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• W2091153250 abstract "We previously demonstrated that the endogenously expressed human intermediate conductance, Ca2+-activated K+ channel (hIK1) was inhibited by arachidonic acid (AA) (Devor, D. C., and Frizzell, R. A. (1998) Am. J. Physiol. 274, C138–C148). Here we demonstrate, using the excised, inside-out patch-clamp technique, that hIK1, heterologously expressed in HEK293 cells, is inhibited 82 ± 27 (n = 16) with 3 ॖm AA, being half-maximally inhibited (IC50) at 1.4 ± 0.7 ॖm. In contrast, AA does not inhibit the Ca2+-dependent, small conductance K+ channel, rSK2, another member of the KCNNgene family. Therefore, we utilized chimeric hIK1/rSK2 channels to define the AA binding domain on hIK1 to the S5-Pore-S6 region of the channel. Subsequent site-directed mutagenesis revealed that mutation of Thr250 to Ser (T250S) resulted in a channel with limited sensitivity to block by AA (8 ± 27, n = 8), demonstrating that Thr250 is a key molecular determinant for the inhibition of hIK1 by AA. Likewise, when Val275 in S6 was mutated to Ala (V275A) AA inhibited only 43 ± 117 (n = 9) of current flow. The double mutation T250S/V275A eliminated the AA sensitivity of hIK1. Introducing the complimentary single amino acid substitutions into rSK2 (S359T and A384V) conferred partial AA sensitivity to rSK2, 21 ± 37 and 31 ± 37, respectively. Further, introducing the double mutation S359T/A384V into rSK2 resulted in a 63 ± 87 (n= 9) inhibition by AA, thereby demonstrating the ability to introduce this inhibitory AA binding site into another member of theKCNN gene family. These results demonstrate that AA interacts with the pore-lining amino acids, Thr250 and Val275 in hIK1, conferring inhibition of hIK1 by AA and that AA and clotrimazole share similar, if not identical, molecular sites of interaction. We previously demonstrated that the endogenously expressed human intermediate conductance, Ca2+-activated K+ channel (hIK1) was inhibited by arachidonic acid (AA) (Devor, D. C., and Frizzell, R. A. (1998) Am. J. Physiol. 274, C138–C148). Here we demonstrate, using the excised, inside-out patch-clamp technique, that hIK1, heterologously expressed in HEK293 cells, is inhibited 82 ± 27 (n = 16) with 3 ॖm AA, being half-maximally inhibited (IC50) at 1.4 ± 0.7 ॖm. In contrast, AA does not inhibit the Ca2+-dependent, small conductance K+ channel, rSK2, another member of the KCNNgene family. Therefore, we utilized chimeric hIK1/rSK2 channels to define the AA binding domain on hIK1 to the S5-Pore-S6 region of the channel. Subsequent site-directed mutagenesis revealed that mutation of Thr250 to Ser (T250S) resulted in a channel with limited sensitivity to block by AA (8 ± 27, n = 8), demonstrating that Thr250 is a key molecular determinant for the inhibition of hIK1 by AA. Likewise, when Val275 in S6 was mutated to Ala (V275A) AA inhibited only 43 ± 117 (n = 9) of current flow. The double mutation T250S/V275A eliminated the AA sensitivity of hIK1. Introducing the complimentary single amino acid substitutions into rSK2 (S359T and A384V) conferred partial AA sensitivity to rSK2, 21 ± 37 and 31 ± 37, respectively. Further, introducing the double mutation S359T/A384V into rSK2 resulted in a 63 ± 87 (n= 9) inhibition by AA, thereby demonstrating the ability to introduce this inhibitory AA binding site into another member of theKCNN gene family. These results demonstrate that AA interacts with the pore-lining amino acids, Thr250 and Val275 in hIK1, conferring inhibition of hIK1 by AA and that AA and clotrimazole share similar, if not identical, molecular sites of interaction. arachidonic acid human embryonic kidney 5,8,11,14-eicosatetraynoic cyclo-oxygenase lipoxygenase Intermediate conductance, Ca2+-activated K+ channels play crucial roles in a wide array of physiological processes, including agonist-mediated transepithelial Cl− secretion across airway and intestinal epithelia (1Dharmsathaphorn K. Cohn J. Beuerlein G. Am. J. Physiol. 