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Identification of Residues in the Drug-binding Domain of Human P-glycoprotein

1999; Elsevier BV; Volume: 274; Issue: 50 Linguagem: Inglês

10.1074/jbc.274.50.35388

1083-351X

Tip W. Loo, David M. Clarke,

Adenosine and Purinergic Signaling

The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe942 and Thr945 in TM11, together with residues previously identified in TM6 (Leu339 and Ala342) and TM12 (Leu975, Val982, and Ala985) (Loo, T. W., and Clarke, D. M. (1997)J. Biol. Chem. 272, 31945–31948) form part of the drug-binding domain of P-gp. The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe942 and Thr945 in TM11, together with residues previously identified in TM6 (Leu339 and Ala342) and TM12 (Leu975, Val982, and Ala985) (Loo, T. W., and Clarke, D. M. (1997)J. Biol. Chem. 272, 31945–31948) form part of the drug-binding domain of P-gp. P-glycoprotein dibromobimane transmembrane The human multidrug resistance P-glycoprotein (P-gp),1 is an ATP-dependent drug pump that can extrude a broad range of structurally diverse compounds from the cell (for recent reviews, see Refs. 1Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1929) Google Scholar, 2Loo T.W. Clarke D.M. Biochem. Cell Biol. 1999; 77: 1-12Crossref PubMed Scopus (88) Google Scholar, 3Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (417) Google Scholar). The physiological role of P-gp is unknown. It is postulated that P-gp may protect us from exogenous and endogenous cytotoxic agents, and this is supported by studies on "knockout" mice (4Schinkel 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 (2089) Google Scholar, 5Schinkel A.H. Wagenaar E. Mol C.A. van Deemter L. J. Clin. Invest. 1996; 97: 2517-2524Crossref PubMed Scopus (1180) Google Scholar). This protective function of P-gp is a major problem during AIDS (6Lee 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 (460) Google Scholar, 7Kim 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 (1040) Google Scholar) and cancer chemotherapy (8Lehnert M. Eur. J. Cancer. 1996; 6: 912-920Abstract Full Text PDF Scopus (195) Google Scholar, 9Chan H.S. Grogan T.M. DeBoer G. Haddad G. Gallie B.L. Ling V. Eur. J. Cancer. 1996; 6: 1051-1061Abstract Full Text PDF Scopus (29) Google Scholar) and contributes to the phenomenon of multidrug resistance. P-gp is a member of the ATP-binding cassette superfamily of transport proteins (10Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3386) Google Scholar). The 1280 amino acids of P-gp are predicted to be organized as two tandem repeats, with each repeat consisting of a hydrophobic domain containing six predicted TM segments followed by a hydrophilic domain containing an ATP-binding site (11Chen C.J. Clark D. Ueda K. Pastan I. Gottesman M.M. Roninson I.B. J. Biol. Chem. 1990; 265: 506-514Abstract Full Text PDF PubMed Google Scholar, 12Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 13Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The mechanism of how P-gp couples ATP hydrolysis to the transport of drug substrates in not known. Earlier studies showed that both halves of the protein are required for activity (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar). Both ATP-binding sites are active (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 15Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) and essential for activity. P-gp activity is completely abolished if either nucleotide-binding site is blocked by mutation (16Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar) or chemical modification (17Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 18Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Understanding the nature of the drug-binding site(s) of P-gp is an important step in determining its mechanism. To this end, we used cysteine-scanning mutagenesis together with a thiol-reactive compound, dibromobimane (dBBn), to identify residues within the transmembrane (TM) domains that form the drug-binding site(s) (19Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The rationale with this approach is that a thiol-reactive compound that is also a substrate of P-gp would covalently bind to an available cysteine residue in the drug-binding site and inhibit drug-stimulated ATPase activity. In initial studies, we showed that residues in TM6 and TM12 of P-gp contribute to the drug-binding domain (19Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In this study, we tested whether TM11 also participates in binding of drug substrates. The residues in TM11 could potentially contribute or form part of the drug-binding site, since a natural mutation (S939F) in mouse mdr3 P-gp was shown to affect the activity and substrate specificity of the enzyme (20Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (190) Google Scholar, 21Kajiji 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, 22Kajiji S. Dreslin J.A. Grizzuti K. Gros P. Biochemistry. 1994; 33: 5041-5048Crossref PubMed Scopus (56) Google Scholar). Most of the residues in TM11 were initially changed to that with a small, nonpolar side chain, and the mutant protein was assayed for changes in activity and substrate specificity. Direct assay for drug binding was then carried out with cysteine-scanning mutagenesis and inhibition of substrate-stimulated ATPase activity by dBBn. A full-length MDR1 cDNA was modified to encode for 10 histidine residues at the COOH end of P-gp to facilitate its purification by nickel-chelate chromatography (23Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Oligonucleotide-directed mutagenesis was carried out as described previously (24Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar). Cysteine residues were introduced into a Cys-less mutant of P-gp that also had a 10-histidine tag at the COOH terminus as described previously (25Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Expression and purification of histidine-tagged P-gp was done as described previously (23Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Briefly, 40 10-cm diameter culture plates of HEK 293 cells were transfected with the mutant cDNA. After 24 h, the medium was replaced with fresh medium containing 10 μmcyclosporin A. P-gp was expressed in the presence of cyclosporin A because drug substrates promote maturation of the protein (26Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The transfected cells were then harvested 24 h later and solubilized with 1% (w/v) n-dodecyl-β-d-maltoside, and the mutant P-gp was isolated by nickel-chelate chromatography. The purified histidine-tagged P-gp was diluted with an equal volume of 10 mg/ml crude sheep brain phosphatidylethanolamine (Sigma, type II, commercial grade) that had been washed with Tris-buffered saline, pH 7.4, to remove traces of inorganic phosphate. The samples were then sonicated in an ice water bath for 45 s at maximum setting using a Branson Sonifier 450 sonicator with a bath-type probe attachment. An aliquot of sonicated P-gp/lipid sample was assayed for drug-stimulated ATPase activity by the addition of an equal volume of buffer containing 100 mm Tris-HCl, pH 7.4, 100 mm NaCl, 20 mm MgCl2, 10 mm ATP, and the desired drug substrate. The samples were incubated for 30 min at 37 °C, and the amount of inorganic phosphate liberated was determined by the method of Chifflet et al. (27Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (418) Google Scholar). For inhibition with dBBn, the P-gp/lipid mixture was preincubated with 2 mm dBBn (Molecular Probes, Inc.) for 5 min at 37 °C. The reaction was stopped by the addition of cysteine, pH 7.5, to a final concentration of 40 mm. Drug-stimulated ATPase activity was then determined as described above. The purified histidine-tagged P-gp was subjected to SDS-PAGE, transferred onto a sheet of nitrocellulose, and probed with a rabbit polyclonal antibody against P-gp, followed by enhanced chemiluminescence (25Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The amino acids (937–957) that are predicted to be in TM11 are shown in Fig. 1. The region encompassing TM11 spans the membrane, since it was shown that residue 967 is on the extracellular side of the membrane. In contrast, residue 931 is located on the cytoplasmic side of the membrane (12Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). We first tested the importance of residues in TM11 by analyzing the effects of mutations on the drug-stimulated ATPase activity of the mutant P-gp. Measurement of drug-stimulated ATPase activity is a useful assay for studying P-gp-drug interactions. Stimulation of wild-type P-gp ATPase activity by drug substrates has a characteristic pattern. In the presence of low concentrations of drug substrates, there is stimulation of ATPase activity. The activity, however, is inhibited in the presence of high concentrations of drug substrates (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 28Germann U.A. Willingham M.C. Pastan I. Gottesman M.M. Biochemistry. 