ABCMdb

PMID: 8639515

Hanna M, Brault M, Kwan T, Kast C, Gros P
Mutagenesis of transmembrane domain 11 of P-glycoprotein by alanine scanning.
Biochemistry. 1996 Mar 19;35(11):3625-35., 1996-03-19 [PubMed]

Sentences

No. Mutations Sentence Comment

32 ABCB1 p.Pro866Ala ABCB1 p.Phe978Ala ABCB1 p.Pro223Ala ABCB1 p.Phe335Ala Finally, elegant studies of Loo and Clarke, in which proline and phenylalanine residues located within TM domains (Loo & Clarke, 1993a,b) as well as glycines in cytoplasmic loops of human MDR1 were systematically replaced by alanines (Loo & Clarke, 1994a), identified several residues within TM 4 (P223A), TM 6 (F335A), TM 10 (P866A), and TM 12 (F978A) and in the intervening cytoplasmic loops where mutations differentially affect the capacity of P-gp to confer resistance to vinblastine (VBL), adriamycin (ADR), colchicine (COL), and actinomycin D (ACT). Login to comment 68 ABCB4 p.Tyr949Ala ABCB4 p.Ile936Ala Untransfected LR73 cells and G418R mass populations of LR73 transfected with WT and I936A, Q942A, Y949A, and F953A mutant mdr3 cDNAs were plated in medium without drug (2.5 × 103 cells) or in medium containing ACT, ADR, COL, or VBL at three different concentrations (2.5 × 104 cells) in 24-well plates (15-mm wells). Login to comment 124 ABCB4 p.Phe947Ala Eight of the mutations (V933A, T937A, F940A, M944A, M945A, F947A, A950G, and A951G) had no significant effect on function, and the corresponding mutant P-gps expressed levels of resistance to each drug similar to that of WT. Login to comment 125 ABCB1 p.Thr941Ala ABCB1 p.Phe938Ala ABCB4 p.Ile936Ala Eleven of the mutations (F934A, G935A, I936A, F938A, S939A, T941A, Q942A, A943G, Y946A, S948A, and C952A) caused a moderate but reproducible 2-3-fold decrease in resistance to one or more of the drugs. Login to comment 126 ABCB4 p.Tyr949Ala Finally, two mutations, Y949A and F953A, caused a more dramatic 5-10-fold loss of resistance to one or more drugs tested. Login to comment 127 ABCB4 p.Ile936Ala Among mutants with reduced activity, only F953A and I936A displayed an overall decrease in resistance to all drugs analyzed. Login to comment 129 ABCB4 p.Tyr949Ala For example, mutants S939A and Q942A showed wild-type activity against VBL and ACT but had reduced activity against ADR and COL, while mutant A943G displayed reduced activity only against ADR, and mutant Y949A had wild-type activity only against VBL. Login to comment 135 ABCB4 p.Phe947Ala Strikingly, seven of the eight mutations which were without consequences on mdr3 function map to the highly hydrophobic face of this helix, with six of them (V933A, T937A, F940A, M944A, F947A, and A951G) clustered in a continuous mutation-insensitive segment (Figure 4). Login to comment 136 ABCB4 p.Phe947Ala Included in this group are two nonconservative substitutions, F940A and F947A, in which a large aromatic side chain is replaced by a methyl group, and the T937A mutant, in which a hydrophilic hydroxyl group is eliminated. Login to comment 145 ABCB4 p.Ile936Ala Among the amino acids on the more hydrophobic side of the helix, I936A showed the greatest sensitivity to alanine substitution, resulting in an overall 2-3-fold reduction in activity against the four drugs tested. Login to comment 146 ABCB4 p.Ile936Ala The somewhat lower level of expression of the I936A mutant in the membrane fraction of transfectants might have partly contributed to the reduced activity of this mutant. Login to comment 154 ABCB4 p.Tyr949Ala Likewise, the Y949A substitution caused the second-most severe loss of function, resulting in almost complete loss of ADR resistance and 2.5-3× loss of resistance to ACT and COL, while VBL resistance was unaffected in this mutant. Login to comment 155 ABCB4 p.Tyr949Ala On a helical wheel projection the F953A and Y949A mutations map very close to each other and symmetrically opposite to the cluster of neutral mutations mapping to the other, more hydrophobic face of TM11 (Figure 4). Login to comment 156 ABCB1 p.Thr941Ala ABCB1 p.Phe938Ala ABCB4 p.Tyr949Ala ABCB4 p.Ile936Ala ABCB4 p.Phe947Ala These two mutations are also predicted to map near the top half of TM11, within the outer lipid leaflet of Table 1: Drug Survival Characteristics