1989; 256: C1224-C1230Crossref PubMed Google Scholar, 2Devor D.C. Frizzell R.A. Am. J. Physiol. 1998; 274: C138-C148Crossref PubMed Google Scholar, 3Warth R. Hamm K. Bleich M. Kunzelmann K. von Hahn T. Schreiber R. Ullrich 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, 4Warhurst G. Higgs N.B. Tonge A. Turnberg L.A. Am. J. Physiol. 1991; 261: G220-G228PubMed Google Scholar, 5Kachintorn U. Vajanaphanich M. Traynorkaplan A.E. Dharmsathaphorn K. Barrett K.E. Br. J. Pharmacol. 1993; 109: 510-517Crossref PubMed Scopus (49) Google Scholar, 6Devor D.C. Singh A.K. Lambert L.C. DeLuca A. Frizzell R.A. Bridges R.J. J. Gen. Physiol. 1999; 113: 743-760Crossref PubMed Scopus (244) Google Scholar). Indeed, Dharmsathaphorn and Pandol (7Dharmsathaphorn K. Pandol S.J. J. Clin. Invest. 1986; 77: 348-354Crossref PubMed Scopus (238) Google Scholar) initially proposed that Ca2+-mediated intestinal Cl− secretion was dependent upon the activation of a basolateral membrane K+conductance in the absence of a change in apical membrane Cl− conductance. As a consequence of the increased K+ conductance there would be a hyperpolarization of the membrane potential, thereby increasing the electrochemical driving force for Cl− exit across the apical membrane via constitutively active Cl− channels (7Dharmsathaphorn K. Pandol S.J. J. Clin. Invest. 1986; 77: 348-354Crossref PubMed Scopus (238) Google Scholar, 8Hamilton K.L. Meads L. Butt A.G. Pfluegers Arch. Eur. J. Physiol. 1999; 439: 158-166PubMed Google Scholar). An increase in intracellular Ca2+ alone can stimulate Cl−secretion across the colonic epithelial cell line, T84 (5Kachintorn U. Vajanaphanich M. Traynorkaplan A.E. Dharmsathaphorn K. Barrett K.E. Br. J. Pharmacol. 1993; 109: 510-517Crossref PubMed Scopus (49) Google Scholar, 9Devor D.C. Singh A.K. Bridges R.J. Frizzell R.A. Am. J. Physiol. 1996; 271: L785-L795PubMed Google Scholar). Yet, there is a disparity between the magnitude and time course of the rise in intracellular Ca2+ and the subsequent Cl−secretory response (1Dharmsathaphorn K. Cohn J. Beuerlein G. Am. J. Physiol. 1989; 256: C1224-C1230Crossref PubMed Google Scholar, 4Warhurst G. Higgs N.B. Tonge A. Turnberg L.A. Am. J. Physiol. 1991; 261: G220-G228PubMed Google Scholar) such that the Cl− secretory response is transient in nature. These data suggest that second messengers other than Ca2+ may down-regulate the secretory response. Various inhibitory second messengers have been proposed, including protein kinase C (5Kachintorn U. Vajanaphanich M. Traynorkaplan A.E. Dharmsathaphorn K. Barrett K.E. Br. J. Pharmacol. 1993; 109: 510-517Crossref PubMed Scopus (49) Google Scholar, 10Traynor-Kaplan A.E. Buranawuti T. Vajanaphanich M. Barrett K.E. Am. J. Physiol. 1994; 267: C1224-C1230Crossref PubMed Google Scholar, 11Vaandrager A.B. Vandenberghe N. Bot A.G.M. Dejonge H.R. Am. J. Physiol. 1992; 262: G249-G256PubMed Google Scholar), inositol tetrakisphosphate (12Kachintorn U. Vajanaphanich M. Barrett K.E. Traynor-Kaplan A.E. Am. J. Physiol. 1993; 264: C671-C676Crossref PubMed Google Scholar, 13Vajanaphanich M. Schultz C. Rudolf M.T. Wasserman M. Enyedi P. Craxton A. Shears S.B. Tsien R.Y. Barrett K.E. Traynor-Kaplan A. Nature. 1994; 371: 711-714Crossref PubMed Scopus (178) Google Scholar), and arachidonic acid (AA)1 (2Devor D.C. Frizzell R.A. Am. J. Physiol. 1998; 274: C138-C148Crossref PubMed Google Scholar, 14Barrett K.E. Cell Signal. 1995; 7: 225-233Crossref PubMed Scopus (7) Google Scholar, 15Lindeman R.P. Chase H.S. Am. J. Physiol. 1992; 263: C140-C146Crossref PubMed Google Scholar). It is proposed that these second messengers may inhibit the K+conductance, thus modulating the Cl− secretory response even in the presence of elevated intracellular Ca2+levels. Ca2+-mediated agonists are known to increase AA levels in a wide range of tissues where hIK1 is expressed, including colon and lung (14Barrett K.E. Cell Signal. 