title: Fourphit: a selective probe for the methylphenidate binding site on the dopamine transporter.
abstract: Fourphit, a phencyclidine derivative containing an isothiocyanate substitution at the 4-position of the piperidine ring, inhibits the binding of the radiolabeled psychomotor stimulant, [3H]methylphenidate, to sites on the dopamine transport complex in membranes prepared from the crude synaptosomal fraction of rat striatal tissue with an IC50 of 7.1 microM. The inhibition caused by Fourphit is irreversible and is associated with a decrease in the Bmax, but not the KD, of [3H]methylphenidate binding. Pretreatment with saturating concentrations of unlabeled methylphenidate effected a modest (but statistically significant) protection of the stimulant binding site from inactivation by Fourphit, indicating that the acylating phencyclidine derivative may act directly at this site. Preincubation with Fourphit rather than vehicle did not alter the dissociation rate of [3H]methylphenidate when measured in the presence of excess amfonelic acid, nor was any difference detected in the off-rate of [3H]methylphenidate when excess Fourphit was substituted for excess unlabeled methylphenidate as the displacing agent. This lack of effect on the dissociation kinetics of [3H]methylphenidate provides further evidence that Fourphit does not act allosterically at the methylphenidate binding site. Unlike Metaphit (an isomer of Fourphit containing the isothiocyanate moiety at the meta position of the aromatic ring), Fourphit can discriminate between the methylphenidate binding site and the phencyclidine binding site associated with the N-methyl-D-aspartate receptor: Metaphit irreversibly inactivates both binding sites, whereas Fourphit binds reversibly to the phencyclidine binding site. The data suggest that Fourphit may be useful as a relatively selective affinity label for the site on the dopamine transport complex recognized by methylphenidate and other psychomotor stimulants.
authors.affiliation: Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia.
authors.forename: M M
authors.initials: MM
authors.lastname: Schweri
authors.forename: A
authors.initials: A
authors.lastname: Thurkauf
authors.forename: M V
authors.initials: MV
authors.lastname: Mattson
authors.forename: K C
authors.initials: KC
authors.lastname: Rice
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D005260:Female; D008297:Male; D008774:Methylphenidate / Q000378:metabolism*; D010622:Phencyclidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: C064277:fourphit; D008774:Methylphenidate; C012058:tenocyclidine; D010622:Phencyclidine