ABCMdb

PMID: 16004994

Rothnie A, Storm J, McMahon R, Taylor A, Kerr ID, Callaghan R
The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment.
FEBS Lett. 2005 Jul 18;579(18):3984-90., [PubMed]

Sentences

No. Mutations Sentence Comment

74 ABCB1 p.Leu339Cys [3 H]-Azidopine labelling of P-glycoprotein Preparations of both cys-less and L339C isoforms of P-gp were labelled to an extent of 90% with CM as described [22]. Login to comment 75 ABCB1 p.Leu339Cys [3 H]-Azidopine labelling of P-glycoprotein Preparations of both cys-less and L339C isoforms of P-gp were labelled to an extent of 90% with CM as described [22]. Login to comment 90 ABCB1 p.Leu339Cys The time-course shown in Fig. 1 was obtained for mutant L339C, although similar profiles were generated for each P-gp isoform. Login to comment 91 ABCB1 p.Leu339Cys The time-course shown in Fig. 1 was obtained for mutant L339C, although similar profiles were generated for each P-gp isoform. Login to comment 93 ABCB1 p.Leu339Cys ABCB1 p.Phe335Cys The half-lives for reaction of introduced cysteine residues with CM varied from 23 &#b1; 1 min obtained for L339C to 52 &#b1; 3 min for the F335C isoform. Login to comment 94 ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys ABCB1 p.Phe335Cys The half-lives for reaction of introduced cysteine residues with CM varied from 23 ± 1 min obtained for L339C to 52 ± 3 min for the F335C isoform. Login to comment 95 ABCB1 p.Leu339Cys The half-life for L339C was significantly shorter (P < 0.05) than that obtained for the other TM6 mutant isoforms. Login to comment 109 ABCB1 p.Leu339Cys The Vmax of hydrolysis in the presence of vinblastine was reduced to 41 &#b1; 5% (P < 0.01) of that observed in the absence of CM for the L339C isoform. Login to comment 110 ABCB1 p.Leu339Cys The Vmax of hydrolysis in the presence of vinblastine was reduced to 41 ± 5% (P < 0.01) of that observed in the absence of CM for the L339C isoform. Login to comment 111 ABCB1 p.Leu339Cys Full Michaelis-Menten plots for the unlabelled and CM-modified L339C isoform of P-gp are shown in Fig. 3. Login to comment 112 ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys Full Michaelis-Menten plots for the unlabelled and CM-modified L339C isoform of P-gp are shown in Fig. 3. Login to comment 113 ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys The data demonstrate that the vinblastine mediated reduction in Vmax was not accompanied by a shift in affinity (Km(ATP)) of the CM labelled L339C (Km = 0.40 ± 0.05 mM) compared to native protein (Km=0.32 ± 0.05 mM). Login to comment 114 ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys Similarly, of the TM6 mutants examined, only L339C displayed a significant reduction in the nicardipine stimulated Vmax (46 ± 4%, P < 0.01) following labelling with CM (Fig. 2(c)). Login to comment 115 ABCB1 p.Leu339Cys In the presence of nicardipine, the Km of unmodified L339C (Km = 0.33 ± 0.03 mM) was not significantly altered (Km = 0.29 ± 0.05 mM) following covalent modification with CM (Fig. 3). Login to comment 117 ABCB1 p.Leu339Cys Coumarin-maleimide labelled 339C P-gp displays modified TMD fi NBD coupling To further examine the perturbed drug stimulation of ATPase activity, full dose-response analyses were generated for native and CM labelled L339C P-gp for several drugs (Fig. 4). Login to comment 118 ABCB1 p.Leu339Cys Coumarin-maleimide labelled 339C P-gp displays modified TMD fi NBD coupling To further examine the perturbed drug stimulation of ATPase activity, full dose-response analyses were generated for native and CM labelled L339C P-gp for several drugs (Fig. 4). Login to comment 122 ABCB1 p.Leu339Cys Stimulation by vinblastine was characterized by a 2.3 &#b1; 0.1 fold increase in basal activity for unlabelled L339C and at a potency of EC50 = 6.8 &#b1; 0.5 lM (Fig. 4(a)). Login to comment 123 ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys Stimulation by vinblastine was characterized by a 2.3 ± 0.1 fold increase in basal activity for unlabelled L339C and at a potency of EC50 = 6.8 ± 0.5 lM (Fig. 4(a)). Login to comment 124 ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys However, for the CM labelled L339C P-gp vinblastine did not stimulate activity, even at concentrations as high as 100 lM. Login to comment 125 ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys The stimulation of unlabelled L339C P-gp (2.3 ± 0.2 fold-basal) by nicardipine was described with a potency of EC50 = 2.3 ± 0.4 lM Fig. 1. Login to comment 126 ABCB1 p.Leu339Cys Time course