1990; 29: 2295-2303Crossref PubMed Scopus (116) Google Scholar). Fig. 2 shows that at low concentrations of drug substrates, there is initial stimulation of wild-type P-gp ATPase activity, with maximal activity at about 0.03 mmvinblastine, 0.3 mm verapamil, and 3 mmcolchicine, respectively. The ATPase activity is then inhibited at higher concentrations of drug substrates. This typical ATPase activity profile provides a relatively simple assay for testing the effect of mutations or chemical modification of P-gp on P-gp-drug substrate interactions. The effect (i.e. the apparent affinity) is quantitated by measuring the concentration of drug substrate required to achieve half-maximal stimulation. It was also recently shown that there is good correlation in the turnover numbers between vinblastine-stimulated ATPase activity and transport of vinblastine out of the cell (29Ambudkar S.V. Cardarelli C.O. Pashinsky I. Stein W.D. J. Biol. Chem. 1997; 272: 21160-21166Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Therefore, vinblastine was used for measurement of drug-stimulated ATPase activity. Verapamil and colchicine were also included because verapamil causes the highest level of stimulation of ATPase activity, whereas colchicine is commonly used in drug resistance profile assays (24Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 30Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 31Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7243-7248Abstract Full Text PDF PubMed Google Scholar, 32Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (127) Google Scholar). The residues in TM11 were initially changed to alanine, except that alanine was changed to valine, glycine to valine, and isoleucine to serine. All of the changes were made to a cDNA of P-gp containing a His10 tag at the COOH terminus. The presence of the tag facilitated purification of the mutant P-gp by nickel-chelate chromatography. The presence of the histidine tag does not affect the activity of P-gp (23Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The mutant P-gps were transiently expressed in HEK 293 cells. Except for mutant G939V, all of the mutants expressed the 170-kDa protein as the major product. The major product in mutant G939V was a 150-kDa core-glycosylated protein that was sensitive to endoglycosidase H (data not shown). In the presence of cyclosporin A, however, all of the mutants, including mutant G939V, yielded the fully mature 170-kDa protein (Fig. 3). It appeared that mutation G939V affected folding and maturation of P-gp. This folding defect was corrected by expression in the presence of drug substrate (26Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The mutant P-gps were isolated by nickel-chelate chromatography, mixed with crude sheep brain phosphatidylethanolamine, sonicated, and then assayed for drug-stimulated ATPase activity in the presence of various concentrations of verapamil, vinblastine or colchicine (Table I). In the presence of verapamil, the majority of the mutants had 80–120% of the maximal activity of wild-type enzyme. The lowest activity was observed in mutant G939V (62% of wild-type activity), while mutant Y953A had the highest (205%) activity. There was moderate stimulation of verapamil-stimulated ATPase activities in mutants T945A (140%), G955V (143%), and F957A (126%).Table IDrug-stimulated ATPase activityMutantDrugVerapamilVinblastineColchicineV maxK mV maxK mV maxK m% of WTaWT, wild type.μm% of WTμm% of WTmmWT100241005.41000.62I937S9422936.11000.69F938A10632965.1960.68G939V628454.01650.26I940S9332935.6930.65T941A100251045.51000.66F942A8893305.1240.80S943A92261005.2850.62F944A93141055.31010.64T945A1401001658.3560.65Q946A101165578.5180.64A947L1051566013.0511.87M948A103231015.91030.62M949A8240965.5610.60Y950A109371195.1990.62F951A9431995.21010.64S952A108361235.1910.69Y953A205110598.51310.67A954L1084413NDbND, not determined due to low activity.8NDG955V143101043.52200.47C956A9724955.31450.63F957A12621474.8321.0a WT, wild type.b ND, not determined due to low activity. Open table in a new tab Several mutants showed changes in the apparent affinity (concentration of substrate required for half-maximal stimulation of ATPase activity) for verapamil (Table I). Wild-type P-gp had an apparent affinity of 24 μm verapamil, while mutants F942A, T945A, Q946A, A947L, and Y953A had decreased apparent affinities of 93, 100, 165, 156, and 110 μm, respectively. One mutant, G939V, showed