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 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >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 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.
Gerlach
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AC
Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Gangopadhyay
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NN
Devor
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DC
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004553:Electric Conductivity; D004847:Epithelial Cells / Q000378:metabolism; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D015640:Ion Channel Gating; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D015221:Potassium Channels / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated; D014981:Xenopus
D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D007476:Ionophores; D010446:Peptide Fragments; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D005576:Colforsin; D015759:Ionomycin; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine