Defining the Drug-binding Site in the Human Multidrug Resistance P-glycoprotein Using a Methanethiosulfonate Analog of Verapamil, MTS-verapamil

Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the drug-binding domain of P-gp. MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a Km value (25 μm) that was similar to that of verapamil. 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil. Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the binding of verapamil. Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the drug-binding domain of P-gp. MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a Km value (25 μm) that was similar to that of verapamil. 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil. Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the binding of verapamil. P-glycoprotein transmembrane dibromobimane methanethiosulfonate ATP-binding cassette The human multidrug resistance P-glycoprotein (P-gp)1 uses ATP to pump a wide variety of cytotoxic compounds out of the cell (1Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (406) Google Scholar, 2Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1907) Google Scholar). Overexpression 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 (3Kim 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 (1028) Google Scholar, 4Lee 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, 5Robert J. Eur. J. Clin. Invest. 1999; 29: 536-545Crossref PubMed Scopus (71) Google Scholar).Although P-gp is normally expressed in many tissues, its physiological function is unknown. The pattern of P-gp expression in tissues and studies on P-gp “knock-out” mice indicate that it may protect the organism from toxic compounds in our diet (6Thiebaut 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 (2545) Google Scholar, 7Cordon-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 (1584) Google Scholar, 8Schinkel 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 (2051) Google Scholar).P-gp is a member of the ABC (ATP-binding cassette) family of transporters (9Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 10Holland I.B. Blight M.A. J. Mol. Biol. 1999; 293: 381-399Crossref PubMed Scopus (487) Google Scholar). Its 1280 amino acids are organized as two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (11Chen 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 (1709) Google Scholar). There are six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site in each repeat (12Loo T.W. Clarke D.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, C. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of how P-gp is P-gp and as a for the of ABC is that of P-gp are for activity T.W. Clarke D.M. J. Biol. Chem. 1994; Full Text PDF PubMed Google and that can and ATP and are for function T.W. Clarke D.M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, M. E. P. Mol. Cell. Biol. 1989; PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, B. S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. Ramachandra M. Ambudkar S.V. Pastan I. Gottesman M.M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google the of it is to the of residues that contribute to the drug-binding that the transmembrane may for binding were from studies and from studies with substrate Gottesman M.M. Pastan I. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, Cardarelli C. Gottesman M.M. Pastan I. Mol. Pharmacol. 1994; Google Scholar, J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. F. K. M. P. Biochemistry. PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was that the were for because a substrates T.W. Clarke D.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google to identify the residues that the drug-binding within the involved the of cysteine-scanning mutagenesis and with a reactive dibromobimane residues in TMs 4, 6, 10, 11, and 12 with that TMs contribute to the binding of substrate 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; Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google is P-gp many binding Eur. J. 1997; PubMed Scopus Google Scholar, K. P. Eur. J. 1999; PubMed Scopus Google Scholar, C. M. S. J. 1998; PubMed Scopus Google Scholar, S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, C. C.F. P. P. Mol. Pharmacol. PubMed Scopus Google Scholar). is to substrates of P-gp with the residues in the predicted drug-binding is a substrate used in P-gp. The of verapamil is that it the to the ATPase activity of P-gp. of a thiol-reactive of verapamil a for the drug-binding domain of because reactive can protected with verapamil. a thiol-reactive methanethiosulfonate analog of verapamil (MTS-verapamil) was synthesized and used to identify residues in the drug-binding domain of mutagenesis and with was used to the M. A. 1992; PubMed Scopus Google Scholar). of used to the and function of many in S. M. J. J. 1998; PubMed Scopus Google Scholar). used in the of the M. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). P-gp a for cysteine-scanning mutagenesis because the Cys-less P-gp in T.W. Clarke D.M. 1999; PubMed Scopus Google Scholar). that insight into the and of P-gp are because of the to thiol-reactive of P-gp substrates as of the mutants for inhibition by MTS-verapamil inhibition by MTS-verapamil. the reactive mutants in TMs 4, 6, 10, 11, and 12 had in TMs and may indicate that TMs 4, 6, 10, 11, and 12 are within the drug-binding domain that region of P-gp is to MTS-verapamil. significantly protected residues S222C (TM4), (TM6), (TM6), and from by MTS-verapamil. that residues are to the the drug-binding domain of we a in the residues in each segments are in The of the segments are on the results of studies that is to TMs 10, 11, and 12 and that is to TMs 4, 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. Full Text Full Text PDF PubMed Scopus Google Scholar). ABC transmembrane it that a may and J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). TMs 4, 6, 10, 11, and 12 to the drug-binding because residues in segments is by the of The of the drug-binding domain within the TMs with the that P-gp substrates that are in the Pastan I. Gottesman M.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. Pastan I. Gottesman M.M. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is that substrates into the and are from the by inhibition of P-gp mutants by by protected from to from because of protected from to from in a with thiol-reactive also indicate that TMs 4, 6, 10, 11, and 12 are to the drug-binding domain T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the inhibition by and MTS-verapamil is in I. There is in the cysteine residues that can with dibromobimane MTS-verapamil. S222C and L339C are inhibited by and MTS-verapamil. residues were protected from inhibition by the of verapamil. residues Y118C (TM2), V125C (TM2), and V982C were inhibited by and MTS-verapamil were protected from inhibition by verapamil. The in inhibition by and MTS-verapamil may we that is a single drug-binding domain in P-gp that is to structurally diverse of substrate binding to P-gp is with that for the that can also a wide variety of The of the of a single drug-binding Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). studies that in P-gp are T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). residues in the TMs residues in the and The of the segments P-gp to compounds of diverse substrate a in the segments that residues in the TMs contribute to its it is that substrates residues to its may the with the binding of and MTS-verapamil. many of residues in the drug-binding domain that can contribute to binding of substrates may P-gp can a wide range of structurally diverse may also P-gp a for each substrate and may for the of drug-binding Eur. J. 1997; PubMed Scopus Google Scholar, K. P. Eur. J. 1999; PubMed Scopus Google Scholar, C. M. S. J. 1998; PubMed Scopus Google Scholar, S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, C. C.F. P. P. Mol. Pharmacol. PubMed Scopus Google Scholar). may also how the segments can the of P-gp for a substrate P. J. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T.W. Clarke D.M. Biochemistry. 1994; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, J.P. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Gottesman M.M. Pastan I. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). to residues in the TMs may the of the that are the drug-binding site that residues are for the of P-gp for a a for studies on the of P-gp. P-gp substrates and can used to of P-gp by and the results is in the binding also to of P-gp by MTS-verapamil. P-gp activity is to its F.J. PubMed Scopus Google Scholar, 1995; PubMed Scopus Google may for and are of Mol. Pharmacol. Google Scholar, S. F. S. D. J. S. J. 1997; Full Text PDF PubMed Scopus Google Scholar, M.R. T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google and H. S. J. Pharmacol. 1999; PubMed Scopus Google Scholar). of the by verapamil is to by of the MTS-verapamil may for the region of The human multidrug resistance P-glycoprotein (P-gp)1 uses ATP to pump a wide variety of cytotoxic compounds out of the cell (1Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (406) Google Scholar, 2Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1907) Google Scholar). Overexpression 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 (3Kim 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 (1028) Google Scholar, 4Lee 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, 5Robert J. Eur. J. Clin. Invest. 1999; 29: 536-545Crossref PubMed Scopus (71) Google Scholar). P-gp is normally expressed in many tissues, its physiological function is unknown. The pattern of P-gp expression in tissues and studies on P-gp “knock-out” mice indicate that it may protect the organism from toxic compounds in our diet (6Thiebaut 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 (2545) Google Scholar, 7Cordon-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 (1584) Google Scholar, 8Schinkel 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 (2051) Google Scholar). P-gp is a member of the ABC (ATP-binding cassette) family of transporters (9Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 10Holland I.B. Blight M.A. J. Mol. Biol. 1999; 293: 381-399Crossref PubMed Scopus (487) Google Scholar). Its 1280 amino acids are organized as two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (11Chen 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 (1709) Google Scholar). There are six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site in each repeat (12Loo T.W. Clarke D.