1995; 7: 225-233Crossref PubMed Scopus (7) Google Scholar, 16Barrett K.E. Bigby T.D. Am. J. Physiol. 1993; 264: C446-C452Crossref PubMed Google Scholar, 17DeRubertis F.R. Craven P.A. Saito R. J. Clin. Invest. 1984; 74: 1614-1624Crossref PubMed Scopus (116) Google Scholar, 18Lawson L.D. Powell D.W. Am. J. Physiol. 1987; 252: G783-G790PubMed Google Scholar). This can occur in several ways (19Abdel-Latif A.A. Pharmacol. Rev. 1986; 38: 227-272PubMed Google Scholar), including: 1) Ca2+ directly activating PLA2, 2) either diacylglycerol itself or protein kinase C activating PLA2, or 3) diacylglycerol lipase directly generating AA from diacylglycerol. Thus, the generation of AA by Ca2+-mediated agonists would be expected to lag behind the rise in intracellular Ca2+. Given this, an effect of AA on hIK1 would be temporally appropriate to explain the dissociation between changes in intracellular Ca2+ and the resultant Cl− secretory response. Likewise, AA is released during inflammatory responses such as asthma (20Calabrese C. Triggiani M. Marone G. Mazzarella G. Allergy. 2000; 55: 27-30Crossref PubMed Scopus (43) Google Scholar) and irritable bowel disease (21Pacheco S. Hillier K. Smith C. Clin. Sci. 1987; 73: 361-364Crossref PubMed Scopus (55) Google Scholar) such that AA may play an important role in modulating ion channels in these diseases. Arachidonic acid has been shown to modulate a wide variety of ion channels, including K+, Na+, Ca2+, and Cl− channels (22Meves H. Prog. Neurobiol. 1994; 43: 175-186Crossref PubMed Scopus (196) Google Scholar, 23Ordway R.W. Singer J.J. Walsh Jr., J.V. Trends Neurosci. 1991; 14: 96-100Abstract Full Text PDF PubMed Scopus (347) Google Scholar, 24Anderson M.P. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7334-7338Crossref PubMed Scopus (96) Google Scholar, 25Wang W.H. Cassola A. Giebisch G. Am. J. Physiol. 1992; 262: F554-F559Crossref PubMed Google Scholar). Indeed, we previously demonstrated that inhibition of cytosolic PLA2 resulted in a potentiated Cl− secretory response to the Ca2+-mediated agonist, carbachol in T84 cells and that AA was a potent negative modulator of the intermediate conductance, Ca2+-dependent K+ channel in these cells (2Devor D.C. Frizzell R.A. Am. J. Physiol. 1998; 274: C138-C148Crossref PubMed Google Scholar). Recently, we (26Gerlach 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) and others (3Warth R. Hamm K. Bleich M. Kunzelmann K. von Hahn T. Schreiber R. Ullrich 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) have confirmed the molecular identity of this colonic epithelial K+channel as being the recently cloned hIK1/hSK4 (27Ishii T.M. Silvia C. Hirschberg B. Bond C.T. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11651-11656Crossref PubMed Scopus (520) Google Scholar, 28Joiner W.J. Wang L.Y. Tang M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11013-11018Crossref PubMed Scopus (318) Google Scholar). hIK1 is a member of the KCNN gene family, exhibiting significant homology with the SK channels (SK1–3), having ∼407 identity at the amino acid level (27Ishii T.M. Silvia C. Hirschberg B. Bond C.T. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11651-11656Crossref PubMed Scopus (520) Google Scholar, 28Joiner W.J. Wang L.Y. Tang M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11013-11018Crossref PubMed Scopus (318) Google Scholar). In the present study, we demonstrate that AA directly inhibits heterologously expressed hIK1, whereas rSK2 is insensitive to AA. Using a series of hIK1/rSK2 chimeras and point mutations we demonstrate that AA inhibits hIK1 via an interaction with two pore-lining amino acids, Thr250 and Val275. Importantly, substitution of these amino acids into rSK2 induces sensitivity to AA, confirming the critical nature of these amino acids in defining the molecular binding site for AA on hIK1. Human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 107 fetal bovine serum and 17 penicillin-streptomycin in a humidified 57 CO2/957 O2 incubator at 37 °C. Cells were transfected using LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. Stable cell lines were generated for all constructs by subjecting cells to antibiotic selection (1 mg/ml G418) 48 h post-transfection. Selection was typically complete within 14 days post-transfection. Following selection, the concentration of G418 was reduced to 0.2 mg/ml. We and others have previously demonstrated that hIK1, heterologously expressed in HEK cells, has identical biophysical, pharmacological, and second messenger-dependent regulatory properties to the endogenously expressed channel (26Gerlach 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, 29Singh S. Syme C.A. Singh A.K. Devor D.C. Bridges R.J. J. Pharmacol. Exp. Ther. 2001; 296: 600-611PubMed Google Scholar, 30Jensen B.S. Strobaek D. Christophersen P. Jorgensen T.D. Hansen C. Silahtaroglu A. Olesen S.P. Ahring P.K. Am. J. Physiol. 1998; 275: C848-C856Crossref PubMed Google Scholar) demonstrating the utility of this heterologous expression system. pBF plasmid containing the cDNAs for full-length hIK1 and rSK2 were kindly provided by J. P. Adelman (Vollum Institute, Oregon Health Sciences University). hIK1 and rSK2 were subcloned into pcDNA3.1+ (Invitrogen) using the EcoRI andXhoI restriction sites. The schematic structures of hIK1 and rSK2 are shown in Figs. 1 and 2, respectively.Figure 2Effect of arachidonic acid on rSK2.A, schematic of rSK2. B, representative current (pA) trace of the effect of arachidonic acid (3 ॖm) on rSK2. rSK2 was heterologously expressed in HEK cells and recorded in excised, inside-out patches at a holding potential of −100 mV. 0 Ca2+ steps occurred at the beginning and the end of the experiment to illustrate the Ca2+ dependence of rSK2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chimera generation and site-directed mutagenesis were performed as described by Gerlach et al. (31Gerlach A.C. Syme C.A. Giltinan L. Adelman J.P. Devor D.C. J. Biol. Chem. 2001; 276: 10963-10970Abstract Full Text Full Text PDF Scopus (57) Google Scholar). Briefly, chimeras between hIK1 (427 amino acids) and rSK2 (580 amino acids) were generated by overlap extension polymerase chain reaction using Pfxpolymerase (Invitrogen). The chimeras 26IK-SK, 200IK-SK, SK-200IK, and SK-287IK were generated (see Fig. 3 for schematic representations of these chimeric constructs). The chimeras were subcloned into the pcDNA 3.1+ vector using EcoR1 andXhoI restriction sites. Point mutations were generated using the QuikChange site-directed mutagenesis strategy using Pfupolymerase (Invitrogen). The fidelity of all constructs utilized in this study were confirmed by sequencing (ABI PRISM 377 automated sequencer, University of Pittsburgh) and subsequent sequence alignment (NCBI BLAST 2.0) with hIK1 (GenBankTM accession number AF022150) and rSK2 (GenBankTM accession numberU69882). Stable cell lines of each of these constructs were established in HEK293 cells as described above. The effects of AA on hIK1, rSK2, chimeric, and point-mutation channels were assessed with excised, inside-out patch-clamp experiments as a functional assay. Currents were recorded using a List EPC-7 amplifier (Medical Systems, Greenvale, NY) and stored on videotape for later analysis. Electrodes were fabricated from thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL), pulled on a vertical puller (Narishige, Long Island, NY), fire-polished, and had a resistance of 1–4 MΩ. During patch-clamp experiments, the bath solution was (in mm) potassium