Objective: In chronic atrial fibrillation (cAF) the potassium current I_K,ACh develops agonist-independent constitutive activity. We hypothesized that abnormal phosphorylation-dependent regulation underlies the constitutive I_K,ACh activity.

Methods: We used voltage-clamp technique and biochemical assays to study I_K,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 μmol/L carbachol)-activated I_K,ACh was smaller in pAF and cAF. In pAF the selective I_K,ACh blocker tertiapin abolished the muscarinic receptor-activated I_K,ACh but excluded agonist-independent constitutive I_K,ACh activity. Blockade of type-2A phosphatase and the subsequent shift to increased muscarinic receptor phosphorylation (and inactivation) reduced muscarinic receptor-activated I_K,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,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,ACh activity results from abnormal channel phosphorylation by PKC but not by PKG or CaMKII, whereas the additional muscarinic receptor-mediated I_K,ACh activation occurs apparently without involvement of these kinases. In cAF, the higher protein level of PKCε but not PKCα, PKCβ₁ or PKCδ is likely to contribute to the constitutive I_K,ACh activity.

Conclusions: The occurrence of constitutive I_K,ACh activity in cAF results from abnormal PKC function, whereas the muscarinic receptor-mediated I_K,ACh activation does not require the contribution of PKG, PKC or CaMKII. Selective drug targeting of constitutively active I_K,ACh channels may be suitable to reduce the ability of AF to become sustained.