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

摘要

To determine the mechanisms responsible for the termination of Ca²⁺-activated Cl⁻ currents (ICl(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 1 mM ATP, ICl(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 ICl(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 II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(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, ICl(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 ICl(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.