gluconate 145, KCl 5, MgCl2 2, HEPES 10, CaCl2 10 ॖm, pH 7.2 (adjusted with KOH). To obtain a 0 Ca2+ bath solution EGTA (1 mm) was added without CaCl2 (estimated free Ca2+ <10 nm). All bath solutions contained 100 ॖm 1-ethyl-2-benzimidazolinone to maximally activate channels and 300 ॖm ATP to prevent channel rundown (31Gerlach A.C. Syme C.A. Giltinan L. Adelman J.P. Devor D.C. J. Biol. Chem. 2001; 276: 10963-10970Abstract Full Text Full Text PDF Scopus (57) Google Scholar). The pipette solution was (in mm) potassium gluconate 140, KCl 5, MgCl2 1, HEPES 10, CaCl2 1, pH 7.2 (adjusted with KOH). All chimeras tested were strictly Ca2+-sensitive as a Ca2+-free bath solution eliminated all channel activity. Generally, a 3-ॖmconcentration of AA was used (except for the concentration-response experiments). Constructs that were not sensitive to block by AA were always tested in parallel with hIK1 or a chimera known to be sensitive to AA as a positive control. All experiments were performed at room temperature. All patches were held at a holding potential of −100 mV. The voltage is referenced to the extracellular compartment, as is the standard method for membrane potentials. Inward currents are defined as the movement of positive charge from the extracellular compartment to the intracellular compartment and are presented as downward deflections from the baseline in all recordings. Channel data were digitized with the Fetchex application within the pCLAMP suite of programs (version 6.04, Axon Instruments, Foster City, CA) using a PC computer. Single channel analysis was performed on records after low pass filtering at 200 Hz and sampling at 500 Hz (Digidata 1200, Axon Instruments). Total channel current was determined using Biopatch software (version 3.3, Bio-Logic). 1-Ethyl-2-benzimidazolinone, AA, 5,8,11,14-eicosatetraynoic (ETYA), clotrimazole, and all general chemicals were obtained from Sigma, unless otherwise stated. ATP was purchased from Roche Applied Science and added directly to a Ringer's solution as a dry powder. 1-Ethyl-2-benzimidazolinone, AA, and clotrimazole were made as 10,000-fold stock solutions in Me2SO. All data are presented as means ± S.E., where n indicates the number of experiments. Statistical analysis was performed using a Student's t test (paired or unpaired) or analysis of variance with Student-Newman-Keuls multiple posttest. A value of p < 0.05 is considered statistically significant and is reported. We previously demonstrated that AA inhibits endogenously expressed hIK1 in T84 cells with high affinity (2Devor D.C. Frizzell R.A. Am. J. Physiol. 1998; 274: C138-C148Crossref PubMed Google Scholar). Therefore, we initially characterized the AA sensitivity of heterologously expressed hIK1 (Fig. 1A) in HEK293 cells in excised, inside-out patches. As shown in a representative experiment in Fig. 1B, following the establishment of a stable current, the addition of AA (3 ॖm) induced a significant reduction in current. The average current prior to AA was 468 ± 60 pA, which was reduced by 82 ± 27 to 81 ± 18 pA in the presence of AA (p < 0.001, n = 16). Arachidonic acid inhibited hIK1 in a concentration-dependent manner, being half-maximal (IC50) at 1.4 ± 0.7 ॖm (n = 5, Fig. 1C), a value similar to what we previously reported for endogenously expressed hIK1 (IC50 = 0.42 ॖm) in T84 cells (2Devor D.C. Frizzell R.A. Am. J. Physiol. 1998; 274: C138-C148Crossref PubMed Google Scholar). Arachidonic acid can elicit its effect by interacting directly with ion channels or indirectly by metabolites produced via cyclo-oxygenase (COX), lipoxygenase (LOX), and/or cytochrome P450 pathways (22Meves H. Prog. Neurobiol. 1994; 43: 175-186Crossref PubMed Scopus (196) Google Scholar, 23Ordway R.W. Singer J.J. Walsh Jr., J.V. Trends Neurosci. 