mV) and outward (0.15±0.01 vs 0.5±0.06 pA/pF at -60 mV) IK1 (N=4, n=12 and N=6, n=13), and significantly reduced INa density (peak currents at -45 mV, -41±6 vs -58±6 pA/pF, respectively; N=3, n=12 and N=4, n=18). Finally, overexpression of Kir2.1(Δ314-315) reduced resting membrane potential (-67±3 vs -79±2 mV) and maximum upstroke velocity (91±32 vs 184±17 V/s), and increased action potential duration to 90% of repolarization (212±36 vs 110±28 ms respectively; N=3, n=8 and N=3, n=8)
Conclusions: The Nav1.5-Kir2.1 macromolecular complex preassembles early in the forward trafficking pathway. Therefore trafficking deficiency of one complex component, in this case Kir2.1(Δ314-315), affects trafficking of both channels. We demonstrate also that NaV1.5 protein is selected as cargo into Golgi export carriers in a signal dependent manner through an AP-1 clathrin adaptor interaction.
Author Disclosures: D. Ponce-Balbuena: None. C.R. Valdivia: None. C.B. Willis: None. F.J. Alvarado: None. G. Guerrero-Serna: None. M. Zarzoso: None. E. Delpón: None. R. Caballero: None. J. Tamargo: None. R.R. Ramirez: None. K. Kaur: None. H.H. Valdivia: None. J. Jalife: None.
- © 2016 by American Heart Association, Inc.