The Journal of Membrane Biology

, Volume 206, Issue 3, pp 173–185 | Cite as

Recent Progress in Understanding the Mechanism of P-Glycoprotein-mediated Drug Efflux

  • T.W. Loo
  • D.M. Clarke
Topical Review


P-glycoprotein (P-gp) is an ATP-dependent drug pump that can transport a broad range of hydrophobic compounds out of the cell. The protein is clinically important because of its contribution to the phenomenon of multidrug resistance during AIDS/HIV and cancer chemotherapy. P-gp is a member of the ATP-binding cassette (ABC) family of proteins. It is a single polypeptide that contains two repeats joined by a linker region. Each repeat has a transmembrane domain consisting of six transmembrane segments followed by a hydrophilic domain containing the nucleotide-binding domain. In this mini-review, we discuss recent progress in determining the structure and mechanism of human P-glycoprotein.


P-glycoprotein ABC transporter Substrate binding pocket ATPase activity Thiol cross-linking Induced-fit mechanism 



1,6-hexanediyl bismethanethiosulfonate;


3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate;


amino-terminal nucleotide binding domain;


carboxy-terminal nucleotide binding domain;




transmembrane domain;





This work was supported by grants from the National Cancer Institute of Canada through the Canadian Cancer Society and from the Canadian Institutes of Health Research. DMC is the recipient of the Canada Research Chair in Membrane Biology.


  1. Al-Shawi M.K., Polar M.K., Omote H., Figler R.A. 2003. Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein. J. Biol. Chem. 278:52629–52640CrossRefPubMedGoogle Scholar
  2. al-Shawi M.K., Senior A.E. 1993. Characterization of the adenosine triphosphatase activity of Chinese hamster P-glycoprotein. J. Biol. Chem. 268:4197–4206PubMedGoogle Scholar
  3. Alien J.D., Brinkhuis R.F., Wijnholds J., Schinkel A.H. 1999. The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. Cancer Res. 59:4237–4241Google Scholar
  4. Allikmets R., Schriml L.M., Hutchinson A., Romano-Spica V., Dean M. 1998. A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance. Cancer Res. 58:5337–5339PubMedGoogle Scholar
  5. Ambudkar S.V., Kimchi-Sarfaty C., Sauna Z.E., Gottesman M.M. 2003. P-glycoprotein: from genomics to mechanism. Oncogene 22:7468–7485CrossRefPubMedGoogle Scholar
  6. Ambudkar S.V., Lelong I.H., Zhang J., Cardarelli C.O., Gottesman M.M., Pastan. 1992. Partial purification and reconstitution of the human multidrug- resistance pump: characterization of the drug-stimulatable ATP hydrolysis. Proc. Natl. Acad. Sci. USA 89:8472–8476PubMedGoogle Scholar
  7. Biedler J.L., Riehm H. 1970. Cellular resistance to actinomycin D in Chinese hamster cells in vitro: cross-resistance, radioautographic, and cytogenetic studies. Cancer Res. 30:1174–1184PubMedGoogle Scholar
  8. Borges-Walmsley M.I., McKeegan K.S., Walmsley A.R. 2003. Structure and function of efflux pumps that confer resistance to drugs. Biochem. J. 376:313–338CrossRefPubMedGoogle Scholar
  9. Bruggemann E.P., Currier S.J., Gottesman M.M., Pastan I. 1992. Characterization of the azidopine and vinblastine binding site of P-glycoprotein. J. Biol. Chem. 267:21020–21026PubMedGoogle Scholar
  10. Chang G. 2003. Structure of MsbA from Vibrio cholera: A Multidrug Resistance ABC transporter Homolog in a Closed Conformation. J. Mol. Biol. 330:419–430CrossRefPubMedGoogle Scholar
  11. Chang G., Roth C.B. 2001. Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters. Science 293:1793–1800CrossRefPubMedGoogle Scholar
  12. Chen C.J., Chin J.E., Ueda K., Clark D.P., Pastan I., Gottesman M.M., Roninson I.B. 1986. Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47:381–389PubMedGoogle Scholar
  13. Chen J.H., Chang X.B., Aleksandrov A.A., Riordan J.R. 2002. CFTR is a monomer: biochemical and functional evidence. J. Membrane Biol. 188:55–71CrossRefGoogle Scholar
