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 Ca2+-dependent gating of the cloned intermediate conductance, Ca2+-dependent K+ channel, hIK1, In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca2+-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca2+-dependent increase in hIK1 activity (NPo). 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 Mg2+ and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI5-24. The Ca2+ dependence of hIK1 activation was best fit with a stimulatory constant (K-s) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NPo at [Ca2+] > 100 nM 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 Mg2+-dependent fashion, being reversed by alkaline phosphatase. Neither PKI5-24 nor CaMKII281-309 or PKC19-31 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 Mg2+- and PKI5-24-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII281-309 and PKC19-31 had no effect on the ATP-dependent activation. The Ca2+-dependent activation (LC, 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 Ca2+-dependent and occurs via an increase in NPo without an effect on either Ca2+ affinity or apparent cooperativity.