HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 46, 44332-44338, November 15, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/46/44332    most recent M208433200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Loo, T. W. Articles by Clarke, D. M. Search for Related Content PubMed PubMed Citation Articles by Loo, T. W. Articles by Clarke, D. M. Social Bookmarking                      What's this?

Location of the Rhodamine-binding Site in the Human Multidrug Resistance P-glycoprotein^*

Tip W. Loo and David M. Clarke

From the Canadian Institutes of Health Research Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Received for publication, August 18, 2002, and in revised form, September 9, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1-379 plus 681-1025) with only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2-12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the "substrate-induced fit" hypothesis for drug binding.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The human multidrug resistance P-glycoprotein (P-gp)¹ is found in the plasma membrane and uses ATP to pump a wide variety of structurally diverse compounds out of the cell (reviewed in Refs. 1 and 2). Expression of P-gp complicates treatment of AIDS and cancer because many of the therapeutic compounds are also substrates of P-gp (3, 4). P-gp also plays an important role in mediating the bioavailability of oral drugs because of its relatively high expression in the intestine, liver, kidney, and brain (5, 6).

P-gp is a member of the ATP-binding cassette family of transporters (7). Its 1280 amino acids are organized in two repeating units of 610 amino acids that are joined by a linker segment of 60 amino acids (8). Each repeat has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (9, 10). The minimum functional unit is a monomer (11), but both halves of the molecule do not have to be covalently linked for function (12, 13). Both ATP-binding sites are required for activity (14-17) and likely function in an alternating mechanism (18).

An important goal in understanding the mechanism of drug transport is the identification of residues that line the drug-binding site. The drug-binding site(s) are within the TM domains of P-gp because a deletion mutant missing both nucleotide-binding domains could still interact with drug substrates (13). A useful method for identifying residues in the TM segments that contribute to drug binding is to use cysteine-scanning mutagenesis and reaction with thiol-reactive substrates. Such an approach is feasible with P-gp because a Cys-less mutant of P-gp is active (9), and most single cysteine mutants retain activity (19). In previous studies we used the thiol-reactive substrates dibromobimane (20-22) and MTS-verapamil (23) to test the reactivity of the single cysteine mutants. These studies showed that residues in TMs 4-6 and 10-12 contributed to the drug-binding site. Some of these residues were common to the binding of both substrates. Although both dibromobimane and verapamil stimulate the ATPase activity of P-gp, it is not known whether both are transported by P-gp. Kinetic studies indicate that verapamil is a noncompetitive inhibitor of cytotoxic substrates transported by P-gp and may occupy separate site(s) from that of transported compounds (24, 25).

It has been shown that all rhodamine compounds are transported by P-gp (26). In this study, we used a thiol-reactive analog of rhodamine, MTS-rhodamine, to identify residues involved in its binding.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Mutants-- The full-length human MDR1 cDNA was obtained by screening a human kidney cortex cDNA library (27, 28). The seven endogenous cysteines at positions 137, 431, 717, 956, 1074, 1125, and 1227 were replaced with alanine during construction of a Cys-less P-gp (9). The Cys-less P-gp was functional. A (His)₁₀ tag was attached at the COOH end of the molecule (Cys-less P-gp(His)₁₀) to facilitate purification of the Cys-less P-gp by nickel-chelate chromatography (29). Cysteine residues were then introduced into the predicted TM segments of Cys-less P-gp(His)₁₀ as described previously (22). The predicted boundaries of the TM segments are Met⁵¹ to Val⁷¹ (TM1), Tyr¹¹⁷ to Ala¹⁴⁰ (TM2), Lys¹⁸⁹ to Thr²⁰⁹ (TM3), Lys²¹³ to Ala²³³ (TM4), Thr²⁹⁴ to Tyr³¹⁶ (TM5), Gln³³⁰ to Pro³⁵⁰ (TM6), Val⁷¹² to Ile⁷³¹ (TM7), Leu⁷⁵⁷ to Ala⁷⁸⁰ (TM8), Arg⁸³² to Phe⁸⁵¹ (TM9), Trp⁸⁵⁵ to Glu⁸⁷⁵ (TM10), His⁹³⁶ to Phe⁹⁵⁷ (TM11), and Val⁹⁷⁴ to Phe⁹⁹⁴ (TM12). The integrity of the mutated cDNA was confirmed by sequencing the entire cDNA (30).

Expression, Purification, and Measurement of Drug-stimulated ATPase Activity of P-gp Mutants-- The histidine-tagged P-gp mutants were expressed and purified as described previously (29). Briefly, fifty 10-cm diameter culture plates of HEK 293 cells were transfected with the mutant cDNA, and the medium was replaced 24 h later with fresh medium containing 10 µM cyclosporin A. Cyclosporin A is a substrate of P-gp and is a powerful chemical chaperone for promoting maturation of P-gp (31, 32). After another 24 h, the transfected cells were harvested and solubilized with 1% (w/v) n-dodecyl--D-maltoside, and the mutant P-gp was isolated by nickel-chelate chromatography (nickel-nitrilotriacetic acid columns; Qiagen, Inc., Mississauga, Canada).

The P-gp(His)₁₀ mutant proteins eluted from the nickel columns (in buffer containing 10 mM Tris-HCl, pH 7.5, 500 mM NaCl, 300 mM imidazole, pH 7.0, 0.1% (w/v) n-dodecyl--D-maltoside and 10% (v/v) glycerol) were mixed with an equal volume of 10 mg/ml sheep brain phosphatidylethanolamine (Type II-S; Sigma-Aldrich) that was washed and suspended in 10 mM Tris-HCl, pH 7.5, and 150 mM NaCl. The P-gp and lipid mixture was then sonicated for 45 s at 4 °C (bath-type probe, maximum setting; Branson Sonifier 450, Branson Ultrasonic, Danbury, CT). An aliquot of the sonicated P-gp and lipid mixture was assayed for drug-stimulated ATPase activity by the addition of an equal volume of buffer containing 100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM MgCl₂, 10 mM ATP, and 2 mM verapamil. The samples were incubated for 30 min at 37 °C, and the amount of inorganic phosphate liberated was determined (33).

For inhibition with MTS-rhodamine (Toronto Research Chemicals, Toronto, Canada), the P-gp and lipid mixture was preincubated with 1 mM MTS-rhodamine for 15 min at 22 °C. Unreacted MTS-rhodamine was removed by gel filtration (Centri.Spin 20 columns, Princeton Separations, Inc., Adelphia, NJ). The columns were pre-equilibrated with nickel column elution buffer containing 5 mg/ml sheep brain phosphatidylethanolamine and 1 mM verapamil or 1 mM rhodamine B. The verapamil- or rhodamine-stimulated ATPase activity in the flow-through fraction was measured as described above.

In the protection experiments, the P-gp and lipid samples were pretreated for 10 min at 22 °C with or without 3 mM rhodamine B. This is the saturating concentration of rhodamine B for stimulation of ATPase activity. We then added 0.3 mM MTS-rhodamine, and the samples were then incubated for 10 min at 22 °C to allow MTS-rhodamine to react with the mutant. Rhodamine B (3 mM) was then added to the sample without rhodamine B so that both samples now had the same amount of rhodamine B and MTS-rhodamine. The unreacted MTS-rhodamine was removed by gel filtration (Centri.Spin 20 columns). Rhodamine B-stimulated ATPase activity was then measured (final rhodamine B concentration was 1 mM).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulation of Cys-less P-gp by Rhodamine Compounds-- Drug-stimulated ATPase activity is a useful measure of drug interaction with P-gp. The ATPase activity of P-gp is increased 2-20-fold when it interacts with most substrates and modulators of P-gp (21). Furthermore, there is good correlation between substrate-stimulated ATPase activity and transport activity because the turnover numbers are quite similar (34).

Rhodamine dyes (Fig. 1) are transported by P-gp (26) and also stimulate the ATPase activity of Cys-less P-gp. Fig. 2 shows rhodamine B stimulation of the ATPase activity of Cys-less P-gp. The ATPase activity was stimulated 10.5-fold with 1 mM rhodamine B, with half-maximal activation occurring at a concentration of 68 µM. Inhibition of ATPase activity was observed at 3 mM rhodamine B. Such inhibition of activity at high concentrations of substrate is characteristic of P-gp (21, 35).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Structures of rhodamine B and MTS-rhodamine.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of rhodamine B and MTS-rhodamine on Cys-less P-gp ATPase activity. Histidine-tagged Cys-less P-gp was expressed in HEK 293 cells and isolated by nickel-chelate chromatography. The isolated P-gp was mixed with lipid and sonicated, and ATPase activity was determined in the presence of various concentrations of rhodamine or MTS-rhodamine.

Fig. 1 (lower panel) shows the structure of a thiol-reactive analog of rhodamine, MTS-rhodamine. An ethyl linker group attaches the methanethiosulfonate group to rhodamine. Alkylthiosulfonates react selectively with cysteines in a protein under relatively mild conditions, resulting in a disulfide attachment of the R group and release of a sulfinic acid byproduct (36, 37). Alkylthiosulfonate compounds react more rapidly with cysteines than other thiol-specific compounds such as maleimides or iodoacetamides (36).

MTS-rhodamine appeared to be useful for identifying residues in P-gp that contribute to the binding site because of its ability to stimulate the ATPase activity of Cys-less P-gp (Fig. 2). Maximal stimulation of activity (6.2-fold) was at a concentration of 1 mM with half-maximal stimulation occurring at 110 µM. The slightly reduced affinity and stimulation observed with MTS-rhodamine could be due to the presence of the methanethiosulfonate group and/or the presence of the sulfate group rather than the carboxyl group present in rhodamine B. These characteristics were similar to those obtained with rhodamine B, indicating that they may occupy similar binding sites in P-gp.

Inhibition of P-gp Single Cysteine Mutants By MTS-rhodamine-- We tested whether rhodamine-like compounds interact with the TM domains with a "drug rescue" assay involving a P-gp deletion mutant that lacked the nucleotide-binding domains. Many P-gp mutations (processing mutations) cause it to be misfolded and trapped in the endoplasmic reticulum as a core-glycosylated intermediate (38, 39). In a drug rescue assay, the misprocessed P-gp mutant is expressed in the presence of a drug substrate (chemical chaperone) that induces the misfolded protein to fold properly. The rescued P-gp bypasses the quality control system present in the endoplasmic reticulum (40) and is further glycosylated in the Golgi and then trafficked to the cell surface in an active form. The drug substrate diffuses into the endoplasmic reticulum, where it may act as a "scaffold" for the partially folded P-gp to adopt a native conformation (32, 41).

The P-gp deletion mutant (TMD1+TMD2; residues 1-379 plus 681-1025) is normally expressed as an immature core-glycosylated intermediate with an apparent mass of 80 kDa. Expression of the TMD1+TMD2 mutant in the presence of a drug substrate or modulator such as cyclosporin A, vinblastine, or capsaicin, however, results in a 100-kDa protein that is detected at the cell surface (13). Accordingly, the TMD1+TMD2 mutant was expressed in HEK 293 cells in the presence of various concentrations of rhodamine B or 10 µM cyclosporin A and subjected to SDS-PAGE. Fig. 3 shows that the 100-kDa protein was not detected in the absence of drug substrate (No drug). By contrast, the 100-kDa product was readily detected when expression was in the presence of (10 µM) cyclosporin A or rhodamine B. The relative amount of the 100-kDa protein increased with increasing concentrations (16-125 µM) of rhodamine B. Higher concentrations (250-500 µM) of rhodamine B caused cell death. These results show that the TM domains alone are sufficient for binding rhodamine B. 

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of rhodamine B on expression of P-gp deletion mutant lacking the nucleotide-binding domains. HEK 293 cells transfected with the cDNA for A52-tagged TMD1+TMD2 (residues 1-379 plus 681-1025) (13) were treated for 48 h with (+) or without () 10 µM cyclosporin A or with the indicated concentrations of rhodamine B. The cells were then solubilized with SDS sample buffer and subjected to immunoblot analysis with monoclonal antibody A52 and enhanced chemiluminescence (38). The positions of the core-glycosylated (80 kDa) and mature (100 kDa) mutant proteins are indicated.

To identify residues in the TM segments that contribute to binding of rhodamine, we tested whether MTS-rhodamine inhibited the activity of single cysteine mutants. A total of 260 histidine-tagged single cysteine mutants were constructed. These mutations covered all of the residues predicted to be within the 12 TM segments of P-gp. When expressed in HEK 293 cells in the presence of cyclosporin A, the major product was mature 170-kDa P-gp on SDS-PAGE (data not shown). Eight mutants were not tested further because of low expression ((V52C(TM1), G54C(TM1), G62C(TM1), G122C(TM2), S344C(TM6), and G989C(TM12)) or low activity ((G722C(TM7) and G763C(TM8)). Six of these mutants involved substitution of a glycine. Replacement of glycine with the larger cysteine residue may have caused structural perturbations. Similar results were observed when glycine residues in the cytoplasmic loops connecting the TM segments were mutated (42).

The remaining 252 histidine-tagged mutants were expressed in HEK 293 cells in the presence of cyclosporin A. The mutant proteins were isolated by nickel-chelate chromatography, mixed with lipid, and reacted with 1 mM MTS-rhodamine for 15 min at 22 °C. Unreacted MTS-rhodamine and sulfinic acid byproducts were removed by passage through a gel filtration spin column. The verapamil-stimulated ATPase activity of the MTS-rhodamine-treated sample was then determined and compared with that of a mock treated sample. Verapamil was used to measure drug-stimulated ATPase activity because it is the most potent stimulator of the ATPase activity of Cys-less P-gp (about 13-fold) (20), and all 252 single cysteine mutants retain verapamil-stimulated ATPase activity (22).

Mutants that showed at least a 25% decrease or a 50% increase in verapamil-stimulated ATPase activities after treatment with MTS-rhodamine are shown in Fig. 4. In TM1 (Fig. 4), MTS-rhodamine had only modest effects because most mutants retained at least 75% of their activity. The activity of M68C, was inhibited by 44%, whereas that of mutant L65C was increased (189%).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Inhibition of verapamil-stimulated ATPase activity by MTS-rhodamine. Histidine-tagged Cys mutants were isolated by nickel-chelate chromatography, mixed with lipid, and sonicated. The samples were then incubated for 15 min at 22 °C with or without 1 mM MTS-rhodamine. Unreacted MTS-rhodamine was removed by gel filtration and verapamil-stimulated ATPase activity determined. The results (percentages) are expressed relative to that of a mock treated sample. Each value is the average of two different purifications. Only mutants that showed significant inhibition (>25%) or activation (>50%) are shown.

Four mutants (Y118C, V125C, V133C, and C137) in TM2 were very sensitive to MTS-rhodamine because they were inhibited 94, 68, 80, and 93%, respectively. Mutant Q132C showed weaker inhibition (32%).

In TM3, the activities of two mutants (K189C and Q195C) were inhibited 76 and 78%, respectively, by MTS-rhodamine.

The activity of one mutant (S222C) in TM4 was strongly inhibited (98%) by MTS-rhodamine. This mutant appears to be particularly sensitive to modification by thiol-reactive reagents because dibromobimane (22) and MTS-verapamil (23) also affect its activity.

In TM5, however, there were significant differences in the inhibition pattern with MTS-rhodamine compared with that with dibromobimane (22) or MTS-verapamil (23). The cysteines in TM5 showed little or no inhibition with dibromobimane or MTS-verapamil. In contrast, four mutants (N296C, G300C, Y310C, and F314C) were inhibited by >70% with MTS-rhodamine.

The activities of two mutants (I340C and A342C) in TM6 were strongly inhibited (87 and 94%, respectively) by MTS-rhodamine. The activity of mutant F343C, however, was increased (347%) after treatment with MTS-rhodamine.

In TMs 7-10, only a few residues were strongly affected by MTS-rhodamine. The activities of mutants A729C(TM7), F759C(TM8), G774C(TM8), A841C(TM9), and G872C(TM10) were inhibited 87, 80, 79, 78, and 76%, respectively, with MTS-rhodamine.

The activities of 10 mutants in TMs 11 and 12 were quite sensitive (>50% inhibition) to inhibition by MTS-rhodamine. Mutants F942C(TM11), S943C(TM11), T945C(TM11), F957C(TM11), L975C(TM12), V981C(TM12), V982C(TM12), G984C(TM12), A985C(TM12), and S993C(TM12) were inhibited 67, 86, 79, 65, 78, 83, 87, 90, 94, and 51%, respectively, with MTS-rhodamine.

Protection from Inhibition by Rhodamine B-- The highly reactive nature of the methanethiosulfonate group allows the compound to react with any accessible cysteine residue in the molecule. To identify cysteines that are within the rhodamine-binding site, we tested whether rhodamine B could protect the reactive cysteine mutants from inhibition by MTS-rhodamine. The rationale is that rhodamine B would occupy the drug-binding site and prevent reaction of MTS-rhodamine with any reactive cysteine residue in the drug-binding site. The presence of a 10-fold excess of rhodamine B (3 mM) combined with the slightly higher apparent affinity of P-gp for rhodamine B (68 µM versus 110 µM for MTS-rhodamine) should protect the mutant from inactivation if the reactive cysteine is within or close to the drug-binding site.

Twenty-eight mutants, Y118C(TM2), V125C(TM2), V133(TM2), C137C(TM2), K189C(TM3), Q195C(TM3), S222C(TM4), N296C(TM5), G300C(TM5), Y310C(TM5), F314C(TM5), I340C(TM6), A342C(TM6), A729C(TM7), F759C(TM8), G774C(TM8), A841C(TM9), G872C(TM10), F942C(TM11), S943C(TM11), T945C(TM11), F957C(TM11), L975C(TM12), V981C(TM12), V982C(TM12), G984C(TM12), A985C(TM12), and S993C(TM12) showed at least 50% inhibition with MTS-rhodamine and were subjected to protection assays with rhodamine B. The mutant proteins were preincubated with or without 3 mM rhodamine B for 10 min at 22 °C and then treated with 0.3 mM MTS-rhodamine for 10 min at 22 °C. The reaction mixtures was applied to a gel filtration column to remove the MTS-rhodamine and then assayed for rhodamine-stimulated ATPase activity and compared with that of mock treated samples. Rhodamine B protected five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12) from inhibition by MTS-rhodamine (Fig. 5). The greatest protection was observed in mutant V981C(TM12). This mutant was inhibited by 80% by MTS-rhodamine but retained about 84% of its activity after treatment with rhodamine B. Lower levels of protection were observed with mutants I340C, A841C, L975C, and V982C (Fig. 5). No significant protection by rhodamine B was seen in the other 23 mutants (data not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Protection of mutants from MTS-rhodamine inhibition by rhodamine. Histidine-tagged Cys-less and mutant P-gp, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and C982C(TM12), were isolated by nickel-chelate chromatography, mixed with lipids, and sonicated. The samples were then preincubated without (A) or with (B) 3 mM rhodamine B for 10 min at 22 °C. The samples were then treated with or without 0.3 mM MTS-rhodamine for 10 min at 22 °C. Unreacted MTS-verapamil was removed by gel filtration, and rhodamine B-stimulated ATPase activity was determined. The results are expressed relative to that of a sample not treated with MTS-rhodamine. Each value is the average of four different experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Rhodamine compounds such as trimethylrosamine, rhodamines I, II, and II, rhodamine B, rhodamine G, and rhodamine 123 are transported by P-gp. These rhodamine dyes and MTS-rhodamine (this study) also stimulate ATPase activity and are considered to be substrates of P-gp (26).

Two mutants, L65C and F343C, showed increased activity after treatment with MTS-rhodamine. Because mutant L65C had only 51% of the verapamil-stimulated ATPase activity relative to that of the Cys-less parent (22), treatment with MTS treatment essentially restored the activity of the mutant to that of the Cys-less P-gp. Perhaps a bulky residue is required at position 56 for full activity. Mutant F343C showed about 3.5-fold increase in activity after treatment with MTS-rhodamine. Because mutant F343C had about 60% of the activity of Cys-less P-gp, reaction with MTS-rhodamine essentially causes a 2-fold increase in activity relative to the Cys-less parent. The presence of a bulky group a position 343 appears to enhance activity. We previously showed that the bulkiness of side chains in TMs 5 and 6 can have large effects on drug-stimulated ATPase activity (29, 43).

MTS-rhodamine inhibited the ATPase activities of 28 of the 252 single cysteine mutants by at least 50%. A cysteine mutant that was sensitive to inhibition was found in all other TM segments except in TM1. Three cysteine mutants (V52C, G54C, and G62C) in TM1 were not tested because of low expression, indicating that these residues must be important for structure and/or function. Therefore, it appears that all of the TMs are important for function because at least one position in each TM segment is sensitive to mutation or inhibition by MTS-rhodamine.

The activity of mutant C137(TM2) was inhibited by 93% by MTS-rhodamine. This residue is interesting in that it is one of seven endogenous cysteines (other cysteines at positions 431, 717, 956, 1074, 1125, and 1227) found in wild-type P-gp. Previous studies on the inhibition of P-gp activity have shown that only the cysteines located in the cytoplasmic Walker A nucleotide-binding regions (Cys⁴³¹ and Cys¹⁰⁷⁴) are inhibited by thiol-reactive compounds (17, 44, 45). Inhibition of the activity of the C137 mutant was quite specific because similar inhibition of activity was not observed when the mutant was treated with the thiol-reactive compounds such as biotin-maleimide (9), N-ethylmaleimide (17), disulfiram (45), dibromobimane (22), or MTS-verapamil (23). The ability to inhibit the activity of mutant C137 with MTS-rhodamine indicates that this residue could be a target for the development of novel inhibitory reagents that covalently modify P-gp.

Rhodamine B significantly protected the activities of mutants I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12) from inhibition by MTS-rhodamine (Fig. 5). This indicates that these residues must be within or close to the rhodamine drug-binding site.

The results from studies involving cysteine-scanning mutagenesis and reaction with structurally diverse thiol-reactive substrates (this study and Refs. 20-23, 46, and 47), from photolabeling studies (48-52), and from mutational studies (27, 53-55) point to the presence of a "common" drug-binding site. A model of such a common drug-binding site in P-gp is shown in Fig. 6. The arrangement of the TM segments (Fig. 6) is also based on the results of disulfide cross-linking studies that show TMs 4-6 to be close to TMs 10-12 during the resting phase (19, 46, 56, 57). The MTS-rhodamine results (this study) show that TM9 must also be close to or within the binding site for rhodamine-type compounds and would be next to TM6 (Fig. 6). Song and Melera (58) showed in hamster P-gp that there is close interaction between TMs 6 and 9 during drug binding. Mutations in TM9 (I837L and N839I) or in TM6 (G388A and A339P) resulted in similar drug resistance profiles with four structurally different drugs (increased resistance to vincristine or actinomycin D but decreased resistance to colchicine or daunorubicin relative to wild-type P-gp). It is interesting that the equivalent residues in human P-gp (Ile⁸⁴⁰ and Asn⁸⁴² in TM9 and Gly³⁴¹ and Ala³⁴² in TM6) are adjacent to the cysteines (A841C(TM9) and I340C(TM6)) the activities of which are protected by rhodamine B from inhibition by MTS-rhodamine (Fig. 5).

View larger version (63K): [in this window] [in a new window]   Fig. 6.   Location of the cysteine residues in the predicted TMs of P-gp that are inhibited by MTS-rhodamine and protected by rhodamine B. TMs 1-12 of P-gp are arranged as -helical wheels. Residues within the TMs are shown and viewed from the cytoplasmic side of the membrane. Circled white letters on a black background indicate residues that are inhibited by MTS-rhodamine and protected by rhodamine B when mutated to cysteine.

Some studies have suggested that P-gp has four different drug interaction sites that substrates occupy different sites during transport or that each substrate has a distinct binding site (25, 59, 60). These results are not incompatible with the model presented in Fig. 6. Although the model shows the presence of a common drug-binding site, it can be used to accommodate results that predict multiple drug-binding sites. We had proposed that substrates can create their own binding sites ("substrate-induced fit" hypothesis) by using a combination of residues from different TMs to form a particular drug-binding site (21). Forming the drug-binding site this way would depend on the TMs being quite mobile. The binding of a particular substrate would result in a more "rigid" P-gp. Evidence that the TM segments are quite mobile comes from disulfide cross-linking studies (57). When thermal motion is reduced (4 °C), only residues at the cytoplasmic side in TMs 4 and 5 were cross-linked with that in TM12. At higher temperatures (21 and 37 °C), these residues as well as residues at the cytoplasmic side in TM 6 were cross-linked with those in TMs 10 and 11. The substrate-induced fit hypothesis would also explain why P-gp binds substrates with different affinities. When a particular substrate induces a particular fit in P-gp, then the combined effects of contributing residues from each TM would determine the affinity. Also, some substrates may share the same residue(s) during binding. For example, the activities of mutants L339C(TM6) and A342(TM6) are protected from inhibition by dibromobimane (21) and MTS-verapamil (23) but not by MTS-rhodamine, whereas that of mutant V982C(TM12) is protected from inhibition by dibromobimane and MTS-rhodamine but not by MTS-verapamil.

An interesting feature of the cysteine mutants that are inhibited with thiol-reactive compounds is that most are located near the middle of each TM segment (Fig. 7). When the drug-binding site is opened as a fan, it appears that the reactive residues form a ring. The substrates may recognize various combinations of residues in this ring during binding. Future work with other structurally diverse thiol substrates will determine whether other residues contribute to this "ring of recognition."

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Location of the cysteine residues that interact with MTS-rhodamine, MTS-verapamil, or dibromobimane. The numbered cylinders represent TM segments 4-6 and 8-12 of P-gp. The TMs forming the drug-binding site are arranged as a fan. The residues that are protected from inhibition by MTS-rhodamine (this study) are in red, those protected by MTS-verapamil (23) are in black, and those protected by dibromobimane (22) are in yellow.

	ACKNOWLEDGEMENTS

We thank Dr. Randal Kaufman (Boston, MA) for pMT21. We thank Claire Bartlett for assistance with tissue culture.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant RO1 CA80900 and grants from the Canadian Institutes for Health Research and the Canadian Cystic Fibrosis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Investigator of the Canadian Institutes of Health Research. To whom correspondence should be addressed: Dept. of Medicine, University of Toronto, Rm. 7342, Medical Sciences Bldg., 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Tel. or Fax: 416-978-1105.

Published, JBC Papers in Press, September 9, 2002, DOI 10.1074/jbc.M208433200

	ABBREVIATIONS

The abbreviations used are: P-gp, P-glycoprotein(s); MTS, methanethiosulfonate; TM, transmembrane; HEK, human embryonic kidney.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				T. W. Loo, M. C. Bartlett, and D. M. Clarke Arginines in the First Transmembrane Segment Promote Maturation of a P-glycoprotein Processing Mutant by Hydrogen Bond Interactions with Tyrosines in Transmembrane Segment 11 J. Biol. Chem., September 5, 2008; 283(36): 24860 - 24870. [Abstract] [Full Text] [PDF]

				J. M. Gow, L. M. Hodges, L. W. Chinn, and D. L. Kroetz Substrate-Dependent Effects of Human ABCB1 Coding Polymorphisms J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 435 - 442. [Abstract] [Full Text] [PDF]

				Y. Zhang, D.-H. Lin, Z.-J. Wang, Y. Jin, B. Yang, and W.-H. Wang K restriction inhibits protein phosphatase 2B (PP2B) and suppression of PP2B decreases ROMK channel activity in the CCD Am J Physiol Cell Physiol, March 1, 2008; 294(3): C765 - C773. [Abstract] [Full Text] [PDF]

				M. F. De Rosa, C. Ackerley, B. Wang, S. Ito, D. M. Clarke, and C. Lingwood Inhibition of Multidrug Resistance by AdamantylGb3, a Globotriaosylceramide Analog J. Biol. Chem., February 22, 2008; 283(8): 4501 - 4511. [Abstract] [Full Text] [PDF]

				T. W. Loo, M. C. Bartlett, and D. M. Clarke Suppressor Mutations in the Transmembrane Segments of P-glycoprotein Promote Maturation of Processing Mutants and Disrupt a Subset of Drug-binding Sites J. Biol. Chem., November 2, 2007; 282(44): 32043 - 32052. [Abstract] [Full Text] [PDF]

				K. F.K. Ejendal, N. K. Diop, L. C. Schweiger, and C. A. Hrycyna The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding. Protein Sci., July 1, 2006; 15(7): 1597 - 1607. [Abstract] [Full Text] [PDF]

				J. Rautio, J. E. Humphreys, L. O. Webster, A. Balakrishnan, J. P. Keogh, J. R. Kunta, C. J. Serabjit-Singh, and J. W. Polli IN VITRO P-GLYCOPROTEIN INHIBITION ASSAYS FOR ASSESSMENT OF CLINICAL DRUG INTERACTION POTENTIAL OF NEW DRUG CANDIDATES: A RECOMMENDATION FOR PROBE SUBSTRATES Drug Metab. Dispos., May 1, 2006; 34(5): 786 - 792. [Abstract] [Full Text] [PDF]

				N. Maki, K. Moitra, P. Ghosh, and S. Dey Allosteric Modulation Bypasses the Requirement for ATP Hydrolysis in Regenerating Low Affinity Transition State Conformation of Human P-glycoprotein J. Biol. Chem., April 21, 2006; 281(16): 10769 - 10777. [Abstract] [Full Text] [PDF]

				W.-H. Wang Regulation of ROMK (Kir1.1) channels: new mechanisms and aspects Am J Physiol Renal Physiol, January 1, 2006; 290(1): F14 - F19. [Abstract] [Full Text] [PDF]

				C. Moreau, F. Gally, H. Jacquet-Bouix, and M. Vivaudou The Size of a Single Residue of the Sulfonylurea Receptor Dictates the Effectiveness of KATP Channel Openers Mol. Pharmacol., April 1, 2005; 67(4): 1026 - 1033. [Abstract] [Full Text] [PDF]

				D.-H. Lin, H. Sterling, Z. Wang, E. Babilonia, B. Yang, K. Dong, S. C. Hebert, G. Giebisch, and W.-H. Wang ROMK1 channel activity is regulated by monoubiquitination PNAS, March 22, 2005; 102(12): 4306 - 4311. [Abstract] [Full Text] [PDF]

				T. W. Loo, M. C. Bartlett, and D. M. Clarke The Dileucine Motif at the COOH Terminus of Human Multidrug Resistance P-glycoprotein Is Important for Folding but Not Activity J. Biol. Chem., January 28, 2005; 280(4): 2522 - 2528. [Abstract] [Full Text] [PDF]