  14. Choi K.H., Chen C.J., Kriegler M., Roninson I.B. 1988. An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdr1 (P-glycoprotein) gene. Cell 53:519–529CrossRefPubMedGoogle Scholar
  15. Dano K. 1972. Cross resistance between vinca alkaloids and anthracyclines in Ehrlich ascites tumor in vivo. Cancer Chemother. Rep. 56:701–708PubMedGoogle Scholar
  16. Dean M., Rzhetsky A., Allikmets R. 2001. The human ATP-binding cassette (ABC) transporter superfamily. Genome Res. 11:1156–1166PubMedGoogle Scholar
  17. Dearden J.C., A1-Noobi A., Scott A.C., Thomson S.A. 2003. QSAR studies on P-glycoprotein-regulated multidrug resistance and on its reversal by phenothiazines. SAR QSAR Environ. Res. 14:447–454PubMedGoogle Scholar
  18. Demeule M., Laplante A., Murphy G.F., Wenger R.M., Beliveau R. 1998. Identification of the cyclosporin-binding site in P-glycoprotein. Biochemistry 37:18110–18118CrossRefPubMedGoogle Scholar
  19. Demmer A., Thole H., Kubesch P., Brandt T., Raida M., Fislage R., Tummler B. 1997. Localization of the iodomycin binding site in hamster P-glycoprotein. J. Biol. Chem. 272:20913–20919CrossRefPubMedGoogle Scholar
  20. Dey S., Ramachandra M., Pastan I., Gottesman M.M., Ambudkar S.V. 1997. Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. Proc. Natl. Acad. Sci. USA 94:10594–10599CrossRefPubMedGoogle Scholar
  21. Doige C.A., Yu X., Sharom F.J. 1992. ATPase activity of partially purified P-glycoprotein from multidrug- resistant Chinese hamster ovary cells. Biochim. Biophys. Acta. 1109:149–160PubMedGoogle Scholar
  22. Dong J., Yang G., McHaourab H.S. 2005. Structural basis of energy transduction in the transport cycle of MsbA. Science 308:1023–1028CrossRefPubMedGoogle Scholar
  23. Doyle L.A., Yang W., Abruzzo L.V., Krogmann T., Gao Y., Rishi A.K., Ross D.D. 1998. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Natl. Acad. Sci. USA 95:15665–15670CrossRefPubMedGoogle Scholar
  24. Druley T.E., Stein W.D., Roninson I.B. 2001. Analysis of mdr1 p-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity. Biochemistry 40:4312–4322PubMedGoogle Scholar
  25. Eckford P.D., Sharom F.J. 2005. The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids. Biochem. J. 389: 517–526PubMedGoogle Scholar
  26. Ekins S., Kim R.B., Leake B.F., Dantzig A.H., Schuetz E.G., Lan L.B., Yasuda K., Shepard R.L., Winter M.A., Schuetz J.D., Wikel J.H., Wrighton S.A. 2002. Application of three-dimensional quantitative structure-activity relationships of P-glycoprotein inhibitors and substrates. Mol. Pharmacol. 61:974–981PubMedGoogle Scholar
  27. Garrigues A., Loiseau N., Delaforge M., Ferte J., Garrigos M., Andre F., Orlowski S. 2002. Characterization of two pharmacophores on the multidrug transporter P-glycoprotein. Mol. Pharmacol. 62:1288–1298CrossRefPubMedGoogle Scholar
  28. Gerlach J.H., Endicott J.A., Juranka P.F., Henderson G., Sarangi F., Deuchars K.L., Ling V. 1986. Homology between P-glycoprotein and a bacterial haemolysin transport protein suggests a model for multidrug resistance. Nature 324:485–489CrossRefPubMedGoogle Scholar
  29. Gottesman M.M., Pastan I. 1988. The multidrug transporter, a double-edged sword. J. Biol. Chem. 263:12163–12166PubMedGoogle Scholar
  30. Gottesman M.M., Pastan I. 1993. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62:385–427CrossRefPubMedGoogle Scholar
  31. Graf G.A., Yu L., Li W.P., Gerard R., Tuma P.L., Cohen J.C., Hobbs H.H. 2003. ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion. J. Biol. Chem. 278:48275–48282CrossRefPubMedGoogle Scholar
  32. Greenberger L.M. 1993. Major photoaffinity drug labeling sites for iodoaryl azidoprazosin in P- glycoprotein are within, or immediately C-terminal to, transmembrane domains 6 and 12. J. Biol. Chem. 268:11417–11425PubMedGoogle Scholar