the largest increase in apparent affinity for verapamil (8 μm). The mutants were then assayed for vinblastine-stimulated ATPase activity (Table I). One mutant, T945A, showed a pronounced increase in activity (165% of wild-type enzyme). By contrast, mutants A954L and F942A had only 13 and 30%, respectively, of the wild-type activity. Moderate decreases in activity (40–50%) were observed for mutants G939V, Q946A, A947L, Y953A, and F957A. The mutant, A947L, also exhibited a decrease in the apparent affinity for vinblastine. It had an apparent affinity of 13 μm vinblastine, while that of wild-type P-gp was 5.4 μm vinblastine. The vinblastine-stimulated ATPase activity of mutant A954L was too low for accurate determination of its apparent affinity. In the presence of colchicine (Table I), mutant G955V showed the largest change in activity. Its maximal colchicine-stimulated ATPase activity was 220% of that of wild-type P-gp, while those of mutants G939V, C956A, and Y953A were moderately increased (165, 145, and 131%, respectively). There were, however, significant decreases in the activity for mutants Q946A (18%), F942A (24%), and F957A (32%). Two mutants had large changes in their apparent affinity for colchicine. Wild-type P-gp had an apparent affinity of 620 μmcolchicine, while those of mutants A947L and G939V were 1870 and 260 μm colchicine, respectively. These results (Table I) show that the activity and apparent affinity of P-gp for verapamil, vinblastine, and colchicine are greatly affected by some mutations in TM11. A potential disadvantage of these indirect assays, however, is that it is difficult to determine if the mutations that affected activity were actually close to the drug-binding domain or whether they affected the global structure of the protein. Therefore, an assay that can directly measure the importance of each residue of TM11 in P-gp-substrate interaction was used. Another direct method for testing whether residues in TM11 contribute to the drug-binding site(s) is to use cysteine-scanning mutagenesis followed by modification of with the thiol-reactive substrate, dBBn. The rationale for using a thiol-reactive substrate is that it will occupy the drug-binding site of P-gp and covalently label any adjacent cysteine residue and thus inhibit drug-stimulated ATPase activity. dBBn is a relatively good substrate of P-gp, since it stimulates the ATPase activity of Cys-less P-gp more than 8-fold at a concentration of 1 mm, and its ability to act as a substrate can be quenched by reaction with cysteine (19Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Accordingly, a series of Cys mutants was constructed using a Cys-less P-gp that had a histidine tag at the COOH terminus of P-gp (12Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 23Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The mutants were transiently expressed in HEK 293 cells to determine whether the mutations had caused misprocessing of the mutant protein. All the mutants, except F957C, yielded the mature 170-kDa protein as the major product (data not shown). Mutant F957C may be unstable and susceptible to proteolytic digestion. Immunoblots of cell extracts of HEK 293 transfected with mutant F957C showed the presence of only degradation products (data not shown). The Cys mutants were purified by nickel-chelate chromatography, mixed with lipid and assayed for verapamil-stimulated ATPase activity. Verapamil was used because it is the most potent stimulator of P-gp ATPase activity. Fig. 4 shows that all of the mutants had ATPase activities that were comparable (70–110%) to that of Cys-less P-gp. Mutant G939C had the lowest activity (70% of that of Cys-less P-gp). To test whether the activity of the Cys mutants could be inhibited by dBBn, each mutant was treated with 1 mm dBBn for 5 min at 37 °C, quenched with cysteine, and then assayed for verapamil-stimulated ATPase activity. The activity of the dBBn-treated sample was compared with that of a mock-treated sample. Fig. 5 shows that the majority of the mutants, except for G939C, F942C, T945C, and Y953C, were not affected by treatment with dBBn. The activities of mutants F943C and Y953C were slightly inhibited by dBBn (38 and 26% respectively). In contrast, the activities of mutants F942C and T945C, were almost completely inhibited by dBBn (80 and 85%, respectively). The activities of mutants F942C and T945C were inhibited to the greatest extent by dBBn. Therefore, they were chosen for