Permanent Activation of the Human P-glycoprotein by Covalent Modification of a Residue in the Drug-binding Site

The human multidrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling of drug binding in the transmembrane domains with ATP hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography. Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels. Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site. Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites. This results in the permanent activation of ATP hydrolysis in the absence of transport. Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the drug-binding site is occupied. The human multidrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling of drug binding in the transmembrane domains with ATP hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography. Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels. Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site. Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites. This results in the permanent activation of ATP hydrolysis in the absence of transport. Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the drug-binding site is occupied. The human multidrug resistance P-glycoprotein (P-gp) 1The abbreviations used are: P-gp, P-glycoprotein; TM, transmembrane; HEK, human embryonic kidney; MTS, methanethiosulfonate; PBS, phosphate-buffered saline; NBD, nucleotide-binding domain; AM, acetoxymethyl ester. uses ATP to transport structurally diverse compounds out of the cell (for recent reviews, see Refs. 1Borst P. Elferink R.O. Annu. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1355) Google Scholar and 2Hrycyna C.A. Semin. Cell Dev. Biol. 2001; 12: 247-256Crossref PubMed Scopus (51) Google Scholar). The protein contributes to the phenomenon of multidrug resistance because many of the drugs used in AIDS and cancer chemotherapy are substrates of P-gp (3Krishna R. Mayer L.D. Eur. J. Pharm. Sci. 2000; 11: 265-283Crossref PubMed Scopus (992) 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 (460) Google Scholar). P-gp is a member of the ATP-binding cassette family of transporters. The 1280 amino acids of P-gp are arranged as two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (5Chen 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 has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (6Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 7Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 8Kast 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 minimum functional unit is a monomer (9Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), but the two halves of the molecule do not have to be covalently linked for function (10Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 11Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Both ATP-binding sites are required for activity (7Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 12Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar, 13Doige C.A. Yu X. Sharom F.J. Biochim. Biophys. Acta. 1992; 1109: 149-160Crossref PubMed Scopus (138) Google Scholar, 14al-Shawi M.K. Urbatsch I.L. Senior A.E. J. Biol. Chem. 1994; 269: 8986-8992Abstract Full Text PDF PubMed Google Scholar) and likely function in an alternating mechanism (15Senior A.E. Gadsby D.C. Semin. Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (129) Google Scholar). Studies on deletion mutants have shown that the TM domains alone are sufficient to mediate drug binding (11Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Studies on the activity of cysteine mutants and their inhibition by different thiol-reactive substrate analogs indicate that residues from multiple TM segments contribute to the drug-binding site (16Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 17Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 18Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 19Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 20Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 2002; 277: 44332-44338Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). To determine the mechanism of P-gp, it is important to understand how drug binding and subsequent efflux are coupled to ATP hydrolysis. A key step in the reaction cycle is activation of ATP hydrolysis when the drug-binding site is occupied. It is not known whether drug-stimulated ATPase activity requires that the drug-binding site alternate between occupied and unoccupied states or whether ATPase activity is permanently activated when the drug-binding site is permanently occupied. In this study we used cysteine-scanning mutagenesis