Simultaneous Binding of Two Different Drugs in the Binding Pocket of the Human Multidrug Resistance P-glycoprotein

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The drug-binding pocket of P-gp is located in the transmembrane domains. Although occupation of the drug-binding pocket by one molecule is sufficient to activate the ATPase activity of P-gp, the drug-binding pocket may be large enough to accommodate two different substrates at the same time. In this study, we used cysteine-scanning mutagenesis to test whether P-gp could simultaneously interact with the thiol-reactive drug substrate, Tris-(2-maleimidoethyl)amine (TMEA) and a second drug substrate. TMEA is a cross-linker substrate of P-gp that allowed us to test for stimulation of cross-linking by a second substrate such as calcein-acetoxymethyl ester, colchicine, demecolcine, cyclosporin A, rhodamine B, progesterone, and verapamil. We report that verapamil induced TMEA cross-linking of mutant F343C(TM6)/V982C(TM12). By contrast, no cross-linked product was detected in mutants F343C(TM6), V982C(TM12), or F343C(TM6)/V982C(TM12) in the presence of TMEA alone. The verapamil-stimulated ATPase activity of mutant F343C(TM6)/V982C(TM12) in the presence of TMEA decreased with increased cross-linking of the mutant protein. These results show that binding of verapamil must induce changes in the drug-binding pocket (induced-fit mechanism) resulting in exposure of residues F343C(TM6)/V982C(TM12) to TMEA. The results also indicate that the common drug-binding pocket in P-gp is large enough to accommodate both verapamil and TMEA simultaneously and suggests that the substrates must occupy different regions in the common drug-binding pocket. The human multidrug resistance P-glycoprotein (P-gp, ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The drug-binding pocket of P-gp is located in the transmembrane domains. Although occupation of the drug-binding pocket by one molecule is sufficient to activate the ATPase activity of P-gp, the drug-binding pocket may be large enough to accommodate two different substrates at the same time. In this study, we used cysteine-scanning mutagenesis to test whether P-gp could simultaneously interact with the thiol-reactive drug substrate, Tris-(2-maleimidoethyl)amine (TMEA) and a second drug substrate. TMEA is a cross-linker substrate of P-gp that allowed us to test for stimulation of cross-linking by a second substrate such as calcein-acetoxymethyl ester, colchicine, demecolcine, cyclosporin A, rhodamine B, progesterone, and verapamil. We report that verapamil induced TMEA cross-linking of mutant F343C(TM6)/V982C(TM12). By contrast, no cross-linked product was detected in mutants F343C(TM6), V982C(TM12), or F343C(TM6)/V982C(TM12) in the presence of TMEA alone. The verapamil-stimulated ATPase activity of mutant F343C(TM6)/V982C(TM12) in the presence of TMEA decreased with increased cross-linking of the mutant protein. These results show that binding of verapamil must induce changes in the drug-binding pocket (induced-fit mechanism) resulting in exposure of residues F343C(TM6)/V982C(TM12) to TMEA. The results also indicate that the common drug-binding pocket in P-gp is large enough to accommodate both verapamil and TMEA simultaneously and suggests that the substrates must occupy different regions in the common drug-binding pocket. The multidrug resistance P-glycoprotein (P-gp 1The abbreviations used are: P-gp, P-glycoprotein; TM, transmembrane; HEK, human embryonic kidney; AM, acetoxymethyl ester; TMEA, tris-(2-maleimidoethyl)amine; ABC, ATP-binding cassette.; MDR1 or ABCB1 gene product) uses ATP to transport structurally diverse compounds out of the cell (recently reviewed in 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 (1948) Google Scholar and 2Hrycyna C.A. Semin. Cell Dev. Biol. 2001; 12: 247-256Crossref PubMed Scopus (52) Google Scholar). Overexpression of P-gp in tumors and in organs such as the liver, kidney, and the blood-brain barrier can undermine cancer and AIDS chemotherapy regimens because many of the therapeutic drugs are substrates of P-gp (3Gottesman M.M. Fojo T. Bates S.E. Nat. Rev. Cancer. 2002; 2: 48-58Crossref PubMed Scopus (4735) Google Scholar, 4Thomas H. Coley H.M. Cancer Control. 