  33. Gros P., Ben Neriah Y.B., Croop J.M., Housman D.E. 1986. Isolation and expression of a complementary DNA that confers multidrug resistance. Nature 323:728–731CrossRefPubMedGoogle Scholar
  34. Heldwein E.E., Brennan R.G. 2001. Crystal structure of the transcription activator BmrR bound to DNA and a drug. Nature 409:378–82CrossRefPubMedGoogle Scholar
  35. Higgins C.F. 1992. ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8:67–113CrossRefPubMedGoogle Scholar
  36. Higgins C.F., Gottesman M.M. 1992. Is the multidrug transporter a flippase? Trends. Biochem. Sci. 17:18–21Google Scholar
  37. Homolya L., Hollo Z., Germann U.A., Pastan I., Gottesman M.M., Sarkadi B. 1993. Fluorescent cellular indicators are extruded by the multidrug resistance protein. J. Biol. Chem. 268:21493–21496PubMedGoogle Scholar
  38. Hopfner K.P., Karcher A., Shin D.S., Craig L., Arthur L.M., Carney J.P., Tainer J.A. 2000. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell 101:789–800CrossRefPubMedGoogle Scholar
  39. Hrycyna C.A. 2001. Molecular genetic analysis and biochemical characterization of mammalian P-glycoproteins involved in multidrug resistance. Semin. Cell Dev. Biol. 12:247–256CrossRefPubMedGoogle Scholar
  40. Hrycyna C.A., Ramachandra M., Ambudkar S.V., Ko Y.H., Pedersen P.L., Pastan I., Gottesman M.M. 1998. Mechanism of action of human P-glycoprotein ATPase activity. Photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites. J. Biol. Chem. 273:16631–16634CrossRefPubMedGoogle Scholar
  41. Hrycyna C.A., Ramachandra M., Germann U.A., Cheng P.W., Pastan I., Gottesman M.M. 1999. Both ATP sites of human P-glycoprotein are essential but not symmetric. Biochemistry 38:13887–13899CrossRefPubMedGoogle Scholar
  42. Janas E., Hofacker M., Chen M., Gompf S., van der Does C., Tampe R. 2003. The ATP hydrolysis cycle of the nucleotide-binding domain of the mitochondrial ATP-binding cassette transporter Mdllp. J. Biol. Chem. 278:26862–26869CrossRefPubMedGoogle Scholar
  43. Juliano R.L., Ling V. 1976. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta 455:152–162PubMedGoogle Scholar
  44. Karpowich N., Martsinkevich O., Millen L., Yuan Y.R., Dai P.L., MacVey K., Thomas P.J., Hunt J.F. 2001. Crystal structures of the MJ1267 ATP binding cassette reveal an induced- fit effect at the ATPase active site of an ABC transporter. Structure (Camb) 9:571–586Google Scholar
  45. Kast C., Canfield V., Levenson R., Gros P. 1996. Transmembrane organization of mouse P-glycoprotein determined by epitope insertion and immunofluorescence. J. Biol. Chem. 271:9240–9248PubMedGoogle Scholar
  46. Lee C.G., Gottesman M.M., Cardarelli C.O., Ramachandra M., Jeang K.T., Ambudkar S.V., Pastan I., Dey S. 1998. HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. Biochemistry 37:3594–3601PubMedGoogle Scholar
  47. Lee J.Y., Urbatsch I.L., Senior A.E., Wilkens S. 2002. Projection structure of P-glycoprotein by electron microscopy. Evidence for a closed conformation of the nucleotide binding domains. J. Biol. Chem. 271:40125–40131Google Scholar
  48. Leith C.P., Kopecky K.J., Chen I.M., Eijdems L., Slovak M.L., McConnell T.S., Head D.R., Weick J., Grever M.R., Appelbaum F.R., Willman C.L. 1999. Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study. Blood 94:1086–1099PubMedGoogle Scholar
  49. Ling V., Thompson L.H. 1974. Reduced permeability in CHO cells as a mechanism of resistance to colchicine. J. Cell Physiol. 83:103–116CrossRefPubMedGoogle Scholar
  50. Locher K.P., Lee A.T., Rees D.C. 2002. The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science 296:1091–1098CrossRefPubMedGoogle Scholar
  51. Loo T.W., Bartlett M.C., Clarke D.M. 2002. The “LSGGQ” motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence. J. Biol. Chem. 277:41303–41306PubMedGoogle Scholar
