Ca2+/calcineurin regulation of cloned vascular K ATP channels: crosstalk with the protein kinase A pathway.
OBJECTIVE
Vascular ATP-sensitive potassium (K(ATP)) 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.


METHODS
Whole-cell recordings were made in HEK-293 cells stably expressing the vascular K(ATP) channel (K(IR)6.1/SUR2B). The effect of intracellular Ca(2+) and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined.


RESULTS
Constitutively active calcineurin A alpha but not A beta 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
Calcineurin A alpha regulates K(IR)6.1/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.
BHF Laboratories, Department of Medicine, University College, London, UK.
N N
NN
Orie
A M
AM
Thomas
B A
BA
Perrino
A
A
Tinker
L H
LH
Clapp
D019703:Calcineurin / Q000502:physiology*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000138:chemical synthesis / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*
D065095:Calcineurin Inhibitors; D007527:Isoenzymes; D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying; D017868:Cyclic AMP-Dependent Protein Kinases; D019703:Calcineurin; D002118:Calcium