1991; 14: 96-100Abstract Full Text PDF PubMed Scopus (347) Google Scholar). We previously demonstrated that inhibitors of COX, LOX, and cytochrome P-450 did not affect the AA-dependent inhibition of endogenously expressed hIK1 in T84 cells (2Devor D.C. Frizzell R.A. Am. J. Physiol. 1998; 274: C138-C148Crossref PubMed Google Scholar). We confirmed these results in the present study by examining the effects of AA in the presence of ETYA (10 ॖm), a blocker of COX, LOX, and P450 pathways (32Holmqvist M.H. Cao J. Knoppers M.H. Jurman M.E. Distefano P.S. Rhodes K.J. Xie Y. An W.F. J. Neurosci. 2001; 21: 4154-4161Crossref PubMed Google Scholar, 33Liu Y. Liu D. Heath L. Meyers D.M. Krafte D.S. Wagoner P.K. Silvia C.P., Yu, W. Curran M.E. Mol. Pharmacol. 2001; 59: 1061-1068Crossref PubMed Scopus (38) Google Scholar) using excised, inside-out patches. As shown in Fig.1D, ETYA had no effect on hIK1 current. Under control conditions channel current was 622 ± 83 pA, and ETYA did not significantly reduce this current (560 ± 94 pA). However, subsequent addition of AA (3 ॖm) reduced channel current by 777 (117 ± 26 pA, p < 0.01,n = 3) in the continued presence of ETYA. These data suggest that oxidative metabolites of AA are not involved in the inhibition of hIK1 observed. Since hIK1 shares ∼407 sequence homology with rSK2 (27Ishii T.M. Silvia C. Hirschberg B. Bond C.T. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11651-11656Crossref PubMed Scopus (520) Google Scholar, 28Joiner W.J. Wang L.Y. Tang M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11013-11018Crossref PubMed Scopus (318) Google Scholar) we examined whether rSK2 could be inhibited by AA in excised, inside-out patches. A representative experiment is shown in Fig.2B in which AA did not reduce channel current in HEK cells heterologously expressing rSK2. The average channel current was 491 ± 169 pA in the absence of AA and 438 ± 136 pA in the presence of AA (n = 4), representing an 8 ± 37 inhibition of current flow. The Ca2+ dependence of rSK2 was subsequently verified by demonstrating that our 0 Ca2+ solution reduced channel current to zero (Fig. 2B). These data demonstrate that even though hIK1 and rSK2 share ∼407 sequence homology AA sensitivity is specific to hIK1. Based on our observation that AA inhibits hIK1, while having no effect on rSK2, we used a chimeric hIK1/rSK2 strategy to identify the region of the channel that confers AA sensitivity to hIK1. Wang and co-workers (34Macica C.M. Yang Y. Lerea K. Hebert S.C. Wang W. Am. J. Physiol. 1998; 274: F175-F181Crossref PubMed Google Scholar) have demonstrated that the NH2 terminus (particularly Ser4) of ROMK1 plays a key role in the determination of the AA effect on that channel. hIK1 has a serine (Ser24) just prior to S1. Thus, we initially generated the chimera 26IK-SK (Met1–Ala26 of hIK1 with Asp137–Ser508 of rSK2; schematic diagram in Fig. 3A) in which only the cytoplasmic NH2 terminus is derived from hIK1. As shown for one experiment in Fig. 3A (right panel), after a sustained current was established, AA had little effect (7 ± 37 inhibition, Fig. 3E) on this construct; however, 0 bath Ca2+ eliminated channel current. In five experiments, the channel current prior to the addition of AA was 296 ± 16 pA and 277 ± 17 pA in the presence of AA. The absence of inhibition of the 26IK-SK channel suggests that the cytoplasmic NH2terminus of hIK1 is insufficient to confer AA sensitivity to rSK2. Kim et al. (35Kim Y. Gnatenco C. Bang H. Kim D. Pfluegers Arch. Eur. J. Physiol. 