Abstract 17494: The KCNQ1 Carboxy Terminus is Differentially Phosphorylated in Response to Short- Versus Long-Term β-adrenergic Stimulation
Background: The slow component of the cardiac delayed rectifier K+ current, I(Ks), is enhanced through phosphorylation of its pore-forming subunit, KCNQ1, during short-term β-adrenergic receptor (β-AR) stimulation. However, the phosphorylation status of KCNQ1and its auxiliary subunit, KCNE1, during sustained β-AR simulation has not been fully elucidated. We hypothesized that the phosphorylation status of these proteins is altered with increasing duration of β-AR activation.
Methods: HEK293 cells, which endogenously express β-ARs, were co-transfected with cDNA encoding human KCNQ1 and KCNE1. Following passage, cells were treated with isoproterenol (ISO; 100 nM) or vehicle for 3 min, 4 hours, or 24 hours (3 experiments per group). A 100 µg aliquot of enriched membrane protein sample was digested and injected in replicate into a linear ion-trap (LTQ) mass spectrometer. The acquired data were searched against the most updated UniProtKB human database using SEQUEST (v. 28 rev. 12) algorithms in Bioworks (v. 3.3). Peptide and protein identifications were validated and protein abundance was determined using LC-MS/MS IdentiQuantXL. Comparisons were made using a one-way ANOVA.
Results: Fourteen unique proteins were identified using phosphorylated peptide standards, including KCNQ1 and KCNE1. Five phosphorylated peptides were identified on the carboxy terminus of KCNQ1. Sustained (≥ 4 hours), but not short-term, ISO exposure significantly altered phosphorylation at thr624 and ser407 residues. Phosphorylation at thr484 and ser482 residues was enhanced following each ISO treatment, with the greatest increase in the 4-hour group (p<0.001). ISO had no significant effect on phosphorylation at ser457. Additionally, one phosphorylated peptide was identified on the carboxy terminus of KCNE1 at position ser128 that was enhanced with ISO exposure in all groups (p<0.01).
Conclusion: Our results are compatible with the notion that the extent of site-specific KCNQ1 phosphorylation varies as a function of duration of β-AR stimulation, suggesting time-dependent changes in phosphorylation and dephosphorylation cycling. This may have implications for the function of I(Ks) during pathological states associated with chronically elevated sympathetic tone.
- © 2012 by American Heart Association, Inc.