We previously demonstrated that hIK1 is activated directly by ATP in excised, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca 2؉ -dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca 2؉ -dependent K ؉ channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His 299 -Lys 427 . Furthermore, truncation of hIK1 at Leu 414 resulted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/ rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATP sensitivity as including amino acids Arg 355 -Ala 413 . An alanine scan of all nonconserved serines and threonines within this region failed to alter the response of hIK1 to ATP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg 355 -Met 368 of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was ϳ50% of that of hIK1. These results demonstrate that amino acids Arg 355 -Ala 413 within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATPdependent phosphorylation of hIK1 or an associated protein is independent of Ca 2؉ .The human intermediate conductance K Ca channel, hIK1, is required for a variety of physiological processes including transepithelial ion transport (2-5), vasodilation (6, 7), T cell activation (8, 9), cell proliferation (9 -11), and regulatory volume decrease (9, 10). In addition to demonstrating modulation by intracellular Ca 2ϩ , we and others have demonstrated that hIK1 activity can be dynamically regulated by phosphorylation (1,9,(12)(13)(14). Several of these studies have demonstrated an ATP-dependent activation of hIK1 in excised, inside-out membrane patches that can be reversed by exogenous phosphatases and/or kinase inhibitors (1,12,13). Based on these observations, we speculated that the phosphorylation-dependent modulation of hIK1 plays a critical role in modulating the physiological processes in which hIK1 is involved.In our previous study we demonstrated, in excised, insideout patches, that addition of ATP (1 mM) resulted in, on average, a 3-fold increase in hIK1 activity (1), having an EC 50 of 50 M.1 The stimulatory effect of ATP exhibited a slow onset, requiring several minutes for the maximal response, was strictly Mg 2ϩ -dependent, and could be mimicked by neither hydrolyzeable (ADP, GTP, UTP, CTP, ITP) nor non-...
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