ABCMdb

PMID: 21177413

Parveen Z, Stockner T, Bentele C, Pferschy S, Kraupp M, Freissmuth M, Ecker GF, Chiba P
Molecular dissection of dual pseudosymmetric solute translocation pathways in human P-glycoprotein.
Mol Pharmacol. 2011 Mar;79(3):443-52. Epub 2010 Dec 21., [PubMed]

Sentences

No. Mutations Sentence Comment

52 ABCB1 p.Gln132Arg ABCB1 p.Gln132Arg ABCB1 p.Gln132Ala ABCB1 p.Gln132Ala ABCB1 p.Gln132Ala ABCB1 p.Gln773Arg ABCB1 p.Gln773Arg ABCB1 p.Gln773Ala ABCB1 p.Gln773Ala ABCB1 p.Gln773Ala Site-directed mutagenesis was performed at positions 132 and 773 of hexahistidine-tagged human P-gp cloned into the entry vector pENTR4-MDR1-His6 to generate the Q132A, Q132R, Q773A, Q773R, and the Q132A/Q773A mutants using the following forward and reverse primers: Q132A: forward, 5Ј-CTG CTT ACA TTG CGG TTT CAT TTT GG-3Ј; reverse, 5Ј-CCA AAA TGA AAC CGC AAT GTA AGC AG-3Ј; Q132R: forward, 5Ј-GCT GCT TAC ATT CGT GTT TCA TTT TG-3Ј; reverse, 5Ј-CAA AAT GAA ACA CGA ATG TAA GCA GC-3Ј; Q773A: forward, 5Ј-CAT TTT TCC TTG CGG GTT TCA CAT TTG GC-3Ј; reverse, 5Ј-GCC AAA TGT GAA ACC CGC AAG GAA AAA TG-3Ј; Q773R: forward, 5Ј-CAT TTT TCC TTC GAG GTT TCA CAT TTG-3Ј; reverse, 5Ј-CAT TTT TCC TTC GAG GTT TCA CAT TTG-3Ј. Login to comment 53 ABCB1 p.Gln132Arg ABCB1 p.Gln132Arg ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg ABCB1 p.Gln773Arg ABCB1 p.Gln773Arg Double mutant Q132R/Q773R was generated by restriction digestion of pENTR4-Q132R and pENTR4-Q773R with SalI and EcoRI and subsequent ligation of the mutated fragment from pENTR4-Q773R into pENTR4-Q132R using T4 DNA ligase (rapid DNA ligation kit; Fermentas, Vienna, Austria). Login to comment 137 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg Rhodamine123 Efflux in Q132R/A and Q773R/A Mutants. Login to comment 138 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg In an initial series of experiments, we monitored rhodamine123 efflux in HEK-293/EBNA cells transiently transfected with either wild-type P-gp or the Q132R and Q773R mutants. Login to comment 140 ABCB1 p.Gln773Arg A representative single experiment for rhodamine123 efflux in the Q773R mutant is shown in Fig. 3A. Login to comment 146 ABCB1 p.Gln773Arg Introduction of an arginine residue at position 132 (green triangles) decreased transport rates to 52 Ϯ 13% of wild-type (green stippled line), whereas replacement of Gln773 by arginine (orange data points, orange stippled line) TABLE 1 Quality of models as assessed by Modeller (Shen et al., 1995; Shen and Sali, 2006; Melo and Sali, 2007), ProQ (Wallner and Elofsson, 2003), and Procheck (Laskowski et al., 1993) The MODELLER DOPE-score is a statistical potential energy-score trained to recognize correct folding by analyzing nonbonding interactions between atoms in a pair-wise fashion. Login to comment 161 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg Effect of Propafenone Analogs on Rhodamine123 Efflux in Q132R/A and Q773R/A Mutants. Login to comment 166 ABCB1 p.Gln773Arg For each concentration of GPV31, a time-dependent decrease in mean cellular fluorescence was monitored over 4 min. A representative experiment for the Q773R mutant is shown in Fig. 4A. Login to comment 172 ABCB1 p.Gln132Arg The IC50 value for wild-type P-gp was found to be 0.11 Ϯ 0.06 ␮M (black), whereas mutant Q132R showed an almost 7-fold higher IC50 value of 0.71 Ϯ 0.46 ␮M (green). Login to comment 173 ABCB1 p.Gln773Arg In contrast, the Q773R mutant showed an IC50 value of 0.02 Ϯ 0.01 ␮M (orange). Login to comment 177 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg IC50 values for wild type (black) and mutants Q132R (green) and Q773R (orange) (Fig. 5, Table 2) were indistinguishable, demonstrating that uncharged P-gp solutes were not affected by introduction of a positive charge in either position 132 or 773. Login