Determining the Dimensions of the Drug-binding Domain of Human P-glycoprotein Using Thiol Cross-linking Compounds as Molecular Rulers

The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments 4–6 and 10–12 form the drug-binding site. We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers were also substrates of P-gp, because they stimulated ATPase activity (2.5- to 10.1-fold). 36 P-gp mutants with pairs of cysteine residues introduced into transmembrane segments 4–6 and 10–12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 °C. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9–25 Å. Vinblastine and cyclosporin A inhibited cross-linking. The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is “funnel” shaped, narrow at the cytoplasmic side, at least 9–25 Å in the middle, and wider still at the extracellular surface. The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments 4–6 and 10–12 form the drug-binding site. We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers were also substrates of P-gp, because they stimulated ATPase activity (2.5- to 10.1-fold). 36 P-gp mutants with pairs of cysteine residues introduced into transmembrane segments 4–6 and 10–12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 °C. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9–25 Å. Vinblastine and cyclosporin A inhibited cross-linking. The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is “funnel” shaped, narrow at the cytoplasmic side, at least 9–25 Å in the middle, and wider still at the extracellular surface. P-glycoprotein methanethiosulfonate transmembrane polyacrylamide gel electrophoresis human embryonic kidney 1,2-ethanediyl bismethanethiosulfonate 1,3-propanediyl bismethanethiosulfonate 1,4-butanediyl bismethanethiosulfonate 1,5-pentanediyl bismethanethiosulfonate 1,6-hexanediyl bismethanethiosulfonate 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate 3,6,9-trioxaundecane-1,11-diyl bismethanethiosulfonate 3,6,9,12-tetraoxatetradecane-1,14-diyl bismethanethiosulfonate 3,6,9,12,15-pentaoxaheptadecane-1,17-diyl bismethanethiosulfonate The human multidrug resistance P-glycoprotein (P-gp)1 uses ATP to pump out of the cell a wide variety of structurally diverse compounds (recently reviewed in Ref. 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 (1911) Google Scholar). Many of these compounds are clinically important in cancer and AIDS chemotherapy (2Kim 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 (1029) Google Scholar, 3Lee 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, 4Robert J. Eur. J. Clin. Invest. 1999; 29: 536-545Crossref PubMed Scopus (71) Google Scholar). Therefore, overexpression of P-gp often leads to multidrug resistance. The pattern of expression in tissues and studies on P-gp knock-out mice indicate that its physiological role may be to protect the organism from toxins in the environment and in the diet (5Thiebaut 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 (2550) Google Scholar, 6Cordon-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 (1587) Google Scholar, 7Schinkel 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 (2055) Google Scholar). P-gp is a member of the ABC (ATP-bindingcassette) family of transporters (8Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3351) Google Scholar). Its 1280 amino acids are organized in two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (9Chen 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 (1713) Google Scholar). Each repeat has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (10Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 11Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). An important goal in determining the mechanism of P-gp is to understand how P-gp can bind so many different compounds and how ATP hydrolysis causes drug transport. The minimal functional unit in P-gp is a monomer (12Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Both nucleotide-binding sites are required for function, because P-gp is inactive when ATP hydrolysis at either site in blocked by mutation or chemical modification (13Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar, 14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 15Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 16Urbatsch 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, 17Hrycyna 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, 18Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (94) Google Scholar). The nucleotide-binding domains may function in an alternating mechanism (19Senior A.E. Gadsby D.C. Semin Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (129) Google Scholar, 20Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Photolabeling and mutational studies indicate that the drug-binding domain is within the TM domains (21Bruggemann E.P. Currier S.J. Gottesman M.M. Pastan I. J. Biol. Chem. 1992; 267: 21020-21026Abstract Full Text PDF PubMed Google Scholar, 22Greenberger L.M. J. Biol. Chem. 1993; 268: 11417-11425Abstract Full Text PDF PubMed Google Scholar, 23Morris 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, 24Zhang X. Collins K.I. Greenberger L.M. J. Biol. Chem. 1995; 270: 5441-5448Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 25Demmer A. Thole H. Kubesch P. Brandt T. Raida M. Fislage R. Tummler B. J. Biol. Chem. 1997; 272: 20913-20919Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 26Demeule M. Laplante A. Murphy G.F. Wenger R.M. Beliveau R. Biochemistry. 1998; 37: 18110-18118Crossref PubMed Scopus (46) Google Scholar, 27Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (190) Google Scholar, 28Kajiji 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, 29Loo 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. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (125) Google Scholar). This is supported by the finding that a deletion mutant lacking both nucleotide-binding domains can still bind drug substrate (32Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Drug binding requires both halves of the TM domains, because drug-stimulated ATPase activity is only observed when both halves are coexpressed (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar). Disulfide cross-linking studies have provided considerable insight into the structure of membrane proteins (33Falke J.J. Koshland Jr., D.E. Science. 