TABLE 1.
Kinetics of drug transport mediated by representative variants from each of the five mutant groups (I-V) (Fig. 1)
The (−) values means reduced efflux (R6G and Nile Red (NR)) and ATPase activities of the mutants as compared to Cdr1p, while (+) values indicate no significant change. Fifty % and above inhibition is considered as significant. The underline means that the respective parameter is responsible for the susceptible phenotype of the mutant variants. The values are the average ± S.D. of three independent experiments. Km and Vmax were calculated using Graphpad Prism 5 Software.
| Kinetic arameters | Cdr1p (WT) | F559A (Group I) | L529A (Group II) | M604A (Group III) | N609A (Group IV) | I1237A (Group V) |
|---|---|---|---|---|---|---|
| R6G efflux (%) | 100 | 48 ± 7 (−) | 29 ± 7 (−) | 25 ± 6.5 (−) | 72 ± 12 (+) | 94 ± 4.7 (+) |
| NR efflux (%) | 100 | 31 ± 6 (−) | 40 ± 5 (−) | 78 ± 10.6 (+) | 43 ± 7 (−) | 81 ± 6 (+) |
| ATPase (%) | 100 | 48 ± 5 (−) | 87 ± 8 (+) | 76 ± 9 (+) | 82 ± 8 (+) | 87 ± 4 (+) |
| ATP hydrolysis Km (mm) | 0.61 ± 0.14 | 0.64 ± 0.12 | 0.60 ± 0.13 | 0.68 ± 0.19 | 0.69 ± 0.13 | 0.76 ± 0.12 |
| ATP hydrolysis Vmax (nmole/min) | 116 ± 8 | 49 ± 4 | 131 ± 10 | 96 ± 17 | 101 ± 10 | 109 ± 17 |
| Kcat (s−1) | 33.2 ± 2.3 × 103 | 23.5 ± 1.7 × 103 | 30.3 ± 2.4 × 103 | 59.2 ± 10.7 × 103 | 32.3 ± 3.2 × 103 | 32.4 ± 5.1 × 103 |
| Kcat /Km (mm−1 sec−1) | 54.5 ± 3.8 × 106 | 36.7 ± 2.7 × 106 | 50.5 ± 4.0 × 106 | 87.1 ± 15.8 × 106 | 46.8 ± 4.7 × 106 | 42.7 ± 6.7 × 106 |
| R6G binding Kd,R6G (μm) | 5.53 ± 0.56 | 5.7 ± 1.0 | 12.46 ± 1.32 | 11.3 ± 0.75 | 5.48 ± 0.78 | 6.63 ± 0.80 |
| R6G efflux rate (nmol/ml) | 0.62 ± 0.07 | 0.23 ± 0.01 | 0.31 ± 0.01 | 0.37 ± 0.05 | 0.57 ± 0.03 | 0.62 ± 0.04 |
| NR binding Kd,NR (μm) | 0.4 ± 0.04 | 0.6 ± 0.06 | 0.48 ± 0.08 | 0.42 ± 0.12 | 1.73 ± 0.09 | 0.52 ± 0.08 |
| NR efflux rate (nmol/ml) | 98.9 ± 1.9 | 51 ± 47 | 24.9 ± 5.5 | 98.78 ± 0.88 | 46 ± 35 | 99.2 ± 1.4 |