ABCMdb

ABCB1 p.Asn296Cys

None has been submitted yet.

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

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

PMID: 12223492 [PubMed] Loo TW et al: "Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein."

No. Sentence Comment

138 In contrast, four mutants (N296C, G300C, Y310C, and F314C) were inhibited by Ͼ70% with MTS-rhodamine. 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

101 Eleven mutants (I293C/F775C, N296C/F770C, N296C/G774C, I297C/F771C, I297C/G774C, I299C/F770C, I299C/G774C, G300C/F767C, G300C/F770C, G300C/F771C, and G300C/G774C) showed relatively strong (Ͼ50%) cross-linking (Table I). Login to comment
105 Six mutants (A295C/ G774C, N296C/F771C, N296C/Q773C, N296C/F775C, I297C/ F770C, and G300C/F775C) showed relatively weak (Ͻ50%) cross-linking (Table I). Login to comment
110 The eleven mutants (I293C/F775C, N296C/F770C, N296C/G774C, I297C/F771C, I297C/G774C, I299C/F770C, I299C/G774C, G300C/F767C, G300C/F770C, G300C/F771C, and G300C/G774C) that showed relatively strong cross-linking at 37 °C were subjected to cross-linking at 22 and 4 °C. Login to comment
111 Only four mutants (N296C/G774C, I299C/F770C, I299C/G774C, and G300C/F770C) still showed cross-linking at 22 and 4 °C. Login to comment
112 Mutants I293C/F775C, N296C/ F770C, I297C/G774C, G300C/F771C, and G300C/G774C were cross-linked only at 22 °C, whereas mutants I297C/F771C and G300C/F767C showed no cross-linking at either 22 or 4 °C. Fig. 3 shows the temperature-dependent cross-linking of mutants N296C/G774C, I299C/F770C, I299C/G774C, and G300C/ F770C. Login to comment
114 All four mutants were cross-linked at a slower rate at 4 °C, with most of the cross-linked product detected by 8 min. Fig. 3 also shows that in mutant N296C/ F770C about 50% of the cross-linked product was detected by 8 min at 22 °C, and cross-linked product was barely detectable after 32 min at 4 °C. Login to comment
115 Mutants N296C/G774C, I299C/F770C, I299C/G774C, and G300C/F770C were selected for further analysis because they were cross-linked at 37, 22, and 4 °C. Login to comment
117 By contrast, no cross-linked product was detected in the single cysteine mutants, N296C, I299C, G300C, F770C, and G774C, when oxidant was added at these temperatures (data not shown). Login to comment
118 To test if the mutants N296C/G774C, I299C/F770C, I299C/ G774C, and G300C/F770C retained the ability to interact with drug substrates, they were expressed in HEK 293 cells, isolated by nickel-chelate chromatography, mixed with lipid, and assayed for drug-stimulated ATPase activity. Login to comment
124 The verapamiland demecolcine-stimulated ATPase activities rel- TABLE I Cross-linking between residues in TMs 5 and 8 TM5 TM 8 F767C I768C T769C F770C F771C L772C Q773C G774C F775C T776C I293C -a - - - - - - - ϩϩb - T294C - - - - - - - - - - A295C - - - - - - - ϩ - - N296C - - - ϩϩb ϩ - ϩ ϩϩc ϩ - I297C - - - ϩd ϩϩ - - ϩϩb - - S298C - - - - - - - - - - I299C - - - ϩϩc - - - ϩϩc - - G300C ϩϩe - - ϩϩc ϩϩb - - ϩϩb ϩ - A301C - - - - - - - - - - A302C - - - - - - - - - - a No cross-linked product detected in SDS-PAGE gels at 37 °C. b Cross-linked product was also detected at 22 °C. c Cross-linked product was also detected at 22 °C and at 4 °C. d Relatively weak cross-linking (Ͻ50% of P-gp cross-linked) at 37 °C. e Relatively strong cross-linking (Ͼ50% of P-gp cross-linked) at 37 °C. FIG. 2. Login to comment
126 Membranes were prepared from HEK 293 cells expressing P-gp mutants I297C (A), F771C (A), I297C/F771C (A), N296C/F770C (B), or I297C/F770C (B). Login to comment
130 ative to that of Cys-less P-gp were 65 and 62%, 68 and 51%, and 57 and 79% for mutants N296C/G774C, I299C/G774C, and G300C/F770C, respectively. Login to comment
131 We then tested whether cross-linking affected the verapamil-stimulated ATPase activities of mutants N296C/G774C, I299C/ F770C, I299C/G774C, and G300C/F770C. Login to comment
134 By contrast, the verapamil-stimulated ATPase activities of mutants N296C/G774C, I299C/ F770C, I299C/G774C, and G300C/F770C were inhibited by 60-80% after treatment with oxidant. These results suggest that cross-linking inhibits conformational changes in P-gp during ATP hydrolysis (47, 50). Login to comment
135 Because there is evidence that TM5 lines the drug binding pocket of P-gp (44), we tested whether drug substrates that stimulate (demecolcine and verapamil) or inhibit (cyclosporin A) (29) ATPase activity would affect the cross-linking pattern observed in mutants N296C/G774C, I299C/F770C, I299C/ G774C, and G300C/F770C. Login to comment
147 Accordingly, the effect of vanadate trapping on cross-linking of mutants N296C/G774C, I299C/ F770C, I299C/G774C, and G300C/F770C was examined. Login to comment
151 Effect of temperature on cross-linking. Membranes were prepared from HEK 293 cells expressing P-gp mutants N296C/G774C, I299C/F770C, I299C/G774C, G300C/F770C, or N296C/F770C. Login to comment
157 Histidine-tagged Cys-less (C-less) P-gp and mutants N296C/ G774C, I299C/F770C, I299C/G774C, or G300C/F770C were isolated by nickel-chelate chromatography. Login to comment
162 Effect of drug substrates on cross-linking. Membranes were prepared from HEK 293 cells expressing P-gp mutants N296C/ G774C, I299C/F770C, I299C/G774C, or G300C/F770C. Login to comment
191 The model may explain why the presence of drug substrates had relatively little effect on cross-linking of mutants N296C/ G774C, I299C/F770C, I299C/G774C, and G300C/F770C. Login to comment
197 Membranes from HEK 293 cells expressing P-gp mutants N296C/G774C, I299C/F770C, I299C/G774C, or G300C/F770C were preincubated at 37 °C for 10 min in the presence (ϩ) or absence (-) of ATP and MgCl2 plus sodium vanadate (ATP/VO4). Login to comment
211 For example, vanadate trapping of nucleotide nearly abolished cross-linking in mutants N296C/ G774C, I299C/F770C, I299C/G774C, and G300C/F770C (Fig. 6), although cross-linking inhibited drug-stimulated ATPase activity. Login to comment

PMID: 14749322 [PubMed] Loo TW et al: "Val133 and Cys137 in transmembrane segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-glycoprotein."

No. Sentence Comment

176 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). Login to comment

PMID: 21182301 [PubMed] Loo TW et al: "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."

No. Sentence Comment

309 It was not surprising that the mutations affected rhodamine B-stimulated ATPase activity because it was previously shown that treatment of mutants W232C, N296C, and T945C (E875C was not tested) with a thiol-reactive analogue of rhodamine inhibited activity (15). Login to comment
368 These results are consistent with those from cysteine mutagenesis studies where modification of mutant T945C by thiol-reactive analogues of verapamil or rhodamine or mutants W232C and N296C by a thiol reactive analogue of rhodamine inhibited their activity (13, 15). Login to comment