Identification of Residues within the Drug-binding Domain of the Human Multidrug Resistance P-glycoprotein by Cysteine-scanning Mutagenesis and Reaction with Dibromobimane

P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds that have diverse structures. Therefore, the drug-binding domain of the human multidrug resistance P-gp likely consists of residues from multiple transmembrane (TM) segments. In this study, we completed cysteine-scanning mutagenesis of all the predicted TM segments of P-gp (TMs 1–5 and 7–10) and tested for inhibition by a thiol-reactive substrate (dibromobimane) to identify residues within the drug-binding domain. The activities of 189 mutants were analyzed. Verapamil-stimulated ATPase activities of seven mutants (Y118C and V125C (TM2), S222C (TM4), I306C (TM5), S766C (TM9), and I868C and G872C (TM10)) were inhibited by more than 50% by dibromobimane. The activities of mutants S222C (TM4), I306C (TM5), I868C (TM10), and G872C (TM10), but not that of mutants Y118C (TM2), V125C (TM2), and S776C (TM9), were protected from inhibition by dibromobimane by pretreatment with verapamil, vinblastine, or colchicine. These results and those from previous studies (Loo, T. W. and Clarke, D. M. (1997)J. Biol. Chem. 272, 31945–31948; Loo, T. W. and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388–35392) indicate that the drug-binding domain of P-gp consists of residues in TMs 4, 5, 6, 10, 11, and 12. P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds that have diverse structures. Therefore, the drug-binding domain of the human multidrug resistance P-gp likely consists of residues from multiple transmembrane (TM) segments. In this study, we completed cysteine-scanning mutagenesis of all the predicted TM segments of P-gp (TMs 1–5 and 7–10) and tested for inhibition by a thiol-reactive substrate (dibromobimane) to identify residues within the drug-binding domain. The activities of 189 mutants were analyzed. Verapamil-stimulated ATPase activities of seven mutants (Y118C and V125C (TM2), S222C (TM4), I306C (TM5), S766C (TM9), and I868C and G872C (TM10)) were inhibited by more than 50% by dibromobimane. The activities of mutants S222C (TM4), I306C (TM5), I868C (TM10), and G872C (TM10), but not that of mutants Y118C (TM2), V125C (TM2), and S776C (TM9), were protected from inhibition by dibromobimane by pretreatment with verapamil, vinblastine, or colchicine. These results and those from previous studies (Loo, T. W. and Clarke, D. M. (1997)J. Biol. Chem. 272, 31945–31948; Loo, T. W. and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388–35392) indicate that the drug-binding domain of P-gp consists of residues in TMs 4, 5, 6, 10, 11, and 12. dibromobimane P-glycoprotein transmembrane The human multidrug resistance P-glycoprotein (P-gp)1 is located in the plasma membrane and uses ATP to pump a wide variety of structurally diverse cytotoxic compounds out of the cell (1Sharom F.J. J. Membr. 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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, 6Schinkel A.H. Semin. Cancer Biol. 1997; 8: 161-170Crossref PubMed Scopus (443) Google Scholar). The protective role of P-gp contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy because many of the therapeutic compounds are also substrates of P-gp (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, 8Lee 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, 9Robert J. Eur. J. Clin. Invest. 1999; 29: 536-545Crossref PubMed Scopus (71) Google Scholar). P-gp is a member of the ATP-binding cassette family of transporters (10Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3386) Google Scholar, 11Holland I.B. Blight M.A. J. Mol. Biol. 1999; 293: 381-399Crossref PubMed Scopus (489) Google Scholar), and its 1280 amino acids are organized as two repeating units joined by a linker region of about 60 amino acids (12Chen 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 (1721) Google Scholar). Each repeat consists of an NH2-terminal hydrophobic domain containing six transmembrane (TM) segments followed by a hydrophilic domain containing an ATP-binding site (13Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 14Kast 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 exact mechanism of how P-gp functions is unknown. It is known, however, that both halves of the molecule are essential for activity (15Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar) and that both nucleotide-binding domains can bind and hydrolyze ATP and are essential for function (15Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 16Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar, 17Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 18Urbatsch 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, 19Hrycyna 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 (123) Google Scholar). The drug-binding domain is located in the TM domains of P-gp because drug substrates will bind to a deletion mutant lacking both nucleotide-binding domains (20Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). An important step in understanding the mechanism of P-gp is to determine the residues in the drug-binding domain. A common method for identifying residues in a membrane transporter that are critical for substrate binding and/or transport