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Defining the Drug-binding Site in the Human Multidrug Resistance P-glycoprotein Using a Methanethiosulfonate Analog of Verapamil, MTS-verapamil

2001; Elsevier BV; Volume: 276; Issue: 18 Linguagem: Inglês

10.1074/jbc.m100407200

ISSN

1083-351X

Autores

Tip W. Loo, David M. Clarke,

Tópico(s)

HIV/AIDS drug development and treatment

Resumo

Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the drug-binding domain of P-gp. MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a Km value (25 μm) that was similar to that of verapamil. 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil. Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the binding of verapamil. Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the drug-binding domain of P-gp. MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a Km value (25 μm) that was similar to that of verapamil. 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil. Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the binding of verapamil. P-glycoprotein transmembrane dibromobimane methanethiosulfonate ATP-binding cassette The human multidrug resistance P-glycoprotein (P-gp)1 uses ATP to pump a wide variety of cytotoxic compounds out of the cell (1Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (406) Google Scholar, 2Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1907) Google Scholar). Overexpression of P-gp contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy, because many of the therapeutic compounds are also substrates of P-gp (3Kim R.B. Fromm M.F. Wandel C. Leake B. Wood A.J. Roden D.M. Wilkinson G.R. J. Clin. Invest. 1998; 101: 289-294Crossref PubMed Scopus (1028) Google Scholar, 4Lee C.G. Gottesman M.M. Cardarelli C.O. Ramachandra M. Jeang K.T. Ambudkar S.V. Pastan I. Dey S. Biochemistry. 1998; 37: 3594-3601Crossref PubMed Scopus (457) Google Scholar, 5Robert J. Eur. J. Clin. Invest. 1999; 29: 536-545Crossref PubMed Scopus (71) Google Scholar).Although P-gp is normally expressed in many tissues, its physiological function is unknown. The pattern of P-gp expression in tissues and studies on P-gp "knock-out" mice indicate that it may protect the organism from toxic compounds in our diet (6Thiebaut F. Tsuruo T. Hamada H. Gottesman M.M. Pastan I. Willingham M.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7735-7738Crossref PubMed Scopus (2545) Google Scholar, 7Cordon-Cardo C. O'Brien J.P. Casals D. Rittman-Grauer L. Biedler J.L. Melamed M.R. Bertino J.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 695-698Crossref PubMed Scopus (1584) Google Scholar, 8Schinkel A.H. Smit J.J. van Tellingen O. Beijnen J.H. Wagenaar E. van Deemter L. Mol C.A. van der Valk M.A. Robanus-Maandag E.C. te Riele H.P. Berns A.J.M. Borst P. Cell. 1994; 77: 491-502Abstract Full Text PDF PubMed Scopus (2051) Google Scholar).P-gp is a member of the ABC (ATP-binding cassette) family of transporters (9Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 10Holland I.B. Blight M.A. J. Mol. Biol. 1999; 293: 381-399Crossref PubMed Scopus (487) Google Scholar). Its 1280 amino acids are organized as two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (11Chen C.J. Chin J.E. Ueda K. Clark D.P. Pastan I. Gottesman M.M. Roninson I.B. Cell. 1986; 47: 381-389Abstract Full Text PDF PubMed Scopus (1709) Google Scholar). There are six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site in each repeat (12Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 13Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar).The exact mechanism of how P-gp functions is unknown, but P-gp has been studied quite extensively and serves as a model for understanding the mechanism of other ABC transporters. It is known, however, that both halves of P-gp are essential for activity (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar) and that both nucleotide-binding domains can bind and hydrolyze ATP and are essential for function (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 15Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar, 16Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 17Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 18Hrycyna C.A. Ramachandra M. Ambudkar S.V. Ko Y.H. Pedersen P.L. Pastan I. Gottesman M.M. J. Biol. Chem. 1998; 273: 16631-16634Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar).In understanding the mechanism of P-gp, it is important to determine the location of residues that contribute to the drug-binding domain. Important clues that the transmembrane domains may be important for drug binding were obtained from mutational studies and from labeling studies with photoactive substrate analogs (19Bruggemann E.P. Currier S.J. Gottesman M.M. Pastan I. J. Biol. Chem. 1992; 267: 21020-21026Abstract Full Text PDF PubMed Google Scholar, 20Greenberger L.M. J. Biol. Chem. 1993; 268: 11417-11425Abstract Full Text PDF PubMed Google Scholar, 21Morris D.I. Greenberger L.M. Bruggemann E.P. Cardarelli C. Gottesman M.M. Pastan I. Seamon K.B. Mol. Pharmacol. 1994; 46: 329-337PubMed Google Scholar, 22Zhang X. Collins K.I. Greenberger L.M. J. Biol. Chem. 1995; 270: 5441-5448Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 23Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (189) Google Scholar, 24Kajiji S. Talbot F. Grizzuti K. Van Dyke-Phillips V. Agresti M. Safa A.R. Gros P. Biochemistry. 1993; 32: 4185-4194Crossref PubMed Scopus (90) Google Scholar, 25Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar). It was later shown that the TM domains alone were sufficient for drug binding, because a deletion mutant lacking both nucleotide-binding domains could still bind drug substrates (27Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar).Initial attempts to identify the residues that line the drug-binding pocket within the TM domains involved the use of cysteine-scanning mutagenesis and reactivity with a thiol reactive substrate, dibromobimane (dBBn). Several residues in TMs 4, 5, 6, 10, 11, and 12 reacted with dBBn, suggesting that these TMs contribute to the binding of substrate (28Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 29Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 30Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar).It is not known if P-gp has many distinct or overlapping binding sites (31Shapiro A.B. Ling V. Eur. J. Biochem. 1997; 250: 130-137Crossref PubMed Scopus (428) Google Scholar, 32Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Crossref PubMed Scopus (280) Google Scholar, 33Pascaud C. Garrigos M. Orlowski S. Biochem. J. 1998; 333: 351-358Crossref PubMed Scopus (139) Google Scholar, 34Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10594-10599Crossref PubMed Scopus (355) Google Scholar, 35Martin C. Berridge G. Higgins C.F. Mistry P. Charlton P. Callaghan R. Mol. Pharmacol. 2000; 58: 624-632Crossref PubMed Scopus (392) Google Scholar). One step toward understanding this problem is to test whether other substrates of P-gp react with the same residues in the predicted drug-binding domain. Verapamil is a particularly important substrate used in studying P-gp. The advantage of using verapamil is that it shows the greatest ability to stimulate the ATPase activity of P-gp. Development of a thiol-reactive derivative of verapamil would be a useful tool for characterizing the drug-binding domain of P-gp, because reactive cysteines can be specifically protected with verapamil. In this study, a thiol-reactive methanethiosulfonate analog of verapamil (MTS-verapamil) was synthesized and used to identify residues in the drug-binding domain of P-gp.DISCUSSIONCysteine-scanning mutagenesis and modification with sulfhydryl-specific agents was first used to probe the acetylcholine receptor channel (44Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar). Modifications of this method have since been used to study the structure and function of many membrane proteins (reviewed in Ref. 45Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (319) Google Scholar). This technique has been extensively used in the study of the bacterial lac permease protein (46Sahin-Toth M. Karlin A. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10729-10732Crossref PubMed Scopus (64) Google Scholar). P-gp has been a very good eukaryotic protein for cysteine-scanning mutagenesis studies, because the Cys-less P-gp remains functional (reviewed in Ref.47Loo T.W. Clarke D.M. Biochim. Biophys. Acta. 1999; 1461: 315-325Crossref PubMed Scopus (88) Google Scholar). Experiments that provide insight into the structure and mechanism of P-gp are possible because of the ability to synthesize thiol-reactive analogs of P-gp substrates such as MTS-verapamil.Fifteen of the 242 Cys mutants tested for inhibition by MTS-verapamil showed significant inhibition by MTS-verapamil. In general, the reactive mutants in TMs 4, 6, 10, 11, and 12 had lower I50values than those in TMs 2, 7, and 9 (TableI). This may indicate that TMs 4, 6, 10, 11, and 12 are within the drug-binding domain or that this region of P-gp is more accessible to MTS-verapamil. Verapamil significantly protected residues S222C (TM4), L339 (TM6), A342 (TM6), and G984 (TM12) from inactivation by MTS-verapamil. This indicates that these residues either line or are close to the verapamil-binding site. To picture the potential drug-binding domain of P-gp, we constructed a model in which the residues in each TM segments are arranged in α-helical wheels (Fig. 5). The arrangement of the TM segments are based on the results of disulfide cross-linking studies that show TM 6 is close to TMs 10, 11, and 12 and that TM12 is close to TMs 4, 5, and 6 (48Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 49Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In another ABC transporter, cystic fibrosis transmembrane conductance regulator, it has been shown that a salt bridge may exist between TM6 and TM8 (50Cotten J.F. Welsh M.J. J. Biol. Chem. 1999; 274: 5429-5435Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). TMs 4, 5, 6, 10, 11, and 12 have been postulated to line the drug-binding domain, because cross-linking between residues in these segments is affected by the presence of drug substrates. The location of the drug-binding domain within the TMs would be consistent with the idea that P-gp removes substrates that are embedded in the lipid bilayer (51Raviv Y. Pollard H.B. Bruggemann E.P. Pastan I. Gottesman M.M. J. Biol. Chem. 1990; 265: 3975-3980Abstract Full Text PDF PubMed Google Scholar, 52Homolya L. Hollo Z. Germann U.A. Pastan I. Gottesman M.M. Sarkadi B. J. Biol. Chem. 1993; 268: 21493-21496Abstract Full Text PDF PubMed Google Scholar). It is thought that substrates diffuse into the lipid bilayer and are then extracted from the bilayer leaflets by P-gp.Table IComparing inhibition of Cys P-gp mutants by dBBn or MTS-verapamilMutantInhibitorProtection by verapamildBBnMTS-verapamil (I50)dBBnMTS-verapamilμmY118C (TM2)+ 1-aInhibition.+ (40)− 1-bNo protection.−V125C (TM2)++ (63)−−S222C (TM4)++ (40)++ 1-cHighly protected from inhibition.++L339C (TM6)++ (29)++++A342C (TM6)+/−+ (14)ND 1-dND, not determined due to little or no inhibition.++A729C (TM7)1-eNo inhibition.−/+ (70)ND−S766C (TM8)+−−NDA841C (TM9)−+ (101)ND−N842C (TM9)−+ (61)ND−I868C (TM10)++ (25)+ 1-fProtection from inhibition.+A871C (TM10)−+ (14)ND−G872C (TM10)+−+NDF942C (TM11)++ (20)++T945C (TM11)++ (8)++L975C (TM12)+−+NDV982C (TM12)++ (12)−−G984C (TM12)ND+ (17)ND++A985C (TM12)++ (38)+−1-g ND, not done because of low expression.1-a Inhibition.1-b No protection.1-c Highly protected from inhibition.1-d ND, not determined due to little or no inhibition.1-e No inhibition.1-f Protection from inhibition. Open table in a new tab Studies with another thiol-reactive substrate, dBBn, also indicate that TMs 4, 5, 6, 10, 11, and 12 are close to the drug-binding domain (30Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). A comparison of the inhibition by dBBn and MTS-verapamil is shown in Table I. There is some overlap in the cysteine residues that can react with either dibromobimane or MTS-verapamil. For example, S222C (TM4) and L339C (TM6) are inhibited by dBBn and MTS-verapamil. Both residues were protected from inhibition by the presence of verapamil. In contrast, residues Y118C (TM2), V125C (TM2), and V982C (TM12) were inhibited by dBBn and MTS-verapamil but were less protected from inhibition by verapamil. The difference in inhibition by dBBn and MTS-verapamil may be explained if we assume that there is a single drug-binding domain in P-gp that is flexible enough to accommodate structurally diverse substrates. This "substrate-induced fit" model of substrate binding to P-gp is consistent with that proposed for the soluble BmrR transcription factor that can also bind a wide variety of compounds. The crystal structure of BmrR indicates the presence of a single drug-binding pocket (53Zheleznova E.E. Markham P.N. Neyfakh A.A. Brennan R.G. Cell. 1999; 96: 353-362Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Indeed, disulfide-cross-linking studies have shown that regions in P-gp are quite mobile (49Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 54Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 19435-19438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In some instances, cross-linking between residues in the TMs or between residues in the nucleotide-binding domains occurs at 37 °C and not at lower temperatures. The relative mobility of the TM segments could allow P-gp to bind compounds of diverse structures. A substrate could induce a conformational change in the TM segments such that different residues in the TMs would contribute to its binding. In this scenario, it is possible that different substrates could have overlapping residues contributing to its binding. This may be the case with the binding of dBBn and MTS-verapamil. Having many combinations of residues in the drug-binding domain that can contribute to binding of different substrates may explain why P-gp can bind such a wide range of structurally diverse compounds. It may also explain why P-gp has a different affinity for each substrate and may account for the reports of multiple drug-binding sites (31Shapiro A.B. Ling V. Eur. J. Biochem. 1997; 250: 130-137Crossref PubMed Scopus (428) Google Scholar, 32Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Crossref PubMed Scopus (280) Google Scholar, 33Pascaud C. Garrigos M. Orlowski S. Biochem. J. 1998; 333: 351-358Crossref PubMed Scopus (139) Google Scholar, 34Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10594-10599Crossref PubMed Scopus (355) Google Scholar, 35Martin C. Berridge G. Higgins C.F. Mistry P. Charlton P. Callaghan R. Mol. Pharmacol. 2000; 58: 624-632Crossref PubMed Scopus (392) Google Scholar). Fig. 5 may also explain how mutations throughout the TM segments can directly or indirectly affect the affinity of P-gp for a particular substrate (23Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (189) Google Scholar, 25Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 55Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (125) Google Scholar, 56Devine S.E. Ling V. Melera P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4564-4568Crossref PubMed Scopus (127) Google Scholar, 57Chen G. Duran G.E. Steger K.A. Lacayo N.J. Jaffrezou J.P. Dumontet C. Sikic B.I. J. Biol. Chem. 1997; 272: 5974-5982Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 58Zhou Y. Gottesman M.M. Pastan I. Mol. Cell. Biol. 1999; 19: 1450-1459Crossref PubMed Scopus (30) Google Scholar). Mutations to residues in the TMs may change the faces of the helices that are exposed toward the drug-binding site such that different residues are available for drug binding, thereby changing the affinity of P-gp for a particular substrate.MTS-verapamil should be a useful compound for future studies on the mechanism of P-gp. Many P-gp substrates and modulators can be used to test if they will prevent modification of P-gp by MTS-verapamil, and the results will reveal if there is overlap in the binding sites. It will also be interesting to test whether membrane fluidity or lipid composition affects modification of P-gp by MTS-verapamil. P-gp activity is highly sensitive to its lipid environment (59Doige C.A., Yu, X. Sharom F.J. Biochim. Biophys. Acta. 1993; 1146: 65-72Crossref PubMed Scopus (168) Google Scholar, 60Urbatsch I.L. Senior A.E. Arch. Biochem. Biophys. 