of Mass Populations Cells Stably Transfected with Wild-Type or Mutant mdr3 cDNAsa mdr3 ACT ADR COL VBL V933A 32 ( 3 (17×) 370 ( 200 (19×) 590 ( 180 (21×) 200 ( 20 (24×) F934A 37 ( 2 (19×) 110 ( 40 (6×) 240 ( 80 (9×) 200 ( 30 (24×) G935A 26 ( 3 (14×) 200 ( 110 (10×) 350 ( 20 (13×) 120 ( 20 (14×) I936A 23 ( 5 (12×) 100 ( 40 (5×) 170 ( 40 (6×) 80 ( 13 (10×) T937A 50 ( 10 (27×) 280 ( 110 (15×) 620 ( 100 (23×) 160 ( 6 (20×) F938A 42 ( 6 (22×) 140 ( 60 (7×) 360 ( 80 (13×) 260 ( 20 (32×) S939A 46 ( 1 (24×) 160 ( 80 (8×) 280 ( 30 (10×) 160 ( 30 (20×) F940A 43 ( 4 (22×) 380 ( 130 (20×) 980 ( 210 (36×) 240 ( 30 (29×) T941A 48 ( 3 (25×) 100 ( 40 (5×) 140 ( 20 (5×) 130 ( 40 (16×) Q942A 60 ( 6 (32×) 100 ( 40 (5×) 190 ( 10 (7×) 270 ( 70 (33×) A943G 41 ( 4 (22×) 90 ( 10 (5×) 350 ( 60 (13×) 380 ( 40 (46×) M944A 29 ( 6 (15×) 450 ( 190 (23×) 730 ( 90 (27×) 200 ( 40 (24×) M945A 56 ( 2 (29×) 180 ( 50 (9×) 340 ( 80 (13×) 210 ( 50 (26×) Y946A 16 ( 2 (8×) 310 ( 160 (16×) 350 ( 30 (13×) 100 ( 8 (13×) F947A 32 ( 2 (17×) 310 ( 100 (16×) 660 ( 120 (24×) 170 ( 30 (20×) S948A 25 ( 3 (13×) 210 ( 80 (11×) 370 ( 60 (13×) 210 ( 40 (26×) Y949A 18 ( 2 (9×) 60 ( 20 (3×) 180 ( 20 (6×) 190 ( 15 (23×) A950G 46 ( 6 (24×) 280 ( 70 (15×) 520 ( 120 (19×) 200 ( 30 (25×) A951G 60 ( 10 (32×) 300 ( 30 (16×) 560 ( 120 (21×) 280 ( 30 (34×) C952A 24 ( 5 (12×) 360 ( 180 (19×) 500 ( 50 (18×) 140 ( 50 (18×) F953A 8 ( 1 (4×) 28 ( 7 (1×) 45 ( 9 (2×) 100 ( 13 (13×) WT 50 ( 10 (25×) 290 ( 80 (15×) 450 ( 100 (16×) 210 ( 40 (25×) LR73 2 ( 0.1 (1×) 19 ( 6 (1×) 27 ( 4 (1×) 8 ( 2 (1×) a The drug survival of Chinese hamster ovary drug-sensitive cells (LR73) and of cell clones transfected with either wild-type or mutant mdr3 is expressed as the D50 (in nanograms per milliliter), or the dose necessary to reduce the plating efficiency of the control and transfected cells by 50%. Login to comment 187 ABCB4 p.Tyr949Ala ABCB4 p.Ile936Ala For this, mass populations of G418R cells cotransfected with individual mutants I936A, Q942A, Y949A, and F953A, showing unique deviations from wild type in their drug resistance profiles (Figure 3), were plated directly in medium containing increasing concentrations of VBL, ADR, COL, and ACT. Login to comment 203 ABCB4 p.Tyr949Ala ABCB4 p.Tyr949Phe ABCB4 p.Tyr949Gln Indeed, we observed that Y949F had characteristics very similar to Y949A, while Y949Q was near wild type with only a 2-fold reduction observed for adriamycin resistance (data not shown). Login to comment 205 ABCB4 p.Ile936Ala As opposed to other mutation-sensitive positions, the I936A mutation caused an overall reduction by a factor of 2-3-fold in activity of the mutant P-gp, reduction which could not be totally explained by a lower amount of protein produced in the clone analyzed (compare expression and activity to that of G935A). Login to comment 216 ABCB4 p.Tyr949Ala ABCB4 p.Ile936Ala Approximately 2.5 × 103 cells (no drug) or 2.5 × 104 cells (with drug) from mass populations of G418R clones stably transfected with either wild-type mdr3 (columns 1) or mdr3 mutants I936A (2), Q942A (3), Y949A (4), F953A (5), or untransfected LR73 control cells (6) were plated in medium containing increasing concentrations of the drugs actinomycin D (ACT), adriamycin (ADR), colchicine (COL), or vinblastine (VBL). Login to comment 220 ABCB4 p.Tyr949Ala ABCB4 p.Ile936Ala Table 2: Drug Survival Characteristics of Mass Populations of G418R Cell Clones Transfected with Mouse mdr3 mutants in TM11a ACT ADM COL VBL REV 7.5 52.6 71.9 8.0 I936A 59.7 (8.0×) 121.3 (2.3×) 367.1 (5.1×) 70.5 (8.8×) Q942A 54.1 (7.2×) 82.6 (1.6×) 128.1 (1.8×) 81.7 (10.2×) Y949A 31.6 (4.2×) 80.4 (1.5×) 163.3 (2.3×) 81.0 (10.1×) F953A 9.4 (1.2×) 51.6 (1×) 85.9 (1.2×) 78.9 (9.9×) WT 67.2 (9.0×) 249.4 (4.7×) 493.7 (6.9×) 67.6 (8.5×) a The drug cytotoxicity for each mass population of G418R cell clones is expressed as the D50 (nanograms per milliliter), or the dose necessary to reduce the plating efficiency of the control and transfected cell clones by 50%. Login to comment 239 ABCB1 p.Phe957Ala In previous work (Loo & Clarke, 1993b), the same mutation was made in the same residue at the equivalent position in P-gp encoded by the human MDR1 isoform (F957A). Login to comment