for the labelling of L339C P-gp with CM. Login to comment 128 ABCB1 p.Leu339Cys (b) The time-course of labelling L339C P-gp with CM obtained from three independent purified protein preparations. Login to comment 129 ABCB1 p.Leu339Cys (b) The time-course of labelling L339C P-gp with CM obtained from three independent purified protein preparations. Login to comment 130 ABCB1 p.Leu339Cys In contrast to the effect seen with vinblastine, the labelled L339C isoform retained stimulation of ATP hydrolysis by nicardipine although the overall extent was reduced by 60% to 1.3 &#b1; 0.1 fold-basal (P < 0.05, n = 4). Login to comment 131 ABCB1 p.Leu339Cys In contrast to the effect seen with vinblastine, the labelled L339C isoform retained stimulation of ATP hydrolysis by nicardipine although the overall extent was reduced by 60% to 1.3 ± 0.1 fold-basal (P < 0.05, n = 4). Login to comment 132 ABCB1 p.Leu339Cys The effect of paclitaxel on ATP hydrolysis in the CM labelled L339C isoform of P-gp was similar to that observed for vinblastine. Login to comment 133 ABCB1 p.Leu339Cys ABCB1 p.Leu339Cys The effect of paclitaxel on ATP hydrolysis in the CM labelled L339C isoform of P-gp was similar to that observed for vinblastine. Login to comment 134 ABCB1 p.Leu339Cys The ATPase activity of unlabelled L339C-P-gp was stimulated 2.1 ± 0.1 fold by paclitaxel (EC50 = 8.6 ± 3.7 lM) and this was abrogated following the attachment of CM (Fig. 4(c)). Login to comment 135 ABCB1 p.Leu339Cys Following the reaction of L339C with CM, XR9576 retained the ability to inhibit ATP hydrolysis to a level of 0.33 &#b1; 0.03 fold-basal. Login to comment 136 ABCB1 p.Leu339Cys Following the reaction of L339C with CM, XR9576 retained the ability to inhibit ATP hydrolysis to a level of 0.33 ± 0.03 fold-basal. Login to comment 138 ABCB1 p.Leu339Cys Altered drug stimulated ATPase activity of CM labelled L339C P-gp is not due to impaired drug binding The effect of labelling 339C on drug modulation of ATPase activity was not identical for the four established [3] distinct drug binding sites, with changes observed in potency or fold stimulation. Login to comment 139 ABCB1 p.Leu339Cys Altered drug stimulated ATPase activity of CM labelled L339C P-gp is not due to impaired drug binding The effect of labelling 339C on drug modulation of ATPase activity was not identical for the four established [3] distinct drug binding sites, with changes observed in potency or fold stimulation. Login to comment 141 ABCB1 p.Leu339Cys The extent of [3 H]- azidopine binding to the CM labelled L339C isoform of P-gp was identical to unlabelled protein. Login to comment 142 ABCB1 p.Leu339Cys The extent of [3 H]- azidopine binding to the CM labelled L339C isoform of P-gp was identical to unlabelled protein. Login to comment 151 ABCB1 p.Leu339Cys The effect of CM labelling of L339C on the Michaelis-Menten parameters describing ATP hydrolysis. Login to comment 152 ABCB1 p.Leu339Cys The effect of CM labelling of L339C on the Michaelis-Menten parameters describing ATP hydrolysis. Login to comment 154 ABCB1 p.Leu339Cys Activity was normalized such that the Vmax obtained for unlabelled L339C P-gp in the presence of nicardipine was 100%. Login to comment 155 ABCB1 p.Leu339Cys Activity was normalized such that the Vmax obtained for unlabelled L339C P-gp in the presence of nicardipine was 100%. Login to comment 166 ABCB1 p.Leu339Cys Dose-dependent drug stimulation of ATP hydrolysis by native and CM labelled L339C. Login to comment 167 ABCB1 p.Leu339Cys Dose-dependent drug stimulation of ATP hydrolysis by native and CM labelled L339C. Login to comment 170 ABCB1 p.Leu339Cys Native protein is shown as filled circles (d), whilst labelled L339C is depicted with open circles (s). Login to comment 171 ABCB1 p.Leu339Cys Native protein is shown as filled circles (d), whilst labelled L339C is depicted with open circles (s). Login to comment 173 ABCB1 p.Leu339Cys [3 H]-Azidopine photoaffinity labelling of native and CM labelled L339C. Login to comment 174 ABCB1 p.Leu339Cys [3 H]-Azidopine photoaffinity labelling of native and CM labelled L339C. Login to comment 177 ABCB1 p.Leu339Cys (a) Autoradiograms obtained for native and unlabelled L339C in the absence (lane i), or presence of vinblastine (ii), nicardipine (iii), paclitaxel (iv) or XR9576 (v). Login to comment 178 ABCB1 p.Leu339Cys (a) Autoradiograms obtained for native and unlabelled L339C in the absence (lane i), or presence of vinblastine (ii), nicardipine (iii), paclitaxel (iv) or XR9576 (v). Login to comment