and reaction with the thiol-reactive analog of verapamil, MTS-verapamil, to test for the presence of a permanently activated P-gp intermediate. Construction of Mutants—A histidine-tagged Cys-less P-gp cDNA was constructed (6Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 22Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar). The presence of the histidine tag facilitated purification of the mutant P-gps by nickel-chelate chromatography (23Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Single cysteine residues were then introduced into the predicted TM segments of Cys-less P-gp(His)10 as described previously (18Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Expression, Reaction with MTS-verapamil, Purification, and Measurement of Drug-stimulated ATPase Activities of P-gp Mutants—The histidine-tagged P-gp mutants were expressed in HEK 293 cells. After 24 h, the medium was replaced with fresh medium containing 10 μm cyclosporin A. Cyclosporin A acts as a specific chemical chaperone to promote maturation of P-gp and increases the yield of P-gp (24Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 25Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). After another 24 h, the transfected cells from 50 10-cm-diameter culture plates were washed three times with phosphate buffered saline (PBS, pH 7.4) and suspended in 1.5 ml of PBS. The cells were solubilized by addition of 1 volume of PBS containing 2% (w/v) n-dodecyl β-d-maltoside. Insoluble material was removed by centrifugation at 16,000 × g for 15 min at 4 °C. Half of the supernatant (1.3 ml) was mixed with 8 μl of Me2SO (control), and the other half was mixed with 8 μl of 50 mm MTS-verapamil (in Me2SO; final concentration, 0.3 mm). The mixtures were incubated for 10 min at 22 °C, and cooled to 4 °C, and then 150 μl of 3 m NaCl and 50 μl of 1 m imidazole (pH 7.0) were added. P-gp(His)10 was then isolated by nickel-chelate chromatography as described previously (23Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The recovery of P-gp was monitored by immunoblot analysis with rabbit anti-P-gp polyclonal antibody (9Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To protect P-gp from labeling with MTS-verapamil, 0.5 mm cyclosporin A, 5 mm demecolcine, 5 mm verapamil, 0.5 mm vinblastine, 10 mm colchicine, or 2 mm calcein-AM was added to the solubilized material before addition of MTS-verapamil. The highest concentrations possible (solubility in aqueous solution) were chosen to maximize protection from modification by MTS-verapamil. The isolated P-gp(His)10 was mixed with lipid and sonicated, and ATPase activity was determined (26Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). A sample of the P-gp-lipid mixture was mixed with an equal volume of buffer containing 100 mm Tris-HCl, pH 7.4, 100 mm NaCl, 20 mm MgCl2, 10 mm ATP, and either no drug, 1.2 mm calcein-AM, 0.3 mm cyclosporin A, 6 mm demecolcine, 1.2 mmtrans-(E)-flupentixol, 2 mm verapamil, or 0.2 mm vinblastine. These concentrations caused maximal stimulation or inhibition of the ATPase activity of Cys-less P-gp. The samples were then incubated for 30 min at 37 °C, and the amount of inorganic phosphate liberated was determined (27Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (418) Google Scholar). Inhibition of ATPase activity by vanadate was done by addition of an equal volume of buffer containing 100 mm Tris-HCl, pH 7.4, 100 mm NaCl, 20 mm MgCl2, 10 mm ATP, and 0.2 mm orthovanadate to the P-gp-lipid mixture (26Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Orthovanadate was prepared from Na3VO4, pH 10 and boiled for 2 min to break down polymeric species before use (28Goodno C.C. Methods Enzymol. 1982; 85: 116-123Crossref PubMed Scopus (181) Google Scholar). Drug transport by P-gp involves the coupling of drug binding with ATP hydrolysis. The transport cycle predicts the existence of an activated intermediate. Therefore, the goal in this study was to determine whether P-gp could be trapped as an activated intermediate if drug substrates could be prevented from leaving the drug-binding site. An approach to keep the drug-binding site occupied would be to permanently attach a drug substrate to a residue in the drug-binding site. Cysteine-scanning mutagenesis and reaction with a thiol-reactive analog of a drug substrate seemed ideal for these studies. We have done cysteine-scanning mutagenesis on all the residues in the TM segments and found that almost all of the single cysteine mutants were active (16Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 17Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 18Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). A useful thiol-reactive drug substrate is MTS-verapamil (Fig. 1). It is an analog of verapamil and contains an alkylthiosulfonate group that reacts selectively with cysteine residues (29Kenyon G.L. Bruice T.W. Methods Enzymol. 