2003; 10: 159-165Crossref PubMed Scopus (853) Google Scholar). P-gp is one of 48 ATP-binding cassette (ABC) transporters in humans (5Dean M. Rzhetsky A. Allikmets R. Genome Res. 2001; 11: 1156-1166Crossref PubMed Scopus (1530) Google Scholar). The 1280 amino acids of P-gp are arranged as two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (6Chen 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 (1780) Google Scholar). Each repeat has six trans-membrane (TM) segments and a hydrophilic domain containing an ATP-binding site (7Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 8Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 9Kast C. Canfield V. Levenson R. Gros P. Biochemistry. 1995; 34: 4402-4411Crossref PubMed Scopus (88) Google Scholar). The minimum functional unit is a monomer (10Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Both halves of the molecule are essential for activity but do not have to be covalently linked for function (11Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 12Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Both ATP-binding sites are required for activity (8Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 13Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (283) Google Scholar, 14Doige C.A. Yu X. Sharom F.J. Biochim. Biophys. Acta. 1992; 1109: 149-160Crossref PubMed Scopus (138) Google Scholar, 15al-Shawi M.K. Urbatsch I.L. Senior A.E. J. Biol. Chem. 1994; 269: 8986-8992Abstract Full Text PDF PubMed Google Scholar), but the TM domains alone are sufficient to mediate drug binding (12Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). An important goal in understanding the mechanism of P-gp is to determine the location and number of drug-binding sites. Studies with thiol-reactive substrate analogs of P-gp and cysteine mutants have shown that residues from multiple TM segments contribute to a common drug-binding pocket (16Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 18Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 20Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 2002; 277: 44332-44338Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). P-gp in the resting state is in the “closed” conformation where the cytoplasmic ends of the TM segments are close to each other but far apart at the extracellular end of the molecule (20Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 23Loo T.W. Clarke D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3511-3516Crossref PubMed Scopus (73) Google Scholar). Covalent binding of a single molecule of the drug substrate verapamil in the drug-binding pocket was sufficient to permanently activate P-gp (24Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2003; 278: 20449-20452Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The dimensions of the P-gp drug-binding pocket as determined with thiol-reactive cross-linker substrates, however, indicated the drug-binding pocket may accommodate more than one substrate at the same time (20Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). To determine whether more than one substrate could bind to P-gp at the same time, we used the drug substrate Tris-(2-maleimidoethyl)amine (TMEA). TMEA is a useful compound for analyzing the P-gp drug-binding pocket because it will cross-link cysteine residues if they are close to the binding site of TMEA. Binding of a second drug substrate will inhibit TMEA cross-linking if there is significant overlap of the binding sites. Similarly, cross-linking could be unaffected or enhanced if the binding site of the second drug substrate does not overlap that of TMEA. In this study, we used cysteine-scanning mutagenesis and reaction with TMEA in the presence of other drug substrates to determine whether P-gp could simultaneously bind to two different drug substrates in the common drug-binding pocket. Construction of Mutants—Cysteine residues were introduced into a histidine-tagged Cys-less P-gp cDNA (25Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Cys-less P-gp was constructed by replacing the seven endogenous cysteines at positions 137, 431, 717, 956, 1074, 1125, and 1227 with alanines (7Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The Cys-less P-gp retained the ability to confer multidrug resistance and exhibited drug-stimulated ATPase activity (7Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The presence of the histidine tag facilitated purification of the mutant P-gps by nickel-chelate chromatography (26Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Treatment of Mutants