Mechanism of the inhibition of Ca2+-activated Cl- currents by phosphorylation in pulmonary arterial smooth muscle cells

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl- currents (I-ClCa) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, I-ClCa was evoked immediately upon membrane rupture but then exhibited marked rundown to similar to 20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5'-(beta,gamma-imido) -triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, I-ClCa was similar to 100% of initial levels. I-ClCa recorded with AMP-PNP-containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in I-ClCa was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater "on" rates, and voltage-dependent closing steps ("off" rates). Our model reproduced well the Ca2+ and voltage dependence of I-ClCa as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl- channel complex influences current generation dramatically through one or more critical voltage-dependent steps.