1977; 47: 407-430Crossref PubMed Scopus (173) Google Scholar, 30Bruice T.W. Kenyon G.L. J. Protein Chem. 1982; 1: 47-58Crossref Scopus (95) Google Scholar). We showed previously that MTS-verapamil is a good substrate of Cys-less P-gp as it stimulated its ATPase activity 10-fold and with a Km of 25 μm (Km for verapamil, 25 μm) (19Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Accordingly a cysteine was introduced at each position in the predicted TM segments of Cys-less P-gp(His)10. A total of 252 mutants were made, expressed in HEK 293 cells, and then solubilized with 1% (w/v) n-dodecyl β-d-maltoside. Insoluble material was removed by centrifugation, and the samples were treated with or without 0.3 mm MTS-verapamil. This concentration of MTS-verapamil caused maximum activation of Cysless P-gp (19Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). P-gp was then immobilized on a nickel-chelate column, washed with buffer to remove unbound MTS-verapamil, and eluted from the column. The ATPase activities of the P-gp treated with and without MTS-verapamil were determined. One mutant, I306C, showed an 8-fold increase in ATPase activity after treatment with MTS-verapamil. Activation by MTS-verapamil was concentration-dependent. Fig. 1 shows that maximal stimulation occurred after treatment of mutant I306C with 0.1–1 mm MTS-verapamil, and half-maximal stimulation was 39 μm. Nickel-chelate chromatography was effective in removing unreacted MTS-verapamil because the activity of Cys-less P-gp remained at basal levels even after pretreatment with 1 mm MTS-verapamil (Fig. 1). To test whether all of mutant I306C had been modified by MTS-verapamil, we assayed for stimulation or inhibition of the ATPase activity by other drug substrates. The rationale is that if a significant amount of unmodified mutant I306C is present, then the presence of other substrates or inhibitors should affect the activity of the mutant. We tested compounds that are potent stimulators (calcein-AM, demecolcine, vinblastine, and verapamil) (17Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 31Ambudkar S.V. Lelong I.H. Zhang J. Cardarelli C.O. Gottesman M.M. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8472-8476Crossref PubMed Scopus (382) Google Scholar, 32Sarkadi B. Price E.M. Boucher R.C. Germann U.A. Scarborough G.A. J. Biol. Chem. 1992; 267: 4854-4858Abstract Full Text PDF PubMed Google Scholar, 33Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2003; 278: 1575-1578Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) or inhibitors (cyclosporin A and trans-(E)-flupentixol) of P-gp ATPase activity (33Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2003; 278: 1575-1578Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 34Tamai I. Safa A.R. J. Biol. Chem. 1991; 266: 16796-16800Abstract Full Text PDF PubMed Google Scholar, 35Dey S. Hafkemeyer P. Pastan I. Gottesman M.M. Biochemistry. 1999; 38: 6630-6693Crossref PubMed Scopus (57) Google Scholar). Fig. 2 shows the effect of various stimulators and inhibitors on the ATPase activity of mutant I306C before and after treatment with MTS-verapamil. Before treatment with MTS-verapamil, the ATPase activity of mutant I306C was stimulated by demecolcine, calcein-AM, verapamil, and vinblastine by 11.9-, 9.8-, 7-, and 3-fold, respectively. By contrast, cyclosporin A and trans-(E)-flupentixol inhibited the ATPase activity (0.9- and 0.5-fold, respectively). When mutant I306C was pretreated with MTS-verapamil, the presence of other stimulators or inhibitors of P-gp had little effect on its ATPase activity (7.7–8.1-fold versus 8-fold increase) (Fig. 2). This inability to further stimulate or inhibit the activity of the MTS-verapamil-treated mutant I306C suggests that most (more than 90%) of the mutant I306C P-gp was modified and that covalent attachment of verapamil in the drug-binding site blocks access of other drug substrates to the drug-binding site. It was possible that reaction of the mutant with MTS-verapamil partially denatured the protein so that the ATP-binding sites are uncoupled from the drug-binding sites. If modified I306C retained a “native” structure, then it should be possible to restore basal levels of ATPase activity if covalent attachment of MTS-verapamil was removed. Accordingly modified I306C was treated with 20 mm dithiothreitol to