2001; 442: 952-960Crossref PubMed Scopus (86) Google Scholar) reported that the COOH terminus is important in the AA sensitivity of TREK-2, a two-pore, four-transmembrane domain K+ channel. To elucidate the role of the COOH terminus in the AA sensitivity of hIK1, we generated the chimeric channel SK-287IK (Met1–Ala395 of rSK2 with Arg287–Lys427 of hIK1; see Fig. 3Bfor schematic) in which only the cytoplasmic COOH terminus is derived from hIK1. As shown in Fig. 3B (right panel), AA did not alter the channel current of the SK-287IK channel. In six excised, inside-out patch-clamp experiments, the average current was 467 ± 169 pA in the absence of AA and 472 ± 176 pA in the presence of AA. The failure of AA to reduce the activity of the SK-287IK channel suggests that the cytoplasmic COOH terminus of hIK1 is not sufficient to confer AA sensitivity to rSK2. To determine whether the S1-S4 or pore region of hIK1 was important in the AA sensitivity, we generated two additional channel constructs that alternated the first half of hIK1 and rSK2. The first construct, 200IK-SK (Met1–Met200 of hIK1 with Thr207–Ser508 of rSK2; see Fig. 3Cfor schematic) is composed of the entire NH2 terminus to the beginning of S5 of hIK1, while the remainder of the channel construct is derived from rSK2. As shown for one representative experiment in Fig. 3C (right panel), AA only modestly reduced current flow through 200IK-SK. AA inhibited the channel current of 200IK-SK by an average of only 5 ± 57, from 239 ± 33 pA to 228 ± 37 pA (n = 8, Fig.3E). The second channel construct was SK-200IK (Met1–Leu306 of rSK2 with Met200–Lys427 of hIK1; see Fig. 3Dfor schematic) in which the NH2 terminus through the beginning of S5 is from rSK2, whereas S5-pore-S6 and the COOH terminus is derived from hIK1. As shown by one representative trace in Fig.3D (right panel), AA significantly reduced the channel current of SK-200IK. In four experiments the average current of SK-200IK was reduced an average of 70 ± 57 (p < 0.05, Fig. 3E) from 430 ± 109 pA in the absence of AA to 127 ± 34 pA in the presence of AA. In light of the data for the SK-287IK channel construct (Fig. 3B), which demonstrated that the COOH terminus was not crucial for the AA inhibition of hIK1, the experimental results of the SK-200IK channel construct confirm that the AA sensitivity of hIK1 lies within the S5-pore-S6 region of hIK1. To identify the specific amino acid residue(s) responsible for the inhibition of hIK1 by AA, we made selected amino acid mutations in hIK1 with their rSK2 amino acid counterparts. The amino acid alignment of the S5 linker-pore region is shown in Fig.4. The amino acids that are different between hIK1 and rSK2 are shown in bold type. Initially, three multiple amino acid mutations of hIK1 were generated. These mutations are shown in Fig. 4 (underlined) and include: GHL (G235S/H236N/L237F), SDTL (S238L/D239G/T240A/L241M), and VGMW (V256M/G259N/M261Y/W262C). Fig. 5illustrates representative current traces for GHL/SNF (5A), SDTL/LGAM (5B) and VGMW/MNYC (5C). For each of these channels, AA significantly (p < 0.05) inhibited current flow (GHL/SNF, 623 ± 199pA to 133 ± 49 pA, n = 5, Fig, 5A; SDTL/LGAM 494 ± 115 pA to 83 ± 19 pA,n = 6, Fig. 5B; VGMW/MNYC 246 ± 85 pA to 53 ± 19 pA, n = 5, Fig. 5C). These data are summarized in the bar graph shown in Fig. 5F and suggest that these amino acids are not important in conferring the AA sensitivity of hIK1.Figure 5Effect of AA on the channel current of mutated hIK1-Thr250 and -Val275 confer AAsensitivity. Representative current (pA) traces of the mutated hIK1 channels with GHL/SNF (A), SDTL/LGAM (B), VGMW/MNYC (C), T250S (D), and V275A (E) in response to 3 ॖm AA. F, percentage inhibition summary data for all experiments for the mutated channels are given (number of experiments shown above thebars). Data for hIK1 are also presented for comparison. Mutated hIK1 channels were heterologously expressed in HEK cells and recorded in excised, inside-out patches at a holding potential of −100 mV. 0 Ca2+ steps occurred at the end of the experiments for the T250S and V275A constructs to illustrate the Ca2+dependence of these channels.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next mutated the threonine (Thr250) just prior to the K+ selectivity filter GYG (Gly-Tyr-Gly) motif of hIK1 to serine (T250S-hIK1), which is present in rSK2 (see Fig. 4). A representative excised patch-clamp experiment of this construct is shown in Fig. 5D. The T250S-hIK1 channel possessed significantly reduced (p< 0.05) AA sensitivity compared with hIK1 (Fig. 1B). Arachidonic acid reduced the channel current of this construct by only 8 ± 27 from 304 ± 59 pA to 280 ± 62 pA (Fig.5F, n = 8). The effect of AA on the T250S-hIK1 channel was not different than that for rSK2 (8 ± 37 inhibition, Fig. 2). These data suggest that Thr250 is the critical amino acid responsible for conferring the AA sensitivity to hIK1. It is interesting to note that Chandy and co-workers (36Wulff H. Gutman G.A. Cahalan M.D. Chandy K.G. J. Biol. Chem. 2001; 276: 32040-32045Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) previously reported that Thr250, in combination with Val275 (within S6), of hIK1 are crucial in conferring the clotrimazole sensitivity of hIK1. Based on the crystal structure of the KCSA K+ channel (37Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5770) Google Scholar), Chandy and co-workers (36Wulff H. Gutman G.A. Cahalan M.D. Chandy K.G. J. Biol. Chem. 2001; 276: 32040-32045Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) postulated that Thr250 and Val275 line the water-filled pocket that lies just below the narrow K+ selectivity filter of hIK1. In light of the data from our T250S-hIK1 construct, it was of interest to determine whether Val275 played a similar role in the AA sensitivity of hIK1. Therefore, we generated separate hIK1 constructs with V275A or the double mutation T250S/V275A to assess the effect of these amino acids on the AA sensitivity of hIK1. A representative experiment for the V275A-hIK1 construct is shown in Fig. 5E. Although AA significantly" @default.
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• W2091153250 title "Molecular Localization of the Inhibitory Arachidonic Acid Binding Site to the Pore of hIK1" @default.
• W2091153250 cites W1542181051 @default.
• W2091153250 cites W1964143284 @default.
• W2091153250 cites W1969601300 @default.
• W2091153250 cites W1972096233 @default.
• W2091153250 cites W1972636196 @default.
• W2091153250 cites W1987993834 @default.
• W2091153250 cites W1991825108 @default.
• W2091153250 cites W1992439002 @default.
• W2091153250 cites W1998163989 @default.
• W2091153250 cites W2020477012 @default.
• W2091153250 cites W2034191690 @default.
• W2091153250 cites W2043353612 @default.
• W2091153250 cites W2045419222 @default.
• W2091153250 cites W2051040059 @default.
• W2091153250 cites W2052062627 @default.
• W2091153250 cites W2054464114 @default.
• W2091153250 cites W2060201036 @default.
• W2091153250 cites W2060852213 @default.
• W2091153250 cites W2063130162 @default.
• W2091153250 cites W2065720378 @default.
• W2091153250 cites W2068142491 @default.
• W2091153250 cites W2084805245 @default.
• W2091153250 cites W2087967361 @default.
• W2091153250 cites W2089208000 @default.
• W2091153250 cites W2089640144 @default.
• W2091153250 cites W2094565808 @default.
• W2091153250 cites W2107267499 @default.
• W2091153250 cites W2125671493 @default.
• W2091153250 cites W2141626158 @default.
• W2091153250 cites W2143070723 @default.
• W2091153250 cites W2152523334 @default.
• W2091153250 cites W2161387630 @default.
• W2091153250 cites W2185280366 @default.
• W2091153250 cites W2249529700 @default.
• W2091153250 cites W2305508472 @default.
• W2091153250 cites W2337024413 @default.
• W2091153250 cites W2337187204 @default.
• W2091153250 cites W2412067085 @default.
• W2091153250 cites W2416118801 @default.
• W2091153250 cites W2417736569 @default.
• W2091153250 cites W2440801244 @default.
• W2091153250 cites W2471020284 @default.
• W2091153250 cites W2473707390 @default.
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