further analysis to determine whether inhibition by dBBn could be prevented by drug substrates such as verapamil, vinblastine, and colchicine. In this assay, the mutant P-gps were pretreated with dBBn, quenched with cysteine, and then assayed for drug-stimulated ATPase activity (Fig. 6 A); or they were preincubated with verapamil, vinblastine, or colchicine, treated with dBBn, quenched with cysteine, and then assayed for drug-stimulated ATPase activity (Fig. 6 B). The activities were compared with that of a mock-treated sample of P-gp. Fig. 6 A shows that Cys-less P-gp was insensitive to inhibition by dBBn. By contrast, the verapamil-, vinblastine-, or colchicine-stimulated ATPase activities of mutants F942C or T945C were inhibited by more than 70% when pretreated with dBBn. We then tested whether the presence of verapamil, vinblastine, or colchicine could protect mutants F942C or T945C from inactivation by dBBn. The mutant was preincubated with 1 mmverapamil, 0.1 mm vinblastine, or 10 mmcolchicine and then treated with 1 mm dBBn. After 5 min, dBBn reactivity was quenched by the addition of cysteine, and the ATPase activity was determined. Again, the Cys-less P-gp did not show any difference when pretreated with substrate (Fig. 6 B). The activities of mutants F942C and T945C, however, were protected by pretreatment with substrate (Fig. 6 B). Vinblastine offered the greatest protection, since the mutants retained about 70–80% of their original activities. Verapamil and colchicine also protected the mutant from inactivation by dBBn. These results suggest that binding of dBBn to P-gp overlaps that of P-gp binding to verapamil, vinblastine, and colchicine and suggest that TM11 forms part of the binding site for dBBn, verapamil, vinblastine, and colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, and Y953A in TM11 had altered apparent affinities for verapamil, vinblastine, or colchicine. Mutation of residue Ala954 to leucine appeared to disrupt P-gp-substrate interactions, since little drug-stimulated ATPase activity could be detected in the presence of vinblastine or colchicine. When the amino acids of TM11 are arranged in an α-helical wheel (Fig. 7), it was found that these sensitive residues are clustered on one face of the TM segment. These observations are consistent with the results of Hanna et al.(33Hanna M. Brault M. Kwan T. Kast C. Gros P. Biochemistry. 1996; 35: 3625-3635Crossref PubMed Scopus (55) Google Scholar), who showed that mutations to the homologous residues in TM11 of mouse mdr3 P-gp had significant effects on the drug resistance profile of P-gp. There is a potential drawback of studying only the effect of mutations on the drug resistance profile of P-gp or of studying the effect of mutations on the ATPase activity of P-gp. It is often difficult to determine whether the mutations that alter the resistance profile or activity of P-gp are due to the residue being critical for drug binding or due to disruption, by the mutation, of the global structure of the enzyme. Previous mutational analysis of mouse, hamster, and human P-gp have shown that changes introduced into all domains such as the TM domains (20Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (190) Google Scholar, 24Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 30Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 33Hanna M. Brault M. Kwan T. Kast C. Gros P. Biochemistry. 1996; 35: 3625-3635Crossref PubMed Scopus (55) Google Scholar, 34Taguchi Y. Morishima M. Komano T. Ueda K. FEBS. Lett. 1997; 413: 142-146Crossref PubMed Scopus (25) Google Scholar, 35Taguchi Y. Kino K. Morishima M. 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Chem. 1994; 269: 7243-7248Abstract Full Text PDF PubMed Google Scholar, 39Kwan T. Gros P. Biochemistry. 1998; 37: 3337-3350Crossref PubMed Scopus (42) Google Scholar, 42Choi K.H. Chen C.J. Kriegler M. Roninson I.B. Cell. 1988; 53: 519-529Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 43Currier S.J. Kane S.E. Willingham M.C. Cardarelli C.O. Pastan I. Gottesman M.M. J. Biol. Chem. 1992; 267: 25153-25159Abstract Full Text PDF PubMed Google Scholar, 44Welker E. Szabo K. Hollo Z. Muller M. Sarkadi B. Varadi A. Biochem. Biophys. Res. Commun. 