Differential phosphorylation-dependent regulation of constitutively active and muscarinic receptor-activated I-K,I-ACh channels in patients with chronic atrial fibrillation

Objective: In chronic atrial fibrillation (cAF) the potassium current I-K,I-ACh develops agonist-independent constitutive activity. We hypothesized that abnormal phosphorylation-dependent regulation underlies the constitutive I-K,I-ACh activity. Methods: We used voltage-clamp technique and biochemical assays to study I-K,I-ACh regulation in atrial appendages from 61 sinus rhythm (SR), 11 paroxysmal AF (pAF), and 33 cAF patients. Results: Compared to SR basal current was higher in cAF only, whereas the muscarinic receptor (2 mu mol/L carbachol)-activated I-K,I-ACh was smaller in pAF and cAF. In pAF the selective I-K,I-ACh blocker tertiapin abolished the muscarinic receptor-activated I-K,I-ACh but excluded agonist-independent constitutive I-K,I-ACh activity. Blockade of type-2A phosphatase and the subsequent shift to increased muscarinic receptor phosphorylation (and inactivation) reduced muscarinic receptor-activated I-K,I-ACh in SR but not in cAF, pointing to an impaired function of G-protein-coupled receptor kinase. Using subtype-selective kinase inhibitors we found that in SR the muscarinic receptor-activated I-K,I-ACh requires phosphorylation by protein kinase G (PKG), protein kinase C (PKC), and calmodulin-dependent protein kinase II (CaMKII), but not by protein kinase A (PKA). In cAF, constitutive I-K,I-ACh activity results from abnormal channel phosphorylation by PKC but not by PKG or CaMKII, whereas the additional muscarinic receptor-mediated I-K,I-ACh activation occurs apparently without involvement of these kinases. In cAF, the higher protein level of PKC epsilon but Dot PKC alpha, PKC beta(1) or PKC delta is likely to contribute to the constitutive I-K,I-ACh activity. Conclusions: The occurrence of constitutive I-K,I-ACh activity in cAF results from abnormal PKC function, whereas the muscarinic receptor-mediated I-K,I-ACh activation does not require the contribution of PKG, PKC or CaMKII. Selective drug targeting of constitutively active-I-K,I-ACh channels may be suitable to reduce the ability of AF to become sustained. (C) 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.