  52. Loo T.W., Bartlett M.C., Clarke D.M. 2003a. Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains. J. Biol. Chem. 278:1575–1578Google Scholar
  53. Loo T.W., Bartlett M.C., Clarke D.M. 2003b. Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites. J. Biol. Chem. 278:50136–50141Google Scholar
  54. Loo T.W., Bartlett M.C., Clarke D.M. 2003c. Permanent activation of the human p-glycoprotein by covalent modification of a residue in the drug-binding site. J. Biol. Chem. 278:20449–20452Google Scholar
  55. Loo T.W., Bartlett M.C., Clarke D.M. 2003d. Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein. J. Biol. Chem. 278:39706–39710Google Scholar
  56. Loo T.W., Bartlett M.C., Clarke D.M. 2003e. Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit Mechanism for drug binding. J. Biol. Chem. 278:13603–13606Google Scholar
  57. Loo T.W., Bartlett M.C., Clarke D.M. 2004a. Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane. J. Biol. Chem. 279:7692–7697Google Scholar
  58. Loo T.W., Bartlett M.C., Clarke D.M. 2004b. The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium. Biochemistry 43:12081–12089CrossRefGoogle Scholar
  59. Loo T.W., Bartlett M.C., Clarke D.M. 2004c. Residues V133 and C137 in transmembrane segment 2 are close to residues A935 and G939 in transmembrane segment 11 of human P-glycoprotein. J. Biol. Chem. 279:18232–18238Google Scholar
  60. Loo T.W., Bartlett M.C., Clarke D.M. 2004d. Thapsigargin or curcumin does not promote maturation of processing mutants of the ABC transporters, CFTR, and P-glycoprotein. Biochem. Biophys. Res. Commun. 325:580–585CrossRefGoogle Scholar
  61. Loo T.W., Bartlett M.C., Clarke D.M. 2005. ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein. Biochemistry 44:10250–10258CrossRefPubMedGoogle Scholar
  62. Loo T.W., Clarke D.M. 1993a. Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein. J. Biol. Chem. 268:19965–19972Google Scholar
  63. Loo T.W., Clarke D.M. 1993b. Functional consequences of proline mutations in the predicted transmembrane domain of P-glycoprotein. J. Biol. Chem. 268:3143–3149Google Scholar
  64. Loo T.W., Clarke D.M. 1994a. Functional consequences of glycine mutations in the predicted cytoplasmic loops of P-glycoprotein. J. Biol. Chem. 269:7243–7248Google Scholar
  65. Loo T.W., Clarke D.M. 1994b. Mutations to amino acids located in predicted transmembrane segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein. Biochemistry 33:14049–14057CrossRefGoogle Scholar
  66. Loo T.W., Clarke D.M. 1994c. Prolonged association of temperature-sensitive mutants of human P- glycoprotein with calnexin during biogenesis. J. Biol. Chem. 269:28683–28689Google Scholar
  67. Loo T.W., Clarke D.M. 1994d. Reconstitution of drug-stimulated ATPase activity following co- expression of each half of human P-glycoprotein as separate polypeptides. J. Biol. Chem. 269:7750–7755Google Scholar
  68. Loo T.W., Clarke D.M. 1995a. Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug- stimulated ATPase activity. J. Biol. Chem. 270:22957–22961CrossRefGoogle Scholar
  69. Loo T.W., Clarke D.M. 1995b. Membrane topology of a cysteine-less mutant of human P-glycoprotein. J. Biol. Chem. 270:843–848CrossRefGoogle Scholar
  70. Loo T.W., Clarke D.M. 1996a. Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates. J. Biol. Chem. 271:27482–27487Google Scholar
  71. Loo T.W., Clarke D.M. 1996b. The minimum functional unit of human P-glycoprotein appears to be a monomer. J. Biol. Chem. 271:27488–27492Google Scholar
  72. Loo T.W., Clarke D.M. 1997a. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators. J. Biol. Chem. 272:709–712Google Scholar
  73. Loo T.W., Clarke D.M. 1997b. Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12. J. Biol. Chem. 272:20986–20989Google Scholar
  74. Loo T.W., Clarke D.M. 1997c. Identification of residues in the drug-binding site of human P- glycoprotein using a thiol-reactive substrate. J. Biol. Chem. 272:31945–31948Google Scholar