1995; 216: 602-609Crossref PubMed Scopus (19) Google Scholar); and the nucleotide-binding domains (45Hoof T. Demmer A. Hadam M.R. Riordan J.R. Tummler B. J. Biol. Chem. 1994; 269: 20575-20583Abstract Full Text PDF PubMed Google Scholar, 46Beaudet L. Gros P. J. Biol. Chem. 1995; 270: 17159-17170Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) can all affect the activity or substrate specificity of P-gp. It is unlikely that all of these residues are involved in binding drug substrates. Indeed, we recently showed by deletion analysis that the nucleotide-binding domains are not required for drug binding (47Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). By contrast, mutations in the nucleotide-binding domains have been reported to alter drug-stimulated ATPase activity of P-gp (45Hoof T. Demmer A. Hadam M.R. Riordan J.R. Tummler B. J. Biol. Chem. 1994; 269: 20575-20583Abstract Full Text PDF PubMed Google Scholar, 46Beaudet L. Gros P. J. Biol. Chem. 1995; 270: 17159-17170Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Mutant G185V is an example of a mutation that probably alters P-gp-drug interactions because of structural perturbations. This mutant was of great interest because it was the first natural mutation in P-gp that altered the substrate specificity of P-gp. It conferred increased resistance to colchicine but reduced resistance to vinblastine when compared with wild-type P-gp (42Choi K.H. Chen C.J. Kriegler M. Roninson I.B. Cell. 1988; 53: 519-529Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 48Kioka N. Tsubota J. Kakehi Y. Komano T. Gottesman M.M. Pastan I. Ueda K. Biochem. Biophys. Res. Commun. 1989; 162: 224-231Crossref PubMed Scopus (172) Google Scholar). Subsequent analysis of this mutant, however, led to the conclusion that G185V alters function of P-gp by affecting the structure of the transporter (49Ramachandra M. Ambudkar S.V. Gottesman M.M. Pastan I. Hrycyna C.A. Mol. Biol. Cell. 1996; 7: 1485-1498Crossref PubMed Scopus (68) Google Scholar). Similarly, it is also likely that some of the mutations in TM11 in this study that resulted in altered drug-stimulated ATPase activity could also be due to structural perturbations, since relatively small residues were replaced with larger ones (G939V, A947L, A954L, and G955V). Indeed, it is not inconceivable that such a single change can cause rather large perturbations in P-gp. An example of an extreme case is mutant G341C (in TM6) that caused complete misfolding of P-gp such that the mutant P-gp was more susceptible to digestion in the first extracellular loop (50Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 32373-32376Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Therefore, a way of circumventing these potential problems was to use a direct assay with dBBn to test whether TM11 residues participate in binding drug substrate. Reaction of dBBn with two mutants, F942C and T945C, significantly inhibited substrate-stimulated ATPase activity. The presence of verapamil, vinblastine, or colchicine protected mutants F942C and T945C from inhibition by dBBn. Fig. 7 shows that both residues Phe942 and Thr945 are separated by one turn of the helix and are on the same face. Taken together, we propose that residues Phe942 and Thr945 form part of the drug-binding domain of P-gp, together with residues Leu339 and Ala342 (TM6) and residues Leu975, Val982, and Ala985 (TM12). dBBn also inhibited the drug-stimulated ATPase activities of these residues when they were mutated to cysteine (19Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Although the residues in TM6 and TM12 are far apart in the linear sequence of P-gp, they are quite close together in the tertiary structure of P-gp. Cross-linking studies between residues in TM6 and TM12 have shown that residues Gly346 (TM6) and Gly989 (TM12) can be oxidatively cross-linked (25Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Cross-linking between these residues is blocked by the presence of verapamil, vinblastine, or colchicine. TM11 also appears to be close to TM6 and TM12 in native P-gp. TM11 is separated from TM12 by a relatively short extracellular loop of only 16 amino acids. Therefore, as a working model, the residues of TM6, TM11, and TM12 are arranged as α-helical wheels, with the residues sensitive to inhibition by dBBn positioned so that they face a central substrate-binding pocket. The helices are also oriented to take into account the cross-linkable nature of residues 346 (TM6) and 989 (TM12) in the mutant G346C/G989C (25Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Future studies will be required to determine the role of residues in other TM segments of P-gp in binding drug substrates. We thank Dr. Randal Kaufman for pMT21.