ABCMdb

PMID: 15192095

Rothnie A, Storm J, Campbell J, Linton KJ, Kerr ID, Callaghan R
The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein.
J Biol Chem. 2004 Aug 13;279(33):34913-21. Epub 2004 Jun 10., 2004-08-13 [PubMed]

Sentences

No. Mutations Sentence Comment

105 ABCB1 p.Ser337Cys RESULTS Introduction of Single Cysteines into TM6 of P-gp Has Little Effect on the ATPase Activity-Expression levels for the various P-gp isoforms were similar (data not shown), and the yield of purified protein from 50 mg of crude membrane was in the range 41 Ϯ 4 ␮g (S337C) to 65 Ϯ 9 ␮g (Cys-less). Login to comment 106 ABCB1 p.Phe335Cys Only the F335C isoform was consistently outside this range (p Ͻ 0.05, n Ͼ 10) and exhibited a yield of 28 Ϯ 6 ␮g of P-gp. Login to comment 130 ABCB1 p.Gly341Cys ABCB1 p.Leu339Cys ABCB1 p.Phe343Cys ABCB1 p.Ser337Cys ABCB1 p.Phe335Cys ABCB1 p.Thr333Cys ABCB1 p.Val331Cys Values refer to the mean Ϯ S.E. obtained from at least eight independent protein purification preparations. P-gp isoform Substrate affinity , Km Maximal activity, Vmax -Fold stimulationBasal Stimulated Basal Stimulated mM ␮mol Pi min-1 mg protein-1 Cys-less 0.58 Ϯ 0.06 0.38 Ϯ 0.04 0.58 Ϯ 0.15 1.46 Ϯ 0.30 2.9 Ϯ 0.3 V331C 0.50 Ϯ 0.06 0.26 Ϯ 0.02 0.45 Ϯ 0.05 1.54 Ϯ 0.20 3.5 Ϯ 0.3 T333C 0.49 Ϯ 0.05 0.23 Ϯ 0.02 0.35 Ϯ 0.04 1.22 Ϯ 0.15 3.3 Ϯ 0.1 F335C 0.40 Ϯ 0.05 0.24 Ϯ 0.03 0.65 Ϯ 0.15 1.61 Ϯ 0.31 2.2 Ϯ 0.2 S337C 0.53 Ϯ 0.06 0.26 Ϯ 0.04 0.59 Ϯ 0.10 1.67 Ϯ 0.23 3.2 Ϯ 0.4 L339C 0.51 Ϯ 0.07 0.31 Ϯ 0.04 0.57 Ϯ 0.07 1.47 Ϯ 0.15 2.9 Ϯ 0.3 G341C 0.40 Ϯ 0.04 0.24 Ϯ 0.02 0.42 Ϯ 0.03 1.12 Ϯ 0.09 3.1 Ϯ 0.5 F343C 0.41 Ϯ 0.04 0.26 Ϯ 0.03 0.47 Ϯ 0.04 1.17 Ϯ 0.15 2.6 Ϯ 0.3 generate stable covalent bonds with thiol groups under physiological conditions. Login to comment 141 ABCB1 p.Gly341Cys ABCB1 p.Thr333Cys Typical time courses for labeling of T333C with FM and G341C with CM are shown in Fig 1, b and c, respectively. Login to comment 142 ABCB1 p.Thr333Cys Panel b shows a time-dependent increase in the labeling of T333C P-gp (lanes i-vii). Login to comment 147 ABCB1 p.Gly341Cys Panel c clearly demonstrates that the labeling of G341C reached significantly higher levels (lanes i-vii) when compared with denatured protein (lane viii), which in this case indicates 100% labeling. Login to comment 149 ABCB1 p.Gly324Cys Mutant G324C, which contains a cysteine in an accessible extracellular loop, was used as a positive control for labeling. Login to comment 153 ABCB1 p.Gly324Cys Nonspecific labeling of Cys-less P-gp by FM was 4 Ϯ 1% (Fig. 2a), whereas G324C labeled to 100% with a half-life of 12 Ϯ 1 min (data not shown). Login to comment 154 ABCB1 p.Gly341Cys ABCB1 p.Leu339Cys ABCB1 p.Ser337Cys ABCB1 p.Phe335Cys ABCB1 p.Thr333Cys ABCB1 p.Val331Cys Isoforms V331C, T333C, F335C, S337C, L339C, and G341C displayed labeling extents in the range 7-12%, and none was significantly different from the Cys-less isoform (ANOVA). Login to