1995; 316: 135-140Crossref PubMed Scopus (130) Google Scholar).MTS-verapamil may be useful for characterizing other proteins. Verapamil and other phenylalkylamines are inhibitors of thel-type calcium channels (61Hargreaves A.C. Gunthorpe M.J. Taylor C.W. Lummis S.C. Mol. Pharmacol. 1996; 50: 1284-1294PubMed Google Scholar, 62Degtiar V.E. Aczel S. Doring F. Timin E.N. Berjukow S. Kimball D. Mitterdorfer J. Hering S. Biophys. J. 1997; 73: 157-167Abstract Full Text PDF PubMed Scopus (8) Google Scholar, 63Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and Kv1.3 potassium channels (64Rauer H. Grissmer S. Br. J. Pharmacol. 1999; 127: 1065-1074Crossref PubMed Scopus (37) Google Scholar). Blocking of the ion channels by verapamil is thought to occur by occlusion of the ion-conducting pore. Therefore, MTS-verapamil may be useful for mapping the pore region of these channel proteins. The human multidrug resistance P-glycoprotein (P-gp)1 uses ATP to pump a wide variety of cytotoxic compounds out of the cell (1Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (406) Google Scholar, 2Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1907) Google Scholar). Overexpression of P-gp contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy, because many of the therapeutic compounds are also substrates of P-gp (3Kim R.B. Fromm M.F. Wandel C. Leake B. Wood A.J. Roden D.M. Wilkinson G.R. J. Clin. Invest. 1998; 101: 289-294Crossref PubMed Scopus (1028) Google Scholar, 4Lee C.G. Gottesman M.M. Cardarelli C.O. Ramachandra M. Jeang K.T. Ambudkar S.V. Pastan I. Dey S. Biochemistry. 1998; 37: 3594-3601Crossref PubMed Scopus (457) Google Scholar, 5Robert J. Eur. J. Clin. Invest. 1999; 29: 536-545Crossref PubMed Scopus (71) Google Scholar). Although P-gp is normally expressed in many tissues, its physiological function is unknown. The pattern of P-gp expression in tissues and studies on P-gp "knock-out" mice indicate that it may protect the organism from toxic compounds in our diet (6Thiebaut F. Tsuruo T. Hamada H. Gottesman M.M. Pastan I. Willingham M.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7735-7738Crossref PubMed Scopus (2545) Google Scholar, 7Cordon-Cardo C. O'Brien J.P. Casals D. Rittman-Grauer L. Biedler J.L. Melamed M.R. Bertino J.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 695-698Crossref PubMed Scopus (1584) Google Scholar, 8Schinkel A.H. Smit J.J. van Tellingen O. Beijnen J.H. Wagenaar E. van Deemter L. Mol C.A. van der Valk M.A. Robanus-Maandag E.C. te Riele H.P. Berns A.J.M. Borst P. Cell. 1994; 77: 491-502Abstract Full Text PDF PubMed Scopus (2051) Google Scholar). P-gp is a member of the ABC (ATP-binding cassette) family of transporters (9Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 10Holland I.B. Blight M.A. J. Mol. Biol. 1999; 293: 381-399Crossref PubMed Scopus (487) Google Scholar). Its 1280 amino acids are organized as two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (11Chen C.J. Chin J.E. Ueda K. Clark D.P. Pastan I. Gottesman M.M. Roninson I.B. Cell. 1986; 47: 381-389Abstract Full Text PDF PubMed Scopus (1709) Google Scholar). There are six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site in each repeat (12Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 13Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The exact mechanism of how P-gp functions is unknown, but P-gp has been studied quite extensively and serves as a model for understanding the mechanism of other ABC transporters. It is known, however, that both halves of P-gp are essential for activity (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar) and that both nucleotide-binding domains can bind and hydrolyze ATP and are essential for function (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 15Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar, 16Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 17Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 18Hrycyna C.A. Ramachandra M. Ambudkar S.V. Ko Y.H. Pedersen P.L. Pastan I. Gottesman M.M. J. Biol. Chem. 1998; 273: 16631-16634Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In understanding the mechanism of P-gp, it is important to determine the location of residues that contribute to the drug-binding domain. Important clues that the transmembrane domains may be important for drug binding were obtained from mutational studies and from labeling studies with photoactive substrate analogs (19Bruggemann E.P. Currier S.J. Gottesman M.M. Pastan I. J. Biol. Chem. 1992; 267: 21020-21026Abstract Full Text PDF PubMed Google Scholar, 20Greenberger L.M. J. Biol. Chem. 1993; 268: 11417-11425Abstract Full Text PDF PubMed Google Scholar, 21Morris D.I. Greenberger L.M. Bruggemann E.P. Cardarelli C. Gottesman M.M. Pastan I. Seamon K.B. Mol. Pharmacol. 