Regulation of a calcium-sensitive K+ channel (cIK1) by protein kinase C.
Ca2+-sensitive K+ channels (IK1 channels) are required for many physiological functions such as cell proliferation, epithelial transport or cell migration. They are regulated by the intracellular Ca2+ concentration and by phosphorylation-dependent reactions. Here, we investigate by means of the patch-clamp technique mechanisms by which protein kinase C (PKC) regulates the canine isoform, cIK1, cloned from transformed renal epithelial (MDCK-F) cells. cIK1 elicits a K+-selective, inwardly rectifying, and Ca2+-dependent current when expressed in HEK293 or CHO cells. It is inhibited by charybdotoxin, clotrimazole, and activated by 1-ethyl-2-benzimidazolone. cIK1 is activated by intracellular application of ATP or ATP[gS]. ATP-dependent activation is reversed by PKC inhibitors (bisindolylmaleimide, calphostin C), while stimulation with ATP[gS] resists PKC inhibition. Stimulation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) leads to the acute activation of cIK1 currents, which are blocked by PKC inhibitors. In contrast, PKC depletion by overnight incubation with PMA prevents ATP-dependent cIK1 activation. Neither single mutations nor the simultaneous mutation of all PKC sites (T101, S178, T329) to alanine alter the acute regulation of cIK1 channels by PKC. However, current amplitudes of CIK1-T329A and the triple mutant are dramatically increased upon long-term treatment with PMA. These mutations thereby disclose an inhibitory effect on cIKl current of the PKC site at T329. Our results indicate that cIK1 channel activity is regulated in two ways. PKC-dependent activation of cIK1 channels occurs indirectly, while the inhibitory effect probably requires a direct interaction with the channel protein.
Wulf
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Physiologisches Institut, Universität Würzburg, Röntgenring 9, D-97070 Würzburg, Germany.
Schwab
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D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D008564:Membrane Potentials / Q000235:genetics* / Q000502:physiology*; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D012680:Sensitivity and Specificity
D024681:Potassium Channels, Calcium-Activated; D000255:Adenosine Triphosphate; D011493:Protein Kinase C