to comment 181 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg Effect of Verapamil and Vinblastine on Rhodamine123 Efflux in Q132R/A and Q773R/A Mutants. Login to comment 182 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg In a third series of experiments, we evaluated the effect of verapamil and vinblastine on rhodamine123 transport in wild type and mutants Q132R and Q773R. Login to comment 200 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg In contrast, the IC50 values for the Q132R and Q773R mutants were 1.24 Ϯ 0.13 and 0.14 Ϯ 0.04 ␮M, respectively. Login to comment 202 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg The respective IC50 values were 2.68 Ϯ 0.93 ␮M for wild type, 0.45 Ϯ 0.16 ␮M for mutant Q773R, and 9.19 Ϯ 1.2 ␮M for the Q132R mutant. Login to comment 220 ABCB1 p.Gln773Arg A, Rhodamine123 efflux is shown for the Q773R mutant as a representative example. Login to comment 231 ABCB1 p.Gln132Ala ABCB1 p.Gln773Ala Mutants Q132A (yellow) and Q773A (magenta) lie close to the stippled black line and therefore show transport rates that are comparable with wild-type. Login to comment 232 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg Q132R (green) and Q773R (orange) show transport rates of 52 Ϯ 13 and 24 Ϯ 6% of wild type, respectively (error propagation accounted for). Login to comment 233 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg The double mutant Q132R/Q773R (cyan) shows flux rates that are equal to simple diffusion (0.7-0.8/s). Login to comment 246 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg ABCB1 p.Gln773Arg Both the Q132R and the Q773R mutant showed reduced transport activity for rhodamine123, which, however, was more pronounced in the Q773R mutant. Login to comment 251 ABCB1 p.Gln773Arg A, for each concentration of GPV31 (none, black; 0.008 ␮M, dark green; 0.02 ␮M, light blue; 0.05 ␮M, cyan; 0.13 ␮M, lavender; 0.32 ␮M, olive; 0.80 ␮M, dark blue; 2.00 ␮M, light green; and 5.00 ␮M, red) a time-dependent decrease in mean cellular fluorescence was monitored over 4 min. A representative experiment for Q773R P-gp is shown. Login to comment 257 ABCB1 p.Gln773Arg B shows the two concentration response curves for one representative experiment performed with the Q773R mutant. Login to comment 265 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg Wild-type P-gp, black curve; Q132R, green curve; Q773R, orange curve. Login to comment 267 ABCB1 p.Gln132Ala ABCB1 p.Gln132Ala ABCB1 p.Gln773Ala ABCB1 p.Gln773Ala Indistinguishable IC50 values were found for either compound in the Q132A, the Q773A, and the Q132A/Q773A mutants. Login to comment 285 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg Wild-type P-gp, black curve; Q132R, green curve; Q773R, orange curve. Login to comment 289 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg Black curve, wild-type P-gp; green curve, Q132R; orange curve, Q773R. Login to comment 295 ABCB1 p.Gln132Arg ABCB1 p.Gln773Arg Black curve, wild-type P-gp; green curve, Q132R; orange curve, Q773R. Login to comment 299 ABCB1 p.Gln132Arg ABCB1 p.Gln132Ala ABCB1 p.Gln132Ala ABCB1 p.Gln773Arg ABCB1 p.Gln773Ala ABCB1 p.Gln773Ala Mutation IC50 Verapamil Vinblastine GPV31 GPV366 ␮M Wild-type P-gp 0.43 Ϯ 0.10 2.68 Ϯ 0.93 0.11 Ϯ 0.06 2.60 Ϯ 0.69 Q132A 0.30 Ϯ 0.07 2.58 Ϯ 0.31 0.10 Ϯ 0.03 2.57 Ϯ 0.69 Q773A 0.42 Ϯ 0.15 2.80 Ϯ 0.12 0.11 Ϯ 0.05 2.40 Ϯ 1.47 Q132A/Q773A 0.44 Ϯ 0.16 2.24 Ϯ 0.43 0.12 Ϯ 0.01 2.87 Ϯ 0.63 Q132R 1.24 Ϯ 0.13* 9.19 Ϯ 1.20* 0.71 Ϯ 0.46* 2.64 Ϯ 0.59 Q773R 0.14 Ϯ 0.04* 0.45 Ϯ 0.16* 0.02 Ϯ 0.01* 2.09 Ϯ 0.90 * P Ͻ 0.05, significantly different from wild-type values. Login to comment 314 ABCB1 p.Gly251Val The 132Arg mutant did not increase trafficking of the G251V background, whereas mutant 773Arg showed a complete trafficking deficiency. Login to comment