  75. Loo T.W., Clarke D.M. 1998. Superfolding of the partially unfolded core-glycosylated intermediate of human P-glycoprotein into the mature enzyme is promoted by substrate- induced transmembrane domain interactions. J. Biol. Chem. 273:14671–14674PubMedGoogle Scholar
  76. Loo T.W., Clarke D.M. 1999a. Identification of residues in the drug-binding domain of human P-glycoprotein: Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane. J. Biol. Chem. 274:35388–35392Google Scholar
  77. Loo T.W., Clarke D.M. 1999b. The transmembrane domains of the human multidrug resistance P- glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface. J. Biol. Chem. 274:24759–24765Google Scholar
  78. Loo T.W., Clarke D.M. 2000a. Drug-stimulated ATPase activity of human P-glycoprotein is blocked by disulfide cross-linking between the nucleotide-binding sites. J. Biol. Chem. 275:19435–19438Google Scholar
  79. Loo T.W., Clarke D.M. 2000b. Identification of residues within the drug-binding domain of the human multidrug resistance P-glycoprotein by cysteine-scanning mutagenesis and reaction with dibromobimane. J. Biol. Chem. 275:39272–39278Google Scholar
  80. Loo T.W., Clarke D.M. 2000c. The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis. J. Biol. Chem. 275:5253–5256Google Scholar
  81. Loo T.W., Clarke D.M. 2001a. Cross-linking of human multidrug resistance P-glycoprotein by the substrate, Tris-(2-maleimidoethyl)amine, is altered by ATP hydrolysis: Evidence for rotation of a transmembrane helix. J. Biol. Chem. 276:31800–31805Google Scholar
  82. Loo T.W., Clarke D.M. 2001b. Defining the drug-binding site in the human multidrug resistance P- glycoprotein using MTS-verapamil. J. Biol. Chem. 276:14972–14919Google Scholar
  83. Loo T.W., Clarke D.M. 2001c. Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linkers as molecular rulers. J. Biol. Chem. 276:36877–36880Google Scholar
  84. Loo T.W., Clarke D.M. 2002a. Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein. J. Biol. Chem. 277:44332–44338Google Scholar
  85. Loo T.W., Clarke D.M. 2002b. Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site. Proc. Natl. Acad. Sci. USA 99:3511–3516Google Scholar
  86. Loo T.W., Clarke D.M. 2005. Do drug substrates enter the common drug-binding pocket of P-glycoprotein through “gates”? Biochem. Biophys. Res. Commun. 329:419–422CrossRefPubMedGoogle Scholar
  87. Lugo M.R., Sharom F.J. 2005. Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site. Biochemistry 44:643–655PubMedGoogle Scholar
  88. Martin C., Walker J., Rothnie A., Callaghan R. 2003. The expression of P-glycoprotein does influence the distribution of novel fluorescent compounds in solid tumour models. Br. J. Cancer 89:1581–1589PubMedGoogle Scholar
  89. Mechetner E.B., Schott B., Morse B.S., Stein W.D., Druley T., Davis K.A., Tsuruo T., Roninson I.B. 1997. P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity. Proc.Natl. Acad. Sci. USA 94:12908–12913CrossRefPubMedGoogle Scholar
  90. Miyake K., Mickley L., Litman T., Zhan Z., Robey R., Cristensen B., Brangi M., Greenberger L., Dean M., Fojo T., Bates S.E. 1999. Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes. Cancer Res. 59:8–13PubMedGoogle Scholar
  91. Moody I.E., Millen L., Binns D., Hunt J.F., Thomas P.J. 2002. Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters. J. Biol. Chem. 277:21111–21114CrossRefPubMedGoogle Scholar
  92. Murakami S., Nakashima R., Yamashita E., Yamaguchi A. 2002. Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419:587–593CrossRefPubMedGoogle Scholar
  93. Omote H., Al-Shawi M.K. 2002. A novel electron paramagnetic resonance approach to determine the mechanism of drug transport by P-glycoprotein. J. Biol. Chem. 277:45688–45694CrossRefPubMedGoogle Scholar