1987; 237: 1596-1600Crossref PubMed Scopus (228) Google Scholar, 34Yu H. Kono M. McKee T.D. Oprian D.D. Biochemistry. 1995; 34: 14963-14969Crossref PubMed Scopus (119) Google Scholar, 35Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (319) Google Scholar, 36Bass R.B. Falke J.J. Structure Fold Des. 1999; 7: 829-840Abstract Full Text Full Text PDF Scopus (59) Google Scholar, 37Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar). Recently, we identified residues in TMs 4, 5, 6, 10, 11, and 12 that contribute to the drug-binding domain (38Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 39Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 40Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 41Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). In this study, we used a series of thiol-specific cross-linkers with spacer arms of various lengths to measure distances between these residues. There are seven endogenous cysteines at positions 137, 431, 717, 956, 1074, 1125, and 1227 in wild-type P-gp. None of the cysteines are needed for activity, because mutation of all cysteines to alanine (Cys-less P-gp) resulted in an active molecule (10Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The Cys-less P-gp cDNA was also modified to code for ten histidine residues at the COOH end of the molecule (Cys-less P-gp(His)10). The histidine tag facilitated purification of the Cys-less P-gp by nickel-chelate chromatography (42Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Cysteine residues were then introduced into the Cys-less P-gp(His)10 as described previously (40Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The integrity of the mutated cDNA was confirmed by sequencing the entire cDNA (43Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52505) Google Scholar). Expression and purification of histidine-tagged P-gp mutants were described previously (42Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Briefly, 50 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 μm cyclosporin A. Cyclosporin A is a substrate of P-gp and is a powerful chemical chaperone for promoting maturation of P-gp (44Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). The transfected cells were harvested 24 h later, solubilized with 1% (w/v)n-dodecyl-β-d-maltoside, and the mutant P-gp was isolated by nickel-chelate chromatography (Ni-NTA columns; Qiagen, Inc., Mississauga, Ontario, Canada). The P-gp-(His)10 mutants were eluted from the column and mixed with an equal volume of 10 mg/ml sheep brain phosphatidylethanolamine (Type II-S; Sigma-Aldrich) that was washed and suspended in 10 mm Tris-HCl, pH 7.5, and 150 mm NaCl. The P-gp:lipid mixture was then sonicated for 45 s at 4 °C. An aliquot of the mixture was assayed for drug-stimulated ATPase activity by addition of an equal volume of buffer containing 100 mm Tris-HCl, pH 7.5, 100 mm NaCl, 20 mm MgCl2, 10 mm ATP, and 2 mm MTS cross-linker. The samples were incubated for 30 min at 37 °C, and the amount of inorganic phosphate liberated was determined (45Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (414) Google Scholar). The mutant P-gps were expressed in HEK 293 cells in the presence of 10 μm cyclosporin A. Membranes were prepared from transfected cells and suspended in Tris-buffered saline (10 mm Tris-HCl, pH 7.4, 150 mm NaCl) and treated with 0.2 mm cross-linker (Toronto Research Chemicals, Toronto, Ontario, Canada; see Fig. 1) for 15 min at 4 °C. At this concentration, the MTS cross-linkers stimulated the ATPase activity of Cys-less P-gp by about 50%. The reactions were stopped by addition of 2× SDS sample buffer containing 10 mm N-ethylmaleimide. In the protection experiments, the membranes were pretreated for 10 min at 4 °C in the presence of 1 mm vinblastine or 1 mmcyclosporin A (saturating conditions). The samples were subjected to SDS-PAGE on 7.5% acrylamide gels and immunoblot analysis with rabbit polyclonal antibody (12Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To measure distances between residues in the NH2 and COOH halves of the drug-binding domain, we constructed P-gp mutants that had a pair of cysteine residues, one in the NH2 half and the other in the COOH half (Table I). The mutants were then tested for cross-linking with thiol-specific cross-linkers. In attempting to measure distances between residues, it would be most useful to use cross-linkers that had similar spacer arms and reactive groups to minimize differences in chemical reactivity with cysteines. A set of thiol-specific cross-linkers with these properties is the MTS cross-linkers shown in with cysteines in a in a of the spacer and of a T.W. 1977; 47: PubMed Scopus Google Scholar, T.W. J. Chem. Scopus (94) Google Scholar). The MTS compounds are reactive with cysteines than other thiol-specific compounds as or T.W. J. Chem. Scopus (94) Google with MTS 6, product in SDS-PAGE using MTS cross-linkers shown in Fig. The the of the spacer of the MTS 11, cross-linked product with MTS 6, 11, 11, 11, 11, product in SDS-PAGE using MTS cross-linkers shown in Fig. The the of the spacer of the MTS cross-linked product with MTS cross-linker. in a In using cross-linkers to distances in the drug-binding domain, it is important that the compounds can the drug-binding site. is to be to measure of these MTS This is because are is the of MTS compounds in T.W. 1977; 47: PubMed Scopus Google and they are also these is to measure of ATPase of most substrates to P-gp its ATPase activity, and is between drug-stimulated ATPase activity and drug S.V. Cardarelli C.O. I. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). all the MTS cross-linkers 1) were assayed for to Cys-less P-gp ATPase Cys-less P-gp has all seven endogenous cysteines replaced with alanine and is as active and drug-stimulated ATPase as wild-type P-gp (10Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Fig. 2 the of Cys-less P-gp ATPase activity in the presence of of MTS cross-linker. the stimulated the ATPase activity of Cys-less P-gp. The most of activity were and lower of were observed with and to and stimulated activity to In the of activity were compounds with spacer arms of lengths (10 to 13 the of P-gp activity, Cys-less P-gp activity about the substrate vinblastine activity about T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). The results that most of the MTS cross-linkers can the drug-binding site of P-gp. Recently, we used (38Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 39Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 40Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (133) Google and T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (184) Google to that cysteines introduced at positions and in the NH2 half of P-gp and at and in the COOH half of P-gp contribute to the drug-binding domain (40Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). To for mutants with one cysteine in the NH2 half and and in the COOH half and were (Table I). The mutants were expressed in HEK 293 cells and were to be to the form of P-gp The 36 mutants (Table were then subjected to cross-linking by the MTS cross-linkers Membranes were prepared from transfected cells and treated with different cross-linkers at 4 °C. The reactions were at 4 °C to in the The reactions were stopped by addition of SDS sample buffer containing and the mixture was analyzed by In studies we had shown the cross-linking between residues in the NH2 and COOH halves of P-gp resulted in the cross-linked product with lower mobility in SDS gels T.W. Clarke D.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). cross-linking by MTS cross-linkers resulted in mobility of the cross-linked product was in 13 and results are shown in Fig. In mutant cross-linking was observed with and was also observed with and evidence of cross-linking was observed with the cross-linkers to to of mutant was because the cross-linked product was after with mm We then tested drug substrates cross-linking. cross-linking between residues in the drug-binding domain, then the presence of substrates as cyclosporin A and vinblastine cross-linking. Cyclosporin A is an of drug resistance V. Eur. J. 1996; Full Text PDF Scopus Google vinblastine is a drug substrate S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1911) Google Scholar). The membranes containing P-gp mutant were with 1 mmcyclosporin A or 1 mm vinblastine for 10 min at 4 °C. The samples were then treated with and subjected to Fig. that cyclosporin A and vinblastine blocked cross-linking of mutant by An of a cross-linking is in mutant lower The cross-linking results of the other mutants are in I. of the mutants were was cross-linked with and mutant was cross-linked with and of the mutants be and were cross-linked with and was cross-linked with and and were cross-linked with and mutants were was cross-linked with only and were cross-linked with and mutant was cross-linked with and mutants and were cross-linked with and cross-linking was with the cross-linking was blocked when the membranes were with 1 mm 1 mm cyclosporin A. cross-linked product was after with The cross-linking results in that residues and a drug-binding site. in TMs and were cross-linked to residues in TMs 10, 11, and was cross-linked with two residues and in In all cross-linking was blocked by substrates as vinblastine or cyclosporin A. Vinblastine and cyclosporin are large structures that they are about Å in at and K. K. J. Chem. Scholar, A. L. A. P. A. J. S. M. B. P. V. Chem. 2000; Scopus Google Scholar). The cross-linking results indicate that the drug-binding site would be large enough to accommodate vinblastine or cyclosporin A. mutants and are only cross-linked with MTS compounds with spacer arms of than 20 Å. The between TMs 4, 5, and and TM 10, 11, and 12 as by these cross-linkers is about Å and between TMs 4 and 10, because mutants and be cross-linked with is to that of the 36 mutants (Table cross-linking with a cross-linker at either or 4 °C was to mutants as the mutants that were cross-linked with were also cross-linked with the cross-linker. is that the TMs of P-gp are T.W. Clarke D.M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google and can or to accommodate different sizes of This mobility of the in different substrates would different residues to the drug-binding site and the of P-gp for a is also that the reactive cysteines may be in a to with cross-linker of a This may be the that all of the mutants with the of MTS cross-linkers. cross-linking was in of the is that may be a Recently, E. Sci. 2001; PubMed Scopus Google that a with using cross-linking as is that they can be compounds can a range of to of the that form the linker There are in the spacer of that the two reactive it is that mutants are with when it is in that the reactive groups other mutants would only with the when it is in the The residues that have identified to contribute to the drug-binding site are in the of the TM segments and to be 9–25 Å studies have shown that the cytoplasmic of the TMs and to the drug-binding site are than because cross-linking between residues in these TMs with a cross-linking T.W. Clarke D.M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The TM segments on the extracellular be A structure of P-gp the presence of a of about 50 Å in diameter on the extracellular M.F. R. C.F. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The emerging picture of P-gp is shown in a in In this the drug-binding domain to be a “funnel” with the at the extracellular and the at the cytoplasmic Drug binding would to the of the ATP hydrolysis would the of P-gp for substrate in the drug-binding domain by T.W. Clarke D.M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google and large of T.W. Clarke D.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). ATPase activity is inhibited are by cross-linking of the TM segments 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. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). would in of the drug substrate and of the extracellular of the We for and for with

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