Putative Cocaine Receptor in Striatum Is a Glycoprotein with Active Thiol Function

Abstract

Dopamine transporters of bovine and rat striata were identified by their specific [3H]cocaine binding and cocaine-sensitive [3H]dopamine [( 3H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes. Inorganic and organic mercury reagents inhibited both [3H]DA uptake and [3H]cocaine binding, though they were all more potent inhibitors of the former. n-Ethylmaleimide inhibited [3H]DA uptake totally but [3H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [3H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [3H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (less than 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (greater than 10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation.

RTI-76, an isothiocyanate derivative of a phenyltropane cocaine analog, as a tool for irreversibly inactivating dopamine transporter function in vitro

“…If occupancy of only one cocaine site is sufficient to block dopamine uptake through the dimer, it would explain the general assumption that inhibition of dopamine uptake by blockers requires no more than one molecule to block transport of one molecule of dopamine, in agreement with the monophasic nature of the inhibition curves observed for most compounds (Richelson and Pfenning 1984;Andersen 1987;Cao et al 1989;Krueger 1990;Reith et al 1996a;Eshleman et al 1999). Thus, for each translocation cycle, dopamine needs to use only one of the two available dopamine recognition sites per dimer, and cocaine will competitively inhibit regardless of which site dopamine used.…”

“…Although inhibition of dopamine uptake by cocaine via the neuronal dopamine transporter in the striatum and nucleus accumbens has been reported to be of a non-or uncompetitive type (Missale et al 1985;McElvain and Schenk 1992;Wheeler et al 1994;Povlock and Schenk 1997), most studies indicate a competitive mechanism (Richelson and Pfenning 1984;Andersen 1989;Cao et al 1989;Krueger 1990;Jones et al 1995;Xu et al 1995;Reith et al 1996a;Garris et al 1998;Wu et al 1998;Eshleman et al 1999). If indeed the mechanism is competitive, there is likely to be a one-to-one relationship between cocaine binding sites and dopamine recognition sites, i.e.…”

Mechanism for Cocaine Blocking the Transport of Dopamine: Insights from Molecular Modeling and Dynamics Simulations

“…Recent studies have shown that cocaine inhibits dopamine reuptake through its binding with the extracellularly-open state of DAT 9,10,11,12,27,28,29,32,35,36,38,39. Starting from the newly refined DAT structure bound with dopamine, herein denoted by DAT-DA, molecular docking was carried out by using AutoDock3.0.5 program42 to explore the mode of cocaine binding with DAT in the presence of dopamine.…”

Multisubtrate mechanism for the inward transport of dopamine by the human dopamine transporter expressed in HEK cells and its inhibition by cocaine

“…Posttranslational modifications to the protein itself have been shown to differ between different anatomical areas, predominately in glycosylation (Vaughan et al, 1996), and Cao et al (1989) have shown that cocaine binding is sensitive to changes in external glycosylation. Thus, glycosylation and other posttranslational modifications may differ in expressions systems relative to native systems and result in different behaviors of DAT.…”