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

To determine the mechanisms responsible for the termination of Ca2+-activated Cl- currents (I-Cl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+](i)) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with I mM ATP, I-Cl(Ca), decayed before the [Ca2+](i) decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+](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 [Ca2+](i). In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase LT (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of I-Cl(Ca) decay was slowed and matched the [Ca2+](i) decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect, When a sustained increase in [Ca2+](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 AMPPNP-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 [Ca2+](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.