Ca 2+/calcineurin regulation of cloned vascular K ATP channels: Crosstalk with the protein kinase a pathway

Abstract

Background and purpose: Vascular ATP-sensitive potassium (KATP) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca 2+-dependent phosphatase, inhibits K ATP channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA. Experimental approach: Whole-cell recordings were made in HEK-293 cells stably expressing the vascular K ATP channel (K R6.1 /SUR2B). The effect of intracellular Ca 2- and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined. Key results: Constitutively active calcineurin Aa but not Aβ significantly attenuated K ATP currents activated by low intracellular Ca 2+, whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal K ATP currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca 2+ (equivalent to increasing calcineurin activity), almost completely inhibited K ATP channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA. Conclusions and Implications: Calcineurin Aa regulates K IR6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the K ATP channel. © 2009 The British Pharmacological Society All rights reserved.