Transport-dependent accessibility of a cytoplasmic loop cysteine in the human dopamine transporter
Transport-dependent accessibility of a cytoplasmic loop cysteine in the human dopamine transporter
复制标题
DOI：
10.1074/jbc.275.3.1608
复制
发表时间：
2000-01-21
影响因子：
4.8
通讯作者：
Justice, JB
中科院分区：
文献类型：
--
作者：
Chen, NH;Ferrer, JV;Justice, JB
The effect of covalent sulfhydryl modification on dopamine uptake by the human dopamine transporter was determined by rotating disc electrode voltammetry. A transporter construct, X5C, with five mutated cysteines (C90A, C135A, C306A, C319F, and C342A) and the constructs into which the wild-type cysteines were substituted back into X5C, one at a time, all showed nearly normal binding affinity for [H-3]CFT and for cocaine, but they displayed significant reductions in K-m and V-max for DA uptake. Reaction of Cys-90 or Cys-306 with impermeant methanethiosulfonate derivatives enhanced dopamine uptake to a similar extent as the previously observed enhancement of [H-3]CFT binding caused by the same reaction, suggesting that cocaine may bind preferentially to a conformation in the transport cycle. m-Tyramine increased the rate of reaction of (2-amino-ethyl)methanethiosulfonate (MTSEA) with X-A342C, the construct with a cytoplasmic loop residue Cys-342 restored. This m-tyramine-induced increase in reactivity appeared to require the inward transport rather than the outward transport or external binding of m-tyramine, and it was prevented by cocaine. Thus, inward translocation of substrates may involve structural rearrangement of hDAT, which likely exposes Cys-342 to reaction with MTSEA, and Cys-342 may be located on a part of the transporter associated with cytoplasmic gating.