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, C. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of how P-gp is P-gp and as a for the of ABC is that of P-gp are for activity T.W. Clarke D.M. J. Biol. Chem. 1994; Full Text PDF PubMed Google and that can and ATP and are for function T.W. Clarke D.M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, M. E. P. Mol. Cell. Biol. 1989; PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, B. S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. Ramachandra M. Ambudkar S.V. Pastan I. Gottesman M.M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the of it is to the of residues that contribute to the drug-binding that the transmembrane may for binding were from studies and from studies with substrate Gottesman M.M. Pastan I. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, Cardarelli C. Gottesman M.M. Pastan I. Mol. Pharmacol. 1994; Google Scholar, J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. F. K. M. P. Biochemistry. PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was that the were for because a substrates T.W. Clarke D.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). to identify the residues that the drug-binding within the involved the of cysteine-scanning mutagenesis and with a reactive dibromobimane residues in TMs 4, 6, 10, 11, and 12 with that TMs contribute to the binding of substrate 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; Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is P-gp many binding Eur. J. 1997; PubMed Scopus Google Scholar, K. P. Eur. J. 1999; PubMed Scopus Google Scholar, C. M. S. J. 1998; PubMed Scopus Google Scholar, S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, C. C.F. P. P. Mol. Pharmacol. PubMed Scopus Google Scholar). is to substrates of P-gp with the residues in the predicted drug-binding is a substrate used in P-gp. The of verapamil is that it the to the ATPase activity of P-gp. of a thiol-reactive of verapamil a for the drug-binding domain of because reactive can protected with verapamil. a thiol-reactive methanethiosulfonate analog of verapamil (MTS-verapamil) was synthesized and used to identify residues in the drug-binding domain of P-gp. mutagenesis and with was used to the M. A. 1992; PubMed Scopus Google Scholar). of used to the and function of many in S. M. J. J. 1998; PubMed Scopus Google Scholar). used in the of the M. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). P-gp a for cysteine-scanning mutagenesis because the Cys-less P-gp in T.W. Clarke D.M. 1999; PubMed Scopus Google Scholar). that insight into the and of P-gp are because of the to thiol-reactive of P-gp substrates as of the mutants for inhibition by MTS-verapamil inhibition by MTS-verapamil. the reactive mutants in TMs 4, 6, 10, 11, and 12 had in TMs and may indicate that TMs 4, 6, 10, 11, and 12 are within the drug-binding domain that region of P-gp is to MTS-verapamil. significantly protected residues S222C (TM4), (TM6), (TM6), and from by MTS-verapamil. that residues are to the the drug-binding domain of we a in the residues in each segments are in The of the segments are on the results of studies that is to TMs 10, 11, and 12 and that is to TMs 4, 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. Full Text Full Text PDF PubMed Scopus Google Scholar). ABC transmembrane it that a may and J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). TMs 4, 6, 10, 11, and 12 to the drug-binding because residues in segments is by the of The of the drug-binding domain within the TMs with the that P-gp substrates that are in the Pastan I. Gottesman M.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. Pastan I. Gottesman M.M. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is that substrates into the and are from the by inhibition of P-gp mutants by by protected from to from because of protected from to from in a with thiol-reactive also indicate that TMs 4, 6, 10, 11, and 12 are to the drug-binding domain T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the inhibition by and MTS-verapamil is in I. There is in the cysteine residues that can with dibromobimane MTS-verapamil. S222C and L339C are inhibited by and MTS-verapamil. residues were protected from inhibition by the of verapamil. residues Y118C (TM2), V125C (TM2), and V982C were inhibited by and MTS-verapamil were protected from inhibition by verapamil. The in inhibition by and MTS-verapamil may we that is a single drug-binding domain in P-gp that is to structurally