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• W2152523334 abstract "Selective and potent triarylmethane blockers of the intermediate conductance calcium-activated potassium channel, IKCa1, have therapeutic use in sickle cell disease and secretory diarrhea and as immunosuppressants. Clotrimazole, a membrane-permeant triarylmethane, blocked IKCa1 with equal affinity when applied externally or internally, whereas a membrane-impermeant derivative TRAM-30 blocked the channel only when applied to the cytoplasmic side, indicating an internal drug-binding site. Introduction of the S5-P-S6 region of the triarylmethane-insensitive small conductance calcium-activated potassium channel SKCa3 into IKCa1 rendered the channel resistant to triarylmethanes. Replacement of Thr250 or Val275 in IKCa1 with the corresponding SKCa3 residues selectively abolished triarylmethane sensitivity without affecting the affinity of the channel for tetraethylammonium, charybdotoxin, and nifedipine. Introduction of these two residues into SKCa3 rendered the channel sensitive to triarylmethanes. In a molecular model of IKCa1, Thr250 and Val275 line a water-filled cavity just below the selectivity filter. Structure-activity studies suggest that the side chain methyl groups of Thr250 and Val275 may lock the triarylmethanes in place via hydrophobic interactions with the π-electron clouds of the phenyl rings. The heterocyclic moiety may project into the selectivity filter and obstruct the ion-conducting pathway from the inside. Selective and potent triarylmethane blockers of the intermediate conductance calcium-activated potassium channel, IKCa1, have therapeutic use in sickle cell disease and secretory diarrhea and as immunosuppressants. Clotrimazole, a membrane-permeant triarylmethane, blocked IKCa1 with equal affinity when applied externally or internally, whereas a membrane-impermeant derivative TRAM-30 blocked the channel only when applied to the cytoplasmic side, indicating an internal drug-binding site. Introduction of the S5-P-S6 region of the triarylmethane-insensitive small conductance calcium-activated potassium channel SKCa3 into IKCa1 rendered the channel resistant to triarylmethanes. Replacement of Thr250 or Val275 in IKCa1 with the corresponding SKCa3 residues selectively abolished triarylmethane sensitivity without affecting the affinity of the channel for tetraethylammonium, charybdotoxin, and nifedipine. Introduction of these two residues into SKCa3 rendered the channel sensitive to triarylmethanes. In a molecular model of IKCa1, Thr250 and Val275 line a water-filled cavity just below the selectivity filter. Structure-activity studies suggest that the side chain methyl groups of Thr250 and Val275 may lock the triarylmethanes in place via hydrophobic interactions with the π-electron clouds of the phenyl rings. The heterocyclic moiety may project into the selectivity filter and obstruct the ion-conducting pathway from the inside. calcium activated K+ channel intermediate KCa small conductance KCa triarylmethane tetraethylammonium charybdotoxin green fluorescent protein The intermediate conductance calcium-activated potassium channel IKCa11 (also known as IK1, KCa4, SK4, KCNN4, and the “Gardos channel”) plays an important role in the physiology of lymphocytes, erythrocytes, and intestinal and airway epithelial cells (1Ishii 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, 2Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 3Vandorpe D.H. Shmukler B.E. Jiang L. Lim B. Maylie J. Adelman J.P. de Franceschi L. Cappellini M.D. Brugnara C. Alper S.L. J. Biol. Chem. 1998; 273: 21542-21553Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 4Ghanshani S. Wulff H. Miller M.J. Rohm H. Neben A. Gutman G.A. Cahalan J. Biol. Chem. 2000; 275: 