1994; 46: 329-337PubMed Google Scholar, 22Zhang X. Collins K.I. Greenberger L.M. J. Biol. Chem. 1995; 270: 5441-5448Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 23Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (189) Google Scholar, 24Kajiji S. Talbot F. Grizzuti K. Van Dyke-Phillips V. Agresti M. Safa A.R. Gros P. Biochemistry. 1993; 32: 4185-4194Crossref PubMed Scopus (90) Google Scholar, 25Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar). It was later shown that the TM domains alone were sufficient for drug binding, because a deletion mutant lacking both nucleotide-binding domains could still bind drug substrates (27Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Initial attempts to identify the residues that line the drug-binding pocket within the TM domains involved the use of cysteine-scanning mutagenesis and reactivity with a thiol reactive substrate, dibromobimane (dBBn). Several residues in TMs 4, 5, 6, 10, 11, and 12 reacted with dBBn, suggesting that these TMs contribute to the binding of substrate (28Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 29Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 30Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). It is not known if P-gp has many distinct or overlapping binding sites (31Shapiro A.B. Ling V. Eur. J. Biochem. 1997; 250: 130-137Crossref PubMed Scopus (428) Google Scholar, 32Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Crossref PubMed Scopus (280) Google Scholar, 33Pascaud C. Garrigos M. Orlowski S. Biochem. J. 1998; 333: 351-358Crossref PubMed Scopus (139) Google Scholar, 34Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10594-10599Crossref PubMed Scopus (355) Google Scholar, 35Martin C. Berridge G. Higgins C.F. Mistry P. Charlton P. Callaghan R. Mol. Pharmacol. 2000; 58: 624-632Crossref PubMed Scopus (392) Google Scholar). One step toward understanding this problem is to test whether other substrates of P-gp react with the same residues in the predicted drug-binding domain. Verapamil is a particularly important substrate used in studying P-gp. The advantage of using verapamil is that it shows the greatest ability to stimulate the ATPase activity of P-gp. Development of a thiol-reactive derivative of verapamil would be a useful tool for characterizing the drug-binding domain of P-gp, because reactive cysteines can be specifically protected with verapamil. In this study, a thiol-reactive methanethiosulfonate analog of verapamil (MTS-verapamil) was synthesized and used to identify residues in the drug-binding domain of P-gp. DISCUSSIONCysteine-scanning mutagenesis and modification with sulfhydryl-specific agents was first used to probe the acetylcholine receptor channel (44Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar). Modifications of this method have since been used to study the structure and function of many membrane proteins (reviewed in Ref. 45Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (319) Google Scholar). This technique has been extensively used in the study of the bacterial lac permease protein (46Sahin-Toth M. Karlin A. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10729-10732Crossref PubMed Scopus (64) Google Scholar). P-gp has been a very good eukaryotic protein for cysteine-scanning mutagenesis studies, because the Cys-less P-gp remains functional (reviewed in Ref.47Loo T.W. Clarke D.M. Biochim. Biophys. Acta. 1999; 1461: 315-325Crossref PubMed Scopus (88) Google Scholar). Experiments that provide insight into the structure and mechanism of P-gp are possible because of the ability to synthesize thiol-reactive analogs of P-gp substrates such as MTS-verapamil.Fifteen of the 242 Cys mutants tested for inhibition by MTS-verapamil showed significant inhibition by MTS-verapamil. In general, the reactive mutants in TMs 4, 6, 10, 11, and 12 had lower I50values than those in TMs 2, 7, and 9 (TableI). This may indicate that TMs 4, 6, 10, 11, and 12 are within the drug-binding domain or that this region of P-gp is more accessible to MTS-verapamil. Verapamil significantly protected residues S222C (TM4), L339 (TM6), A342 (TM6), and G984 (TM12) from inactivation by MTS-verapamil. This indicates that these residues either line or are close to the verapamil-binding site. To picture the potential drug-binding domain of P-gp, we constructed a model in which the residues in each TM segments are arranged in α-helical wheels (Fig. 5). The arrangement of the TM segments are based on the results of disulfide cross-linking studies that show TM 6 is close to TMs 10, 11, and 12 and that TM12 is close to TMs 4, 5, and 6 (48Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 49Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In another ABC transporter, cystic fibrosis transmembrane conductance regulator, it has been shown that a salt bridge may exist between TM6 and TM8 (50Cotten J.F. Welsh M.J. J. Biol. Chem. 1999; 274: 5429-5435Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). TMs 4, 5, 6, 10, 11, and 12 have been postulated to line the drug-binding domain, because cross-linking between residues in these segments is affected by the presence of drug substrates. The location of the drug-binding domain within the TMs would be consistent with the idea that P-gp removes substrates that are embedded in the lipid bilayer (51Raviv Y. Pollard H.B. Bruggemann E.P. Pastan I. Gottesman M.M. J. Biol. Chem. 1990; 265: 3975-3980Abstract Full Text PDF PubMed Google Scholar, 52Homolya L. Hollo Z. Germann U.A. Pastan I. Gottesman M.M. Sarkadi B. J. Biol. Chem. 1993; 268: 21493-21496Abstract Full Text PDF PubMed Google Scholar). It is thought that substrates diffuse into the lipid bilayer and are then extracted from the bilayer leaflets by P-gp.Table IComparing inhibition of Cys P-gp mutants by dBBn or MTS-verapamilMutantInhibitorProtection by verapamildBBnMTS-verapamil (I50)dBBnMTS-verapamilμmY118C (TM2)+ 1-aInhibition.