ATP-dependent Activation of the Intermediate Conductance, Ca2+-activated K+ Channel, hIK1, Is Conferred by a C-terminal Domain

Abstract

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 Ca2+-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 Ca2+-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 His299–Lys427. Furthermore, truncation of hIK1 at Leu414 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 Arg355–Ala413. An alanine scan of all non-conserved 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 Arg355–Met368 of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was ∼50‥ of that of hIK1. These results demonstrate that amino acids Arg355–Ala413within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca2+. 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 Ca2+-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 Ca2+-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 His299–Lys427. Furthermore, truncation of hIK1 at Leu414 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 Arg355–Ala413. An alanine scan of all non-conserved 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 Arg355–Met368 of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was ∼50‥ of that of hIK1. These results demonstrate that amino acids Arg355–Ala413within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca2+. adenosine 5′-(β,γ-imino)triphosphate adenosine 5′-(β,γ-methylenetriphosphate) The human intermediate conductance KCa channel, hIK1, is required for a variety of physiological processes including transepithelial ion transport (2Devor D.C. Frizzell R.A. Am. J. Physiol. 1993; 265: C1271-C1280Crossref PubMed Google Scholar, 3Devor D.C. Singh A.K. Frizzell R.A. Bridges R.J. Am. J. Physiol. 1996; 271: L775-L784PubMed Google Scholar, 4Warth R. Hamm K. Bleich M. Kunzelmann K. von Hahn T. Schreiber R. Ulrich E. Mengel M. Trautmann N. Kindle P. Schwab A. Greger R. Pfluegers Arch. Eur. J. Physiol. 1999; 438: 437-444Crossref PubMed Scopus (117) Google Scholar, 5Dharmsathaphorn K. Pandol S.J. J. Clin. Invest. 1986; 77: 348-354Crossref PubMed Scopus (238) Google Scholar), vasodilation (6Rapacon M. Mieyal P. McGiff J.C. Fulton D. Quilley J. Br. J. Pharmacol. 1996; 118: 1504-1508Crossref PubMed Scopus (37) Google Scholar, 7Mieyal P. Fulton D. McGiff J.C. Quilley J. J. Pharmacol. Exp. Ther. 1998; 285: 659-664PubMed Google Scholar), T cell activation (8Jensen B.S. Odum N. Jorgensen N.K. Christophersen P. Olesen S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10917-10921Crossref PubMed Scopus (98) Google Scholar, 9Khanna R. Chang M.C. Joiner W.J. Kaczmarek L.K. Schlichter L.C. J. Biol. Chem. 1999; 274: 14838-14849Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), cell proliferation (9Khanna R. Chang M.C. Joiner W.J. Kaczmarek L.K. Schlichter L.C. J. Biol. Chem. 1999; 274: 14838-14849Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 10Vandorpe D.H. Shmulker B.E. Jiang L. Lim B. Maylie J. Adelman J.P. De Franceschi L. Capellini M.D. Brugnara C. Alper S.L. J. Biol. Chem. 1998; 273: 21542-21553Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 11Wulff H. Miller M.J. Hansel W. Grissmer S. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8151-8156Crossref PubMed Scopus (532) Google Scholar), and regulatory volume decrease (9Khanna R. Chang M.C. Joiner W.J. Kaczmarek L.K. Schlichter L.C. J. Biol. Chem. 1999; 274: 14838-14849Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 10Vandorpe D.H. Shmulker B.E. Jiang L. Lim B. Maylie J. Adelman J.P. De Franceschi L. Capellini M.D. Brugnara C. Alper S.L. J. Biol. Chem. 1998; 273: 21542-21553Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). In addition to demonstrating modulation by intracellular Ca2+, we and others have demonstrated that hIK1 activity can be dynamically regulated by phosphorylation (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 9Khanna R. Chang M.C. Joiner W.J. Kaczmarek L.K. Schlichter L.C. J. Biol. Chem. 1999; 274: 14838-14849Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar,12Pellegrino M. Pellgrini M. Pfluegers Arch. Eur. J. Physiol. 1998; 436: 749-756Crossref PubMed Scopus (42) Google Scholar, 13Tabcharani J.A. Boucher A. Eng J.W. Hanrahan J.W. J. Membr. Biol. 1994; 142: 255-266PubMed Google Scholar, 14Roch B. Baro I. Hongre A.