TABLE 3.
Binding and uptake characteristics for histidine mutants introduced into the DAT H193K background
Experiments performed on transient transfected COS7 cells expressing the Zn2+-insensitive DAT H193K background or mutants herein. The Kd and Bmax values of CFT binding and the Km and Vmax for DA uptake were calculated from nonlinear regression analysis of accumulated [3H]radioligand in the presence increasing concentrations of unlabeled ligand (CFT or DA) using 11 consecutive concentrations performed in triplicate. Nonspecific [3H]DA uptake or [3H]CFT binding were determined with 1 μm nomifensine. The IC50 values used in the estimation of Kd and Km values were calculated from means of pIC50 values and the S.E. interval from the pIC50 ± S.E. The Bmax and Vmax values are shown as means ± S.E. ND, not detectable.
| DAT construct | CFT |
DA |
||||
|---|---|---|---|---|---|---|
| Bmax | Kd | N | Vmax | Km | N | |
| fmol/105 cells | nm | fmol/105 cells/min | μm | |||
| H193K | 191 ± 12 | 15 (12; 190) | 3 | 6300 ± 1100 | 1.7 (0.9; 3.5) | 4 |
| H193K/R85H | 209 ± 28 | 36 (25; 51) | 3 | 430 ± 119 | 3.1 (1.7; 5.6) | 5 |
| H193K/D476H | 205 ± 28 | 44 (36; 54) | 3 | 1421 ± 131 | 2.0 (1.3; 3.1) | 3 |
| H193K/R85H/D476H | 89 ± 11 | 13 (11; 16) | 4 | 463 ± 33 | 3.7 (2.5; 5.4) | 8 |
| H193K/R85H/D476H + 200 μm Zn2+ | 201 ± 21 | 17 (15; 19) | 4 | ND | ND | 3 |