Table 1.
Kinetic properties of VMAT2 mutated at positions 342 and 400
| Strain | Serotonin transport | TBZ binding (Kd)) (nM) | Displacement of TBZ binding by MPP+
(IC50) (μM) |
|
| Km (nM) | Vmax (pmoles/min per microgram protein) | |||
| Wild type | 178 ± 43 | 9 ± 0.4 | 10 ± 1 | 69 ± 2 |
| D400E | 372 ± 69 | 2 ± 1 | 29 ± 5 | 145 ± 44 |
| D400S | — | — | 18 ± 2 | 273 ± 40 |
| Y342H | 165 ± 97 | 7 ± 1.5 | 5 ± 2 | — |
| Y342H- D400E | — | — | 81 ± 6 | — |
For Kd measurements, 1.5 μL membrane (5–10 μg total protein) produced from HEK293 cells transfected with wild-type or mutated rVMAT2 was incubated in the presence of various concentrations of [3H]-TBZOH for 20 min. For Km measurements, liposomes (1 µL) were incubated for 5 min with various concentrations of [3H]-serotonin. For determination of TBZ binding and displacement by MPP+, membranes prepared from HEK293 cells expressing wild type, D400E, or D400S rVMAT2 were incubated in the presence of various concentrations of MPP+ for 20 min followed by 20-min incubation with [3H]-TBZOH (5 nM) at pH 8.