+ (40)− 1-bNo protection.−V125C (TM2)++ (63)−−S222C (TM4)++ (40)++ 1-cHighly protected from inhibition.++L339C (TM6)++ (29)++++A342C (TM6)+/−+ (14)ND 1-dND, not determined due to little or no inhibition.++A729C (TM7)1-eNo inhibition.−/+ (70)ND−S766C (TM8)+−−NDA841C (TM9)−+ (101)ND−N842C (TM9)−+ (61)ND−I868C (TM10)++ (25)+ 1-fProtection from inhibition.+A871C (TM10)−+ (14)ND−G872C (TM10)+−+NDF942C (TM11)++ (20)++T945C (TM11)++ (8)++L975C (TM12)+−+NDV982C (TM12)++ (12)−−G984C (TM12)ND+ (17)ND++A985C (TM12)++ (38)+−1-g ND, not done because of low expression.1-a Inhibition.1-b No protection.1-c Highly protected from inhibition.1-d ND, not determined due to little or no inhibition.1-e No inhibition.1-f Protection from inhibition. Open table in a new tab Studies with another thiol-reactive substrate, dBBn, also indicate that TMs 4, 5, 6, 10, 11, and 12 are close to the drug-binding domain (30Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). A comparison of the inhibition by dBBn and MTS-verapamil is shown in Table I. There is some overlap in the cysteine residues that can react with either dibromobimane or MTS-verapamil. For example, S222C (TM4) and L339C (TM6) are inhibited by dBBn and MTS-verapamil. Both residues were protected from inhibition by the presence of verapamil. In contrast, residues Y118C (TM2), V125C (TM2), and V982C (TM12) were inhibited by dBBn and MTS-verapamil but were less protected from inhibition by verapamil. The difference in inhibition by dBBn and MTS-verapamil may be explained if we assume that there is a single drug-binding domain in P-gp that is flexible enough to accommodate structurally diverse substrates. This "substrate-induced fit" model of substrate binding to P-gp is consistent with that proposed for the soluble BmrR transcription factor that can also bind a wide variety of compounds. The crystal structure of BmrR indicates the presence of a single drug-binding pocket (53Zheleznova E.E. Markham P.N. Neyfakh A.A. Brennan R.G. Cell. 1999; 96: 353-362Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Indeed, disulfide-cross-linking studies have shown that regions in P-gp are quite mobile (49Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 54Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 19435-19438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In some instances, cross-linking between residues in the TMs or between residues in the nucleotide-binding domains occurs at 37 °C and not at lower temperatures. The relative mobility of the TM segments could allow P-gp to bind compounds of diverse structures. A substrate could induce a conformational change in the TM segments such that different residues in the TMs would contribute to its binding. In this scenario, it is possible that different substrates could have overlapping residues contributing to its binding. This may be the case with the binding of dBBn and MTS-verapamil. Having many combinations of residues in the drug-binding domain that can contribute to binding of different substrates may explain why P-gp can bind such a wide range of structurally diverse compounds. It may also explain why P-gp has a different affinity for each substrate and may account for the reports of multiple drug-binding sites (31Shapiro A.B. Ling V. Eur. J. Biochem. 1997; 250: 130-137Crossref PubMed Scopus (428) Google Scholar, 32Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Crossref PubMed Scopus (280) Google Scholar, 33Pascaud C. Garrigos M. Orlowski S. Biochem. J. 1998; 333: 351-358Crossref PubMed Scopus (139) Google Scholar, 34Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10594-10599Crossref PubMed Scopus (355) Google Scholar, 35Martin C. Berridge G. Higgins C.F. Mistry P. Charlton P. Callaghan R. Mol. Pharmacol. 2000; 58: 624-632Crossref PubMed Scopus (392) Google Scholar). Fig. 5 may also explain how mutations throughout the TM segments can directly or indirectly affect the affinity of P-gp for a particular substrate (23Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (189) Google Scholar, 25Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 55Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (125) Google Scholar, 56Devine S.E. Ling V. Melera P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4564-4568Crossref PubMed Scopus (127) Google Scholar, 57Chen G. Duran G.E. Steger K.A. Lacayo N.J. Jaffrezou J.P. Dumontet C. Sikic B.I. J. Biol. Chem. 1997; 272: 5974-5982Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 58Zhou Y. Gottesman M.M. Pastan I. Mol. Cell. Biol. 1999; 19: 1450-1459Crossref PubMed Scopus (30) Google Scholar). Mutations to residues in the TMs may change the faces of the helices that are exposed toward the drug-binding site such that different residues are available for drug binding, thereby changing the affinity of P-gp for a particular substrate.MTS-verapamil should be a useful compound for future studies on the mechanism of P-gp. Many P-gp substrates and modulators can be used to test if they will prevent modification of P-gp by MTS-verapamil, and the results will reveal if there is overlap in the binding sites. It will also be interesting to test whether membrane fluidity or lipid composition affects modification of P-gp by MTS-verapamil. P-gp activity is highly sensitive to its lipid environment (59Doige C.A., Yu, X. Sharom F.J. Biochim. Biophys. Acta. 1993; 1146: 65-72Crossref PubMed Scopus (168) Google Scholar, 60Urbatsch I.L. Senior A.E. Arch. Biochem. Biophys. 1995; 316: 135-140Crossref PubMed Scopus (130) Google Scholar).MTS-verapamil may be useful for characterizing other proteins. Verapamil and other phenylalkylamines are inhibitors of thel-type calcium channels (61Hargreaves A.C. Gunthorpe M.J. Taylor C.W. Lummis S.C. Mol. Pharmacol. 1996; 50: 1284-1294PubMed Google Scholar, 62Degtiar V.E. Aczel S. Doring F. Timin E.N. Berjukow S. Kimball D. Mitterdorfer J. Hering S. Biophys. J. 1997; 73: 157-167Abstract Full Text PDF PubMed Scopus (8) Google Scholar, 63Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and Kv1.3 potassium channels (64Rauer H. Grissmer S. Br. J. Pharmacol. 1999; 127: 1065-1074Crossref PubMed Scopus (37) Google Scholar). Blocking of the ion channels by verapamil is thought to occur by occlusion of the ion-conducting pore. Therefore, MTS-verapamil may be useful for mapping the pore region of these channel proteins. Cysteine-scanning mutagenesis and modification with sulfhydryl-specific agents was first used to probe the acetylcholine receptor channel (44Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar). Modifications of this method have since been used to study the structure and function of many membrane proteins (reviewed in Ref. 45Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (319) Google Scholar). This technique has been extensively used in the study of the bacterial lac permease protein (46Sahin-Toth M. Karlin A. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10729-10732Crossref PubMed Scopus (64) Google Scholar). P-gp has been a very good eukaryotic protein for cysteine-scanning mutagenesis studies, because the Cys-less P-gp remains functional (reviewed in Ref.47Loo T.W. Clarke D.M. Biochim. Biophys. Acta. 1999; 1461: 315-325Crossref PubMed Scopus (88) Google Scholar). Experiments that provide insight into the structure and mechanism of P-gp are possible because of the ability to synthesize thiol-reactive analogs of P-gp substrates such as MTS-verapamil. Fifteen of the 242 Cys mutants tested for inhibition by MTS-verapamil showed significant inhibition by MTS-verapamil. In general, the reactive mutants in TMs 4, 6, 10, 11, and 12 had lower I50values than those in TMs 2, 7, and 9 (TableI). This may indicate that TMs 4, 6, 10, 11, and 12 are within the drug-binding domain or that this region of P-gp is more accessible to MTS-verapamil. Verapamil significantly protected residues S222C (TM4), L339 (TM6), A342 (TM6), and G984 (TM12) from inactivation by MTS-verapamil. This indicates that these residues either line or are close to the verapamil-binding site. To picture the potential drug-binding domain of P-gp, we constructed a model in which the residues in each TM segments are arranged in α-helical wheels (Fig. 5). The arrangement of the TM segments are based on the results of disulfide cross-linking studies that show TM 6 is close to TMs 10, 11, and 12 and that TM12 is close to TMs 4, 5, and 6 (48Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 49Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In another ABC transporter, cystic fibrosis transmembrane conductance regulator, it has been shown that a salt bridge may exist between TM6 and TM8 (50Cotten J.F. Welsh M.J. J. Biol. Chem. 1999; 274: 5429-5435Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). TMs 4, 5, 6, 10, 11, and 12 have been postulated to line the drug-binding domain, because cross-linking between residues in these segments is affected by the presence of drug substrates. The location of the drug-binding domain within the TMs would be consistent with the idea that P-gp removes substrates that are embedded in the lipid bilayer (51Raviv Y. Pollard H.B. Bruggemann E.P. Pastan I. Gottesman M.M. J. Biol. Chem. 1990; 265: 3975-3980Abstract Full Text PDF PubMed Google Scholar, 52Homolya L. Hollo Z. Germann U.A. Pastan I. Gottesman M.M. Sarkadi B. J. Biol. Chem. 1993; 268: 21493-21496Abstract Full Text PDF PubMed Google Scholar). It is thought that substrates diffuse into the lipid bilayer and are then extracted from the bilayer leaflets by P-gp. 1-g ND, not done because of low expression. Studies with another thiol-reactive substrate, dBBn, also indicate that TMs 4, 5, 6, 10, 11, and 12 are close to the drug-binding domain (30Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). A comparison of the inhibition by dBBn and MTS-verapamil is shown in Table I. There is some overlap in the cysteine residues that can react with either dibromobimane or MTS-verapamil. For example, S222C (TM4) and L339C (TM6) are inhibited by dBBn and MTS-verapamil. Both