is to use alanine-scanning mutagenesis. This has been used successfully for transporters such as bacteriorhodopsin (21Subramaniam S. Curr. Opin. Struct. Biol. 1999; 9: 462-468Crossref PubMed Scopus (43) Google Scholar) and the SERCA1 calcium pump (22Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (471) Google Scholar). Recent crystal structures of these two transporters showed that the amino acids involved in ligand binding are in agreement with those identified through mutational analyses (23Luecke H. Schobert B. Richter H.T. Cartailler J.P. Lanyi J.K. J. Mol. Biol. 1999; 291: 899-911Crossref PubMed Scopus (1310) Google Scholar, 24Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar). The residues in P-gp that are involved in drug binding, however, have been difficult to characterize because a large number of mutations throughout the molecule can alter the substrate specificity (25Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 27Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7243-7248Abstract Full Text PDF PubMed Google Scholar, 28Taguchi Y. Morishima M. Komano T. Ueda K. FEBS Lett. 1997; 413: 142-146Crossref PubMed Scopus (25) Google Scholar, 29Beaudet L. Gros P. J. Biol. Chem. 1995; 270: 17159-17170Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 30Hanna M. Brault M. Kwan T. Kast C. Gros P. Biochemistry. 1996; 35: 3625-3635Crossref PubMed Scopus (55) Google Scholar, 31Kwan T. Gros P. Biochemistry. 1998; 37: 3337-3350Crossref PubMed Scopus (42) Google Scholar). It has been difficult to determine whether mutations that affected activity were actually close to the drug-binding site or whether they affected the global structure of the protein (32Ramachandra M. Ambudkar S.V. Gottesman M.M. Pastan I. Hrycyna C.A. Mol. Biol. Cell. 1996; 7: 1485-1498Crossref PubMed Scopus (68) Google Scholar). To avoid these difficulties, we used a direct assay involving cysteine-scanning mutagenesis and modification with a thiol-reactive substrate to identify residues in the TM segments that are critical for drug binding. The rationale is that the thiol-reactive substrate, dibromobimane (dBBn), will enter the drug-binding site of P-gp, covalently label any adjacent cysteine residue, and inhibit activity. Inhibition by dBBn should be preventable by pretreatment with other substrates such as verapamil, colchicine, and vinblastine if the reactive residue is in the drug-binding domain. We have used this method and identified the residues important for drug binding in TMs 6, 11, and 12 (33, 34). In this study, we identify important residues in the remaining TM segments (TMs 1–5 and 7–10) that are important for activity. Wild-type P-gp has cysteine residues at positions 137, 431, 717, 956, 1074, 1125, and 1227. None of these cysteines are important for activity because mutation of all cysteines to alanine (Cys-less P-gp) resulted in an active molecule (13Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). The Cys-less P-gp cDNA to for residues at the of the molecule (Cys-less This of the Cys-less P-gp by T.W. Clarke D.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). residues were the Cys-less as T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the cDNA by the cDNA F. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Expression and of P-gp mutants were as T.W. Clarke D.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). of cells were with the mutant the with containing A. P-gp in the of A because is a drug substrate that of the protein T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The cells were and with and the mutant P-gp by The mutants were from the with containing and and with an of brain that and in and The for at An of the for ATPase activity by of an of containing and the drug substrates verapamil, vinblastine, or The were for at and the of by the method of S. A. R. S. PubMed Scopus Google Scholar). inhibition with the with dBBn for at The by the of to a of ATPase activity as In the the were with vinblastine, or for at the of dBBn of dBBn or These were substrate for of ATPase is for by dBBn T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The were with or dBBn for at and the by the of ATPase activity with the drug used for were verapamil, vinblastine, or The P-gp to a of and with a P-gp, followed by T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). is that the drug-binding site is likely to be within the TM segments (20Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, Y. Pastan I. Gottesman M.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. Pastan I. Gottesman M.M. B. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, A. H. P. T. M. R. B. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (42) Google Scholar, G. J.P. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The amino acids predicted to be in TM segments are in We that residues within TMs 6, 11, and 12 were important for drug binding T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In this study, we used cysteine-scanning mutagenesis of the remaining TM segments and inhibition by dBBn to identify residues that to the drug-binding domain. residue in TMs 4, 5, and to and we whether the mutant protein The mutant were in by and for ATPase activity. We as the substrate because the of the ATPase activity of Cys-less P-gp T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). ATPase activity has been to with transport because the are S.V. Cardarelli C.O. I. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the ATPase activity of mutant with dibromobimane. A of 189 mutants were analyzed. (TM2), and (TM10), were not because of expression or activity. The mutants and to be in and were not with Cys-less of cell of cells these mutants showed of the of P-gp not Expression of these mutants in the of A of the protein to the P-gp, but the were to of ATPase activity. The remaining P-gp mutants were as as Cys-less of these mutants and than of the ATPase activity of Cys-less (TM2), and and of the ATPase activity of Cys-less It is that mutants and positions in of We that mutation of in the substrate specificity of P-gp (25Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google of ATPase activity by mutants were by with and for at with or The by the of and the ATPase activity The results of are to that of a The activity of mutant to Cys-less is in the Each is the of two The from the is the activities of mutants in TMs 4, 5, and 10, not because of The mutants that expression to that of Cys-less than of the ATPase activity of Cys-less were tested for inhibition by is a substrate of P-gp, and its with a cysteine within the drug-binding site be to in the inhibition of ATPase activity. The mutant were by with and for at with The by the of cysteine to the of dBBn and to a that a substrate of P-gp T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The ATPase activities of the were and with that of The results for TM are in In the active mutants more than of ATPase activity with mutants in were inhibited by Y118C and V125C were inhibited and by The results for were to those in in that all of the mutants more than of activity with In the activity of inhibited by to In were in activity for mutants and the remaining mutants more than of activity with mutant I306C of the ATPase activity of Cys-less its ATPase activity with dBBn not ATPase activity also to with the TM in the of P-gp, showed results to those for in that all the mutants were to inhibition by dBBn than of the pattern of inhibition in mutants to that with mutants the residues at the and resulted in in the ATPase activities and the residues at the V125C and S766C resulted in mutants that were to inhibition by dBBn and The mutants in were relatively to inhibition by with the and about I868C and G872C in were to inhibition by activities were inhibited by and The of dBBn to 50% inhibition of ATPase activity for mutants Y118C (TM2), V125C (TM2), S222C (TM4), S766C I868C (TM10), and G872C were and of cells these mutants showed that the expression to that of Cys-less P-gp In all the the the results identified residues within the predicted TM segments that were to inhibition by that these cysteines were to dBBn and not be close to the drug-binding To that inhibition to inhibition of a residue in the drug-binding important to that the of a substrate protect the mutant P-gp from inhibition by verapamil, vinblastine, and colchicine, were for the because used in the inhibition and showed the of activity. and were also because they are structurally from and have been used to mutants of P-gp (25Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 27Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7243-7248Abstract Full Text PDF PubMed Google Scholar, T.W. Clarke D.M. Biochemistry. 1994; PubMed Scopus Google Scholar). We have used and vinblastine to characterize the residues in TMs 6, 11, and 12 that were to inhibition by dBBn T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). mutants that were inhibited by Y118C (TM2), V125C (TM2), (TM4), S766C I868C (TM10), and G872C (TM10), were tested for to be protected from inhibition by dBBn in the of I306C also because its ATPase activity with dBBn not A that dBBn also inhibited the and ATPase activities of the with the of mutant inhibited the ATPase activity of the and activities were and To for by drug the mutants were with vinblastine or and with dBBn for at and the with The mutants and that were more to of dBBn were with the and were with ATPase activity with the drug used for The results are in B. by substrate for mutant S222C The mutant protected from by dBBn in the of vinblastine protected mutant G872C from by dBBn because more than of the activity G872C protected by verapamil, substrates also showed of mutant I868C from dBBn inhibition by vinblastine for mutant or by any of the drug substrates for mutants Y118C (TM2), V125C (TM2), and S766C We were not to determine whether the ATPase activity of mutants or S766C inhibited dBBn because the ATPase activities were To whether in membrane affected the drug of the the were at The were with verapamil, vinblastine, or for at and with dBBn for at and the ATPase activity at The results were to those in not We drug-binding by inhibition of ATPase activity of P-gp mutants containing a Cys-less P-gp is a because the mutant resistance to a wide variety of cytotoxic substrates (13Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), and about of the ATPase activity of T.W. Clarke D.