Abstract 20459: Calcium/Calmodulin-Dependent Protein Kinase II Regulates KCNQ1 to Reduce the Slow Component of the Delayed Rectifier Potassium Current, IKs
Introduction: Sustained β-adrenergic receptor (β-AR) activation during heart failure has pathologic consequences including enhanced calcium/calmodulin-dependent protein kinase II (CaMKII) activity, which is arrhythmogenic. We previously identified phosphorylated threonine (T) and serine (S) residues (T482, S484, S457) that are in close proximity to the calmodulin-binding domain on the carboxy-terminus of KCNQ1, the pore-forming subunit responsible for IKs. The objective of this study was to assess the functional impact of phosphorylation at these sites and to investigate the potential for CaMKII to regulate IKs via phosphorylation of these sites.
Methods & Results: Dephosphorylated (alanine; A) and phosphorylated (aspartic acid; D) mimics were introduced on KCNQ1 at T482, S484 and S457. Whole-cell, voltage-clamp experiments were performed in HEK293 cells co-expressing wild-type (WT) or mutant hKCNQ1 and the auxiliary subunit hKCNE1. Sustained β-AR stimulation with isoproterenol (ISO; 100 nM) exposure for 12-24 hours reduced peak IKs (ISO: 21.5 ± 15.1 pA/pF [mean ± SD], n=14 vs. control: 45.7 ± 35.3, n=20; p<0.05) similar to the tri-phosphorylated (triple D) mimic (24.5 ± 15.3) versus the tri-dephosphorylated (triple A) mimic (50.0 ± 27.4; p<0.05). The CaMKII inhibitor KN-93 (500 nM; n=17) reversed ISO-associated reductions in IKs versus KN-92 control (n=14; p<0.05). The phospho-mimics at S484D and S457D (n≥16; p<0.05) but not T482D reduced peak IKs relative to their dephosphorylated (A) mimics. Lentiviral-mediated CaMKII overexpression reduced IKs similar to ISO in both WT (n=15; p<0.05) and dephosphorylated S457A (n=10; p<0.05) but not the dephosphorylated S484A mutant (n=10; p>0.05). KCNQ1 peptide fragments were synthesized to identify CaMKII phosphorylation sites. The S484 peptide fragment was the strongest phospho-acceptor site for CaMKII whereas the S457 peptide fragment was not phosphorylated by CaMKII.
Conclusion: CaMKII phosphorylates S484 on KCNQ1 during sustained β-AR stimulation to reduce IKs. Phosphorylation at S457 reduces IKs function but is not regulated via sustained β-AR stimulation or CaMKII. Functional deficits in IKs during sustained β-AR stimulation may be mediated through enhanced CaMKII signaling.
Author Disclosures: T. Shugg: None. M. Shao: None. A. Chawla: None. M. Rubart-von der Lohe: None. A. Hudmon: None. B.R. Overholser: None.
- © 2016 by American Heart Association, Inc.