-S. Escande D. Pfluegers Arch. Eur. J. Physiol. 1995; 426: 355-363Crossref Scopus (26) Google Scholar). 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 (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 12Pellegrino M. Pellgrini M. Pfluegers Arch. Eur. J. Physiol. 1998; 436: 749-756Crossref PubMed Scopus (42) Google Scholar, 13Tabcharani J.A. Boucher A. Eng J.W. Hanrahan J.W. J. Membr. Biol. 1994; 142: 255-266PubMed Google Scholar). 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, inside-out patches, that addition of ATP (1 mm) resulted in, on average, a 3-fold increase in hIK1 activity (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), having an EC50 of 50 μm. 1A. C. Gerlach and D. C. Devor, unpublished observations. 1A. C. Gerlach and D. C. Devor, unpublished observations.The stimulatory effect of ATP exhibited a slow onset, requiring several minutes for the maximal response, was strictly Mg2+-dependent, and could be mimicked by neither hydrolyzeable (ADP, GTP, UTP, CTP, ITP) nor non-hydrolyzeable (AMP-PNP,2 AMP-PCP) ATP analogs. The ATP-dependent stimulation of hIK1 could be reversed by both alkaline and acid phosphatases, suggesting that ATP activates hIK1 via a membrane-delimited kinase. In patches both from T84 cells, which natively express hIK1, (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 2Devor D.C. Frizzell R.A. Am. J. Physiol. 1993; 265: C1271-C1280Crossref PubMed Google Scholar, 4Warth R. Hamm K. Bleich M. Kunzelmann K. von Hahn T. Schreiber R. Ulrich E. Mengel M. Trautmann N. Kindle P. Schwab A. Greger R. Pfluegers Arch. Eur. J. Physiol. 1999; 438: 437-444Crossref PubMed Scopus (117) Google Scholar) and from theXenopus oocyte heterologous expression system, inhibitors of cAMP-dependent protein kinase could partially reverse that ATP-dependent activation. However, mutation of the only cAMP-dependent protein kinase consensus phosphorylation site, serine 334, as well as of all four protein kinase C consensus phosphorylation sites (Thr101, Ser178, Thr329, Ser388) resulted in channels that remained sensitive to phosphorylation. These results suggested that hIK1 was itself not the target for phosphorylation. In this article we demonstrate that unlike hIK1, the SKCachannels, which share both sequence and functional homology to hIK1, fail to respond to ATP in excised, inside-out patches. Therefore, we used a chimeric hIK1/rSK2 strategy to define an amino acid region, arginine 355 through alanine 413, within the C terminus of hIK1 that confers complete ATP dependence. In addition, we demonstrate that the first 14 amino acids of this region, residues Arg355–Met368, which have been shown to interact with calmodulin in a Ca2+-dependent manner, are critical to the ATP-dependent modulation of hIK1. X. laevis care and handling procedures were in accordance with University of Pittsburgh guidelines. X. laevis were obtained from either Xenopus 1 (Dexter, MI) or Nasco (Fort Atkinson, WI). Frogs were anesthetized with 3-aminobenzoic acid ethyl ester, ovaries were surgically removed, and oocytes were dissected in modified Barth's solution containing the following: 88 mm NaCl, 2.4 mmNaHCO3, 1 mm KCl, 0.82 mmMgSO4, 0.33 mm Ca(NO3), 0.41 mm CaCl2, 10 mm HEPES, and 1‥ penicillin-streptomycin. The oocyte follicular cells were removed by incubation in 5 mg/ml collagenase (Life Technologies, Inc.) plus 0.5 mg/ml trypsin inhibitor in Ca2+-free ND-96 (in mm concentrations: 96 NaCl, 1 KCl, 1 MgCl2, 5 HEPES; pH adjusted to 7.5 with NaOH) at room temperature for ∼60 min. The oocytes were then incubated in 100 mmK2HPO4 (pH adjusted to 6.5 with HCl) containing 1 mg/ml bovine serum albumin for 30 min to remove any remaining follicular cells. Stage 5 and 6 oocytes were pre-sorted and allowed to incubate overnight in modified Barth's solution at 20 °C prior to injection of cRNA. All cDNAs were subcloned into the oocyte expression vector pBF containing both 5′ and 3′ untranslated regions of the Xenopus β-globin gene flanking the multi-cloning site. The plasmid was linearized using eitherPvuI or MluI (Roche Molecular Biochemicals), and 5′ capped cRNAs were generated using SP6 polymerase (mMESSAGE mMACHINE™ in vitro transcription kit, Ambion). cRNAs were evaluated both spectrophotometrically and by agarose gel electrophoresis with