JBC PeproTech; Our Business is Cytokines!

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M400229200 on January 28, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18232-18238, April 30, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/18/18232    most recent
M400229200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Loo, T. W.
Right arrow Articles by Clarke, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Loo, T. W.
Right arrow Articles by Clarke, D. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Val133 and Cys137 in Transmembrane Segment 2 Are Close to Arg935 and Gly939 in Transmembrane Segment 11 of Human P-glycoprotein*

Tip W. Loo, M. Claire Bartlett, and David M. Clarke{ddagger}

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

Received for publication, January 9, 2004 , and in revised form, January 25, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
P-glycoprotein (P-gp; ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The protein has two homologous halves joined by a linker region. Each half consists of a transmembrane (TM) domain with six TM segments and a nucleotide-binding domain. The drug substrate-binding pocket is at the interface between the TM segments in each half of the protein. Preliminary studies suggested that the arrangement of the two halves of P-gp shows rotational symmetry (i.e. "head-to-tail" arrangement). Here, we tested this model by determining whether the cytoplasmic ends of TM2 and TM3 in the N-terminal half are in close contact with TM11 in the C-terminal half. Mutants containing a pair of cysteines in TM2/TM11 or TM3/TM11 were subjected to oxidative cross-linking with copper phenanthroline. Two of the 110 TM2/TM11 mutants, V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C (TM11), were cross-linked at 4 °C, when thermal motion is reduced. Cross-linking was specific since no cross-linked product was detected in the 100 double Cys TM3/TM11 mutants. Vanadate trapping of nucleotide or the presence of some drug substrates inhibited cross-linking of mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). Cross-linking of TM2 and TM11 also blocked drug-stimulated ATPase activity. The close proximity of TM2/TM11 and TM5/TM8 (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2004) J. Biol. Chem. 279, 7692–7697) indicates that these regions between the two halves must enclose the drug-binding pocket at the cytoplasmic side of P-gp. They may form the "hinges" required for conformational changes during the transport cycle.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human multidrug resistance P-glycoprotein (P-gp1; ABCB1) uses ATP to transport a wide variety of structurally unrelated compounds of different sizes from the cell. The physiological function of P-gp is unknown. It is present in relatively higher levels in some organs such as the intestine, kidney, and blood-brain/testes barrier and therefore may function to protect the organism from toxins in the diet and the environment (1–3). Its relatively high expression in these organs can affect the therapeutic efficacy of oral drugs, whereas overexpression of P-gp in some tumor cells can complicate cancer chemotherapy regimens (4, 5).

P-gp is a member of the ATP-binding cassette family of transporters (6). Its 1280 amino acids are arranged as two homologous halves with 43% amino acid identity. A linker region of ~60 amino acids connects the two halves of the protein (7). Each half has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (8, 9). The protein functions as a monomer (10). Each half of P-gp has basal ATPase activity, but drug-stimulated ATPase activity or conferral of drug resistance requires the presence of both halves of the protein (11, 12). Both halves do not have to be covalently linked for function (11). Both nucleotide-binding domains (NBDs) are essential for activity (13–15), and the two ATP molecules likely interact at the interface of the Walker A site in one NBD and the LSGGQ consensus site in the other NBD (16). Drug substrates that stimulate or inhibit ATPase activity cause these sequences to come closer or to move farther apart, respectively (17).

The TM segments likely interact with drug substrates in the lipid bilayer (18–20). Studies on P-gp deletion mutants show that the transmembrane domains (TMDs) alone can bind drug substrates (12). Drug substrates bind at distinct regions in a common drug-binding pocket that is formed by the interface between the TMDs of both halves of P-gp (21–25). Disulfide cross-linking studies and labeling of cysteine mutants with thiol-reactive drug substrates indicate that the two halves are arranged in a "head-to-tail" arrangement (26), with TM4–6 of TMD1 (the N-terminal TMD containing TM1–6) and TM9–12 of TMD2 (the C-terminal TMD containing TM7–12) forming the drug-binding pocket (25, 27). Drug binding involves an induced fit mechanism (28), and only binding in some orientations can allow for conformational changes to induce ATP hydrolysis (29, 30). Therefore, knowledge about the packing of the TM segments between the two halves of P-gp is important for understanding the mechanism of P-gp.

The drug-binding pocket is "funnel-shaped," narrow at the cytoplasmic side and wider at the extracellular end (27). We recently showed that the homologous halves of P-gp are in close contact at the cytoplasmic ends of TM5 and TM8 (31). We predicted the presence of another contact point between TM2 or TM3 with TM11 in the two halves of P-gp for the formation of the funnel-shaped drug-binding pocket. In this study, we used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine the position of the cytoplasmic side of TM11 in TMD2 relative to that of TM2 and TM3 in TMD1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Mutants—An active Cys-less P-gp was constructed by replacing the seven endogenous cysteines at positions 137, 431, 717, 956, 1074, 1125, and 1227 with alanines (8). A 10-histidine tag was attached to the C-terminal end of the Cys-less P-gp cDNA. The presence of a histidine tag facilitates purification of the P-gp mutants by nickel chelate chromatography (32). Pairs of cysteines were reintroduced into the histidine-tagged Cys-less P-gp cDNA near the cytoplasmic ends of TM2 (positions 128–138), TM3 (positions 188–197), and TM11 (positions 935–944).

Disulfide Cross-linking Analysis—Human embryonic kidney (HEK) 293 cells were transfected with the mutant cDNAs. The medium was replaced with fresh medium after 24 h, and the cells were grown for another 48 h at 27 °C. The cells were harvested and washed once with phosphate-buffered saline (PBS; 10 mM sodium phosphate and 150 mM NaCl), pH 7.4, and the membranes were prepared as described previously (33). The membranes were suspended in PBS, pH 7.4, and samples were cross-linked by incubation with 1 mM Cu(II)(phenanthroline)3 as the oxidant for various times at 4, 22, or 37 °C as described in the figure legends.

To test for the effect of drug substrates on cross-linking, the membranes were preincubated for 10 min at 22 °C in the presence of no drug, 1 mM verapamil, 1 mM demecolcine, 1 mM rhodamine B, 0.5 mM cis-(Z)-flupenthixol, 0.5 mM trans-(E)-flupenthixol, 0.1 mM cyclosporin A, 0.5 mM Hoechst 33342, 0.5 mM progesterone, 5 mM colchicine, or 0.2 mM vinblastine before addition of oxidant for 10 min at 22 °C. To test for the effect of nucleotides or vanadate trapping of nucleotide on cross-linking, the membranes were suspended in Tris-buffered saline (10 mM Tris-HCl, pH 7.4, and 150 mM NaCl), pH 7.4. Samples were incubated for 10 min at 22 °C in the presence of 10 mM ATP, 20 mM MgCl2, and 0.2 mM sodium vanadate. Sodium vanadate was boiled for 3 min to break polymeric species (34) and chilled in an ice bath before use. A sample of membrane was also incubated for 10 min at 22 °C in the presence of 20 mM MgCl2 with 10 mM ATP, 0.2 mM sodium vanadate, 10 mM ADP, or 10 mM AMP-PNP. The samples were incubated at 22 °C for 10 min before cross-linking at 22 °C for 10 min. The reactions were stopped by addition of SDS sample buffer (125 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, and 4% (w/v) SDS) containing 50 mM EDTA and no reducing agent. The reaction mixtures were subjected to SDS-PAGE (7.5% polyacrylamide gels) and immunoblot analysis with a rabbit polyclonal antibody against P-gp (35).

Purification and Measurement of Drug-stimulated ATPase Activity of P-gp Mutants—Fifty plates (10-cm diameter) of HEK 293 cells were transfected with mutant cDNA. After 24 h at 37 °C, the medium was replaced with fresh medium containing 10 µM cyclosporin A. Cyclosporin A, a substrate of P-gp, acts as a potent chemical chaperone in increasing the yield of P-gp by promoting proper folding and packing of the TM segments (12, 36–38). After another 24 h at 37 °C, the cells were harvested and washed three times with PBS, pH 7.4, and suspended in PBS. The cells were solubilized at 4 °C with 1 volume (0.75 ml) of PBS containing 2% (w/v) n-dodecyl {beta}-D-maltoside. After 15 min at 4 °C, insoluble material was removed by centrifugation at 16,000 x g for 15 min at 4 °C. The supernatant was passed through a DNA miniprep microcentrifuge column (QIAGEN Inc.) to remove any DNA. The flowthrough material was subjected to nickel chelate chromatography as described previously (32). The recovery of P-gp was monitored by immunoblot analysis with rabbit anti-P-gp polyclonal antibody (35) and enhanced chemiluminescence (Pierce). An aliquot of the isolated P-gp-His10 was mixed with an equal volume of 10 mg/ml sheep brain phosphatidylethanolamine (Type II-S, Sigma) that had been washed and suspended in 10 mM Tris-HCl, pH 7.5, and 150 mM NaCl. The P-gp and lipid mixture was sonicated. A sample of the P-gp/lipid mixture was incubated with an equal volume of ATPase buffer containing 100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM MgCl2, 10 mM ATP, and either no drug substrate or 2 mM verapamil. These concentrations caused maximal stimulation of the ATPase activity of Cys-less P-gp. The samples were incubated at 37 °C for 30 min, and the amount of inorganic phosphate liberated was determined (39).

To test for the effect of cross-linking on ATPase activity, membranes were prepared from cells expressing histidine-tagged Cys-less P-gp or mutant C137C(TM2)/A935C(TM11) and treated with or without oxidant at 22 °C for 10 min. The reaction was stopped by addition of EDTA to 1 mM. The membranes were then solubilized by addition of an equal volume of PBS containing 2% (w/v) n-dodecyl {beta}-D-maltoside detergent. The P-gps were isolated by nickel chelate chromatography. The isolated P-gps were mixed with lipid and sonicated, and ATPase assays were performed as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The 12 predicted TM segments of P-gp are shown in Fig. 1A. Each TMD contains six TM segments that can interact with each other when expressed as separate polypeptides or when the NBDs are deleted (35). Cysteine-scanning mutagenesis of the TM segments and reaction of the cysteine mutants with thiol-reactive drug substrates indicate that TM4–6 in the N-terminal half and TM9–12 in the C-terminal half contribute residues to the common drug-binding pocket (25, 29, 40). This pocket is shaped like a funnel, with the narrowest part at the cytoplasmic ends of the TM segments (27). We recently showed that two halves of P-gp are close together at N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C (TM8), and G300C(TM5)/F770C(TM8), suggesting that the packing of the two halves shows rotational symmetry (i.e. head-to-tail arrangement) (31). Since the two halves of P-gp share 43% amino acid identity, we predicted that TM2 and/or TM3 could come into contact with TM11 to complete the narrow part of the funnel (Fig. 1B). Accordingly, we tested this prediction by using cysteine-scanning mutagenesis and oxidative cross-linking analysis. Pairs of cysteines were introduced at the cytoplasmic halves of either TM2 (positions 128–138) or TM3 (positions 188–197) and TM11 (positions 935–944) of the Cysless P-gp mutant. These positions are at the narrowest part of the funnel, and the probability of cross-linking with a zero-length cross-linker (copper phenanthroline) would likely be greatest at these positions. The Cys-less P-gp mutant has been useful in cross-linking experiments because it still confers multidrug resistance in transfected cells (8), and the presence of a disulfide bond between the TM segments in the two halves causes the protein to migrate with reduced mobility on SDS-polyacrylamide gels (41). The 210 double cysteine TM2/TM11 and TM3/TM11 mutants were expressed in HEK 293 cells. Membranes were prepared and treated with oxidant (copper phenanthroline) at 37 °C, and the presence of cross-linked product was detected by SDS-PAGE and immunoblot analysis. Three of the 110 TM2/TM11 mutants (V133C(TM2)/G939C(TM11), C137C(TM2)/A935C(TM11), and L138C(TM2)/A935C(TM11)) showed relatively strong cross-linking (>50%), whereas 12 mutants (Y130C(TM2)/G939C(TM11), Y130C(TM2)/I940C(TM11), Y130C(TM2)/F942C(TM11), Y130C(TM2)/S943C(TM11), V133C(TM2)/F938C(TM11), V133C(TM2)/F942C(TM11), V133C(TM2)/S943C(TM11), S134C(TM2)/A935C(TM11), S134C(TM2)/H936C(TM11), S134C(TM2)/G939C(TM11), S134C(TM2)/I940C(TM11), and C137C(TM2)/G939C(TM11)) showed relatively weak cross-linking (<50%) (Table I). When cross-linking was carried out at 22 °C, only mutants V133C(TM2)/G939C(TM11), C137C(TM2)/A935C (TM11), and L138C(TM2)/A935C(TM11) formed detectable cross-linked products. Examples of relatively strong cross-linking (mutants V133C(TM2)//G939C(TM11) and C137C(TM2)/A935C(TM11)) are shown in Fig. 2. No cross-linked product was detected when the single cysteine mutants V133C(TM2), G939C(TM11), C137C(TM2), and A935C(M11) were treated with copper phenanthroline. These results indicate that intramolecular cross-linking was responsible for the reduced mobility of the cross-linked product. No cross-linked product was detected in the 100 TM3/TM11 mutants (data not shown).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 1.
Orientation of the TM segments of P-gp. A, the four domains of P-gp are shown as a linear model. TMD1 consists of six TM segments (cylinders 1–6) from the N-terminal half of P-gp, followed by the N-terminal NBD (NBD1). TMD2 consists of six TM segments (cylinders 7–12) from the C-terminal half of P-gp, followed by the C-terminal NBD (NBD2). The branched lines represent glycosylation sites. B, the predicted orientation of the TM segments surrounding the common drug-binding pocket is shown.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Cross-linking between residues in TM2 and TM11

–, no cross-linked product detected on SDS-polyacrylamide gels at 37 °C; +, relatively weak cross-linking (<50% of P-gp cross-linked) at 37 °C; ++, relatively strong cross-linking (>50% of P-gp cross-linked) at 37 °C; *, cross-linked product also detected at 22 °C; **, cross-linked product also detected at 22 and 4 °C.

 



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 2.
Disulfide cross-linking of P-gp mutants. Membranes were prepared from HEK 293 cells expressing P-gp mutant V133C(TM2), G939C(TM11), C137C(TM2), A935C(TM11), V133C(TM2)/G939C (TM11), or C137C(TM2)/A935C(TM11). The membranes were treated with (+) or without (–) oxidant (copper phenanthroline (CuP)) for 10 min at 37 °C, and the reactions were stopped by addition of SDS sample buffer containing EDTA and no reducing agent. The mixtures were subjected to immunoblot analysis. The positions of the cross-linked (X-link) product and mature (170-kDa) P-gps are indicated.

 
When mutants V133C(TM2)/G939C(TM11), C137C(TM2)/A935C(TM11), and L138C(TM2)/A935C(TM11) were subjected to oxidative cross-linking for various times at 22 °C, cross-linked products were rapidly detected in mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11) within 1 min, but only after 16 min in mutant L138C(TM2)/A935C(TM11) (Fig. 3). At 4 °C, time-dependent cross-linking was observed in mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C (TM11), but not in mutant L138C(TM2)/A935C(TM11) even after 32 min (Fig. 3). Therefore, only mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11) were used for further analysis.



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 3.
Effect of temperature on cross-linking of mutants. Membranes were prepared from HEK 293 cells expressing P-gp mutant V133C(TM2)/G939C(TM11), C137C(TM2)/A935C(TM11), or L138C-(TM2)/A935C(TM11). The membranes were treated with 1 mM oxidant (copper phenanthroline (CuP)) at 22 and 4 °C for the indicated times. The reactions were stopped by addition of SDS sample buffer containing EDTA and no reducing agent. The mixtures were subjected to immunoblot analysis. The positions of the cross-linked (X-link) product and mature (170-kDa) P-gps are indicated.

 
To determine whether mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11) retained activity, we assayed for drug-stimulated ATPase activity. Drug-stimulated ATPase activity is a useful assay because it has been shown that the turnover numbers for transport and drug-stimulated ATPase activity are comparable (42). In addition, there is good correlation between drug resistance and ATPase assays (43). Accordingly, histidine-tagged mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11) were expressed in HEK 293 cells, isolated by nickel chelate chromatography, mixed with lipid, and assayed for verapamil-stimulated ATPase activity. Verapamil was used, as it highly stimulates (>10-fold) the ATPase activity of Cys-less P-gp (17). Under saturating concentrations of verapamil (1 mM), mutant C137C(TM2)/A935C(TM11) showed about the same amount of ATPase activity as Cys-less P-gp, whereas mutant V133C(TM2)/G939C(TM11) had 70% of the activity of Cys-less P-gp.

The foregoing results of cross-linking indicate that TM2 and TM11 are close to each other at their cytoplasmic ends. It is possible that conformational changes between the two TMDs of P-gp are critical for coupling drug binding and/or release to ATP hydrolysis. Accordingly, we tested whether blocking movement between TM2 and TM11 by cross-linking mutant C137C(TM2)/A935C(TM11) affected drug-stimulated ATPase activity. Mutant C137C(TM2)/A935C(TM11) was selected since its verapamil-stimulated ATPase activity was similar to that of Cys-less P-gp. Membranes prepared from HEK 293 cells expressing mutant C137C(TM2)/A935C(TM11) were treated with or without 0.2 mM copper phenanthroline for 15 min at 22 °C, and the reaction was stopped by addition of EDTA to 1 mM.An equal volume of PBS containing 2% (w/v) n-dodecyl {beta}-D-maltoside detergent was added to the treated membranes, and the P-gp mutants were isolated by nickel chelate chromatography. The proteins were mixed with lipid, sonicated, and assayed for verapamil-stimulated ATPase activity. Fig. 4 shows that cross-linking of mutant C137C(TM2)/A935C(TM11) inhibited ATPase activity by 92%, whereas the activity of the Cys-less mutant was relatively unaffected by treatment with oxidant. Therefore, conformational changes or movement between TM2 and TM11 are required for coupling drug binding to ATPase activity.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 4.
Effect of cross-linking on verapamil-stimulated ATPase activity of Cys-less P-gp and mutant C137C(TM2)/A935C(TM11). Membranes were prepared from cells expressing histidine-tagged Cysless (C-less) P-gp or mutant C137C(TM2)/A935C(TM11) and treated with (+) or without (–) oxidant (copper phenanthroline (CuP)) at 22 °C for 10 min. The P-gps were then isolated by nickel chelate chromatography. Samples were mixed with lipids and sonicated, and verapamil-stimulated ATPase activities were determined. The activities are expressed relative to that of a sample that was mock-treated with oxidant and are the average of two different experiments.

 
We then tested whether ATPase activity affected the cross-linking pattern of mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). P-gp is an interesting ATPase because exposure to vanadate during ATP hydrolysis allows the protein to be trapped in a transition state (15). Vanadate traps ADP at either NBD by mimicking the transition state of the {gamma}-phosphate of ATP during ATP hydrolysis. Vanadate trapping at one NBD inhibits ATP hydrolysis at the second site (15). Accordingly, membranes were prepared from HEK 293 cells expressing mutant V133C(TM2)/G939C(TM11) or C137C-(TM2)/A935C(TM11) and preincubated with or without ATP plus vanadate, ATP, vanadate, ADP, or the non-hydrolyzable ATP analog AMP-PNP. AMP-PNP was included because it has been reported that the binding of AMP-PNP by P-gp causes large conformational changes in the TMDs (44). The membranes were treated with oxidant, and the samples were subjected to immunoblot analysis. Fig. 5 shows that vanadate trapping of nucleotide almost completely abolished cross-linking in both mutants. This result indicates that TM2 and TM11 move apart in the transition state. Alternatively, there could be rotation of one or both TM segments. By contrast, the presence of ATP, ADP, vanadate alone, or AMP-PNP did not affect cross-linking of the mutant. The presence of ATP did not inhibit cross-linking probably because the protein only transiently adopts the transition state conformation during ATP hydrolysis. Since cross-linking was not inhibited by AMP-PNP, it appears that occupation of the ATP-binding sites by nucleotides is unlikely to cause large conformational changes between TM2 and TM11.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 5.
Effect of nucleotides and vanadate trapping of nucleotide on cross-linking of P-gp mutants. Membranes from HEK 293 cells expressing mutant V133C(TM2)/G939C(TM11) or C137C(TM2)/A935C(TM11) containing 20 mM MgCl2 were preincubated with no additions (None) or with ATP plus vanadate, ATP, sodium vanadate, ADP, or AMP-PNP. The samples were then treated with (+) or without (–) oxidant (copper phenanthroline (CuP)) for 10 min at 22 °C. The reactions were stopped by addition of SDS sample buffer containing EDTA and no reducing agent. The mixtures were subjected to immunoblot analysis. The positions of the cross-linked (X-link) product and mature (170-kDa) P-gps are indicated.

 
We have shown that residues in TM11 line the drug-binding pocket of P-gp (22). We then tested whether drug substrates would affect the cross-linking pattern of mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). The "substrate-induced" fit mechanism for drug binding (28) predicts that the packing of the TM segments would be changed by the presence of different drug substrates. Membranes were prepared from HEK 293 cells expressing mutant V133C(TM2)/G939C(TM11) or C137C(TM2)/A935C(TM11) and preincubated with the drug substrates verapamil, demecolcine, rhodamine B, the cis- and trans-isomers of flupenthixol, cyclosporin A, Hoechst 33342, progesterone, colchicine, and vinblastine before cross-linking with oxidant. Verapamil and demecolcine were selected because they highly stimulate P-gp ATPase activity. Rhodamine B, Hoechst 33342, colchicine, and vinblastine are commonly used P-gp substrates, whereas the isomers of flupenthixol, cyclosporin A, and progesterone are P-gp modulators. Fig. 6 shows that cross-linking was not significantly inhibited (<25% change) in the presence of verapamil, demecolcine, cyclosporin A, progesterone, or colchicine in either mutant. The cross-linking of both mutants, however, was inhibited by the presence of rhodamine B. Three differences were observed in the presence of the isomers of flupenthixol (~2–4-fold reduction in cross-linking), Hoechst 33342, or vinblastine. Mutant V133C(TM2)/G939C(TM11) was protected from cross-linking by Hoechst 33342, vinblastine, or the isomers of flupenthixol, whereas mutant C137C(TM2)/A935C(TM11) was not. These results suggest that Hoechst 33342, vinblastine, or the isomers of flupenthixol induced a conformational change that moved Cys133 in TM2 and Cys939 in TM11 far enough apart to inhibit cross-linking. Cys137 in TM2 and Cys935 in TM11 remained close enough to be cross-linked because they are closer to the cytoplasmic ends of the helices. When the sequences of the homologous halves of P-gp are compared, Val133 in TM2 corresponds to Gly774 in TM8, whereas Gly939 in TM11 corresponds to Asn296 in TM5 (Fig. 7A). This is interesting because both mutants V133C(TM2)/G939C(TM11) (this study) and N296C(TM5)/G774C(TM8) (31) were cross-linked. Therefore, we tested the effects of drug substrates on the cross-linking pattern of mutant N296C(TM5)/G774C(TM8). Some differences were observed when the mutants were compared. Cross-linking of mutant V133C(TM2)/G939C(TM11), but not mutant N296C(TM5)/G77C(TM8), was inhibited by Hoechst 33342 and vinblastine. Progesterone inhibited some cross-linking in mutant N296C(TM5)/G774C(TM8) (~50% decrease), but not in mutant V133C(TM2)/G939C(TM11) (Fig. 6). Although the cross-linkable sites are located at equivalent positions in a rotational symmetry (head-to-tail arrangement) of the TM segments in the two halves, the same drugs induced different conformational changes at both sites.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 6.
Effect of drug substrates on cross-linking. A, membranes were prepared from HEK 293 cells expressing P-gp mutant V133C(TM2)/G939C(TM11), C137C(TM2)/A935C(TM11), or N296C-(TM5)/G774C(TM8). The membranes were preincubated at 22 °C for 10 min in the presence of no drug substrate (None), 1 mM verapamil (Ver), 1 mM demecolcine (Deme), 1 mM rhodamine B (Rhod), 0.5 mM cis-(Z)-flupenthixol (C-Flu), 0.5 mM trans-(E)-flupenthixol (T-Flu), 0.1 mM cyclosporin A (Cyclo), 0.5 mM Hoechst 33342 (Hoechst), 0.5 mM progesterone (Prog), 5 mM colchicine (Colch), or 0.2 mM vinblastine (Vin). The samples were treated with (+) or without (–) 0.1 mM oxidant (copper phenanthroline (CuP)) for 10 min at 22 °C. The reactions were stopped by addition of SDS sample buffer containing EDTA and no reducing agent. The mixtures were subjected to immunoblot analysis. The positions of the cross-linked (X-link) product and mature (170-kDa) P-gps are indicated. B, cross-linking was quantitated by scanning the gel lanes, followed by analysis with a Macintosh computer using the NIH Image program (available at rsb.info.nih.gov/nih-image/). The amount of cross-linking is expressed relative to the sample cross-linked in the absence of drug substrate.

 



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 7.
Model of interaction between TM2 and TM11. A, the residues of homologous TM segments in each half of P-gp are aligned: TM5 (Ala311–Thr294) and TM11 (Ala954–Ile937); and TM2 (Tyr118–Phe135) and TM8 (Phe759–Thr776). Residues that occupy equivalent positions in the TM segments and that are cross-linked (V133C(TM2)/G939C(TM11) and N296C(TM5)/G774C(TM8), respectively) are boxed. B, TM2 and TM11 are shown as {alpha}-helices, with the amino acid positions shown as white circles. The lines between the residues in TM2 and TM11 represent positions that, when mutated to cysteine, will cross-link when treated with oxidant at 4 °C.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our original model of TM segment packing, we predicted that each TMD lined one side of the drug-binding pocket (26). The TM segments in each TMD were in a linear head-to-tail arrangement such that TM1 was close to TM12, whereas TM6 was close to TM7. This arrangement of the TM segments resulted in rotational symmetry around the drug-binding pocket (26). The recent crystal structure of the related MsbA lipid A half-transporter from Escherichia coli (45) and Vibrio cholera (46) indicates that the TM segments in their TMD are not arranged in a linear order. If P-gp showed a similar organization, then TM5 and TM2 in the N-terminal half would lie close to TM8 and TM11 in the C-terminal half of P-gp, respectively (Fig. 1B). In a previous study, we showed that the TM5/TM8 (but not TM6/TM8 and TM6/TM7) cytoplasmic ends are indeed close together (31).

In this study, cross-linking analysis of the residues on the cytoplasmic side of TM2 or TM3 and TM11 indicated that TM2 in TMD1 is in close proximity to TM11 in TMD2. Two mutants, V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11), were particularly interesting because they were cross-linked at 4 °C, when molecular motion is low. Copper phenanthroline is very efficient in promoting the formation of the disulfide bond between thiol groups if their {beta}-carbons are a maximal distance of 7 Å from each other, with the average being 5–6 Å, or when the sulfhydryl groups are brought close together transiently because of molecular motion (47). This would account for the large number of mutants that were cross-linked at 37 °C (Table I).

The observation that Cys133 and Cys137 in TM2 can form disulfide bonds with Cys935 and Cys939 in TM11, respectively, in the presence of oxidant at 4 °C suggests that these residues are within 5 Å of each other. When the residues in TM2 and TM11 are modeled as {alpha}-helices (Fig. 7B), the predicted locations of residues 133 and 137 in TM2 and residues 935 and 939 in TM11 are consistent with the experimental results. Residues 133 and 137 are predicted to lie on one face of the helix, whereas residues 935 and 939 are separated by one turn of the helix, but are on the same face of TM11. These results, as well as those from previous cross-linking studies (26, 31), indicate that the two TMDs are in close proximity at the cytoplasmic side (Fig. 1B). This arrangement of the P-gp TMDs is incompatible with the E. coli MsbA crystal structure (45) and with the P-gp model proposed by Seigneuret and Garnier-Suillerot (48). In both structures, the TMDs are arranged such that they are close at the extracellular ends, whereas the cytoplasmic ends of the TMDs as well as the NBDs are relatively far apart. In contrast, a projection structure of P-gp in a lipid bilayer shows that the protein adopts a more compact structure, with the two NBDs close together (49). Such a structure would also bring the cytoplasmic ends of the TMDs close together. Indeed, cross-linking analysis of the NBDs shows that the Walker A consensus sequence in one NBD is close to the LSGGQ sequence in the other NBD (16). A compact P-gp structure in which the two NBDs and the cytoplasmic ends of the two TMDs are close together is more consistent with the V. cholera MsbA structure (46).

Recently, Stenham et al. (50) proposed two detailed models of P-gp founded on disulfide cross-linking studies and homology modeling based on the E. coli MsbA structure. Both models incorporated the idea that the two NBDs form a consensus interface (16, 51, 52), but greatly differed in their TMD1/TMD2 interfaces. The first model maintained the NBD/TMD interface as seen with E. coli MsbA. In the second model, the NBDs were rotated with respect to their cognate TMDs. The second model was found to be more compatible with our cross-linking data (26, 27, 53, 54). The authors also cross-linked residues at the extracellular ends of TM2, TM5, TM8, and TM11 with linkers of 6–16 Å at 37 °C. They showed cross-linking between TM5 and TM8 and between TM2 and TM11. Our results showing cross-linking between the cytoplasmic ends of TM5 and TM8 (31) and between TM2 and TM11 (this study) are consistent with the second model.

The contact points between V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11) are not static, but appear to undergo considerable motion during the transport cycle. Conformational changes at the TM2/TM11 interface appear to be an important requirement during the transport cycle because cross-linking inhibited ATPase activity.

The orientations of TM2 and TM11 were sensitive to ATP hydrolysis, as vanadate trapping of nucleotide inhibited cross-linking of both mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). Nucleotide binding alone was not sufficient to block cross-linking since no detectable reduction in cross-linking was observed in the presence of ATP, ADP, or AMP-PNP.

Conformational changes were also observed with some substrates. Some substrates such as rhodamine B inhibited cross-linking of both mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). Perhaps binding of rhodamine B caused TM2 and TM11 to move apart. Alternatively, binding of rhodamine B may have caused at least one of the TM segments to rotate in P-gp (28, 40, 55). Other drug substrates such as Hoechst 33342 and vinblastine inhibited cross-linking of only mutant V133C(TM2)/G939C(TM11). Residues 133 and 939 are predicted to be closer to the middle of the lipid bilayer. This may explain why mutant V133C(TM2)/G939C(TM11), but not mutant C137C(TM2)/A935C(TM11), was inhibited. Hoechst 33342 or vinblastine may induce a "hinge-like" motion at TM2 and TM11 so that only the extracellular ends of these TM segments move farther apart. Not all drug substrates inhibited cross-linking. No detectable effect on cross-linking was observed with drug substrates such as verapamil, demecolcine, cyclosporin A, and colchicine (Fig. 6). The observation that different drug substrates have different effects on cross-linking would be consistent with the idea of the substrate-induced fit mechanism for drug binding (28). In this mechanism, packing of the TM segments is rearranged when P-gp binds to a particular drug substrate.

In summary, the close proximity of TM2 and TM11 is consistent with models of P-gp that place the cytoplasmic ends of the TMDs as well as the NBDs close together (Fig. 1B). The conformational sensitivity of the TM2/TM11 and TM5/TM8 interfaces to ATP hydrolysis and drug binding suggests that these regions of the drug-binding pocket may form hinges that allow conformation changes to occur during the transport cycle.


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

{ddagger} Recipient of the Canada Research Chair in Membrane Biology. To whom correspondence should be addressed: Dept. of Medicine, University of Toronto, Medical Sciences Bldg., Rm. 7342, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel./Fax: 416-978-1105; E-mail: david.clarke{at}utoronto.ca.

1 The abbreviations used are: P-gp, P-glycoprotein; TM, transmembrane (segment); NBD, nucleotide-binding domain; TMD, transmembrane domain; HEK, human embryonic kidney; PBS, phosphate-buffered saline; AMP-PNP, adenosine 5'-({beta},{gamma}-imino)triphosphate. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gottesman, M. M., Fojo, T., and Bates, S. E. (2002) Nat. Rev. Cancer 2, 48–58[CrossRef][Medline] [Order article via Infotrieve]
  2. Hrycyna, C. A. (2001) Semin. Cell Dev. Biol. 12, 247–256[CrossRef][Medline] [Order article via Infotrieve]
  3. Loo, T. W., and Clarke, D. M. (1999) Biochem. Cell Biol. 77, 11–23[CrossRef][Medline] [Order article via Infotrieve]
  4. Lee, C. G., Gottesman, M. M., Cardarelli, C. O., Ramachandra, M., Jeang, K. T., Ambudkar, S. V., Pastan, I., and Dey, S. (1998) Biochemistry 37, 3594–3601[CrossRef][Medline] [Order article via Infotrieve]
  5. Thomas, H., and Coley, H. M. (2003) Cancer Control 10, 159–165[Medline] [Order article via Infotrieve]
  6. Dean, M., Rzhetsky, A., and Allikmets, R. (2001) Genome Res. 11, 1156–1166[Abstract/Free Full Text]
  7. Chen, C. J., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B. (1986) Cell 47, 381–389[CrossRef][Medline] [Order article via Infotrieve]
  8. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 843–848[Abstract/Free Full Text]
  9. Kast, C., Canfield, V., Levenson, R., and Gros, P. (1996) J. Biol. Chem. 271, 9240–9248[Abstract/Free Full Text]
  10. Loo, T. W., and Clarke, D. M. (1996) J. Biol. Chem. 271, 27488–27492[Abstract/Free Full Text]
  11. Loo, T. W., and Clarke, D. M. (1994) J. Biol. Chem. 269, 7750–7755[Abstract/Free Full Text]
  12. Loo, T. W., and Clarke, D. M. (1999) J. Biol. Chem. 274, 24759–24765[Abstract/Free Full Text]
  13. Azzaria, M., Schurr, E., and Gros, P. (1989) Mol. Cell. Biol. 9, 5289–5297[Abstract/Free Full Text]
  14. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 22957–22961[Abstract/Free Full Text]
  15. Urbatsch, I. L., Sankaran, B., Weber, J., and Senior, A. E. (1995) J. Biol. Chem. 270, 19383–19390[Abstract/Free Full Text]
  16. Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 41303–41306[Abstract/Free Full Text]
  17. Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2003) J. Biol. Chem. 278, 1575–1578[Abstract/Free Full Text]
  18. Homolya, L., Hollo, Z., Germann, U. A., Pastan, I., Gottesman, M. M., and Sarkadi, B. (1993) J. Biol. Chem. 268, 21493–21496[Abstract/Free Full Text]
  19. Raviv, Y., Pollard, H. B., Bruggemann, E. P., Pastan, I., and Gottesman, M. M. (1990) J. Biol. Chem. 265, 3975–3980[Abstract/Free Full Text]
  20. Omote, H., and Al-Shawi, M. K. (2002) J. Biol. Chem. 277, 45688–45694[Abstract/Free Full Text]
  21. Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945–31948[Abstract/Free Full Text]
  22. Loo, T. W., and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388–35392[Abstract/Free Full Text]
  23. Loo, T. W., and Clarke, D. M. (2000) J. Biol. Chem. 275, 39272–39278[Abstract/Free Full Text]
  24. Loo, T. W., and Clarke, D. M. (2001) J. Biol. Chem. 276, 14972–14979[Abstract/Free Full Text]
  25. Loo, T. W., and Clarke, D. M. (2002) J. Biol. Chem. 277, 44332–44338[Abstract/Free Full Text]
  26. Loo, T. W., and Clarke, D. M. (2000) J. Biol. Chem. 275, 5253–5256[Abstract/Free Full Text]
  27. Loo, T. W., and Clarke, D. M. (2001) J. Biol. Chem. 276, 36877–36880[Abstract/Free Full Text]
  28. Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2003) J. Biol. Chem. 278, 13603–13606[Abstract/Free Full Text]
  29. Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2003) J. Biol. Chem. 278, 20449–20452[Abstract/Free Full Text]
  30. Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2003) J. Biol. Chem. 278, 50136–50141[Abstract/Free Full Text]
  31. Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2004) J. Biol. Chem. 279, 7692–7697[Abstract/Free Full Text]
  32. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 21449–21452[Abstract/Free Full Text]
  33. Loo, T. W., and Clarke, D. M. (1994) J. Biol. Chem. 269, 7243–7248[Abstract/Free Full Text]
  34. Goodno, C. C. (1982) Methods Enzymol. 85, 116–123[Medline] [Order article via Infotrieve]
  35. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 21839–21844[Abstract/Free Full Text]
  36. Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 709–712[Abstract/Free Full Text]
  37. Loo, T. W., and Clarke, D. M. (1998) J. Biol. Chem. 273, 14671–14674[Abstract/Free Full Text]
  38. Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 27585–27588[Abstract/Free Full Text]
  39. Chifflet, S., Torriglia, A., Chiesa, R., and Tolosa, S. (1988) Anal. Biochem. 168, 1–4[CrossRef][Medline] [Order article via Infotrieve]
  40. Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2003) J. Biol. Chem. 278, 39706–39710[Abstract/Free Full Text]
  41. Loo, T. W., and Clarke, D. M. (1996) J. Biol. Chem. 271, 27482–27487[Abstract/Free Full Text]
  42. Ambudkar, S. V., Cardarelli, C. O., Pashinsky, I., and Stein, W. D. (1997) J. Biol. Chem. 272, 21160–21166[Abstract/Free Full Text]
  43. Loo, T. W., and Clarke, D. M. (1998) Methods Enzymol. 292, 480–492[Medline] [Order article via Infotrieve]
  44. Rosenberg, M. F., Velarde, G., Ford, R. C., Martin, C., Berridge, G., Kerr, I. D., Callaghan, R., Schmidlin, A., Wooding, C., Linton, K. J., and Higgins, C. F. (2001) EMBO J. 20, 5615–5625[CrossRef][Medline] [Order article via Infotrieve]
  45. Chang, G., and Roth, C. B. (2001) Science 293, 1793–1800[Abstract/Free Full Text]
  46. Chang, G. (2003) J. Mol. Biol. 330, 419–430[CrossRef][Medline] [Order article via Infotrieve]
  47. Katz, B. A., and Kossiakoff, A. (1986) J. Biol. Chem. 261, 15480–15485[Abstract/Free Full Text]
  48. Seigneuret, M., and Garnier-Suillerot, A. (2003) J. Biol. Chem. 278, 30115–30124[Abstract/Free Full Text]
  49. Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125–40131[Abstract/Free Full Text]
  50. Stenham, D. R., Campbell, J. D., Sansom, M. S., Higgins, C. F., Kerr, I. D., and Linton, K. J. (2003) FASEB J. 17, 2287–2289[Abstract/Free Full Text]
  51. Hopfner, K. P., Karcher, A., Shin, D. S., Craig, L., Arthur, L. M., Carney, J. P., and Tainer, J. A. (2000) Cell 101, 789–800[CrossRef][Medline] [Order article via Infotrieve]
  52. Smith, P. C., Karpowich, N., Millen, L., Moody, J. E., Rosen, J., Thomas, P. J., and Hunt, J. F. (2002) Mol. Cell 10, 139–149[CrossRef][Medline] [Order article via Infotrieve]
  53. Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986–20989[Abstract/Free Full Text]
  54. Loo, T. W., and Clarke, D. M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3511–3516[Abstract/Free Full Text]
  55. Loo, T. W., and Clarke, D. M. (2001) J. Biol. Chem. 276, 31800–31805[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. Keusekotten, R. M. Leonhardt, S. Ehses, and M. R. Knittler
Biogenesis of Functional Antigenic Peptide Transporter TAP Requires Assembly of Pre-existing TAP1 with Newly Synthesized TAP2
J. Biol. Chem., June 30, 2006; 281(26): 17545 - 17551.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
H. Omote and M. K. Al-Shawi
Interaction of Transported Drugs with the Lipid Bilayer and P-Glycoprotein through a Solvation Exchange Mechanism
Biophys. J., June 1, 2006; 90(11): 4046 - 4059.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
T. W. Loo and D. M. Clarke
Using a cysteine-less mutant to provide insight into the structure and mechanism of CFTR
J. Physiol., April 15, 2006; 572(2): 312 - 312.
[Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. A. Eisenhauer, S. Shames, P. D. Pawelek, and J. W. Coulton
Siderophore Transport through Escherichia coli Outer Membrane Receptor FhuA with Disulfide-tethered Cork and Barrel Domains
J. Biol. Chem., August 26, 2005; 280(34): 30574 - 30580.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
K. Pleban, S. Kopp, E. Csaszar, M. Peer, T. Hrebicek, A. Rizzi, G. F. Ecker, and P. Chiba
P-Glycoprotein Substrate Binding Domains Are Located at the Transmembrane Domain/Transmembrane Domain Interfaces: A Combined Photoaffinity Labeling-Protein Homology Modeling Approach
Mol. Pharmacol., February 1, 2005; 67(2): 365 - 374.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. F. Rosenberg, R. Callaghan, S. Modok, C. F. Higgins, and R. C. Ford
Three-dimensional Structure of P-glycoprotein: THE TRANSMEMBRANE REGIONS ADOPT AN ASYMMETRIC CONFIGURATION IN THE NUCLEOTIDE-BOUND STATE
J. Biol. Chem., January 28, 2005; 280(4): 2857 - 2862.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Y. Chen, M. C. Bartlett, T. W. Loo, and D. M. Clarke
The {Delta}F508 Mutation Disrupts Packing of the Transmembrane Segments of the Cystic Fibrosis Transmembrane Conductance Regulator
J. Biol. Chem., September 17, 2004; 279(38): 39620 - 39627.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. W. Loo, M. C. Bartlett, and D. M. Clarke
Processing Mutations Located throughout the Human Multidrug Resistance P-glycoprotein Disrupt Interactions between the Nucleotide Binding Domains
J. Biol. Chem., September 10, 2004; 279(37): 38395 - 38401.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/18/18232    most recent
M400229200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal