Thermodynamic analyses of the binding of substrates and uptake inhibitors on the neuronal carrier of dopamine labeled with [3H]GBR 12783 or [3H]mazindol

Abstract

We have investigated the thermodynamic properties of the binding of substrates and uptake blockers to the specific sites labeled with a tritiated dopamine uptake inhibitor (i.e., 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([3H] GBR 12783) or [3H]mazindol) using striatal membrane preparations. Raising the incubation temperature from 0 degrees C to 25 degrees C or 37 degrees C resulted in an increase in the dissociation constant of both [3H]mazindol and [3H]GBR 12783 for their specific sites of binding present in membrane suspensions obtained from either rabbit or rat striatum. However, maximal concentrations of binding sites were not affected by temperature. At all tested temperatures, both substrates and carrier blockers competed with either [3H]mazindol or [3H]GBR 12783 in a monophasic fashion, with Hill coefficients close to unity. Raising the temperature induced little or no increase in inhibition constants (Ki) for substrates (Ki ratio 37/0 degrees C less than 2.5). This is consistent with the mild increase of the Michaelis constant of dopamine for the neuronal uptake system when the incubation temperature was raised from 12.5 to 37 degrees C (from 126 to 406 nM). In contrast, increasing the temperature resulted in a more important increase in the Ki of uptake inhibitors (33 greater than Ki ratio greater than 5). Thermodynamic calculations showed that the binding of substrates is generally characterized by a mild decrease in enthalpy (range, -2- -6 kcal/mol) associated with an increase in entropy, whereas binding of uptake inhibitors led to a decrease of both parameters.(ABSTRACT TRUNCATED AT 250 WORDS)