comment 155 ABCB1 p.Gly324Cys ABCB1 p.Phe343Cys In contrast, F343C in the native protein conformation was accessible to labeling with FM as adjudged by the 81 Ϯ 2% labeling extent (Lext), which was characterized by a half-life (t1/2 ϭ 47 Ϯ 8 min) almost 4 times slower than observed for G324C. Login to comment 158 ABCB1 p.Gly324Cys The level of nonspecific interaction with the Cys-less P-gp was higher (Lext ϭ 25 Ϯ 4%), and G324C was also labeled by CM (Lext ϭ 102 Ϯ 4%), although the half-life (t1/2 ϭ 31 Ϯ 4 min) was almost 3 times slower than observed for FM. Login to comment 160 ABCB1 p.Leu339Cys Only the L339C isoform was significantly different with more rapid reaction kinetics (t1/2 ϭ 23 Ϯ 1 min, p Ͻ 0.05, n ϭ 3). Login to comment 162 ABCB1 p.Gly324Cys Nonspecific interaction with Cys-less protein was 19 Ϯ 1% and G324C labeled fully (Lext ϭ 97 Ϯ 3%) with a half-life approximately double (t1/2 ϭ 23 Ϯ 2 min) that obtained for FM. Login to comment 163 ABCB1 p.Gly341Cys ABCB1 p.Ser337Cys ABCB1 p.Phe335Cys ABCB1 p.Thr333Cys Isoforms T333C, F335C, S337C, and G341C did not label with BM since the Lext values (19-27%) were not signif- TABLE II Potency of drugs that affect the ATPase activity of purified reconstituted single cysteine mutants of P-gp Pure, reconstituted P-gp (0.3 ␮g) was incubated in the presence of ATP (2 mM) and varying concentrations of nicardipine, vinblastine, or vanadate. Login to comment 166 ABCB1 p.Gly341Cys ABCB1 p.Leu339Cys ABCB1 p.Phe343Cys ABCB1 p.Ser337Cys ABCB1 p.Phe335Cys ABCB1 p.Thr333Cys ABCB1 p.Val331Cys Values refer to the mean Ϯ S.E. obtained from a minimum of three independent protein purification preparations. P-gp isoform Potency of drug effect Nicardipine, EC50 Vinblastine, EC50 Vanadate, IC50 ␮M ␮M ␮M Cys-less 3.2 Ϯ 0.3 4.2 Ϯ 0.6 4.0 Ϯ 0.4 V331C 3.3 Ϯ 0.4 7.2 Ϯ 1.7 3.2 Ϯ 0.4 T333C 2.3 Ϯ 0.2 4.6 Ϯ 0.4 3.9 Ϯ 0.8 F335C 2.3 Ϯ 0.4 4.2 Ϯ 0.8 5.5 Ϯ 1.1 S337C 2.7 Ϯ 0.5 4.1 Ϯ 1.0 5.8 Ϯ 0.8 L339C 2.1 Ϯ 0.3 5.1 Ϯ 0.8 4.2 Ϯ 0.7 G341C 3.9 Ϯ 0.5 4.0 Ϯ 0.6 6.8 Ϯ 1.3 F343C 2.1 Ϯ 0.3 5.6 Ϯ 2.7 2.7 Ϯ 0.8 FIG. 1. Login to comment 168 ABCB1 p.Gly341Cys ABCB1 p.Thr333Cys Structures of maleimide-containing probes (a) and the fluorescence profiles of SDS-PAGE gels obtained for time courses of fluorescein maleimide labeling of T333C (b) and coumarin maleimide labeling of G341C (c). Login to comment 182 ABCB1 p.Val331Cys The V331C isoform was labeled with BM (Lext ϭ 62 Ϯ 4%), although the half-life for this labeling was considerably slower (t1/2 ϭ 143 Ϯ 21 min, p Ͻ 0.05) than obtained for any other reaction. Login to comment 183 ABCB1 p.Leu339Cys ABCB1 p.Val331Cys L339C was also efficiently labeled with BM (Lext ϭ 71 Ϯ 2%); however, the reaction kinetics were faster (t1/2 ϭ 49 Ϯ 7 min) than observed with V331C. Login to comment 184 ABCB1 p.Phe343Cys Surprisingly, mutant F343C, which was the only isoform labeled by the hydrophilic compound FM, reacted with the amphiphilic probe BM (Lext ϭ 68 Ϯ 11%) with a half-life of only 18 Ϯ 4 min. Login to comment 189 ABCB1 p.Phe343Cys Isoform F343C was the only protein to label with the hydrophilic probe FM, and the labeling was significantly reduced by both AMP-PNP binding (Lext ϭ 29 Ϯ 3%) and vanadate trapping (Lext ϭ 26 Ϯ 4%). Login to comment 191 ABCB1 p.Phe343Cys The residual labeled protein is likely to arise from the proportion of F343C P-gp that was not completely bound with AMP-PNP or vanadate-trapped. Login to comment 192 ABCB1 p.Phe343Cys This was confirmed by similar labeling half-lives compared with nucleotide free F343C P-gp. Login to comment 193 ABCB1 p.Phe343Cys F343C was the only isoform whose labeling with FM was affected by the catalytic cycle because all other mutant P-gp proteins remained inaccessible to labeling with this hydrophilic molecule. Login to comment 195 ABCB1 p.Leu339Cys Similarly, the kinetics of the reaction were also unchanged (t1/2 values of 45-65 min) with the exception of isoform L339C. Login to comment 200 ABCB1 p.Phe343Cys ABCB1 p.Thr333Cys Isoforms T333C (t1/2 ϭ 16 Ϯ 3 min) and F343C (t1/2 ϭ 18 Ϯ 5 min) labeled with CM with significantly increased rates compared with the values observed in either basal conditions or in the presence of AMP-PNP, reflecting an increased accessibility of the introduced cysteine residues. Login to comment 201 ABCB1 p.Leu339Cys In the vanadate-trapped state, L339C labeling (t1/2 ϭ 28 Ϯ 3 min) regained the rapid reaction kinetics with CM that were observed in the basal state but lost in the nucleotide-bound protein. Login to comment 203 ABCB1 p.Leu339Cys ABCB1 p.Phe343Cys ABCB1 p.Val331Cys Whereas under basal conditions V331C, L339C, and F343C were accessible to BM, only the latter was labeled (Lext ϭ 90 Ϯ 8%) after AMP-PNP binding to the protein (Fig. 4b). Login to comment 205 ABCB1 p.Phe343Cys This was further confirmed by the observation that the half-life of labeling with BM in F343C was unaffected by the binding of AMP-PNP (t1/2 ϭ 18 Ϯ 1 min) compared with the basal state. Login to comment 206 ABCB1 p.Phe343Cys Vanadate trapping of the F343C isoform also failed to alter either the extent (Lext ϭ 98 Ϯ 3%) or FIG. 4. Login to comment 212 ABCB1 p.Thr333Cys Isoform T333C, which was inaccessible to BM under basal conditions, remained so after nucleotide binding and only labeled to a significant extent in the vanadate-trapped state (Lext ϭ 85 Ϯ 2%). Login to comment 213 ABCB1 p.Val331Cys In contrast, V331C was affected in the opposite manner with accessibility only apparent in the basal state. Login to comment 215 ABCB1 p.Leu339Cys The effects on L339C accessibility to BM paralleled the observations with CM. Login to comment 217 ABCB1 p.Gly341Cys ABCB1 p.Phe335Cys Two isoforms, F335C and G341C, could not be labeled by BM under any conditions and presumably lie in a region with low accessibility due to proximity of other structural elements. Login to comment