residues were protected from inhibition by the presence of verapamil. In contrast, residues Y118C (TM2), V125C (TM2), and V982C (TM12) were inhibited by dBBn and MTS-verapamil but were less protected from inhibition by verapamil. The difference in inhibition by dBBn and MTS-verapamil may be explained if we assume that there is a single drug-binding domain in P-gp that is flexible enough to accommodate structurally diverse substrates. This "substrate-induced fit" model of substrate binding to P-gp is consistent with that proposed for the soluble BmrR transcription factor that can also bind a wide variety of compounds. The crystal structure of BmrR indicates the presence of a single drug-binding pocket (53Zheleznova E.E. Markham P.N. Neyfakh A.A. Brennan R.G. Cell. 1999; 96: 353-362Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Indeed, disulfide-cross-linking studies have shown that regions in P-gp are quite mobile (49Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 54Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 19435-19438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In some instances, cross-linking between residues in the TMs or between residues in the nucleotide-binding domains occurs at 37 °C and not at lower temperatures. The relative mobility of the TM segments could allow P-gp to bind compounds of diverse structures. A substrate could induce a conformational change in the TM segments such that different residues in the TMs would contribute to its binding. In this scenario, it is possible that different substrates could have overlapping residues contributing to its binding. This may be the case with the binding of dBBn and MTS-verapamil. Having many combinations of residues in the drug-binding domain that can contribute to binding of different substrates may explain why P-gp can bind such a wide range of structurally diverse compounds. It may also explain why P-gp has a different affinity for each substrate and may account for the reports of multiple drug-binding sites (31Shapiro A.B. Ling V. Eur. J. Biochem. 1997; 250: 130-137Crossref PubMed Scopus (428) Google Scholar, 32Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Crossref PubMed Scopus (280) Google Scholar, 33Pascaud C. Garrigos M. Orlowski S. Biochem. J. 1998; 333: 351-358Crossref PubMed Scopus (139) Google Scholar, 34Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10594-10599Crossref PubMed Scopus (355) Google Scholar, 35Martin C. Berridge G. Higgins C.F. Mistry P. Charlton P. Callaghan R. Mol. Pharmacol. 2000; 58: 624-632Crossref PubMed Scopus (392) Google Scholar). Fig. 5 may also explain how mutations throughout the TM segments can directly or indirectly affect the affinity of P-gp for a particular substrate (23Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (189) Google Scholar, 25Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 55Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (125) Google Scholar, 56Devine S.E. Ling V. Melera P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4564-4568Crossref PubMed Scopus (127) Google Scholar, 57Chen G. Duran G.E. Steger K.A. Lacayo N.J. Jaffrezou J.P. Dumontet C. Sikic B.I. J. Biol. Chem. 1997; 272: 5974-5982Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 58Zhou Y. Gottesman M.M. Pastan I. Mol. Cell. Biol. 1999; 19: 1450-1459Crossref PubMed Scopus (30) Google Scholar). Mutations to residues in the TMs may change the faces of the helices that are exposed toward the drug-binding site such that different residues are available for drug binding, thereby changing the affinity of P-gp for a particular substrate. MTS-verapamil should be a useful compound for future studies on the mechanism of P-gp. Many P-gp substrates and modulators can be used to test if they will prevent modification of P-gp by MTS-verapamil, and the results will reveal if there is overlap in the binding sites. It will also be interesting to test whether membrane fluidity or lipid composition affects modification of P-gp by MTS-verapamil. P-gp activity is highly sensitive to its lipid environment (59Doige C.A., Yu, X. Sharom F.J. Biochim. Biophys. Acta. 1993; 1146: 65-72Crossref PubMed Scopus (168) Google Scholar, 60Urbatsch I.L. Senior A.E. Arch. Biochem. Biophys. 1995; 316: 135-140Crossref PubMed Scopus (130) Google Scholar). MTS-verapamil may be useful for characterizing other proteins. Verapamil and other phenylalkylamines are inhibitors of thel-type calcium channels (61Hargreaves A.C. Gunthorpe M.J. Taylor C.W. Lummis S.C. Mol. Pharmacol. 1996; 50: 1284-1294PubMed Google Scholar, 62Degtiar V.E. Aczel S. Doring F. Timin E.N. Berjukow S. Kimball D. Mitterdorfer J. Hering S. Biophys. J. 1997; 73: 157-167Abstract Full Text PDF PubMed Scopus (8) Google Scholar, 63Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and Kv1.3 potassium channels (64Rauer H. Grissmer S. Br. J. Pharmacol. 1999; 127: 1065-1074Crossref PubMed Scopus (37) Google Scholar). Blocking of the ion channels by verapamil is thought to occur by occlusion of the ion-conducting pore. Therefore, MTS-verapamil may be useful for mapping the pore region of these channel proteins. We thank Dr. Randal Kaufman (Boston) for pMT21, and we thank Claire Bartlett for assistance with tissue culture.

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