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). A of this is that the assay inhibition of ATPase activity and not drug transport activity. ATPase activity to transport activity because the number for ATPase activity with vinblastine transport out of the cell S.V. Cardarelli C.O. I. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). This that residues S222C (TM4), I306C (TM5), I868C (TM10), and G872C be important for drug binding. We have that residues and in and in and and in are important for drug binding T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In to how residues that are from other the drug-binding we have the residues in TM as In 5, we have the residues that are to inhibition by dBBn such that they the of the It is to that the residues also on of the also the results from studies that that TMs and are close to T.W. Clarke D.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). and were protected from inhibition by dBBn by verapamil, vinblastine, and colchicine. This that and be common to the binding of all In residue (TM10), on the as (TM10), protected by and vinblastine but not by and be involved in the binding of and be important for drug binding because of the of dBBn on the ATPase activity of mutant inhibited the activity of this but not its or activity. also protected the mutant from by of the drug-binding domain. in and activities of residues I306C with dBBn also be an that is close to or within the drug-binding It is also that modification of a residue within or close to the drug-binding domain to inhibition or of ATPase activity. and on of in the and be to residues in T.W. Clarke D.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of to residues in inhibited by vinblastine and by and colchicine. of TMs 4, 5, and as of the drug-binding domain of P-gp is with the results from mutation and studies with of of at positions in and large in the substrate specificity of the protein (25Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar). The mutants and in of T. Gros P. Biochemistry. 1998; 37: 3337-3350Crossref PubMed Scopus (42) Google Scholar) and T. S. Dey S. Pastan I. Gottesman M.M. Mol. Pharmacol. 1998; Google Scholar) and or in of human P-gp also the substrate specificity of the transporter T.W. Clarke D.M. 1998; PubMed Scopus Google Scholar). The of human P-gp is It is also an ATP-binding cassette and many of the substrates of P-gp are also substrates of E. M. S. L. A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). mutagenesis studies on showed that residue in predicted important for with R. A. D. K. Mol. Biol. Cell. 1998; 9: PubMed Scopus (134) Google Scholar). This residue with residue of human P-gp and the drug-binding domain in TMs and are also by of A. H. P. T. M. R. B. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and Cardarelli C. Gottesman M.M. Pastan I. Mol. Pharmacol. 1994; Google Scholar). also inhibited P-gp by with cysteines in (Y118C and and The drug substrates verapamil, vinblastine, and not protect these mutants from inhibition by It is that of these residues not within the drug-binding site for these compounds because relatively high of dBBn were to inhibit 50% of the activity of mutants Y118C and V125C and is that modification of mutant or S766C an essential during of drug binding to ATPase activity. close to a residue, T.W. Clarke D.M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). It is also that these residues within drug-binding site or The results from studies that P-gp to drug S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, C. M. S. J. 1998; PubMed Scopus Google Scholar, K. P. V. Eur. J. 1999; PubMed Scopus Google Scholar). In of the substrates two drug-binding during transport S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). In other substrates with binding K. P. V. Eur. J. 1999; PubMed Scopus Google Scholar). In both ATP to drug It has been that two to of ATP are for molecule of vinblastine S.V. Cardarelli C.O. I. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of how P-gp can transport such a large variety of structurally compounds is to that residues in the TMs to the binding of a This be because of the of the TM segments. have that the TMs are at because results indicate that residues in can residues in TMs 10, 11, and residues in can residues in 4, 5, and T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). It is that binding of a an binding site by the of the TMs and residues to the binding of a an binding site has also been for the that can bind a wide variety of compounds Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). This is also with the that structurally diverse drug substrates can of mutants that have mutations in all of the molecule T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The drug substrates can this by of the transmembrane domains T.W. Clarke D.M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The to mutants with substrates that is at site or site that can be by This is by the that residues and T.W. Clarke D.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google and T.W. Clarke D.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) can be protected from inhibition by dBBn with verapamil, vinblastine, and colchicine. with other substrates and compounds that are not by P-gp will be to whether P-gp drug-binding site with specificity or multiple with We for We for with

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