diverse of substrate binding to P-gp is with that for the that can also a wide variety of The of the of a single drug-binding Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). studies that in P-gp are T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). residues in the TMs residues in the and The of the segments P-gp to compounds of diverse substrate a in the segments that residues in the TMs contribute to its it is that substrates residues to its may the with the binding of and MTS-verapamil. many of residues in the drug-binding domain that can contribute to binding of substrates may P-gp can a wide range of structurally diverse may also P-gp a for each substrate and may for the of drug-binding Eur. J. 1997; PubMed Scopus Google Scholar, K. P. Eur. J. 1999; PubMed Scopus Google Scholar, C. M. S. J. 1998; PubMed Scopus Google Scholar, S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, C. C.F. P. P. Mol. Pharmacol. PubMed Scopus Google Scholar). may also how the segments can the of P-gp for a substrate P. J. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T.W. Clarke D.M. Biochemistry. 1994; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, J.P. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Gottesman M.M. Pastan I. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). to residues in the TMs may the of the that are the drug-binding site that residues are for the of P-gp for a a for studies on the of P-gp. P-gp substrates and can used to of P-gp by and the results is in the binding also to of P-gp by MTS-verapamil. P-gp activity is to its F.J. PubMed Scopus Google Scholar, 1995; PubMed Scopus Google may for and are of Mol. Pharmacol. Google Scholar, S. F. S. D. J. S. J. 1997; Full Text PDF PubMed Scopus Google Scholar, M.R. T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google and H. S. J. Pharmacol. 1999; PubMed Scopus Google Scholar). of the by verapamil is to by of the MTS-verapamil may for the region of mutagenesis and with was used to the M. A. 1992; PubMed Scopus Google Scholar). of used to the and function of many in S. M. J. J. 1998; PubMed Scopus Google Scholar). used in the of the M. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). P-gp a for cysteine-scanning mutagenesis because the Cys-less P-gp in T.W. Clarke D.M. 1999; PubMed Scopus Google Scholar). that insight into the and of P-gp are because of the to thiol-reactive of P-gp substrates as MTS-verapamil. of the mutants for inhibition by MTS-verapamil inhibition by MTS-verapamil. the reactive mutants in TMs 4, 6, 10, 11, and 12 had in TMs and may indicate that TMs 4, 6, 10, 11, and 12 are within the drug-binding domain that region of P-gp is to MTS-verapamil. significantly protected residues S222C (TM4), (TM6), (TM6), and from by MTS-verapamil. that residues are to the the drug-binding domain of we a in the residues in each segments are in The of the segments are on the results of studies that is to TMs 10, 11, and 12 and that is to TMs 4, 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. Full Text Full Text PDF PubMed Scopus Google Scholar). ABC transmembrane it that a may and J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). TMs 4, 6, 10, 11, and 12 to the drug-binding because residues in segments is by the of The of the drug-binding domain within the TMs with the that P-gp substrates that are in the Pastan I. Gottesman M.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. Pastan I. Gottesman M.M. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is that substrates into the and are from the by P-gp. because of with thiol-reactive also indicate that TMs 4, 6, 10, 11, and 12 are to the drug-binding domain T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the inhibition by and MTS-verapamil is in I. There is in the cysteine residues that can with dibromobimane MTS-verapamil. S222C and L339C are inhibited by and MTS-verapamil. residues were protected from inhibition by the of verapamil. residues Y118C (TM2), V125C (TM2), and V982C were inhibited by and MTS-verapamil were protected from inhibition by verapamil. The in inhibition by and MTS-verapamil may we that is a single drug-binding domain in P-gp that is to structurally diverse of substrate binding to P-gp is with that for the that can also a wide variety of The of the of a single drug-binding Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). studies that in P-gp are T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.W. Clarke D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). residues in the TMs residues in the and The of the segments P-gp to compounds of diverse substrate a in the segments that residues in the TMs contribute to its it is that substrates residues to its may the with the binding of and MTS-verapamil. many of residues in the drug-binding domain that can contribute to binding of substrates may P-gp can a wide range of structurally diverse may also P-gp a for each substrate and may for the of drug-binding Eur. 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