COCAINE ALTERS THE ACCESSIBILITY OF ENDOGENOUS CYSTEINES IN PUTATIVE EXTRACELLULAR AND INTRACELLULAR LOOPS OF THE HUMAN DOPAMINE TRANSPORTER

Cocaine and other psychostimulants act by blocking the dopamine transp orter,]Binding of the cocaine analog, [H-3] 2-beta-carbomethoxy-3-beta -(4-fluorophenyl) tropane (CFT) to the dopamine transporter is sensiti ve to polar sulfhy-dryl-specific derivatives of methanethiosulfonate ( MTS), These reagents preferentially react with water-accessible, reduc ed cysteines, The human dopamine transporter has 13 cysteines. Their t opology is not completely determined. We sought to identify those cyst eine residues the modification of which affects CFT binding and to det ermine the topology of these reactive cysteines, We mutated each of th e cysteines, one at a time and in various combinations, to residues th at preserved binding and transport, and we tested the sensitivity of e ach of the mutant transporters to the reagents, One construct, X5C, ha d five mutated cysteines (C90A, C135A, C306A, C319F, and C342A), Using a membrane preparation in which both extracellular and intracellular cysteines could be accessible, we found that CFT binding in X5C, as co mpared with wild-type transporter, was two orders of magnitude less se nsitive to MTS ethylammonium (MTSEA), The wild-type cysteines were sub stituted back into X5C, one at a time, and these constructs were teste d in cells and in membranes, Cys-90 and Cys-306 appear to be extracell ular, and Cys-135 and Cys-342 appear to be intracellular, Each of thes e residues is predicted to be in extramembranous loops. The binding of cocaine increases the sate of reaction of MTSEA and MTS ethyltrimethy lammonium with the extracellular Cys-90 and therefore acts by inducing a conformational change, Cocaine decreases the rate of reaction of MT SEA with Cys-135 and Cys-342, acting either directly or indirectly on these intracellular residues.