with TMEA—HEK 293 cells (10 10-cm diameter plates) were transfected with the mutant cDNAs. After 24 h, the media were replaced with fresh media and the cells grown for 72 h at 27 °C. Membranes were prepared and suspended in 200 μl of Tris-buffered saline (10 mm Tris-HCl, pH 7.4, 150 mm NaCl) as described previously (26Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 27Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar) and used for disulfide cross-linking analysis (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To test the effect of drug substrates on cross-linking by TMEA, the membranes were pre-incubated with no drug or saturating levels of calcein-AM (1 mm), colchicine (10 mm), demecolcine (2 mm), cyclosporin A (0.2 mm), progesterone (2 mm), rhodamine B (2 mm), verapamil (1 mm), R(+)-verapamil (1 mm) or S(–)-verapamil (1 mm) (Sigma) for 10 min at 21 °C. The mixtures were then incubated for 15 min at 21 °C in the presence of 0, 0.1, or 1 mm TMEA. The reactions were stopped by addition of 300 mm cysteine, pH 7.0, to a final concentration of 30 mm. After another 10 min at 21 °C, SDS sample buffer (125 mm Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, and 4%(v/v) 2-mercaptoethanol) was added. The mixtures were subjected to immunoblot analysis with rabbit polyclonal anti-P-gp antibody (28Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21839-21844Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and enhanced chemiluminescence (Pierce). To monitor the time course of verapamil-stimulated TMEA cross-linking, the membranes were pre-incubated with 1 mm verapamil for 10 min at 21 °C. The membranes were then incubated for 0–32 min in the presence of 0.1 mm TMEA. The reactions were stopped by addition of SDS sample buffer containing 30 mm cysteine. To monitor the dependence of cross-linking on TMEA concentration, membranes were pre-incubated with 1 mm verapamil for 10 min at 21 °C and then treated with various concentrations (0–1 mm) of TMEA for 15 min at 21 °C. The reactions were stopped by addition of SDS sample buffer containing 30 mm cysteine. The dependence of TMEA cross-linking on verapamil concentration was determined by pre-incubating the membranes in the presence of various concentrations (0–1 mm) of verapamil for 10 min at 21 °C followed by treatment with 0.1 mm TMEA for 15 min at 21 °C. The reactions were stopped by addition of SDS sample buffer containing 30 mm cysteine. Expression, Purification, and Measurement of Drug-stimulated ATPase Activity of P-gp Mutants—HEK 293 cells were transfected with the mutant cDNAs. The medium was replaced after 24 h with fresh medium containing 10 μm cyclosporin A. Cyclosporin A is a substrate of P-gp and acts as a powerful chemical chaperone in promoting maturation of P-gp and increases the yield of mature P-gp (29Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 30Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 31Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 32373-32376Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 32Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 27585-27588Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). After another 24 h, the transfected cells were harvested, and the mutants were isolated by nickel-chelate chromatography. The isolated mutant P-gps were mixed with lipid (sheep brain lipid, type IIs, Sigma) and sonicated as described previously (26Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). An aliquot of the P-gp/lipid mixture was incubated with 1 mm verapamil for 10 min at 21 °C and then treated with various concentrations (0–1 mm) of TMEA for 15 min at 21 °C. The reactions were stopped by addition of cysteine, pH 7.0, to a final concentration of 30 mm. After another 10 min at 21 °C, the samples were mixed with an equal volume of ATPase buffer containing 100 mm Tris-HCl, pH 7.4, 100 mm NaCl, 20 mm MgCl2, 10 mm ATP and no verapamil or 1 mm verapamil. The samples were incubated for 30 min at 37 °C, and the amount of inorganic phosphate liberated was determined (33Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (425) Google Scholar). We had shown that the P-gp drug-binding pocket is shaped liked a “funnel,” i.e. narrow at the cytoplasmic side, at least 9 Å in the middle, and even wider at the extracellular side (20Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). it that the drug-binding pocket was large enough to simultaneously accommodate more than one substrate. To determine whether two different substrates could bind at the same time, we cysteine mutants for drug-stimulated cross-linking with the thiol-reactive cross-linker TMEA for The was that binding of the drug substrate a in the drug-binding pocket that it cross-linking of cysteine residues by TMEA if the drug-binding site of the substrate not overlap that of TMEA. in P-gp is because the cross-linked product with on T.W. Clarke D.