ABCMdb

ABCB1 p.Leu339Cys

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Publications

PMID: 11598005 [PubMed] Rosenberg MF et al: "Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle."

No. Sentence Comment

136 The mutant proteins were active, with a drug-stimulated ATPase activity comparable to that of cysteine-less P-gp (except for L329C and L339C, which were discounted) (data not shown). Login to comment

PMID: 12391288 [PubMed] Gruol DJ et al: "Evidence for the locations of distinct steroid and Vinca alkaloid interaction domains within the murine mdr1b P-glycoprotein."

No. Sentence Comment

172 However, the human L339C mutation produced a nearly complete loss of the ability of methanethiosulfonate-verapamil to induce ATPase activity. Login to comment

PMID: 15192095 [PubMed] Rothnie A et al: "The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein."

No. Sentence Comment

130 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
154 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
160 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
166 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
183 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
195 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
201 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 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
215 The effects on L339C accessibility to BM paralleled the observations with CM. Login to comment

PMID: 15379547 [PubMed] Loo TW et al: "The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium."

No. Sentence Comment

212 Samples of mutants L339C/(TM6)/F942C(TM11), I306C- (TM5)/I868C(TM10), and S222C(TM4)/G872C(TM10) were then treated with (+) or without (-) M17M cross-linker for 15 min at 22 °C. Mutant G300C(TM5)/F770C(TM8) was cross-linked with 1 mM copper (phenanthroline)3 (CuP) for 15 min at 22 °C. In panel B, whole cells expressing mutants L531C(NBD1)/C1074C(NBD2), I306C(TM5)/I868C(TM10), L339C(TM6)/F942C(TM11), or S222C- (TM4)/G872C(TM10) were incubated for 10 min at 22 °C in the presence of 2.5 mM MTSEA, 10 mM MTSES, or 1 mM MTSET. Login to comment

PMID: 17636884 [PubMed] Loo TW et al: "Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments."

No. Sentence Comment

55 In the presence of ATP, however, mutant F343C(TM6)/V982C(TM12), but not mutant L339C- (TM6)/V982C(TM12), was cross-linked with TMEA (31). Login to comment
74 The positions of the catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) and the cysteine mutations in the TM segments used in the disulfide cross-linking studies (L332C, L339C, F343C, F728C, L975C, and V982C) are shown. Login to comment
81 Cross-linking with TMEA was inhibited by the presence of ATP in both mutants L339C(TM6)/V982C(TM12) and L339C- (TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Login to comment
106 Membranes were prepared from HEK 293 cells expressing P-gp mutants L339C- (TM6)/V982C(TM12) or L339C(TM6)/V982C(TM12)/E556Q- (NBD1)/E1201Q(NBD2) (A) and F343C(TM6)/V982C(TM12) or F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) (B). Login to comment
167 Samples were then subjected to immunoblot analysis. Figure 7C shows that there was little difference in the concentration-dependent cross-linking of mutant L339C- (TM6)/F728C(TM7)/E556Q(NBD1)/E1201Q(NBD2) in the presence or absence of ATP. Login to comment
171 Therefore, we confirmed that mutant L339C- (TM6)/F728C(TM7)/E556Q(NBD1)/E1201Q(NBD2) did not exhibit ATP hydrolysis by subjecting it to vanadate trapping of nucleotide. Login to comment

PMID: 17696319 [PubMed] Storm J et al: "Residue G346 in transmembrane segment six is involved in inter-domain communication in P-glycoprotein."

No. Sentence Comment

293 A similar conclusion was demonstrated for the Q347C and S349C isoforms (Tables 3 and 4) and was also shown for the L339C isoform (29). Login to comment

PMID: 17848563 [PubMed] Loo TW et al: "Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites."

No. Sentence Comment

86 Disulfide Cross-linking Analysis-The double cysteine mutants L339C(TM6)/F728C(TM7), L65R(TM1)/L339C(TM6)/ F728C(TM7), T199R(TM3)/L339C(TM6)/F728C(TM7), I306R (TM5)/L339C(TM6)/F728C(TM7), or F343R(TM6)/L339C (TM6)/F728C(TM7) were transiently expressed in HEK 293 cells (32). Login to comment
193 Accordingly, mutants L65R(TM1)/L339C(TM6)/F728C(TM7), T199R(TM3)/L339C (TM6)/F728C(TM7), I306R(TM5)/L339C(TM6)/F728C(TM7), and F343R(TM6)/L339C(TM6)/F728C(TM7) were constructed and expressed in HEK 293 cells. Login to comment

PMID: 18003606 [PubMed] De Rosa MF et al: "Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog."

No. Sentence Comment

270 C, panel i, membranes prepared from HEK 293 cells expressing mutant L339C/F728C were preincubated for 15 min at 22 °C with different concentrations of adaGb3 (0-500 ␮M) (lanes 2-6). Login to comment
291 Modification of the L339C residue altered signal transduction from several distinct MDR1 drug-binding sites (73), suggesting adaGb3 should prove effective against many MDR1 substrates. Login to comment

PMID: 18596043 [PubMed] Loo TW et al: "Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11."

No. Sentence Comment

194 TABLE 2 Concentrations of drug substrates required to inhibit cross-linking by 50% Mutant Vinblastine Cyclosporin A Rhodamine B ␮M ␮M ␮M L339C/F728C 0.5 Ϯ 0.2a 0.8 Ϯ 0.3 52 Ϯ 14 G64R/L339C/F728C 53 Ϯ 15b 1.5 Ϯ 0.4 61 Ϯ 10 M68R/L339C/F728C 266 Ϯ 62b 1.3 Ϯ 0.3 57 Ϯ 7 V71R/L339C/F728C 0.6 Ϯ 0.2 0.8 Ϯ 0.2 57 Ϯ 10 a Each value is the mean Ϯ S.D. (n ϭ 3-4 separate cross-linking experiments). 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
78 No cross-linked product was observed for mutants F336C/S979C and L339C/V982C. Login to comment
80 We also tested mutants F335C/L976C, L339C/S979C, F343C/F983C, G347C/A987C, and S351C/ V991C for cross-linking since they were predicted to lie on opposing faces of TM6 and TM12 modeled in a right-handed coiled-coil. Login to comment
107 Mutants S979C/F336C or L339C/V982C did not yield any cross-linked product even in the presence of ATP or drug substrates (data not shown). Login to comment
124 Cross-linking was not observed between F336C/S979C or L339C/V982C, even in the presence of ATP or drug substrates FIG. 2. 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

21 We show that the drug-stimulated ATPase activities of mutants L339C and A342C (TM6) and L975C, V982C, and A985C (TM12) were particularly sensitive to inhibition by dBBn and that the inhibition was prevented by various drug substrates. Login to comment
98 In contrast, mutants L339C, A342C, L975C, V982C, and A985C were significantly inhibited by dBBn, because they retained only 10, 40, 13, 25, and 32% of their activities, respectively. Login to comment
99 The concentration of dBBn required to give 50% inhibition of ATPase activity for mutants L339C, L975C, V982C, A985C, and A342C were 90, 112, 320, 480, and 700 ␮M, respectively. Login to comment
111 The P-glycoproteins(His)10 of Cys-less and mutants L339C, A342C, L975C, V982C, and A985C were mixed with lipid and then preincubated for 15 min at 4 °C without drug or in the presence of 2 mM verapamil (Ver. Login to comment
124 Similarly, mutants L339C, L975C, and V982C were also protected from dBBn inactivation by various drug substrates. Login to comment
126 Colchicine was also very effective in protecting mutant L339C from dBBn inactivation because it retained about 80% of its colchicine-stimulated ATPase activity. Login to comment
129 It offered little or no protection for mutant V982C and only moderately protected mutants L339C and L975C. Login to comment

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

No. Sentence Comment

211 In contrast, two other potential pairs that lie between the first and second of the four cross-linked pairs within TMs 6 and 12 (Loo & Clarke, 1997), namely F336C/S979C and L339C/V982C, failed to form cross-links. Login to comment

PMID: 22700974 [PubMed] Loo TW et al: "The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together."

No. Sentence Comment

244 Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51). Login to comment
237 Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51). Login to comment

PMID: 16545467 [PubMed] Shilling RA et al: "New light on multidrug binding by an ATP-binding-cassette transporter."

No. Sentence Comment

78 Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,2]. Login to comment
76 Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,25]. Login to comment

PMID: 16004994 [PubMed] Rothnie A et al: "The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment."

No. Sentence Comment

75 [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
91 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
94 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 The half-life for L339C was significantly shorter (P < 0.05) than that obtained for the other TM6 mutant isoforms. Login to comment
110 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
112 Full Michaelis-Menten plots for the unlabelled and CM-modified L339C isoform of P-gp are shown in Fig. 3. Login to comment
113 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 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 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
118 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
123 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 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 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 Time course for the labelling of L339C P-gp with CM. Login to comment
129 (b) The time-course of labelling L339C P-gp with CM obtained from three independent purified protein preparations. Login to comment
131 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
133 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 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
136 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
139 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
142 The extent of [3 H]- azidopine binding to the CM labelled L339C isoform of P-gp was identical to unlabelled protein. Login to comment
152 The effect of CM labelling of L339C on the Michaelis-Menten parameters describing ATP hydrolysis. Login to comment
155 Activity was normalized such that the Vmax obtained for unlabelled L339C P-gp in the presence of nicardipine was 100%. Login to comment
167 Dose-dependent drug stimulation of ATP hydrolysis by native and CM labelled L339C. Login to comment
171 Native protein is shown as filled circles (d), whilst labelled L339C is depicted with open circles (s). Login to comment
174 [3 H]-Azidopine photoaffinity labelling of native and CM labelled L339C. Login to comment
178 (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
74 [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 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 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
109 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
111 Full Michaelis-Menten plots for the unlabelled and CM-modified L339C isoform of P-gp are shown in Fig. 3. Login to comment
117 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 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
128 (b) The time-course of labelling L339C P-gp with CM obtained from three independent purified protein preparations. Login to comment
130 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
132 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
135 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
138 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 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 The effect of CM labelling of L339C on the Michaelis-Menten parameters describing ATP hydrolysis. Login to comment
154 Activity was normalized such that the Vmax obtained for unlabelled L339C P-gp in the presence of nicardipine was 100%. Login to comment
166 Dose-dependent drug stimulation of ATP hydrolysis by native and CM labelled L339C. Login to comment
170 Native protein is shown as filled circles (d), whilst labelled L339C is depicted with open circles (s). Login to comment
173 [3 H]-Azidopine photoaffinity labelling of native and CM labelled L339C. Login to comment
177 (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

PMID: 25600711 [PubMed] Pan L et al: "Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics."

No. Sentence Comment

196 Specifically, ATP binding inhibited the crosslink of pairs of human Pgp between TM6 and TM12 at L339C-V982C (mouse L334-V978) and L332C-L975C (mouse L328-L971) but promoted the crosslink of F343C-V982C (mouse F339-V978). Login to comment