ethidium bromide staining. Oocytes were injected with 10–50 ng of cRNA 2–4 days prior to recording. Site-directed mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Chimeras between hIK1 and rSK2 were generated by overlap extension polymerase chain reaction using eitherTaq (Stratagene) or Pfx (Life Technologies, Inc.) polymerase. The chimeras were subcloned into the pBF vector using theEcoRI and XhoI restriction sites. The fidelity of all mutations and chimeras was confirmed by sequencing (ABI PRISM 377 automated sequencer, University of Pittsburgh) and subsequent sequence alignment (NCBI Blast 2.0) with hIK1 (GenBankTMaccession number AF022150) and/or rSK2 (GenBankTM number To the chimera the we the as an In the of the is from amino acids of Based on sequence C. B. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), amino acid of rSK2 to of hIK1. A chimeric is between amino acid residues rSK2 and the of hIK1, in the amino acids from rSK2, at which the hIK1 residues are The oocyte membrane was dissected prior to in a solution containing the (in mm 20 KCl, 1 MgCl2, 10 and 10 (pH adjusted to with were in the inside-out using an and on for were from and to of The solution the (in mm 5 KCl, MgCl2, 10 HEPES, and 1 (pH adjusted to with The the (in 5 KCl, MgCl2, 10 HEPES, and 1 (pH adjusted to with was to the by University of with Ca2+, was from the and was at 1 mm 10 chimera was strictly Ca2+-free all that were not activated by ATP (Roche Molecular were in with a chimera to be by ATP as a In addition, not were on oocyte in with that did express All were at a of The was to the as is the for membrane were using with a of and a of 1 were using were by in of the All are as the number of was using a A of is and is We previously demonstrated that ATP activates hIK1 via a (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). hIK1 sequence homology with the of channels C. B. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, W.J. M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), we the channels hSK1, and could similarly be by ATP in excised, inside-out patches shown for in the of addition of ATP (1 mm) failed to activate rSK2 and However, subsequent addition of the hIK1 and rSK2 D.C. Singh A.K. Frizzell R.A. Bridges R.J. Am. J. Physiol. 1996; 271: L775-L784PubMed Google Scholar, B.S. D. Christophersen P. Jorgensen C. A. Olesen S.P. A.K. Am. J. Physiol. 1998; 275: PubMed Google Scholar, Gerlach A.C. Singh A.K. Devor D.C. Am. J. Physiol. 2000; PubMed Google Scholar), a increase in In to our results with the the activity of hIK1 in response to ATP addition as previously (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The for the was as rSK2, hSK1, then an of to a as rSK2, hSK1, addition of ATP (1 mm) failed to activate the channels hSK1, whereas addition of resulted in a activation hSK1, In to these results on the to hIK1 activity from to with the subsequent addition of ATP to These demonstrate that sequence homology and in the of Ca2+-dependent the channels and hIK1, ATP-dependent modulation is for hIK1. Based on the that ATP activates hIK1 effect on the of we used a chimeric strategy between hIK1 and rSK2 to define a within hIK1 that confers ATP-dependent activation. we generated the chimera in in which the through amino acid of the was from hIK1, and the of the was from rSK2 acids shown for in addition of ATP (1 mm) to an excised, inside-out failed to whereas the subsequent addition of In the 20 by ATP whereas the subsequent addition of resulted in an increase in to The of ATP to activate the that the C-terminal of hIK1 is critical for ATP-dependent activation. To the role of the C-terminal in the ATP-dependent activation of hIK1 we generated the chimeric in which only amino acids were from hIK1 for ATP activated this as shown for a in B. average, of ATP a increase in the activity of this chimera from a of The for this activation was to that for hIK1. The increase in of this chimera in response to ATP did not from the for hIK1 suggesting that the region that the ATP of hIK1 is within the C-terminal In an to define the of hIK1 responsible for the phosphorylation-dependent we a truncation truncation of only amino acids of hIK1 resulted in channels that failed to express of we 14 amino acids were functional channels shown for in was activated by ATP. In of ATP from a of These demonstrate that hIK1 the of the C-terminal for functional and amino acids are not required for ATP-dependent activation. truncations of hIK1 failed to express functional we generated and in which of the C terminus of hIK1 were demonstrating the ATP-dependent activation of and are shown in of these chimeras was activated by ATP (1 from a activity of from a activity of and from a of The increase in for these chimeras did not from the increase for hIK1 and These that the acid region, of hIK1 the responsible for ATP dependence. The amino acid sequence of this region is shown in In to these the chimera to in which the C-terminal of rSK2 acids was of hIK1, failed to express functional channels and could not be to our this that the C terminus of hIK1 is to the functional expression of these The amino acid sequence between residues and of hIK1 consensus phosphorylation the does 10 serines and of these residues are between hIK1 and have been shown to ion channels at we these non-conserved residues to in in ATP (1 mm) activated the from an of 10 to ATP activated the mutation from a activity of to Finally, the mutations into hIK1 effect on the of ATP to activate the channel, ATP from 6 to These demonstrate that ATP-dependent modulation of hIK1 is independent of non-conserved serines and threonines within amino acids of hIK1. To the of hIK1 responsible for ATP-dependent regulation, we generated chimeras in which of the C terminus of hIK1 were These and overlap with the region of hIK1 that is critical for the Ca2+-dependent of calmodulin S. H. K. J. K. Chandy K.G. Cahalan M.D. J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) and rSK2 B. A. T. T. B. S. Maylie J. Adelman J.P. 1998; PubMed Scopus Google Scholar, R. T. A. A. B. Adelman J.P. Maylie J. J. 1999; PubMed Google Scholar). is for this that these did not express functional channels as well as the chimeras To increase our we these chimeras at 10 Ca2+, we have previously shown that ATP activates hIK1 at this of (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In 10 failed to respond to ATP, having an of which to to in the of ATP. a the to to failed to respond to ATP (1 a increase in 5 In the which was not by ATP whereas to These that the 14 amino acids of hIK1, Arg355–Met368, are for the ATP-dependent regulation of the channel. To define the role of this acid in the ATP-dependent activation of hIK1, we generated a chimera in which we amino acids Arg355–Met368 of hIK1 into the homologous region of rSK2 acids acid region is shown in shown for in these 14 amino acids from hIK1 into rSK2 ATP sensitivity to In 10 the in 10 and this in response to ATP activation was ∼50‥ of that for hIK1 in response to ATP These results demonstrate within amino acids of hIK1, the acid region Arg355–Met368 is critical to ATP-dependent activation. We generated chimeras in which we regions of rSK2 into the C terminus of hIK1 in an to demonstrate of ATP-dependent activation in hIK1. to we both with hIK1 truncations and the these failed to express functional we demonstrated that phosphorylation hIK1 activity only in the of 100 However, these studies did not the of the ATP-dependent activation of hIK1 to To this we the for which a is shown in into a containing 10 Ca2+, hIK1 to a of activity Ca2+-free was for 1 min to complete of Ca2+. that activity to demonstrating that to the for activity this Ca2+-free containing ATP (1 mm) was for min. of is to maximal activation in the of and that this failed to increase demonstrating that ATP activate hIK1 in the of Ca2+. with ATP, Ca2+-free ATP was for 1 min. this 10 and was to the to complete of ATP from the Finally, the solution containing 10 was in a activation of hIK1 to The of this is with an ATP-dependent phosphorylation in the of Ca2+, the in 10 subsequent to ATP was the In the to and this was of Ca2+. of 10 Ca2+, to ATP in Ca2+-free the to increase in activity is not from that hIK1 was by ATP in the of These demonstrate that ATP-dependent phosphorylation in the of Ca2+. We previously demonstrated that ATP activates hIK1 in a protein kinase A inhibitor in excised, inside-out patches. These results suggested a role for phosphorylation in the regulation of this KCa channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In the we hIK1/rSK2 chimeras and hIK1 truncations to amino acids as and for the ATP-dependent regulation of hIK1. The is of consensus phosphorylation and mutation of all serines and threonines within this region that are in hIK1 and not rSK2 resulted in channels that remained sensitive to ATP. The first 14 amino acids of this region, Arg355–Met368, a critical role in the ATP-dependent modulation of hIK1, substitution of these amino acids into rSK2 an ATP-dependent increase in that was ∼50‥ of the hIK1 Several could for the of the in the ATP-dependent regulation of hIK1. is that ATP directly this region of the channel. could then a in the phosphorylation of an hIK1 of the be we to be consensus ATP sites. previously hIK1 can be activated by ATP all consensus phosphorylation sites are (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). this phosphorylation at a site or at the stimulatory effect of ATP could phosphorylation of residues within the We this to be substitution of the Arg355–Met368 region of hIK1 into rSK2 confers ATP region does a single serine mutation of this in hIK1 to alanine does not the sensitivity of the to ATP. we that the phosphorylation-dependent regulation of hIK1 is via an that with amino acids Arg355–Ala413. The role of in the regulation of ion channels is well these confer cell the sensitivity of the KCa to cAMP-dependent protein kinase is dependent with In the of the activity is by cAMP-dependent protein whereas in the of activity is M.C. Chang J. 1996; PubMed Google Scholar). The that a be for hIK1. We previously demonstrated a role for cAMP-dependent protein phosphorylation of hIK1 in both and heterologous expression However, hIK1 was in cells, the was via cAMP-dependent protein phosphorylation (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). (9Khanna R. Chang M.C. Joiner W.J. Kaczmarek L.K. Schlichter L.C. J. Biol. Chem. 1999; 274: 14838-14849Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) phosphorylation-dependent modulation of hIK1. These natively in T hIK1 activity was via In contrast, hIK1 was in cells, kinase inhibitors effect on our results are of the of a in the ATP-dependent regulation of hIK1, this that Xenopus oocytes express this we have that Xenopus oocytes express this truncations only amino acids of the hIK1 C terminus failed to express functional In contrast, been that C-terminal truncations in of 100 amino acids of rSK2 have functional B. A. T. T. B. S. Maylie J. Adelman J.P. 1998; PubMed Scopus Google Scholar). In our chimeric in which of rSK2 were or hIK1 resulted in channels that failed to express functional whereas chimeras in which of hIK1 were either or rSK2 on these that unlike rSK2, the region that confers phosphorylation hIK1 is required for the functional expression of the channel. first by Joiner W.J. M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), this region of both hIK1 and rSK2 a and in are to a variety of B. J. Sci. 1994; Full Text PDF PubMed Scopus Google Scholar), be to the role of this in the ATP-dependent modulation and functional expression of hIK1. We previously demonstrated that phosphorylation does not increase hIK1 activity in the of (1Gerlach A.C. Gangopadhyay N.N. Devor D.C. J. Biol. Chem. 2000; 275: 585-598Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Here we demonstrate that ATP-dependent phosphorylation in the of Ca2+, this phosphorylation is to activate the is the required for These a role for Ca2+-dependent as in the ATP-dependent activation of hIK1. These results are with our previous demonstrating that the inhibitor of kinase kinase inhibitor failed to alter the ATP-dependent activation of hIK1. the region we define as critical for phosphorylation-dependent activation with the region of hIK1 that calmodulin in a Ca2+-dependent manner, our results demonstrate that hIK1 not be in either a or a for phosphorylation to In using a series of truncations and hIK1/rSK2 we have defined amino acids as and for the ATP-dependent modulation of hIK1. The first 14 amino acids of this region, Arg355–Met368, are critical ATP to of all serines and threonines within the that are not in rSK2 results in channels that sensitive to ATP-dependent phosphorylation. the ATP-dependent activation of hIK1 be independent of phosphorylation of the hIK1