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, I.L. K. S. Gros P. Senior A.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). TMEA was as the cross-linker because it is a P-gp substrate (25Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google it is Å and it has thiol-reactive side that can membranes from cells P-gp mutants with one cysteine in the TM domain and a second cysteine in the TM domain were pre-incubated with saturating concentrations of the drug substrates calcein-AM (1 mm), colchicine (10 mm), demecolcine (2 mm), cyclosporin A (0.2 mm), progesterone (2 mm), rhodamine B (2 mm), verapamil (1 mm), R(+)-verapamil (1 mm), or S(–)-verapamil (1 The membranes were then treated with 0.1 mm TMEA for 15 min at 21 °C. The reactions were at 21 °C because at T.W. 47: PubMed Scopus Google Scholar), at a results in increased in The reactions were stopped by addition of cysteine and the samples subjected to immunoblot mutant F343C(TM6)/V982C(TM12) cross-linking with TMEA. and S(–)-verapamil cross-linking of P-gp with TMEA By contrast, of the drug substrates cross-linking in mutant or mutant B and Similarly, the drug substrates not cross-linking in mutant F343C(TM6)/V982C(TM12) in the of TMEA results were cross-linking was out with 1 mm TMEA not of drugs on cross-linking of P-gp Membranes were prepared from 293 cells P-gp mutants F343C(TM6)/V982C(TM12) and or The membranes were pre-incubated at 21 °C for 10 min with no drug calcein-AM colchicine demecolcine cyclosporin A rhodamine B progesterone verapamil R(+)-verapamil or S(–)-verapamil The reaction mixtures were then treated with or 0.1 mm TMEA for 15 min at 21 °C. The reactions were stopped by addition of cysteine and SDS sample The mixtures were subjected to immunoblot The positions of the cross-linked product and mature P-gps are Both of R(+)-verapamil and TMEA cross-linking of mutant F343C(TM6)/V982C(TM12) is with that both were equal in ability to the ATPase activity of P-gp about 30 F343C(TM6)/V982C(TM12) a in the of cross-linking In the presence of 0.1 mm TMEA and 1 mm cross-linked product was detected after min at 21 °C. After the of the mutant was The dependence of cross-linking on TMEA concentration was also Membranes from mutant F343C(TM6)/V982C(TM12) were pre-incubated with 1 mm verapamil and then with various concentrations (0–1 mm) of TMEA. cross-linked product was detected in the presence of mm TMEA. Similarly, the dependence of TMEA cross-linking on the verapamil concentration was determined by pre-incubating membranes with various concentrations of verapamil to 1 mm) followed by treatment with 0.1 mm TMEA for 15 min at 21 °C. product was detected at μm verapamil. The amount of cross-linked product increased with concentrations of with of the mutant cross-linked in the presence of or 1 mm verapamil. To determine whether the of the mutant F343C(TM6)/V982C(TM12) was with TMEA, we for of drug-stimulated ATPase The was that cross-linking of activity because activity is on of TM segments P-gp T.W. Clarke D.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). histidine-tagged F343C(TM6), V982C(TM12), and F343C(TM6)/V982C(TM12) mutant P-gps were isolated by nickel-chelate mixed with lipid, and samples incubated for 10 min at 21 °C in the presence of 1 mm verapamil. The samples were then treated for 15 min at 21 °C in the presence of various concentrations (0–1 mm) of TMEA. The reactions were by addition of cysteine, and verapamil-stimulated ATPase were The activity of the Cys-less P-gp was not by TMEA not (25Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). was however, the concentration of TMEA required for cross-linking mm and the concentration to 1 mm) required for more than of the activity of mutant F343C(TM6)/V982C(TM12). at μm TMEA. The mutants and were also for by TMEA. We previously that the activity of mutant at μm TMEA incubated with TMEA in the of verapamil (25Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In the study, of mutant with TMEA in the presence of 1 mm verapamil a of with at μm TMEA a in to TMEA verapamil was TMEA had effect on the activity of mutant in the of verapamil of mutant in the presence of 1 mm however, a large in of TMEA. The activity of the mutant was by more than in the presence of verapamil and 1 mm TMEA. of verapamil-stimulated ATPase activity at μm TMEA. that the presence of verapamil increased the or of to TMEA. In a (25Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), we that TMEA alone could cross-link mutant In this mutant cross-linking was by the presence of verapamil. By contrast, cross-linking in mutant F343C(TM6)/V982C(TM12) by TMEA in the presence of verapamil 1 and An for is that binding of verapamil changes in resulting in changes in the positions of residues and changes for the mechanism of We had shown previously T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) that drug substrates such as colchicine, cyclosporin A, demecolcine, and progesterone can cross-linking cysteine residues in the TM segments the drug-binding pocket with a cross-linker was that progesterone cross-linking in mutants and cyclosporin A cross-linking in mutant and colchicine and demecolcine cross-linking in mutant is as an residues and are on the same of the but by a of the Binding of verapamil a of the extracellular to to a more to with TMEA. of such have with TM segments that the site in the ATPase C. H. 2002; PubMed Scopus Google Scholar). The of with and in and of about a Å the of the The ability of drug substrates to the of a has also for other The is a from can be induced by structurally diverse The of with six different drugs were M.C. S. 2001; PubMed Scopus Google Scholar). a and one A of 1 has also for other such as and M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, A. K. J. 1999; PubMed Google Scholar). binding induced large changes in the drug-binding pocket of and indicated the presence of two binding sites a single pocket that was by of the drug-binding pocket of P-gp that it may many with In the of drug substrates, the six TM segments and that contribute residues to the drug-binding site are close to each other because cysteines in can be cross-linked to cysteines in with (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Binding of drug substrates increases the and to at least Å (20Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). is that are with of the from J. Mol. Biol. 2003; PubMed Scopus Google Scholar). The of was in the has to P-gp and two to the minimum functional The ability of verapamil to cross-linking of mutant F343C(TM6)/V982C(TM12) with TMEA that both verapamil and TMEA can simultaneously occupy the drug-binding pocket. The results also that binding of one than two TMEA because cross-linking by TMEA was in mutant F343C(TM6)/V982C(TM12) in the presence of verapamil. Binding of two TMEA have in The cross-linking of mutant F343C(TM6)/V982C(TM12) in the presence of verapamil TMEA suggests that single of verapamil and TMEA are to sites that are close to each other in the drug-binding pocket. and that P-gp has to different drug-binding sites V. J. Biochem. 1997; PubMed Scopus Google Scholar, M. S. J. Biochem. 1997; PubMed Scopus Google Scholar, S. Ramachandra M. Pastan I. Gottesman M.M. S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). of results is a that the drug-binding pocket is located at the the TM domains from both halves of this binding pocket are regions that bind to drug substrates sites could results the presence of two sites the drug-binding pocket. TMEA is a substrate of P-gp because it can the ATPase activity of Cys-less P-gp (25Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Although TMEA cross-linked and this may not be the TMEA drug-binding site that to ATPase and The location could not be determined because cross-linking of mutant an protein. The site is close but because verapamil changes in P-gp, the site may also have in a that the TMEA molecule to as a of a of promoting cross-linking and Binding of verapamil to the extracellular a of the results in exposure of that it can be cross-linked to with TMEA. In the results from this for the mechanism for drug binding and that two different substrates can bind simultaneously in the same drug-binding pocket.

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