  94. Omote H., Figler R.A., Polar M.K., Al-Shawi M.K. 2004. Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation. Biochemistry 43:3917–3928CrossRefPubMedGoogle Scholar
  95. Pascaud C., Garrigos M., Orlowski S. 1998. Multidrug resistance transporter P- glycoprotein has distinct but interacting binding sites for cytotoxic drugs and reversing agents. Biochem. J. 333:351–8PubMedGoogle Scholar
  96. Penzotti J.E., Lamb M.L., Evensen E., Grootenhuis P.D. 2002. A computational ensemble pharmacophore model for identifying substrates of P-glycoprotein. J. Med. Chem. 45:1737–1740CrossRefPubMedGoogle Scholar
  97. Pleban K., Kopp S., Csaszar E., Peer M., Hrebicek T., Rizzi A., Ecker G.F., Chiba P. 2005. P-glycoprotein substrate binding domains are located at the transmembrane domain/transmembrane domain interfaces: a combined photoaffinity labeling-protein homology modeling approach. Mol. Pharmacol. 67:365–374PubMedGoogle Scholar
  98. Poruchynsky M.S., Ling V. 1994. Detection of oligomeric and monomeric forms of P-glycoprotein in multidrug resistant cells. Biochemistry 33:4163–4174PubMedGoogle Scholar
  99. Qu Q., Chu J.W., Sharom F.J. 2003. Transition state P-glycoprotein binds drugs and modulators with unchanged affinity, suggesting a concerted transport mechanism. Biochemistry 42:1345–1353PubMedGoogle Scholar
  100. Qu Q., Sharom F.J. 2001. FRET analysis indicates that the two ATPase active sites of the P- glycoprotein multidrug transporter are closely associated. Biochemistry 40:1413–1422CrossRefPubMedGoogle Scholar
  101. Qu Q., Sharom F.J. 2002. Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet. Biochemistry 41:4744–4752CrossRefPubMedGoogle Scholar
  102. Ramachandra M., Ambudkar S.V., Chen D., Hrycyna C.A., Dey S., Gottesman M.M., Pastan I. 1998. Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state. Biochemistry 37:5010–5019CrossRefPubMedGoogle Scholar
  103. Ramjeesingh M., Li C., Kogan I., Wang Y., Huan L.J., Bear C.E. 2001. A monomer is the minimum functional unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator. Biochemistry 40:10700–10706CrossRefPubMedGoogle Scholar
  104. Raviv Y., Pollard H.B., Bruggemann E.P., Pastan I., Gottesman M.M. 1990. Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells. J. Biol. Chem. 265:3975–3980PubMedGoogle Scholar
  105. Reyes C.L., Chang G. 2005. Structure of the ABC transporter MsbA in complex with ADP-vanadate and lipopolysaccharide. Science 308:1028–1031CrossRefPubMedGoogle Scholar
  106. Riordan J.R., Ling V. 1985. Genetic and biochemical characterization of multidrug resistance. Pharmacol. Ther. 28:51–75CrossRefPubMedGoogle Scholar
  107. Romsicki Y., Sharom F.J. 2001. Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter. Biochemistry 40:6937–6947CrossRefPubMedGoogle Scholar
  108. Rosenberg M.F., Callaghan R., Ford R.C., Higgins C.F. 1997. Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. J. Biol. Chem. 272:10685–10694CrossRefPubMedGoogle Scholar
  109. Rosenberg M.F., Callaghan R., Modok S., Higgins C.F., Ford R.C. 2005. Three-dimensional structure of P-glycoprotein: the transmembrane regions adopt an asymmetric configuration in the nucleotide-bound state. J. Biol. Chem. 280:2857–2862PubMedGoogle Scholar
  110. Rosenberg M.F., Kamis A.B., Callaghan R., Higgins C.F., Ford R.C. 2003. Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding. J. Biol. Chem. 278:8294–8299CrossRefPubMedGoogle Scholar
  111. Rosenberg M.F., Velarde G., Ford R.C., Martin C., Berridge G., Kerr I.D., Callaghan R., Schmidlin A., Wooding C., Linton K.J., Higgins C.F. 2001. Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle. EMBO J. 20:5615–5625CrossRefPubMedGoogle Scholar
  112. Rothnie A., Storm J., Campbell J., Linton K.J., Kerr I.D., Callaghan R. 2004. The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein. J. Biol. Chem. 279:34913–34921CrossRefPubMedGoogle Scholar
  113. Sarkadi B., Price E.M., Boucher R.C., Germann U.A., Scarborough G.A. 1992. Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J. Biol. Chem. 267:4854–4858PubMedGoogle Scholar
  114. Sauna Z.E., Ambudkar S.V. 2000. Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein. Proc. Natl. Acad. Sci. USA 97:2515–2520CrossRefPubMedGoogle Scholar
  115. Sauna Z.E., Andrus M.B., Turner T.M., Ambudkar S.V. 2004. Biochemical basis of polyvalency as a strategy for enhancing the efficacy of P-glycoprotein (ABCB1) modulators: stipiamide homodimers separated with defined-length spacers reverse drug efflux with greater efficacy. Biochemistry 43:2262–2271CrossRefPubMedGoogle Scholar
  116. Sauna Z.E., Muller M., Peng X.H., Ambudkar S.V. 2002. Importance of the conserved Walker B glutamate residues, 556 and 1201, for the completion of the catalytic cycle of ATP hydrolysis by human P-glycoprotein (ABCB1). Biochemistry 41:13989–4000CrossRefPubMedGoogle Scholar
  117. Schinkel 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.G., te Riele H.P., Berns A.J.M., Borst P. 1994. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77:491–502CrossRefPubMedGoogle Scholar
  118. Schumacher M.A., Miller M.C., Grkovic S., Brown M.H., Skurray R.A., Brennan R.G. 2001. Structural mechanisms of QacR induction and multidrug recognition. Science 294:2158–2163CrossRefPubMedGoogle Scholar
  119. Schwab M., Eichelbaum M., Fromm M.F. 2003. Genetic polymorphisms of the human MDR1 drug transporter. Annu. Rev. Pharmacol. Toxicol. 43:285–307CrossRefPubMedGoogle Scholar
  120. See Y.P., Carlsen S.A., Till J.E., Ling V. 1974. Increased drug permeability in Chinese hamster ovary cells in the presence of cyanide. Biochim. Biophys. Acta 373:242–252PubMedGoogle Scholar
  121. Seelig A. 1998. A general pattern for substrate recognition by P-glycoprotein. Eur. J. Biochem. 251:252–261CrossRefPubMedGoogle Scholar
  122. Senior A.E., al-Shawi M.K., Urbatsch I.L. 1995. The catalytic cycle of P-glycoprotein. FEBS Lett. 377:285–289CrossRefPubMedGoogle Scholar
  123. Shapiro A.B., Ling V. 1997. Extraction of Hoechst 33342 from the cytoplasmic leaflet of the plasma membrane by P-glycoprotein. Eur. J. Biochem. 250:122–129PubMedGoogle Scholar
  124. Sharom F.J. 1997. The P-glycoprotein efflux pump: how does it transport drugs? J. Membrane Biol. 160:161–175CrossRefGoogle Scholar
  125. Sharom F.J., Yu X., Doige C.A. 1993. Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein. J. Biol. Chem. 268:24197–24202PubMedGoogle Scholar
  126. Shen D.W., Cardarelli C., Hwang J., Corawell M., Richert N., Ishii S., Pastan I., Gottesman M.M. 1986. Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins. J. Biol. Chem. 261:7762–7770PubMedGoogle Scholar
  127. Smith P.C., Karpowich N., Millen L., Moody I.E., Rosen J., Thomas P.J., Hunt J.F. 2002. ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol. Cell 10:139–149CrossRefPubMedGoogle Scholar
  128. Sonveaux N., Vigano C., Shapiro A.B., Ling V., Ruysschaert J.M. 1999. Ligand-mediated tertiary structure changes of reconstituted P-glycoprotein. A tryptophan fluorescence quenching analysis. J. Biol. Chem. 274:17649–17654CrossRefPubMedGoogle Scholar
  129. Stenham D.R., Campbell J.D., Sansom M.S., Higgins C.F., Kerr I.D., Linton K.J. 2003. An atomic detail model for the human ATP binding cassette transporter P-glycoprotein derived from disulfide cross-linking and homology modeling. FASEB J. 17:2281–2289PubMedGoogle Scholar
  130. Tang-Wai D.F., Kajiji S., DiCapua F., de Graaf D., Roninson I.E., Gros P. 1995. Human (MDR1) and mouse (mdr1, mdr3) P-glycoproteins can be distinguished by their respective drug resistance profiles and sensitivity to modulators. Biochemistry 34:32–39CrossRefPubMedGoogle Scholar