37137-37149Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). IKCa1 channels are voltage-independent, and their activation is steeply sensitive to rises in intracellular Ca2+, calcium sensitivity being conferred by calmodulin tightly complexed to the C terminus of the channel (5Fanger C.M. Ghanshani S. Logsdon N.J. Rauer H. Kalman K. Zhou J. Beckingham K. Chandy K.G. Cahalan M.D. Aiyar J. J. Biol. Chem. 1999; 274: 5746-5754Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Pharmacologically, IKCa1 channels are distinguished from SKCa (SKCa1–3) channels by their sensitivity to clotrimazole and charybdotoxin and by their insensitivity to apamin. In mitogen-activated human lymphocytes, IKCa1 regulates membrane potential and calcium signaling, and selective inhibitors of IKCa1 suppress lymphocyte proliferation and cytokine secretion by attenuating Ca2+ influx (4Ghanshani S. Wulff H. Miller M.J. Rohm H. Neben A. Gutman G.A. Cahalan J. Biol. Chem. 2000; 275: 37137-37149Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 6Khanna 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, 7Jensen 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 (99) Google Scholar, 8Wulff H. Miller M.J. Hänsel W. Grissmer S. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8151-8156Crossref PubMed Scopus (536) Google Scholar, 9Fanger C.M. Rauer H. Neben A.L. Miller M.J. Rauer H. Wulff H. Rosa J.C. Ganellin C.R. Chandy K.G. Cahalan M.D. J. Biol. Chem. 2001; 276: 12249-12256Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In red blood cells, IKCa1 participates in volume control. Erythrocyte dehydration in sickle cell disease can be attributed partly to excessive K+ loss through IKCa1 channels that are activated by a rise in intracellular Ca2+ during sickling (10Brugnara C. Current Opin. Hematol. 1997; 4: 122-127Crossref PubMed Scopus (57) Google Scholar). Clotrimazole and ICA-15451, two potent triarylmethane (TRAM) blockers of this channel, are in clinical trials for the treatment of sickle cell disease (11Brugnara C. Gee B. Armsby C.C. Kurth S. Sakamoto M. Rifai N. Alper S.L. Platt O.S. J. Clin. Invest. 1996; 97: 1227-1234Crossref PubMed Scopus (207) Google Scholar, 12Coghlan M.J. Carrol W.A. Gopalakrishnan M. J. Med. Chem. 2001; 44: 1-27Crossref PubMed Scopus (218) Google Scholar). In intestinal and airway epithelium cells, basolateral expression of the IKCa1 channel modulates apical water and Cl− secretion (13Rufo P.A. Jiang L. Moe S.J. Brugnara C. Alper S.L. Lencer W.I. J. Clin. Invest. 1996; 98: 2066-2075Crossref PubMed Scopus (60) Google Scholar, 14Devor D.C. Singh A.K. Lambert L.C. DeLuca A. Frizzel R.A. Bridges R.J. J. Gen. Physiol. 1999; 113: 743-760Crossref PubMed Scopus (244) Google Scholar), and blockade of this channel by clotrimazole has been reported to ameliorate secretory diarrhea (15Rufo P.A. Merlin D. Riegler M. Ferguson-Maltzman M.H. Dickinson B.L. Brugnara C. Alper S.L. Lencer W.I. J. Clin. Invest. 1997; 100: 3111-3120Crossref PubMed Scopus (105) Google Scholar). However, the therapeutic usefulness of clotrimazole is seriously limited by its nanomolar affinity for cytochrome P450-dependent enzymes, especially CYP3A4 (the major xenobiotic metabolizing enzyme in human liver), which leads to a high incidence of side effects (11Brugnara C. Gee B. Armsby C.C. Kurth S. Sakamoto M. Rifai N. Alper S.L. Platt O.S. J. Clin. Invest. 1996; 97: 1227-1234Crossref PubMed Scopus (207) Google Scholar, 16Sawyer P.R. Brogden R.N. Pinder R.M. Speight T.M. Avery G.S. Drugs. 1975; 9: 424-447Crossref PubMed Scopus (123) Google Scholar). Starting with clotrimazole as a template, we recently designed and synthesized TRAM analogs that block IKCa1 potently and specifically without inhibiting P450-dependent enzymes (8Wulff H. Miller M.J. Hänsel W. Grissmer S. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8151-8156Crossref PubMed Scopus (536) Google Scholar). The most potent of these, TRAM-34, blocks IKCa1 with a K d of 20 nm and exhibits exquisite selectivity for the channel. Related TRAMs containing tetrazole (TRAM-84) or acetonitrile (TRAM-39) in place of the pyrazole are also selective and potent inhibitors of IKCa1. Although applied externally, the TRAMs are readily membrane-permeant because of their high lipophilicity (logP value 3.5–5.0) and may interact with a site on the inner face of the channel. Furthermore, a membrane-impermeant clotrimazole analog has been reported previously to block the native IKCa channel in erythrocytes only when applied to the cytoplasmic surface, suggesting an internal binding site (17Dunn P.M. J. Membr. Biol. 1998; 165: 133-143Crossref PubMed Scopus (41) Google Scholar). In the present study, we have used molecular chimeras and mutagenesis to identify the channel residues that interact with the TRAM inhibitors. Clotrimazole, econazole, ketoconazole, nifedipine, and TEA were purchased from Sigma. Charybdotoxin (ChTX) was from Bachem (King of Prussia, PA). TRAM-34 ([1-(2-chlorophenyl)diphenyl)methyl]-1H-pyrazole), TRAM-3 ((2-chlorophenyl)diphenylmethanol), and TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile) were synthesized as described previously (8Wulff H. Miller M.J. Hänsel W. Grissmer S. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8151-8156Crossref PubMed Scopus (536) Google Scholar). TRAM-30 was prepared by refluxing clotrimazole (2.00 g, 5.81 mmol) with an excess of methyl iodide in butanone for 36 h. The reaction mixture was evaporated to dryness. On addition of petroleum ether to the resulting oily residue and intensive cooling and scratching, the material began to solidify and was then washed thoroughly with acetone and petroleum ether (mp: 139 °C; 1H NMR (300 MHz, Me2SO): δ 9.13 (s, 1H, -(2-H), 7.72 (s, 1H), 7.48–7.41 (m, 9H), 7.27–7.21 (m, 5H), 6.96 (s, 1H), 4.26 (s, 3H, N+-CH3); MS (70eV)m/z 277 (52), 254 (13Rufo P.A. Jiang L. Moe S.J. Brugnara C. Alper S.L. Lencer W.I. J. Clin. Invest. 1996; 98: 2066-2075Crossref PubMed Scopus (60) Google Scholar), 241 (42), 226 (8Wulff H. Miller M.J. Hänsel W. Grissmer S. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8151-8156Crossref PubMed Scopus (536) Google Scholar), 199 (14Devor D.C. Singh A.K. Lambert L.C. DeLuca A. Frizzel R.A. Bridges R.J. J. Gen. Physiol. 1999; 113: 743-760Crossref PubMed Scopus (244) Google Scholar), 165 (100), 127 (11Brugnara C. Gee B. Armsby C.C. Kurth S. Sakamoto M. Rifai N. Alper S.L. Platt O.S. J. Clin. Invest. 1996; 97: 1227-1234Crossref PubMed Scopus (207) Google Scholar), 119 (30), 106 (10Brugnara C. Current Opin. Hematol. 1997; 4: 122-127Crossref PubMed Scopus (57) Google Scholar), 82 (47), 63 (10), 54 (20). C23H20CClN2 Calculated:C56.75%H4.14%N5.75%Found:C56.56%H4.12%N5.75% The cloning of human IKCa1(GenBankTM accession number AF033021) and humanSKCa3 containing 19 polyglutamines in the N terminus (GenBankTM accession numbers AF031815 and AJ251016) have been reported previously (2Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 18Ghanshani S. Coleman M. Gustavsson P. Wu A.C. Gargus J.J. Gutman G.A. Dahl N. Mohrenweiser H. Chandy K.G. Genomics. 1998; 51: 160-161Crossref PubMed Scopus (30) Google Scholar, 19Chandy K.G. Fantino E. Wittekindt O. Kalman K. Tong L.L. Ho T.H. Gutman G.A. Crocq M.A. Ganguli R. Nimgaonkar V. Morris- Rosendahl D.J. Gargus J.J. Mol. Psychiatry. 