Kinase-dependent Regulation of the Intermediate Conductance, Calcium-dependent Potassium Channel, hIK1

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca 2+ -dependent gating of the cloned intermediate conductance, Ca 2+ -dependent K + channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca 2+ -dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca 2+ -dependent increase in hIK1 activity (NP o ). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg 2+ and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI 5–24 . The Ca 2+ dependence of hIK1 activation was best fit with a stimulatory constant ( K s ) of 350 n m and a Hill coefficient ( n ) of 2.3. ATP increased NP o at [Ca 2+ ] >100 n m while having no effect on K s or n . Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg 2+ -dependent fashion, being reversed by alkaline phosphatase. Neither PKI 5–24 nor CaMKII 281–309 or PKC 19–31 affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg 2+ - and PKI 5–24 -dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII 281–309 and PKC 19–31 had no effect on the ATP-dependent activation. The Ca 2+ -dependent activation ( K s and n ) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca 2+ -dependent and occurs via an increase in NP o without an effect on either Ca 2+ affinity or apparent cooperativity.