  131. Thiebaut F., Tsuruo T., Hamada H., Gottesman M.M., Pastan I., Willingham M.C. 1987. Cellular localization of the multidrug-resistance gene product P- glycoprotein in normal human tissues. Proc. Natl. Acad. Sci. USA 84:7735–7738PubMedGoogle Scholar
  132. Tombline G., Bartholomew L., Gimi K., Tyndall G.A., Senior A.E. 2004a. Synergy between conserved ABC signature Ser residues in P-glycoprotein catalysis. J. Biol. Chem. 279:5363–5373Google Scholar
  133. Tombline G., Bartholomew L.A., Tyndall G.A., Gimi K., Urbatsch I.L., Senior A.E. 2004b. Properties of P-glycoprotein with mutations in the “catalytic carboxylate” glutamate residues. J. Biol. Chem. 279:46518–46526Google Scholar
  134. Tombline G., Bartholomew L.A., Urbatsch I.L., Senior A.E. 2004c. Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly. J. Biol. Chem 279:31212–31220Google Scholar
  135. Tran T.T., Mittal A., Aldinger T., Polli J.W., Ayrton A., Ellens H., Bentz J. 2005. The elementary mass action rate constants of P-gp transport for a confluent monolayer of MDCKII-hMDR1 cells. Biophys. J. 88:715–738PubMedGoogle Scholar
  136. Urbatsch I.L., al-Shawi M.K., Senior A.E. 1994. Characterization of the ATPase activity of purified Chinese hamster P- glycoprotein. Biochemistry 33:7069–7076CrossRefPubMedGoogle Scholar
  137. Urbatsch I.L., Gimi K., Wilke-Mounts S., Lerner-Marmarosh N., Rousseau M.E., Gros P., Senior A.E. 2001. Cysteines 431 and 1074 are responsible for inhibitory disulfide cross- linking between the two nucleotide-binding sites in human P- glycoprotein. J. Biol. Chem. 276:26980–26987CrossRefPubMedGoogle Scholar
  138. Urbatsch I.L., Sankaran B., Bhagat S., Senior A.E. 1995a. Both P-glycoprotein nucleotide-binding sites are catalytically active. J. Biol. Chem. 270:26956–26961Google Scholar
  139. Urbatsch I.L., Sankaran B., Weber J., Senior A.E. 1995b. P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site. J. Biol. Chem. 270:19383–19390Google Scholar
  140. van der Kolk D.M., de Vries E.G., van Putten W.J., Verdonck L.F., Ossenkoppele G.J., Verhoef G.E., Vellenga E. 2000. P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia. Clin. Cancer Res. 6:3205–3214PubMedGoogle Scholar
  141. Vigano C., Julien M., Carrier I., Gros P., Ruysschaert J.M. 2002. Structural and functional asymmetry of the nucleotide-binding domains of P-glycoprotein investigated by attenuated total reflection Fourier transform infrared spectroscopy. J. Biol. Chem. 277:5008–5016CrossRefPubMedGoogle Scholar
  142. Wang R.B., Kuo C.L., Lien L.L., Lien E.J. 2003. Structure-activity relationship: analyses of p-glycoprotein substrates and inhibitors. J. Clin. Pharm. Ther. 28:203–228CrossRefPubMedGoogle Scholar
  143. Watkins R.E., Wisely G.B., Moore L.B., Collins J.L., Lambert M.H., Williams S.P., Willson T.M., Kliewer S.A., Redinbo M.R. 2001. The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science 292:2329–33CrossRefPubMedGoogle Scholar
  144. Wiese M., Pajeva I.K. 2001. Structure-activity relationships of multidrug resistance reversers. Curr. Med. Chem. 8:685–713PubMedGoogle Scholar
  145. Yu E.W., McDermott G., Zgurskaya H.I., Nikaido H., Koshland D.E., Jr. 2003. Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump. Science 300:976–980PubMedGoogle Scholar
  146. Yuan Y.R., Blecker S., Martsinkevich O., Millen L., Thomas P.J., Hunt J.F. 2001. The crystal structure of the MJ0796 ATP-binding cassette. Implications for the structural consequences of ATP hydrolysis in the active site of an ABC transporter. J. Biol. Chem. 276:32313–32321PubMedGoogle Scholar
  147. Zhang Z.R., Cui G., Liu X., Song B., Dawson D.C., McCarty N.A. 2005 Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel. One polypeptide forms one pore. J. Biol. Chem. 280:458–468PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  1. 1.Department of Medicine and Department of BiochemistryUniversity of TorontoTorontoCanada

Personalised recommendations