1998; 3: 32-37Crossref PubMed Scopus (184) Google Scholar). Both channel genes were subcloned into the pEGFP-C1 vector (CLONTECH) in frame with GFP. Mutant IKCa1 channels were generated by either one- or two-step polymerase chain reaction. Polymerase chain reaction products were digested with HindIII/BbrPI and cloned intoHindIII/BbrPI-cut GFP-IKCa1. ForSKCa3 mutations, KpnI and a C-terminal-introducedBamHI site were used. All clones were verified by sequencing. The DNA for transfection was prepared with the Qiagen (Valencia, CA) mini-prep kit. COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mm glutamine, 1 mm Na+ pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin. The cells were transiently transfected with GFP-tagged wild-type or mutantIKCa1 and SKCa3 cDNAs using FuGene™ 6 (Roche) according to manufacturer protocol. Cells were trypsinized 16–72 h after transfection, plated onto polylysine-coated coverslips, and used within the next 4 h. Whole-cell patch clamp recordings (EPC-9, HEKA Elektronik) were made at a holding potential of −80 mV with a pipette solution containing 145 mm K+aspartate, 2 mm MgCl2, 10 mm HEPES, 10 mm K2EGTA, and 8.5 mmCaCl2 (1 µm free [Ca2+]), pH 7.2, 290–310 mosm. To reduce currents from the native chloride channels in COS-7, Na+ aspartate Ringer was used as an external solution containing 160 mm Na+aspartate, 4.5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES, pH 7.4, 290–310 mosm. Currents were elicited by 200-ms voltage ramps from −120 to 40 mV applied every 10 s, and the reduction of slope conductance at −80 mV by drug was taken as a measure of channel block. The recordings in Fig. 1were done on excised inside-out patches with the external solution described above as pipette solution. K+ aspartate solutions containing 1 µm and 50 nm free [Ca2+] were applied to the cytoplasmic side of the patch. IKCa1 channels are potently blocked by clotrimazole and related TRAMs with a Hill coefficient of unity. These highly lipophilic compounds readily cross the plasma membrane and block the channel with a slow onset. An earlier study (17Dunn P.M. J. Membr. Biol. 1998; 165: 133-143Crossref PubMed Scopus (41) Google Scholar) that used a membrane-impermeant clotrimazole analog on the native IKCa channel in erythrocytes reported a drug-binding site on the cytoplasmic surface of the channel. To confirm this prediction on the cloned channel we generated a membrane-impermeant quaternary TRAM (TRAM-30) and evaluated its ability to block IKCa1 when added from the outside (in the whole-cell configuration) or the intracellular side in inside-outside patches. As a control we studied clotrimazole (logP = 3.5), which is freely membrane-permeant. Currents (10–200 pA) in inside-out patches from IKCa1-expressing COS-7 cells excised into 1 µm Ca2+ disappeared when the Ca2+ concentration was reduced to 50 nm and were restored after the re-addition of 1 µmCa2+ (Fig. 1 A). This Ca2+-activated current was completely and rapidly blocked by 1 µm clotrimazole (Fig. 1, A andB), and its K+ selectivity was confirmed by the shift in reversal potential when the Na+ aspartate pipette solution was replaced by K+ aspartate (Fig.1 C). These IKCa1 currents were also reversibly blocked by the membrane-impermeant quaternary compound, TRAM-30 (Fig. 1,D and E) with a K d of 700 nm (Fig. 1 F, Δ), which was almost ineffective (K d = 350 µm, ▴) when applied to the external surface of the channel in the whole-cell configuration. In contrast, the membrane-permeant clotrimazole blocked the channel with equal potency when applied to either side of the membrane (Fig.1 F, ▪ and ■). The 10-fold lower potency of TRAM-30 compared with clotrimazole is caused by the bulk of the additional methyl group on the imidazole ring, consistent with the published pharmacophore of the TRAMs (8Wulff H. Miller M.J. Hänsel W. Grissmer S. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8151-8156Crossref PubMed Scopus (536) Google Scholar). These results suggest that TRAMs interact with a site on the intracellular surface of the channel. To localize the region on the channel to which the TRAMs bind, we generated a chimera between IKCa1 and the phylogenetically related TRAM-insensitive small conductance SKCa3 channel (Fig.2A). In whole-cell recordings, IKCa1 currents were potently blocked by externally applied TRAM-34 (Fig. 2 A, left), whereas SKCa3 was insensitive (Fig. 2 A, middle). A chimera containing the SKCa3 S5-P-S6 region in the IKCa1 body was insensitive to 1 µmTRAM-34 (Fig. 2 A, right). Together with the data obtained in inside-out experiments (see above), these results suggest that the TRAM-binding site is situated on the cytoplasmic side of the S5-P-S6 region of IKCa1. An alignment of the IKCa1 and SKCa3 amino acid sequences through this region reveals 45 differences (Fig. 2 B). To define the residues involved we replaced IKCa1 residues with the corresponding ones from SKCa3. Mutations were made in the cytoplasmic half of S5, the intracellular portion of the P-loop and the entire S6 segment, and as a control we also mutated residue 239 that had been shown previously to be involved in ChTX binding (20Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Mutations involving Thr250 (P245S + T250S; or T250S) or Val275(V275A +C276G; or V275A) abolished sensitivity to TRAM-34 and clotrimazole, whereas all other mutants showed unchanged TRAM-34 sensitivity (Fig. 2 B, TableI). The V272I mutant could not be investigated because it was nonfunctional. The T250S and V275A mutants were studied in more detail.Table IKd values of TRAM-34 for wild-type (WT) and mutant IKCa1 channelsK d for TRAM-34WT-IKCa120 nmThr250P245S + T250S>20 µmT250S>20 µmD239K + P245S20 nmVal275V272I + V275A + C276G + L281Vnot functionalD239K + P245S + L281V28 nmV272Inot functionalV275A + C276G>20 µmV275A>20 µmOthersY199L + N201T + T202I + H203C + R206T + L207V25 nmG259H + M261Y + W262C120 nmI265G30 nmC269L20 nmF291L + N292T20 nmThr250: Asp239 and Pro245 are not part of the TRAM-binding site, because mutations at these positions do not alter TRAM sensitivity. Mutations involving Thr250 abolish TRAM binding. Val275: Leu281 is not part of the binding site, because a mutation involving this residue did not alter TRAM sensitivity. Mutations involving Val272 could not be studied, because they were nonfunctional. Mutations involving Val275completely abolished TRAM sensitivity. The C276G point mutant was not generated, because Cys276 faces away from the pore in a published model of IKCa1 (20Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Others: these mutants did not alter TRAM sensitivity significantly with the exception of the G259H + 261Y + W262C mutant, which reduced TRAM sensitivity 6-fold. These residues are buried in the IKCa1 model, and the small change in TRAM sensitivity resulting from their substitution with SKCa3 residues may be caused by an indirect effect via alteration of the shape of the protein. Open table in a new tab Thr250: Asp239 and Pro245 are not part of the TRAM-binding site, because mutations at these positions do not alter TRAM sensitivity. Mutations involving Thr250 abolish TRAM binding. Val275: Leu281 is not part of the binding site, because a mutation involving this residue did not alter TRAM sensitivity. Mutations involving Val272 could not be studied, because they were nonfunctional. Mutations involving Val275completely abolished TRAM sensitivity. The C276G point mutant was not generated, because Cys276 faces away from the pore in a published model of IKCa1 (20Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Others: these mutants did not alter TRAM sensitivity significantly with the exception of the G259H + 261Y + W262C mutant, which reduced TRAM sensitivity 6-fold. These residues are buried in the IKCa1 model, and the small change in TRAM sensitivity resulting from their substitution with SKCa3 residues may be caused by an indirect effect via alteration of the shape of the protein. The T250S mutant produced a K+-selective current equally sensitive to block by ChTX (K d = 5 ± 1 nm), TEA (K d = 20 ± 4 mm), and nifedipine (K d = 5 ± 1 µm) as the wild-type IKCa1 channel (4Ghanshani S. Wulff H. Miller M.J. Rohm H. Neben A. Gutman G.A. Cahalan J. Biol. Chem. 2000; 275: 37137-37149Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 8Wulff H. Miller M.J. Hänsel W. Grissmer S. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8151-8156Crossref PubMed Scopus (536) Google Scholar, 20Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), whereas it was completely insensitive to TRAM-34 (Fig. 3, A–D). Similar results were obtained with the V275A mutant (Fig. 3,E–H). Thus, these two point mutations selectively abolished TRAM-34 sensitivity without altering other pharmacological properties of the channel or its K+ selectivity, indicating that the overall conformation of these mutant channels had not been grossly perturbed. It also shows that the TRAM-binding site is distinct from those of ChTX, TEA, and nifedipine. The TRAM pharmacophore consists of two parts, a triphenyl moiety and either a small heterocycle or another π-electron-rich substituent such as an acetonitrile or hydroxyl group. To identify the part of the molecule that interacts with the channel, we first examined four TRAMs that contain the triphenyl moiety but different substituents, TRAM-34 (pyrazole), TRAM-39 (acetonitrile), TRAM-3 (hydroxyl), and clotrimazole (imidazole). All four compounds were inactive on the T250S and V275A mutant channels (Fig. 4). In contrast, two azole inhibitors of IKCa1 (econazole and ketoconazole) containing an imidazole moiety such as clotrimazole but lacking the triphenyl group blocked both mutant channels as effectively as the wild-type channel (Figs. 3 H and 4). Collectively, these results suggest that the triphenyl moiety interacts with Thr250 and Val275. If Thr250and Val275 constitute the TRAM-binding site, introduction of these residues into the SKCa3 channel should confer sensitivity to the TRAMs. The SKCa3-S507T+A532V mutant produced a K+-selective channel (Fig.5A) that was blocked by apamin with a potency roughly equal to the wild-type SKCa3 channel (Fig.5 B). Unlike SKCa3, which was relatively insensitive to TRAM-34 (K d = 28 µm), the S507T+A532V-SKCa3 mutant was substantially blocked by 1 and 5 µm TRAM-34 (K d = 680 ± 70 nm, Fig. 5, C and D). The positions of Thr250 and Val274 are highlighted in a published model of the IKCa1 pore (20Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) based on the KcsA crystal structure (Fig. 6). Both residues line a large water-filled cavity that lies just below the narrow selectivity filter of the channel, which is formed by the conserved GYG motif. If this cavity in IKCa1 is as wide as the 10-Å cavity in KcsA, it should easily accommodate clotrimazole and TRAM-34, both of which have dimensions of 9.5 × 9.5 × 8.6 Å. An AM-1-optimized structure of TRAM-34 drawn to scale is shown in Fig. 6 next to the channel model. A highly lipophilic membrane-permeant compound like TRAM-34 (logP = 4.0) could easily reach the internal cavity either by traversing the ion conduction pathway from the inside or through crevices and tunnels in the protein similar to those recently described in sodium channels (21Sato C. Ueno Y. Asai K. Takahashi K. Sato M. Engel A. Fujiyoshi Y. Nature. 