Mutant | [3H]dopamine uptake | [3H]CFT binding | Dopamine competition for [3H]CFT binding (Ki)1-a | KD/KMratio | KD/Ki ratio | ||
---|---|---|---|---|---|---|---|
KM | Vmax | KD | Bmax | ||||
μM | fmol/μg/min | nM | fmol/μg | μM | |||
WT | 2.2 ± 0.5 | 278.9 ± 33.3 | 22.8 ± 2.0 | 4.1 ± 0.5 | 0.89 ± 0.13 | 1.00 | 1.00 |
1W84A | 2.5 ± 0.4 | 112.2 ± 11.3 | 7.7 ± 0.6 | 3.2 ± 0.6 | 2.02 ± 0.461-150 | 0.30 | 0.15 |
W162A | 7.3 ± 3.4 | 7.7 ± 3.1 | 25.1 ± 16.8 | 1.4 ± 0.5 | 0.0089 ± 0.0011-160 | 0.33 | 110 |
4W255A | 36.4 ± 4.8 | 33.2 ± 9.1 | 33.7 ± 6.6 | 1.3 ± 0.2 | 1.98 ± 0.541-150 | 0.1 | 0.66 |
5W266A | 0.96 ± 0.40 | 56.6 ± 6.2 | 22.3 ± 6.2 | 3.3 ± 1.1 | 3.46 ± 1.051-150 | 2.2 | 0.25 |
W310A | 12.2 ± 2.1 | 5.8 ± 1.2 | 6.3 ± 2.3 | 0.2 ± 0.1 | 0.0038 ± 0.00061-165 | 0.05 | 64.7 |
8W406A1-b | 0.36 ± 0.04 | 9.2 ± 1.2 | 78.3 ± 5.6 | 4.2 ± 1.2 | 0.17 ± 0.031-165 | 21.0 | 18.0 |
10W496A | 5.9 ± 1.0 | 63.2 ± 7.5 | 130.5 ± 18.0 | 5.4 ± 3.5 | 1-c | 2.1 | |
11W519A | 1.1 ± 0.2 | 98.6 ± 29.3 | 36.4 ± 2.0 | 3.8 ± 0.6 | 0.86 ± 0.29 | 3.2 | 1.65 |
11W523A | 0.63 ± 0.01 | 21.5 ± 7.1 | 90.4 ± 11.0 | 4.2 ± 0.8 | 1.00 ± 0.23 | 13.8 | 3.53 |
12W555A | 2.3 ± 0.5 | 231.2 ± 6.0 | 62.3 ± 6.7 | 5.9 ± 0.6 | 2.74 ± 0.721-150 | 2.6 | 0.89 |
12W561A1-b | 3.7 ± 0.7 | 265.2 ± 26.1 | 31.6 ± 8.6 | 4.7 ± 0.8 | 3.47 ± 0.491-165 | 0.8 | 0.36 |
Performances of [3H]dopamine uptake, [3H]CFT binding, and dopamine competition for [3H]CFT binding site(s) on wild-type and mutant DAT proteins, determinations ofK M, V max, K D, and B max values and calculation ofK i value are all described in Materials and Methods. Statistical analyses of the [3H]dopamine uptake and [3H]CFT binding data are shown in the bar graph presentation of the same data sets (see Fig. 2).K D/K M ratios (also see Fig. 5) are listed here for comparison withK D/K i ratios.
↵1-a Data presented as means ± S.E.M. (n = 3–5).
↵1-b Tryptophan not conserved in other Na+/Cl−-dependent monoamine transporters.
↵1-c Not determined due to large variability.
t tests:
↵1-150 , P < .05;
↵1-160 , P < .01;
↵1-165 ,P < .001; otherwise P > .05 compared with WT. One-way ANOVA: P < .05 for W266A;P < .01 for 12W561A compared with WT.