Nh. Chen et al., Transport-dependent accessibility of a cytoplasmic loop cysteine in the human dopamine transporter, J BIOL CHEM, 275(3), 2000, pp. 1608-1614
The effect of covalent sulfhydryl modification on dopamine uptake by the hu
man dopamine transporter was determined by rotating disc electrode voltamme
try. A transporter construct, X5C, with five mutated cysteines (C90A, C135A
, C306A, C319F, and C342A) and the constructs into which the wild-type cyst
eines were substituted back into X5C, one at a time, all showed nearly norm
al binding affinity for [H-3]CFT and for cocaine, but they displayed signif
icant reductions in K-m and V-max for DA uptake. Reaction of Cys-90 or Cys-
306 with impermeant methanethiosulfonate derivatives enhanced dopamine upta
ke to a similar extent as the previously observed enhancement of [H-3]CFT b
inding caused by the same reaction, suggesting that cocaine may bind prefer
entially to a conformation in the transport cycle. m-Tyramine increased the
rate of reaction of (2-amino-ethyl)methanethiosulfonate (MTSEA) with X-A34
2C, the construct with a cytoplasmic loop residue Cys-342 restored. This m-
tyramine-induced increase in reactivity appeared to require the inward tran
sport rather than the outward transport or external binding of m-tyramine,
and it was prevented by cocaine. Thus, inward translocation of substrates m
ay involve structural rearrangement of hDAT, which likely exposes Cys-342 t
o reaction with MTSEA, and Cys-342 may be located on a part of the transpor
ter associated with cytoplasmic gating.