2001; 409: 1047-1051Crossref PubMed Scopus (228) Google Scholar). Using a combination of electrophysiology and mutagenesis we have defined the TRAM-binding site on IKCa1. Our data suggest that the binding site is located on the cytoplasmic side of the S5-P-S6 region of the channel. First, a membrane-impermeant analog blocks the channel with 500-fold greater potency when added from the insideversus the outside, and second, introduction of the S5-P-S6 region of the TRAM-resistant SKCa3 into IKCa1 renders the channel insensitive to these compounds. Single and multiple replacements of IKCa1 residues in the S5-P-S6 region with the corresponding residues in SKCa3 identified Thr250 in the P-region and Val275 in S6 as being critical for TRAM binding. Based on our IKCa1 model (20Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), both these residues lie in close proximity to each other in the water-filled cavity below the ion selectivity filter. The introduction of these two residues into SKCa3 made this channel TRAM-sensitive. Given the tetrameric nature of K+ channels, the TRAM-binding site could involve Thr250 and Val275 from more than one subunit. The exquisite selectivity of IKCa1 for the TRAMs is most likely caused by the tight interactions between the propeller-shaped TRAMs and the Thr250 and Val275 of multiple IKCa1 subunits. The three SKCa channels contain serine and alanine at positions equivalent to Thr250 and Val275, and these residues contribute to their TRAM insensitivity. Consistent with this idea, the introduction of Thr250 and Val275 into SKCa3 (SK3-S507T+A532V) produces an ∼40-fold enhancement in TRAM-34 sensitivity compared with the wild-type SKCa3 channel (Fig. 5). Other TRAM-resistant Kv (Kv1.1–1.5, 3.1, 4.2), Kir (Kir2.1), and BKCa (Slo) channels contain threonine and valine at positions homologous to Thr250 and Val275, although they share little sequence similarity with IKCa1 in the residues neighboring the threonine and valine (22Doyle 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). The threonine and valine residues in these channels may be oriented differently from Thr250 and Val275 in IKCa1, resulting in a less than optimal interaction with the phenyl rings of TRAM-34. Differences in the shape of the internal cavities of these channels compared with IKCa1 may also contribute to TRAM insensitivity. Bulky residues in the vicinity of the threonine or valine in these channels may also sterically interfere with their ability to interact with TRAMs. In an earlier study, we proposed that the TRAM-binding site had dimensions of roughly 9.5 × 9.5 × 8.6 Å based on structure-activity relationships of the TRAMs; introduction of bulky substituents on the triphenyl moiety or on the heterocycle abolished affinity (8Wulff H. Miller M.J. Hänsel W. Grissmer S. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8151-8156Crossref PubMed Scopus (536) Google Scholar). Consistent with this earlier report, our heuristic model suggests that TRAM-34 could fit snugly into the internal cavity just below the selectivity filter with each of the phenyl rings locked in place by interactions with Thr250 and Val275from three of the four IKCa1 subunits. We propose that the π-electron cloud of the phenyl rings of TRAM-34 form hydrophobic interactions with the positively polarized hydrogen atoms of the methyl group of Thr250. The conservative substitution of serine in place of Thr250 abolishes TRAM sensitivity, possibly because serine lacks the side chain methyl group of threonine. Val275could also form a hydrophobic interaction with the phenyl rings. This interaction must be size-dependent, because the introduction of the smaller alanine (88 Å3) in place of the valine (140 Å3) abolishes TRAM sensitivity. Although each hydrophobic interaction in itself is weak, taken together they may well account for the 20 nm K d value of TRAM-34. Our model also predicts that the heterocyclic moiety could project upwards into the selectivity filter and occlude the ion conduction pathway. We would like to thank Ray Norton and Mark Lanigan for the generation of the IKCa1 model and Annabelle Chialing Wu and Hilda Fernandez for excellent technical assistance." @default.
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