ABCMdb

ABCB1 p.Pro350Cys

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Publications

PMID: 10681495 [PubMed] Loo TW et al: "The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis."

No. Sentence Comment

35 If the TM segments of P-gp possess enough flexibility to accommodate substrates of varying sizes, then it was reasonable to assume that residues in other TMs could be cross-linked to P350C or S993C. Login to comment
51 Printed in U.S.A. This paper is available on line at http://www.jbc.org atUniversityofNorthCarolinaatChapelHill,onSeptember29,2011www.jbc.orgDownloadedfrom ation was that P350C (TM6) and S993C (TM12) are predicted to be close to the cytoplasmic side of the membrane. Login to comment
53 Accordingly, mutants were constructed that had either P350C (TM6) and a cysteine residue introduced into predicted TMs 7 to 11, or between S993C (TM12) and a cysteine introduced into predicted TMs 1 to 5. Login to comment
64 No cross-linked product was detected between S993C (TM12) and cysteines introduced into TMs 1, 2, or 3 or between P350C (TM6) and cysteines introduced into TMs 7, 8, or 9 (Table I). Login to comment
65 Twelve of the 125 P-gp mutants (TM4/TM12 constructs L227C/S993C, V231C/S993C, W232C/S993C, A233C/S993C, I235C/S993C, and L236C/S993C; TM5/TM12 constructs A295C/S993C and I299C/S993C; TM10/TM6 constructs V874C/P350C, E875C/ P350C, and M876C/P350C; and TM11/TM6 construct G939C/ P350C), however, had slower mobilities in SDS-PAGE after treatment with oxidant. Login to comment
66 A representative result (mutant G939C/P350C) is shown in Fig. 1A. Login to comment
74 Table II shows the minimum temperature required for cross-linking was 4 °C for mutants A233C(TM4)/ S993C(TM12), I235C(TM4)/S993C(TM12), L236C(TM4)/ S993C(TM12), and I299C(TM5)/S993(TM12); 21 °C for mutants V231C(TM4)/S993C(TM12), W232C(TM4)/S993C(TM12), A295C(TM5)/S993C(TM12), V874C (TM10)/P350C (TM6), M876C(TM10)/P350C(TM6), and G939C(TM11)/P350C(TM6); and 37 °C for mutants L227C (TM4)/S993C/(TM12) and E875C(TM10)/P350C(TM6). Login to comment
77 In these cross-linking experiments, the amount of oxidant was lowered by 10-fold (0.2 mM), and the minimum temperature required to induce cross-TABLE I Cross-linking analysis of P-gp Cross-linking of S993C (TM12) with residues in the following TM: TM1 TM2 TM3 TM4 TM5 M51C -a Y130C - G185C - G226C - I293C - V52C - I131C - I186C - L227C ϩb T294C - V53C - Q132C - G187C - S228C - A295C ϩ G54C - V133C - D188C - A229C - N296C - T55C - S134C - K189C - A230C - I297C - L56C - F135C - I190C - V231C ϩ S298C - A57C - W136C - G191C - W232C ϩ I299C ϩ A58C - C137C - M192C - A233C ϩ G300C - I59C - L138C - F193C - K234C - A301C - I60C - A139C - F194C - I235C ϩ A302C - H61C - A140C - Q195C - L236C ϩ F303C - G141C - S196C - S237C - L304C - Cross-linking of P350C (TM6) with residues in the following TM: TM7 TM8 TM9 TM10 TM11 F711C - F770C - A828C - I867C - A935C - V712C - F771C - I829C - I868C - H936C - V713C - L772C - G830C - A869C - I937C - G714C - Q773C - S831C - I870C - F938C - V715C - G774C - R832C - A871C - G939C ϩ F716C - F775C - L833C - G872C - I940C - C717C - T776C - A834C - V873C - T941C - A718C - F777C - V835C - V874C ϩ F942C - I719C - G778C - I836C - E875C ϩ S943C - I720C - K779C - T837C - M876C ϩ F944C - N721C - A780C - Q838C - K877C - T945C - G722C - G781C - N839C - M878C - Q946C - G723C - E782C - I840C - L879C - A947C - I783C - a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE. Login to comment
79 A representative result (mutant G939C/P350C) is shown in Fig. 1C. Login to comment
99 Mutants L227C/S993C, V231C/ S993C, W232C/S993C, A233C/S993C, I235C/S993C, L236C/ S993C, A295C/S993C, I299C/S993C, V874C/P350C, E875C/ P350C, M876C/P350C, and G939C/P350C were inhibited by 81, 88, 90, 89, 93, 81, 78, 87, 87, 77, 70, and 78%, respectively. Login to comment
110 Oxidative cross-linking of P-gp mutant G939C/P350C. Login to comment
111 A, membranes from HEK 293 cells transfected with vector alone (Control), mutant G939C/P350C, or Cys-less (C-less) P-gp cDNA were treated for 10 min at 37 °C in the presence (ϩ) or absence (-) of 2 mM Cu2ϩ (phenanthroline)3. Login to comment
115 C, membranes from HEK 293 cells expressing mutant G939C/P350C were treated with 0.2 mM Cu2ϩ (phenanthroline)3 in the presence of no drug (None), 1 mM colchicine (Colch), 0.15 mM verapamil (Ver), 10 ␮M cyclosporin A (Cyclo) or 35 ␮M vinblastine (Vin) or in the presence of no addition (None), 5 mM ATP (ATP), 5 mM ATP and 0.1 mM vanadate (ATP/VO4) or 0.1 mM vanadate (VO4). Login to comment
118 TABLE II Minimum temperature required for cross-linking Residues TM segments 4 °C 21 °C 37 °C L227C/S993C 4/12 -a - ϩ V231C/S993C 4/12 - ϩ ϩ W232C/S993C 4/12 - ϩ ϩ A233C/S993C 4/12 ϩb ϩ ϩ I235C/S993C 4/12 ϩ ϩ ϩ L236C/S993C 4/12 ϩ ϩ ϩ A295C/S993C 5/12 - ϩ ϩ I299C/S993C 5/12 ϩ ϩ ϩ V874C/P350C 10/6 - ϩ ϩ E875C/P350C 10/6 - - ϩ M876C/P350C 10/6 - ϩ ϩ G939C/P350C 11/6 - ϩ ϩ a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE. Login to comment

PMID: 12609990 [PubMed] Loo TW et al: "Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding."

No. Sentence Comment

30 These concentrations gave maximal stimulation of the mutant P-gp mutant P350C. Login to comment
51 These mutants contained P350C(TM6) and another cysteine on the cytoplasmic half of TMs 7-12 (i.e. P350C ϩ another cysteine at positions: 711-723 (TM7), 770 -783 (TM8), 828 - 840 (TM9), 867-879 (TM10), 935-947 (TM11), and 986-994 (TM12)) or S993C(TM12) with another cysteine in the cytoplasmic half of TMs 1-6 (i.e. S993C ϩ another cysteine at positions: 51-61 (TM1), 130-141 (TM2), 185-196 (TM3), 226-237 (TM4), 293-304 (TM5), and 343-351 (TM6)). Login to comment
70 An example was mutant P350C- (TM6)/G939C(TM11). Login to comment
108 Their activities were compared with that of mutant P350C (parent). Login to comment
109 We previously showed that mutant P350C exhibited high levels of drug-stimulated ATPase activity (11). Login to comment
119 The activities of the mutant P-gps are expressed relative to mutant P350C. Login to comment
124 In the presence of drug substrate (progesterone), TM segments 11 and 12 undergo rotational and/or lateral movements so that cross-linking can occur between P350C and V991C (black ball) in TM12 and with A935C (red ball) and G939C (turquoise ball) in TM11. Login to comment
133 Substrate-induced Cross-linking between TM Segments of P-gp atUniversityofNorthCarolinaatChapelHill,onSeptember29,2011www.jbc.orgDownloadedfrom substrates such as progesterone are effective in promoting new cross-links with P350C (Fig. 2). Login to comment
134 In the absence of drug substrate, residue P350C in TM6 can be cross-linked to S993C in TM12. Login to comment
139 Therefore, the presence of drug substrate (progesterone) likely causes a slight rotation/rearrangement of TM12 relative to TM6 such that residue V991C comes closer to P350C. Login to comment
144 Cyclosporin A also promoted cross-linking between TM6 and TM11, but only between P350C and G939C. Login to comment
145 It is possible that cyclosporin A altered the tilt or distance between TM6 and TM11 such that only A939C but not A935C were close enough to P350C to be cross-linked. Login to comment

PMID: 14670948 [PubMed] Loo TW et al: "Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane."

No. Sentence Comment

221 In previous cross-linking studies on the packing of the TM segments, we showed that a cysteine residue in TM6 (P350C) could be cross-linked to a cysteine at the cytoplasmic ends in TMs 10, 11, and 12, whereas a cysteine in TM12 (S993C) could be cross-linked to cysteines at the cytoplasmic ends of TMs 4, 5, and 6 (33). Login to comment

PMID: 9261097 [PubMed] Loo TW et al: "Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12."

No. Sentence Comment

68 To test these predictions, we introduced pairs of cysteines into a Cys-less mutant of P-glycoprotein to create the mutants F336C/S979C, L339C/V982C, F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment
76 Fig. 1D shows that a product with reduced mobility on SDS-PAGE gels was present when mutants F343C/M986C, G346C/G989C, and P350C/ S993C were treated with oxidant. Login to comment
77 The highest level of cross-linking occurred in mutant P350C/S993C, since most of the protein migrated with reduced mobility after treatment with oxidant (Fig. 1D, lane 14). Login to comment
100 The effect of nucleotides on cross-linking was also tested on mutants F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment
103 To test the effect of drug substrates, cross-linking of mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/ S993C was done in the presence of verapamil, cyclosporin A, vinblastine, or colchicine. Login to comment
105 By contrast, all the drug substrates were effective in blocking cross-linking of mutants F343C/M986C and G346C/G989C (Fig. 3, B and C), but were less effective in preventing cross-linking of mutant P350C/S993C (Fig. 3D). Login to comment
106 In mutant P350C/S993C, verapamil, cyclosporin A, and vinblastine were more effective than colchicine in inhibiting cross-linking. Login to comment
108 Effect of Cross-linking on Drug-stimulated ATPase Activity- Mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/S993C were still active since they retained about 90, 30, 10, and 70%, respectively, of the verapamil-stimulated ATPase activity of the Cys-less P-glycoprotein. Login to comment
109 Cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C, but not L332C/L975C, was reversed by treatment with dithiothreitol (Fig. 4A). Login to comment
111 Mutant P350C/S993C was tested because it exhibited the greatest degree of cross-linking (Fig. 1D). Login to comment
112 In addition, mutant P350C/S993C had the highest activity of those mutants whose cross-linked products were sensitive to dithiothreitol. Login to comment
113 Membranes prepared from HEK 293 cells expressing Cys-less or mutant P350C/S993C P-glycoprotein- (His)10 were treated with or without 2 mM copper phenanthroline for 10 min at 37 °C. Login to comment
116 Fig. 4B (lane 4) shows that the cross-linked mutant P350C/S993C was also efficiently recovered, indicating that the histidine tag remained accessible after cross-linking. Login to comment
119 Mutant P350C/S993C, however, showed approximately 75% reduction of the verapamil-stimulated ATPase activity after cross-linking. Login to comment
121 This result suggested that dithiothreitol broke the disulfide bond between residues P350C and S993C and allowed movement to occur between TM6 and TM12 during drug-stimulated ATPase activity. Login to comment
144 Drug substrates inhibited cross-linking of mutants F343C/ M986C, G346C/G989C, and P350C/S993C. Login to comment

PMID: 9405384 [PubMed] Loo TW et al: "Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate."

No. Sentence Comment

141 Cross-linking between residues F343C/M986C, G346C/G989C, and P350C/S993C was prevented by the presence of drug substrates. Login to comment

PMID: 9841738 [PubMed] Jones PM et al: "A new structural model for P-glycoprotein."

No. Sentence Comment

204 Four cross-linked pairs, namely L332C/L975C, F343C/M986C, G346C/G989C and P350C/S993C, were generated in separate mutant molecules. Login to comment
207 Cross-linking of P350C to S993C severely impaired drug-stimulated ATPase activity, suggesting that the two regions of which they are components must be free to move independently for proper function. Login to comment