ABCMdb

ABCB1 p.Gly251Val

Predicted by SNAP2:	A: D (80%), C: D (75%), D: D (91%), E: D (91%), F: D (91%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (80%), P: D (91%), Q: D (91%), R: D (91%), S: D (75%), T: D (85%), V: D (91%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Suppressor mutations in the transmembrane segments... J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11. Loo TW, Bartlett MC, Clarke DM
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. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11., 2007-11-02 [PMID:17848563]

Abstract [show]
Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.

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94 Limited Proteolysis with Trypsin-HEK 293 cells expressing mutants G251V, L65R(TM1)/G251V, or wild-type P-gp were incubated in the presence or absence of 10 ␮M cyclosporin A for 24 h. The membranes were then prepared as described previously (37) and suspended in Tris-buffered saline. Login to comment
112 The positions of ⌬Y490 and G251V processing mutations are shown as gray circles. Login to comment
165 Processing mutant G251V was chosen because it yields very low levels of mature 170-kDa protein and could be rescued by the presence of arginine in TM5 (I306R) or in TM6 (F343R) (21). Login to comment
166 Accordingly, mutants L65R(TM1)/G251V, T199R(TM3)/G251V, I306R(TM5)/G251V (positive control), and A342R(TM6)/G251V (negative control) were constructed and expressed in HEK 293 cells. Whole cell SDS extracts were then subjected to immunoblot analysis. Fig. 4A shows that the presence of an arginine residue at positions 65(TM1), 199(TM3), and 306(TM5) promoted maturation of mutant G251V. Login to comment
185 Arginines Disrupt Subset of P-gp Drug-binding Sites NOVEMBER 2, 2007•VOLUME 282•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 32047 By contrast, the presence of an arginine at position 342(TM6) did not rescue mutant G251V. Login to comment
207 Effect of arginine mutations on maturation of the G251V and ⌬Y490 processing mutants. Login to comment
208 Wild-type, G251V (A), or ⌬Y490 (B) P-gps containing mutations L65R(TM1), T199R(TM3), I306R(TM5), or A342R(TM6) were expressed in HEK 293 cells. Whole cell SDS extracts were subjected to SDS-PAGE on 5.5% gels and immunoblot analysis. Login to comment
215 The positions of mature (170 kDa) and cross-linked (X-link) P-gps are indicated. Arginines Disrupt Subset of P-gp Drug-binding Sites 32048 prepared from cells expressing mutant G251V that were grown in the absence or presence of cyclosporin A, mutant L65R(TM1)/G251V, or wild-type P-gp. Login to comment
227 Arginines Disrupt Subset of P-gp Drug-binding Sites NOVEMBER 2, 2007•VOLUME 282•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 32049 by immunoblot analysis. Fig. 7 shows that the mature 170-kDa protein in mutant G251V expressed in the presence of cyclosporin A was 100-fold more resistant to trypsin compared with the mutant expressed in the absence of drug substrate. Login to comment
228 Similarly, the L65R(TM1)/G251V mutant and wild-type mature proteins were 100-fold more resistant to trypsin when compared with the 150-kDa protein of mutant G251V. Login to comment
229 These results indicate that the L65R mutation converts mutant G251V into a more protease-resistant conformation. Login to comment
240 Protease sensitivity of mutants G251V and L65R(TM1)/ G251V. Login to comment
241 Membranes prepared from HEK 293 cells expressing mutant G251V that were grown in the absence or presence (ϩCyclo A) of cyclosporin A, mutant L65R(TM1)/G251V or wild-type P-gps were treated with various concentrations of TPCK-trypsin. Login to comment

[hide] Arginines in the first transmembrane segment promo... J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2. Loo TW, Bartlett MC, Clarke DM
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. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PMID:18596043]

Abstract [show]
A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.

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2 To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). Login to comment
7 Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Login to comment
21 It was found that the L65R mutation promoted maturation of P-gp processing mutants such as ⌬Y490 and G251V (9). Login to comment
45 Accordingly,wefirstperformedarginine-scanningmutagenesis on TM1 of a P-gp processing mutant (G251V) to test their effects on maturation of the mutant. Login to comment
53 Mutations were introduced into wild-type or processing mutant G251V (13) P-gp cDNAs as described previously (6, 15). Login to comment
54 To perform arginine-scanning mutagenesis of TM1, the cDNA of mutant G251V P-gp was modified to create a set of mutants that contained one arginine at positions 52-72. Login to comment
71 RESULTS Effect of TM1 Arginines on Maturation of Processing Mutant G251V-The crystal structure of the bacterial ABC drug transporter Sav1866 showed that the 12 TM segments of the transport complex surrounded a funnel-shaped chamber exposed to the extracellular environment (24). Login to comment
91 Therefore, processing mutant G251V was selected for analysis as it shows inefficient maturation relative to wild-type P-gp (33). Login to comment
92 The G251V mutation is located in the first intracellular loop (see Fig. 1A). Login to comment
97 Immunoblot analysis of the G251V processing mutant, however, showed that the major product was the 150-kDa immature protein along with a minor amount (about 20%) of mature 170-kDa product (Fig. 2). Login to comment
98 The most common effect of introducing arginines into mutant G251V was to reduce the level of mature P-gp (12 of 21 mutants). Login to comment
100 Fig. 2 shows that the presence of an arginine at these positions reduced the level of mature product in mutant G251V from about 20% to less than 5% (Fig. 2B). Login to comment
101 Introduction of arginines at positions 52 or 61 had little effect on maturation of G251V P-gp. Login to comment
102 Introduction of arginines at the extracellular half of TM1 (positions 64, 65, 68, and 71), however, increased maturation of mutant G251V. Login to comment
118 Mutants G251V, V53R/G251V (inhibited maturation) and M68R/G251V (enhanced maturation) were subjected endoglycosidase H or F digestion to determine whether the alterations in mobility of the mutant P-gps were indeed due to changes in the glycosylation state of the protein. Login to comment
125 Because the M68R mutation was most efficient in promoting maturation of the G251V processing mutant, we tested whether it could also promote maturation of a different processing mutant. Login to comment
131 Drug Rescue of P-gp G251V Arginine Mutants That Do Not Show Enhanced Maturation-If the arginine mutants that showed reduced maturation relative to the G251V (such as FIGURE 2. Login to comment
132 Effect of TM1 arginine mutations on maturation of the G251V processing mutant. Login to comment
133 A, immunoblot analysis of whole cell extracts of HEK 293 cells expressing no P-gp (Control), wild-type P-gp (Wild-type), or a G251V processing mutant containing the indicated TM1 arginine mutations. Login to comment
138 Whole cell SDS extracts of HEK 293 cells expressing A52-tagged mutants G251V, V53R/G251V, or M68R/G251V were treated with endoglycosidase Hf (H),peptideN-glycosidaseF(F)ornoendoglycosidase(-).Equivalentamountsweresubjectedtoimmunoblot analysis. Login to comment
142 Therefore, mutants showing reduced (T55R/G251V) or similar maturation efficiencies (H61R/G251V, M69R/G251V) to the G251V parent were expressed in the presence of 5 ␮M cyclosporin A, 30 ␮M verapamil, 5 ␮M vinblastine, or 30 ␮M rhodamine B. Login to comment
143 These drug concentrations promoted rescue of the G251V parent so that mature (170 kDa) protein was the major product in the treated cells (Fig. 5). Login to comment
144 By contrast, a mutant where the introduced arginine inhibited maturation of G251V (mutant T55R) could not be rescued with any of the drug substrates (Fig. 5). Login to comment
146 For example, only cyclosporin A rescued mutant H61R/G251V (Fig. 5). Login to comment
147 By contrast, all drugs except verapamil rescued mutant M69R/G251V (Fig. 5). Login to comment
148 The effects of drug substrates on the expression of the other arginine/G251V mutants are summarized in Table 1. Login to comment
150 Mutants V52R, L67R, and L70R resembled the parent (G251V) P-gp, as expression in any of the drug substrates promoted efficient maturation (mature P-gp became the major product) of the protein. Login to comment
155 Effect of Suppressor Arginine Mutations on P-gp-Drug Interactions-Because mutant M68R/G251V showed efficient maturation (about 85%) when expressed in the absence of drug substrates (Fig. 2), we tested whether Arg-68 affected P-gp- drug interactions in disulfide protection assays using mutant L339C(TM6)/F728C(TM7) as described previously (9). Login to comment
156 Because the G64R and V71R changes also promoted maturation of the G251V processing mutant (Fig. 2), we also introduced these mutations into the L339C(TM6)/F728C(TM7) mutant. Login to comment
163 HEK 293 cells expressing mutants G251V, T55R/G251V, H61R/G251V, or M69R/G251V were treated with no drug (None), 5 ␮M cyclosporin A (Cyclo), 30 ␮M verapamil (Ver), 5 ␮M vinblastine (Vin), or 30 ␮M rhodamine B (Rhod) for 24 h. Whole cell extracts were then subjected to immunoblot analysis. Login to comment
165 TABLE 1 Effects of drug substrates on the maturation of TM1 arginine mutants containing the G251V mutation Mutation (G251V ؉) No drug Cyclosporin A Verapamil Vinblastine Rhodamine None - 111a,c 111 111 11 V52R -a,b 111 111 111 11 V53R 2 - - - - G64R 2 - - - - T55R 2 - - - - L56R 2 - - - - A57R 2 - - - - A58R 2 - - - - I59R 2 - - - - I60R 2 - - - - H61R - 111 1 1 1 G62R 2 - - - - A63R 2 - - - - G64R 1 111 1 1 11 L65R 11 111 11 11 11 P66R 2 1 - - - L67R - 111 111 111 11 M68R 111 111 111 111 111 M69R - 111 - 11 111 L70R - 111 111 111 11 V71R 1 111 111 111 11 F72R 2 1 1 1 1 a Change in the amount of mature (170 kDa) protein in the presence of drug substrate relative to that in the absence of drug substrate. Login to comment
167 c Change in the amount of mature (170 kDa) protein relative to the mutant G251V expressed in the absence of drug substrate. Login to comment
208 Effect of TM11 Mutations on Rescue of Mutant G251V Containing the L65R and M68R Changes-One reason that Arg-68 caused the largest increase in maturation of mutant G251V is that the arginine residue may promote packing of the TM segments. Login to comment
214 To test if Tyr-950 or Tyr-953 influences the ability of the M68R mutation to promote maturation of the G251V mutant, we introduced the Y950A or Y953A mutations into mutant M68R/ G251V mutant. Login to comment
216 Fig. 8 shows the maturation efficiencies compared with the M68R/G251V parent. Login to comment
218 Not all mutations in TM11, however, affected maturation of the M68R/G251V mutant. Login to comment
219 The presence of mutations Q946A, M948A, M949A, F951A, or S952A did not appear to affect the maturation of the M68R/ G251V mutant (Fig. 8A, lanes 2-5, 7, and 8). Login to comment
220 Because the L65R mutation also promoted maturation of mutant G251V, we tested whether the Y950A or Y953A mutations would have any effect on maturation of the L65R/G251V mutant. Login to comment
222 In contrast, both Y950A and Y953A affected the maturation efficiency of the M68R/G251V mutant. Login to comment
223 The Y950A and Y953A mutations did not affect maturation of the (G251V) parent (Fig. 8A, lanes 13 and 14) or wild-type P-gp (data not shown). Login to comment
224 These results suggest that interactions between Arg-65(TM1) and Tyr-950(TM11), Arg-68(TM1) and Tyr-950(TM11), or Arg-68 (TM1) and Tyr953(TM11) may be responsible for the enhanced maturation of the G251V mutant. Login to comment
229 Mutants L65R/G251V/Y950F, M68R/G251V/ Y950A/Y953A, and M68R/G251V/Y950F/Y953F were constructed, and the cDNAs were expressed in HEK 293 cells. Login to comment
230 Only the Y950F change was introduced into the L65R/G251V mutant since the Y953A change did not affect maturation of the mutant (Fig. 8B, lane 4). Login to comment
231 Immunoblot analysis shows the Y950A/Y953A and Y950F/Y953F changes in M68R/G251V (Fig. 8A, lanes 11 and 12) or Y950F change in L65R/G251V (Fig. 8B, lane 5) reduced maturation to levels similar to that observed in the G251V parent. Login to comment
237 changes into the G251V parent did not affect its maturation efficiency (Fig. 8, A, lane 15, and B, lane 6). Login to comment
239 DISCUSSION It was found that maturation of mutant G251V was not inhibited when arginines were introduced at many positions on the extracellular half of TM1. Login to comment
261 Cyclosporin A and rhodamine B but not vinblastine or verapamil promoted maturation of the mutant G64R/ G251V (Table 1). Login to comment
262 The H61R mutation appeared to perturb vinblastine interactions as expression in the presence of vinblastine did not promote maturation of the mutant H61R/G251V (Fig. 5). Login to comment
264 Effect of TM11 mutations on maturation of M68R/G251V and L65R/G251V mutants. Login to comment
265 A, HEK 293 cells were transfected with mutants M68R/G251V (lanes 2-12) or G251V (lanes 1, 13-15) containing the indicated TM11 mutations. Login to comment
267 B, HEK 293 cells were transfected with mutants M65R/G251V (lanes 2-5) or G251V (lanes 1, 6) containing the indicated TM11 mutations. Login to comment
276 Mutants H61R/G251V or M69R/G251V showed little drug rescue with verapamil (Fig. 5), and mutants G64R and M68R in a wild-type background showed reductions in apparent affinity for verapamil in ATPase assays (Fig. 7). Login to comment
291 Shading on the P-gp amino acids represents the effects of arginine substitutions on maturation of mutant G251V; the white, gray, and black-filled circles represent inhibition, no effect, or promotion of maturation, respectively. Login to comment
298 Some mutations such as G64R, L65R, M68R, and V71R actually promoted maturation of the G251V mutant. Login to comment
299 The L65R and M68R mutations were particularly effective in promoting maturation as they increased maturation efficiency of the G251V mutant from about 20 to 60-85%. Login to comment
302 Processing mutations such as G251V may alter the orientation of one or more TM segments in the membrane to interfere with subsequent packing of the TM segments at the interface between TMD1 and TMD2. Login to comment
304 An alternative possibility to explain why the L65R and M68R mutations were particularly effective in promoting maturation of the G251V mutant is that they promoted packing of the TM segments by forming hydrogen bonds with Tyr-950 and/or Tyr-953 located in TM11. Login to comment
306 Processing mutations such as G251V appear to act as thermodynamic hurdles to inhibit packing of the TM segments between TMD1 and TMD2 (6). Login to comment
308 This possibility is supported by the observations that the conservative Y950F and Y953F mutations reduced maturation of the M68R/G251V mutant to levels observed with the G251V parent. Login to comment
309 An arginine introduced at position 65 in TM1 only appeared to form a hydrogen bond with Tyr-950 as the Y950F mutation but not the Y953F change reduced maturation of the L65R/G251V mutant to levels observed with the G251V parent. Login to comment

[hide] Molecular dissection of dual pseudosymmetric solut... Mol Pharmacol. 2011 Mar;79(3):443-52. Epub 2010 Dec 21. Parveen Z, Stockner T, Bentele C, Pferschy S, Kraupp M, Freissmuth M, Ecker GF, Chiba P
Molecular dissection of dual pseudosymmetric solute translocation pathways in human P-glycoprotein.
Mol Pharmacol. 2011 Mar;79(3):443-52. Epub 2010 Dec 21., [PMID:21177413]

Abstract [show]
The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by high-resolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.

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314 The 132Arg mutant did not increase trafficking of the G251V background, whereas mutant 773Arg showed a complete trafficking deficiency. Login to comment

[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]

Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.

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42 In this study, the G251V processing mutant was used as a reporter molecule because it exhibits partial maturation (about 15% mature). Login to comment
57 The red balls show the locations of Trp232 and the processing mutations at positions 251 (G251V), 490 (ΔY490), 709 (P709A), 722 (G722A), and 1260 (L1260A). Login to comment
58 (B) HEK 293 cells were transfected with A52-tagged wild-type P-gp, mutants G251V, G251V/W232R, and G251V/ W232A,orplasmidvector(control).Wholecellextractsweresubjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment
67 Mutations were introduced into wild-type P-gp or processing mutants containing processing mutations in different domains (G251V in TMD1, ΔY490 in NBD1, P709A in the linker region, G722A in TMD2, or L1260A in NBD2) as described previously (28). Login to comment
84 Baby hamster kidney (BHK) cells expressing A52-tagged wild-type P-gp or mutants G251V, G251V/W232R, W232R, W232A, N296A, E875A, or T945A were generated as described previously (25). Login to comment
91 The G251V or L1260A processing mutations were introduced into Cys-less P-gp containing pairs of cysteines in different domains (L443C(NBD1)/ S909C(TMD2), L531C(NBD1)/C1074(NBD2), or C137(TMD1)/A935C(TMD2)) with or without the W232R mutation. Login to comment
112 The W232R suppressor mutation that promotes maturation of the G251V processing mutant is located in TM4 of TMD1 (Figure 1A). Login to comment
114 We tested whether a W232A change would also promote maturation of the G251V mutant. Login to comment
117 The amount of mature P-gp relative to total (mature plus immature) was 70 ( 6% for mutant W232R/G251V and 92 ( 5% for wild-type P-gp. Login to comment
118 By contrast, immature protein was the major product in mutants G251V (10 ( 4% mature) and G251V/W232A (13 ( 5% mature). Login to comment
119 The results indicate that introduction of the arginine at 232 rather than removal of the tryptophan was responsible for the increase in maturation in mutant G251V. Login to comment
123 We previously observed that W232R could rescue the ΔY490 (NBD1) and G251V (TMD1) P-gp processing mutants (42). Login to comment
136 Therefore, some of the other Trp to Arg changes to flanking tryptophans could also promote maturation of G251V P-gp by helping to anchor other TM segments in the membrane. Login to comment
137 Accordingly, mutants G251V/W212R(TM3), G251V/W315R- (TM5), G251V/W708R(TM7), and G251V/W855R(TM10) were constructed. Login to comment
138 The W136R mutation inhibited maturation of G251V as shown previously (42), and the mutant was included in this study as a control (Figure 2B, lanes 8 and 9). Login to comment
141 Mutant G251V/T55R(TM1) (Figure 2B, lanes 3 and 4) was included as a negative control because its maturation was unaffected by the presence of cyclosporin A (41). Login to comment
143 It was found that none of the other Trp to Arg mutations promoted maturation of the G251V processing mutant in the absence of cyclosporin A (Figure 2B, lanes 8, 10, 14, 16, and 18). Login to comment
155 To test if the W232R mutation restored domain-domain contacts, it was introduced into the G251V or L1260A processing mutants that also contained pairs of cysteines at the TMD1-TMD2 (C137(TMD1)/A935C(TMD2), NBD1-NBD2 (L531C(NBD1)/C1074(NBD2), or NBD1-TMD2 (L443C(NBD1)/S909C(TMD2) interfaces. Login to comment
156 The G251V and L1260A parents were used because the G251V mutation is in the same domain as W232R (TMD1) whereas L1260A is in another domain (NBD2). Login to comment
158 Introduction of the W232R mutation into all of the G251V (Figure 3A, lanes 3, 7, and 11) or L1260A (Figure 3B, lanes 3, 7, and 11) double-cysteine processing mutants restored maturation. Login to comment
170 (B) Cells transfected with vector (control), A52-tagged wild-type P-gp (WT), mutant G251V cDNA containingT55R, or the indicated Trp to Arg mutation were expressed in the absence (-) or presence (þ) of 10 μM cyclosporin A (cyclo A). Login to comment
181 To test for evidence of hydrogen bond interactions of W232R with residues in TM segments 5, 6, or 12, potential hydrogen bond partners (Thr294(TM5), Asn296(TM5), Ser298(TM5), Ser344(TM6), Gln347(TM6), Ser349(TM6), Ser351(TM6)), (Gln990(TM12), Ser992(TM12), or Ser993(TM12)) (see Figure 4A,B) were mutated to alanine in a G251V/W232R background. Login to comment
184 It was observed that only one mutation in TM5 (N296A) inhibited maturation of the G251V/W232R mutant when it was expressed in the absence of cyclosporin A (Figure 4C, lane 7). Login to comment
185 By contrast, mutation of potential hydrogen-bonding residues in TM6 (Ser344, Q347, S349, or Q351) or TM12 (Gln990, Ser992, or S993) to alanine did not affect maturation of G251V/W232R (Figure 4D). Login to comment
186 Introduction of the N296A mutation into G251V/W232R(TM4) caused the mutant to behave in a fashion similar to the original FIGURE 3: Effect of W232R on cross-linking between domains of processing mutants. Login to comment
187 Membranes were prepared from cells expressing P-gp processing mutants G251V ( W232R (A) or L1260A ( W232R (B) that also contained pairs of cysteines in various domains (L443C(NBD1)/S909C(TMD2), L531C(NBD1)/C1074(NBD2), C137(TMD1)/A935C(TMD2)). Login to comment
192 FIGURE 4: Effect of mutating residues capable of forming hydrogen bonds with W232R on maturation of G251V. Login to comment
194 (C) HEK 293 cells transfected with A52-tagged mutant G251V or G251V/ W232R containing alanine mutations in potential hydrogen-bonding residues in TM5 (T294A, N296A, S298A) or G251V/N296A were expressed in the absence (-) or presence (þ) of cyclosporin A (cyclo A). Login to comment
196 (D) HEK 293 cells transfected with A52-tagged mutant G251V/W232R(TM4) containing mutations in potential hydrogen-bonding residues in TM6 (S344A, Q347A, S349A, S351A) or TM12 (Q990A, S992A, S993A) were cultured in the absence (-) or presence (þ) of cyclosporin A (cyclo A). Login to comment
199 G251V parent. Login to comment
200 Like the G251V parent (Figure 4C, lanes 1 and 2), the G251V/W232R/N296A mutant (Figure 4C, lane 7) showed about 15% maturation efficiency when expressed in the absence of drug substrates, and the mature 170 kDa P-gp was the major product when expressed in the presence of cyclosporin A (Figure 4C, lane 8). Login to comment
201 Therefore, the presence of the N296A mutation prevented the ability of W232R to promote maturation of the G251V mutant. Login to comment
202 Figure 4A (lanes 11 and 12) shows that the N296A change alone had little effect on the maturation characteristics of the G251V parent because it could still be rescued with cyclosporin A. Login to comment
203 These results suggest that Arg232(TM4) promotes maturation of the G251V mutant through hydrogen bond interactions with Asn296(TM5) because the N296A mutation inhibited the ability of W232R to promote maturation of G251V (Figure 4C, lane 7). Login to comment
207 To test if other mutations to Trp232 would promote maturation, it was changed to other residues capable (Asp, Asn, Ser, Tyr) or incapable (Ala, Gly, Ile, Phe) of forming hydrogen bonds in a G251V background. Login to comment
210 No significant increase in maturation efficiency of G251V was observed when Trp232 was changed to amino acids that do not form hydrogen bonds (Ala, Gly, Ile, or Phe). Login to comment
211 The pattern of rescue of the G251V/W232X mutants was consistent with the prediction that hydrogen bond interactions were responsible for the increase in maturation efficiency. Login to comment
223 It was found that the FIGURE 5: Effect of different W232 mutations on maturation of G251V. Login to comment
224 (A) HEK 293 cells expressing A52-tagged G251V containing mutations in W232 (-, no change) capable (Arg(R), Asp(D), Asn- (N), Ser(S), Tyr(Y)) or incapable (Ala(A), Gly(G), Ile(I), Phe(F)) of forming hydrogen bonds were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment
226 (B) The amount of mature P-gp (percent mature) relative to total P-gp (170 plus 150 kDa) in mutant G251V/W232X (X = W, R, D, N, S, Y, A, G, I, or F) is shown. Login to comment
227 The results are the average values from three separate transfections ( SD. An asterisk indicates significant difference (P < 0.05) from parent (G251V). Login to comment
238 A G251V/T945R/E875A mutant was constructed to test if removal of the negative charge would affect maturation. Login to comment
243 Therefore, we tested the effect of reversing the charges by constructing mutant G251V/T945E/ E875R. Login to comment
244 It was found that the T945E/E875R combination but not individual T945E or E875R mutations promoted maturation of G251V (Figure 7D). Login to comment
256 FIGURE 7: Effect of mutations in Thr945 or Glu875 on the rescue of G251V P-gp or mutant TMD1 þ 2. Login to comment
258 (C) Whole cell extracts from cells expressing A52-tagged mutant G251V (none) or G251V/T945R (T945R) with the indicated changes topotential hydrogen bondpartnersinTM10(lane4) orTM12(lanes 7-9) were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment
260 (D) Whole cell extracts of cells expressing A52-tagged G251V (none) or G251V mutants containing opposite charges at positions 945 and 875 (T945E/E875, T954E, or E875R) were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment
270 To test if rescue by an arginine suppressor mutation yielded an active transporter, we examined the ability of mutants G251V and G251V/W232R to confer resistance to cytotoxic compounds colchicine, paclitaxel, and vinblastine. Login to comment
272 Stable BHK cell lines expressing wild-type P-gp or mutants G251V, W232R, or G251V/W232R were generated by cotransfecting P-gp cDNAs with pWL-neo vector followed by selection with G418. Login to comment
279 Cells expressing mutant G251V showed almost no increase (P<0.05) in drug resistance compared to control cells. Login to comment
280 This is to be expected since mutant G251V is a processing mutant in which the majority of P-gp is misfolded in the cell. Login to comment
281 Introduction of the W232R mutation into G251V, however, yielded a functional P-gp. Login to comment
282 Cells expressing mutant G251V/W232R were more resistant to colchicine (12-fold; P < 0.05) compared to cells expressing wild-type P-gp (9-fold) but were less resistant to vinblastine (3-fold (P < 0.05) versus 17-fold) and paclitaxel (18-fold (P < 0.05) versus 29-fold). Login to comment
283 These results show that the W232R mutation restored maturation of mutant G251V P-gp to yield a functional transporter at the cell surface. Login to comment
297 (A) Stable BHK cell lines expressing no P-gp (control), equivalent levels of wild type, mutants G251V, W232R, or G251V/W232R P-gp were incubated for 6 days in the presence of various concentrations of drug substrates vinblastine (gray bars), colchicine (white bars), or paclitaxel (black bars). Login to comment
314 It also restored maturation of the G251V processing mutant to yield a functional transporter at the cell surface (Figure 9A). Login to comment
386 Evidence for mobility of the TM segments is suggested by the large number of cross-links observed between TM segments in a cysteine mutagenesis study (51), that drug binding occurs through an induced-fit mechanism (93), that ATP hydrolysis can cause rotation of one or more helices (94), and that other residues at position 232 whose side chains are of different sizes and capable of forming hydrogen bonds (Asp, Ser, Tyr) could rescue G251V (Figure 5). Login to comment

[hide] Correction of defective protein kinesis of human P... J Biol Chem. 1997 Jan 10;272(2):709-12. Loo TW, Clarke DM
Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.
J Biol Chem. 1997 Jan 10;272(2):709-12., 1997-01-10 [PMID:8995353]

Abstract [show]
There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

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64 In addition to the mutants G268V and ⌬Y490, we were able to facilitate processing of P-glycoproteins with mutations in the predicted transmembrane segments (TM1, G54V; TM5, G300V; TM7, A718L; and TM9, A841L), in the extracellular loops between transmembrane segments (G854V), in the cytoplasmic loops (G251V and W803A), in the nucleotide-binding domains (G427C and S434C), and in the linker region connecting the two halves of the molecule (E707A) (data not shown). Login to comment
140 Another interesting observation is that misfolded mutants that are temperatureand glycerol-insensitive, such as G251V, G268V, and E707A could also be rescued by these drug substrates when expressed in either HEK 293 or NIH 3T3 cells (data not shown). Login to comment

[hide] Thapsigargin or curcumin does not promote maturati... Biochem Biophys Res Commun. 2004 Dec 10;325(2):580-5. Loo TW, Bartlett MC, Clarke DM
Thapsigargin or curcumin does not promote maturation of processing mutants of the ABC transporters, CFTR, and P-glycoprotein.
Biochem Biophys Res Commun. 2004 Dec 10;325(2):580-5., [PMID:15530432]

Abstract [show]
Misprocessed plasma membrane proteins of CFTR and P-glycoprotein (P-gp) are retained in the endoplasmic reticulum (ER) by molecular chaperones. Depletion of the calcium stores in the ER by the SERCA calcium pump inhibitors thapsigargin or curcumin inhibits these interactions and allows the protein to be trafficked to the plasma membrane [Nat. Med. 8 (2002) 485; Science 304 (2004) 600]. We tested this hypothesis by treating various cell lines expressing misprocessed mutants of CFTR or P-gp with thapsigargin or curcumin. Conversion of the immature core-glycosylated protein to mature product was detected by immunoblot analysis of whole cell extracts. Mature product was not detected in any of the misprocessed mutants. By contrast, all misprocessed P-gp mutants were rescued by the chemical chaperone/drug substrate cyclosporin A in a dose-dependent manner. These results show that thapsigargin or curcumin is not effective in rescuing misprocessed mutants of P-gp and CFTR.

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40 HEK293 cells expressing A52-tagged wild-type or mutant G251V P-gp were grown at 37 °C. Login to comment
58 Mutations G251V and F804A are in the intracellular loops of the NH2- and COOH-terminal halves of P-gp, respectively. Login to comment
70 Although the processing mutations (G251V, G300V, DY490, P709A, G722A, F804A, and P1194A) are located in different segments of P-gp, all the mutants could be rescued when expressed with drug substrates such as cyclosporin A. Login to comment
71 A representative example is shown with mutant G251V. Login to comment
72 HEK cells expressing mutant G251V were grown in the presence of various concentrations of cyclosporin A for 18 h at 37 °C. Login to comment
76 To confirm that the cyclosporin A acted as a chemical chaperone to stabilize the mutant G251V protein resulting in decreased turnover, we performed pulse-chase studies (Fig. 2B). Login to comment
77 HEK293 cells expressing mutant G251V were incubated with 35 [S]methionine for 20 min. Login to comment
82 When synthesis of mutant G251V is carried out in the presence of 10 lM cyclosporin A, however, mature 170 kDa protein was readily detected after 1 h. Login to comment
85 To determine if the mature 170 kDa protein of mutant G251V P-gp was active after rescue, we expressed the histidine-tagged wild-type or mutant G251V P-gp in HEK293 cells in the presence of 10 lM cyclosporin A for 18 h at 37 °C. Login to comment
88 The mature form of mutant G251V was still active since it retained >90% of wild-type P-gp activity (not shown). Login to comment
90 We then compared the abilities of cyclosporin A, thapsigargin, and curcumin to induce maturation of P-gp processing mutants G251V, G300V, DY490, P709A, G722A, F804A, and P1194A. Login to comment
102 Accordingly, we stably expressed mutant G251V in baby hamster kidney (BHK) cells because it has been reported that curcumin can induce maturation Fig. 2. Effect of cyclosporin A on maturation of P-gp processing mutant G251V. Login to comment
103 (A) HEK293 cells expressing the A52 epitope-tagged mutant G251V were incubated for 18 h at 37 °C in the presence of various concentrations of cyclosporin A (Cyclo A). Login to comment
105 (B) HEK293 cells were transfected with A52-tagged G251V P-gp cDNA. Login to comment
111 The BHK cells expressing mutant G251V were incubated with 10 lM cyclosporin A or various concentrations of thapsigargin (0-10 lM) or curcumin (0-30 lM). Login to comment
112 Fig. 3B shows that in the absence of drug substrates, the major product of mutant G251V in BHK cells was the 150 kDa immature protein. Login to comment
138 (B) BHK cells stably expressing A52-tagged mutant G251V were incubated for 18 h at 37 °C in the presence of 10 lM cyclosporin A (Cyclo) or various concentrations (lM) of thapsigargin or curcumin. Login to comment

[hide] Mutation of Glu521 or Glu535 in cytoplasmic loop 5... J Biol Chem. 2012 Mar 2;287(10):7543-55. Epub 2012 Jan 9. Iram SH, Cole SP
Mutation of Glu521 or Glu535 in cytoplasmic loop 5 causes differential misfolding in multiple domains of multidrug and organic anion transporter MRP1 (ABCC1).
J Biol Chem. 2012 Mar 2;287(10):7543-55. Epub 2012 Jan 9., [PMID:22232552]

Abstract [show]
The polytopic 5-domain multidrug resistance protein 1 (MRP1/ABCC1) extrudes a variety of drugs and organic anions across the plasma membrane. Four charged residues in the fifth cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 are critical for stable expression of MRP1 at the plasma membrane. Thus Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) all cause misfolding of MRP1 and target the protein for proteasome-mediated degradation. Of four chemical chaperones tested, 4-phenylbutyric acid (4-PBA) was the most effective at restoring expression of MRP1 mutants K513A, K516A, E521A, and E535A. However, although 4-PBA treatment of K513A resulted in wild-type protein levels (and activity), the same treatment had little or no effect on the expression of K516A. On the other hand, 4-PBA treatment allowed both E521A and E535A to exit the endoplasmic reticulum and be stably expressed at the plasma membrane. However, the 4-PBA-rescued E535A mutant exhibited decreased transport activity associated with reduced substrate affinity and conformational changes in both halves of the transporter. By contrast, E521A exhibited reduced transport activity associated with alterations in the mutant interactions with ATP as well as a distinct conformational change in the COOH-proximal half of MRP1. These findings illustrate the critical and complex role of CL5 for stable expression of MRP1 at the plasma membrane and more specifically show the differential importance of Glu(521) and Glu(535) in interdomain interactions required for proper folding and assembly of MRP1 into a fully transport competent native structure.

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261 Thus, the P-glycoprotein processing mutant G251V/T55R is not rescued by any drug substrates, whereas another mutant G251V/H61R can be rescued by only a single substrate (cyclosporine A) (39). Login to comment

[hide] Mutations in intracellular loops 1 and 3 lead to m... J Biol Chem. 2013 Nov 8;288(45):32622-36. doi: 10.1074/jbc.M113.498980. Epub 2013 Sep 24. Kapoor K, Bhatnagar J, Chufan EE, Ambudkar SV
Mutations in intracellular loops 1 and 3 lead to misfolding of human P-glycoprotein (ABCB1) that can be rescued by cyclosporine A, which reduces its association with chaperone Hsp70.
J Biol Chem. 2013 Nov 8;288(45):32622-36. doi: 10.1074/jbc.M113.498980. Epub 2013 Sep 24., [PMID:24064216]

Abstract [show]
P-glycoprotein (P-gp) is an ATP binding cassette transporter that effluxes a variety of structurally diverse compounds including anticancer drugs. Computational models of human P-gp in the apo- and nucleotide-bound conformation show that the adenine group of ATP forms hydrogen bonds with the conserved Asp-164 and Asp-805 in intracellular loops 1 and 3, respectively, which are located at the interface between the nucleotide binding domains and transmembrane domains. We investigated the role of Asp-164 and Asp-805 residues by substituting them with cysteine in a cysteine-less background. It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of P-gp, which thus failed to transport the drug substrates. The misfolded protein could be rescued to the cell surface by growing the cells at a lower temperature (27 degrees C) or by treatment with substrates (cyclosporine A, FK506), modulators (tariquidar), or small corrector molecules. We also show that short term (4-6 h) treatment with 15 muM cyclosporine A or FK506 rescues the pre-formed immature protein trapped in the endoplasmic reticulum in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70, and the treatment with cyclosporine A reduces the association of mutant P-gp, thus allowing it to be trafficked to the cell surface. The function of rescued cell surface mutant P-gp is similar to that of wild-type protein. These data demonstrate that the Asp-164 and Asp-805 residues are not important for ATP binding, as proposed earlier, but are critical for proper folding and maturation of a functional transporter.

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402 Also G251V, which is in ICL1 and lies spatially close to Asp-164, is a well known processing mutant of P-gp (37). Login to comment

[hide] Drug rescue distinguishes between different struct... Biochemistry. 2013 Oct 15;52(41):7167-9. doi: 10.1021/bi401269m. Epub 2013 Oct 2. Loo TW, Clarke DM
Drug rescue distinguishes between different structural models of human P-glycoprotein.
Biochemistry. 2013 Oct 15;52(41):7167-9. doi: 10.1021/bi401269m. Epub 2013 Oct 2., [PMID:24083983]

Abstract [show]
There is no high-resolution crystal structure of the human P-glycoprotein (P-gp) drug pump. Homology models of human P-gp based on the crystal structures of mouse or Caenorhabditis elegans P-gps show large differences in the orientation of transmembrane segment 5 (TM5). TM5 is one of the most important transmembrane segments involved in drug-substrate interactions. Drug rescue of P-gp processing mutants containing an arginine at each position in TM5 was used to identify positions facing the lipid or internal aqueous chamber. Only the model based on the C. elegans P-gp structure was compatible with the drug rescue results.

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15 Drug rescue of TM5 and TM9 G251V P-gp arginine mutants. Login to comment
18 An asterisk indicates a significant difference from the amount of the mature form observed when the G251V parent was expressed without cyclosporine A (~5% mature). Login to comment
23 Accordingly, the ability of drug substrates to promote maturation of a processing mutant (G251V) containing an arginine at each position in TM5 was used to map the locations of residues that faced the lipid bilayer (would prevent rescue) or the aqueous channel (would be rescued). Login to comment
24 The rationale for using this assay was that drug substrates such as cyclosporine A can promote maturation of a P-gp processing mutant (G251V).14 The G251V mutation is located in the second intracellular loop (ICL2) (Figure 1A) and appears to trap P-gp in a partially folded conformation as a 150 kDa core-glycosylated protein. Login to comment
25 Expression in the presence of a drug substrate induces G251V to complete the folding process to yield an active mature 170 kDa protein.15 Introduction of an arginine onto the lipid face of TM5 would inhibit drug rescue. Login to comment
27 Examples of drug rescue of G251V and TM5 mutants G251V/I297R and G251V/S298R are shown in Figure 1B. When processing mutant G251V is expressed in the absence of cyclosporine A, the major product was the immature 150 kDa protein (~95% of total P-gp). Login to comment
30 Arginine mutations were then introduced into each position of TM5 or TM9 in the G251V background. Login to comment

[hide] Multiple transport-active binding sites are availa... PLoS One. 2013 Dec 5;8(12):e82463. doi: 10.1371/journal.pone.0082463. eCollection 2013. Chufan EE, Kapoor K, Sim HM, Singh S, Talele TT, Durell SR, Ambudkar SV
Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).
PLoS One. 2013 Dec 5;8(12):e82463. doi: 10.1371/journal.pone.0082463. eCollection 2013., [PMID:24349290]

Abstract [show]
P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.

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220 The same residues studied in the present work were mutated to arginine by Loo and coworkers [38]; they found these mutations promote or have a neutral effect on maturation of a Pgp processing mutant (G251V) defective in folding, also confirming that these residues are in the drug translocation pathway and/or drug-binding pocket of Pgp. Login to comment