Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

To determine the mechanisms responsible for the termination of Ca(2+)-activated Cl(−) currents (I(Cl(Ca))), simultaneous measurements of whole cell currents and intracellular Ca(2+) concentration ([Ca(2+)](i)) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, I(Cl(Ca)) decayed before the [Ca(2+)](i) decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca(2+)](i). Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of I(Cl(Ca)), resulting in a rate of current decay similar to that of the fall in [Ca(2+)](i). In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of I(Cl(Ca)) decay was slowed and matched the [Ca(2+)](i) decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca(2+)](i) was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, I(Cl(Ca)) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial I(Cl(Ca)), and little or no current (despite a normal [Ca(2+)](i) transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.