reduce the disulfide bond between P-gp and MTS-verapamil, and then P-gp was reisolated by nickel-chelate chromatography. Fig. 3 shows that reduction of the disulfide bond by dithiothreitol reduced the activity of the mutant as its ATPase activity was only slightly higher (1.5-fold) than the untreated I306C mutant. Mutant I306C remained active after removal of the covalently bound MTS-verapamil since it retained the ability to be stimulated by verapamil (6.9-fold). The characteristics of the ATPase activity of modified I306C mutant were also examined. We tested for vanadate trapping of nucleotide. P-gp traps nucleotides in the presence of vanadate plus Mg-ATP and results in the formation of an inhibitory transition state that inhibits ATP hydrolysis at the second ATP site (36Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Fig. 3 shows that mutant I306C that had been labeled with MTS-verapamil was still inhibited by vanadate. In addition, the modified mutant I306C had a Km for ATP (1.1 mm) that was very to that of Cys-less P-gp mm) not If MTS-verapamil occupied the drug-binding site in mutant I306C, then pretreatment of the mutant with other drug substrates should protect it from labeling by MTS-verapamil if a common drug-binding site. Accordingly mutant I306C was treated with or without the drug substrates calcein-AM, demecolcine, verapamil, vinblastine, cyclosporin A, or colchicine before treatment with MTS-verapamil for 10 min at 22 °C. P-gp was isolated by nickel-chelate and ATPase activity was determined. Fig. 4 shows that all of the drug substrates mutant I306C from labeling with MTS-verapamil since prevented modification and activation of I306C by MTS-verapamil by These results suggest that the compounds tested likely a common drug-binding site. of mutant I306C with MTS-verapamil by removal of unreacted MTS-verapamil caused the mutant to in an activated ATP is at a without ATP hydrolysis is uncoupled from transport. Addition of other stimulators or inhibitors of P-gp could not the ATPase activity of the activated mutant. An for this is that covalent attachment of MTS-verapamil to the cysteine residue at position in in the permanent of the drug-binding site in an to that of P-gp with unmodified that MTS-verapamil to I306C the of the mutant with verapamil is that verapamil and MTS-verapamil caused activation of ATPase activity In addition, activation of I306C by MTS-verapamil was by and the mutant could Both MTS-verapamil and verapamil to the drug-binding site because the activity of MTS-verapamil-treated I306C was not by verapamil or other stimulators and inhibitors of P-gp, and mutant I306C be from labeling by verapamil and the other substrates and A to the activated state of P-gp is shown in Fig. In the state P-gp ATP with but ATPase activity is because the are from the A sites in the nucleotide-binding domains (33Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2003; 278: 1575-1578Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of verapamil in the TM segments in the to the and A and promote ATP hydrolysis T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). ATP hydrolysis then the of the TM segments (26Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, T.W. Clarke D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar) to drug of verapamil from the drug-binding site the and A to and P-gp to the state T.W. Clarke D.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, Ambudkar S.V. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Covalent attachment of verapamil, results in the permanent of the drug-binding site The inability of MTS-verapamil to the activated state the and A and in a that hydrolysis of ATP and between states and (Fig. It is possible that Ile-306 may be part of the mechanism that that the drug-binding site is occupied and then in the predicts that covalent modification of the drug-binding site in permanent activation of ATP hydrolysis in the absence of transport because hydrolysis of ATP and of inorganic phosphate before of the drug In this ATP-binding cassette to be different from the such as the In the ATP hydrolysis is linked to in the but is not released after transport of C. Rev. PubMed Scopus Google Scholar, C. C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). This study also shows that contribute residues to the drug-binding site since other substrates and inhibitors could protect I306C from labeling by MTS-verapamil. We had predicted that I306C likely the drug-binding site because it could be to other in and with thiol-reactive substrates T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar).