ABCMdb

PMID: 14670948

Loo TW, Bartlett MC, Clarke DM
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.
J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10., 2004-02-27 [PubMed]

Sentences

No. Mutations Sentence Comment

74 ABCB1 p.Ile306Cys Transmembrane segments 5 and 6 are particularly interesting because labeling of mutants I306C (TM5) and F343C(TM6) with methanethiosulfonate (MTS)-verapamil or MTS-rhodamine, respectively, caused permanent activation of P-gp ATPase activity (23, 44). Login to comment 92 ABCB1 p.Phe343Cys ABCB1 p.Ile306Cys Residues that react with the MTS derivatives of drug substrates verapamil (I306C) and rhodamine (F343C) to stimulate ATPase activity are shown as closed circles. Login to comment 101 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe775Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Ile297Cys ABCB1 p.Ile297Cys ABCB1 p.Phe771Cys ABCB1 p.Phe771Cys ABCB1 p.Asn296Cys ABCB1 p.Asn296Cys ABCB1 p.Ile293Cys ABCB1 p.Phe767Cys 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 ABCB1 p.Ala295Cys ABCB1 p.Gly774Cys ABCB1 p.Phe775Cys ABCB1 p.Phe775Cys ABCB1 p.Gln773Cys ABCB1 p.Phe770Cys ABCB1 p.Ile297Cys ABCB1 p.Phe771Cys ABCB1 p.Asn296Cys ABCB1 p.Asn296Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe775Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Ile297Cys ABCB1 p.Ile297Cys ABCB1 p.Phe771Cys ABCB1 p.Phe771Cys ABCB1 p.Asn296Cys ABCB1 p.Asn296Cys ABCB1 p.Ile293Cys ABCB1 p.Phe767Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe775Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Ile297Cys ABCB1 p.Ile297Cys ABCB1 p.Phe771Cys ABCB1 p.Phe771Cys ABCB1 p.Asn296Cys ABCB1 p.Asn296Cys ABCB1 p.Ile293Cys ABCB1 p.Phe767Cys 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 ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 123 ABCB1 p.Ile299Cys ABCB1 p.Phe770Cys Mutant I299C/F770C had about the same amount of ATPase activity as Cys-less P-gp. Login to comment 124 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Ala295Cys ABCB1 p.Ala295Cys ABCB1 p.Ala302Cys ABCB1 p.Ala302Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe775Cys ABCB1 p.Phe775Cys ABCB1 p.Thr294Cys ABCB1 p.Thr294Cys ABCB1 p.Gln773Cys ABCB1 p.Gln773Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Leu772Cys ABCB1 p.Leu772Cys ABCB1 p.Thr776Cys ABCB1 p.Thr776Cys ABCB1 p.Ile297Cys ABCB1 p.Ile297Cys ABCB1 p.Phe771Cys ABCB1 p.Phe771Cys ABCB1 p.Ala301Cys ABCB1 p.Ala301Cys ABCB1 p.Ser298Cys ABCB1 p.Ser298Cys ABCB1 p.Asn296Cys ABCB1 p.Asn296Cys ABCB1 p.Ile293Cys ABCB1 p.Ile293Cys ABCB1 p.Phe767Cys ABCB1 p.Phe767Cys ABCB1 p.Ile768Cys ABCB1 p.Ile768Cys ABCB1 p.Thr769Cys ABCB1 p.Thr769Cys 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 ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Ile297Cys ABCB1 p.Ile297Cys ABCB1 p.Ile297Cys ABCB1 p.Phe771Cys ABCB1 p.Phe771Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 137 ABCB1 p.Gly774Cys The samples were then cooled to 4 °C and treated with oxidant for 10 min. Fig. 5 shows that the drug substrates had little or no effect on the cross-linking pattern of mutant N296/G774C. Login to comment 138 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys Mutant I299C/F770C showed a small decrease in cross-linking with cyclosporin A and demecolcine, whereas mutant I299C/ G774C was only slightly affected by the drug substrates. Login to comment 139 ABCB1 p.Phe770Cys Mutants G300C/F770C showed the largest decrease in cross-linking with cyclosporin A. Login to comment 147 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 ABCB1 p.Ile299Cys ABCB1 p.Ile299Cys ABCB1 p.Gly774Cys ABCB1 p.Gly774Cys ABCB1 p.Phe770Cys ABCB1 p.Phe770Cys ABCB1 p.Asn296Cys 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 221 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys 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 227 ABCB1 p.Gly341Cys We had shown that mutations in TM6 such as G341C promoted proteolytic cleavage of P-gp between residues Arg-113 and Tyr-114 in ECL1 in the endoplasmic reticulum (62). Login to comment 228 ABCB1 p.Gly341Cys Mutant G341C was particularly sensitive to cleavage at R113 since no full-length protein was detected in cells expressing the mutant. Login to comment