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

J. J. Bonnet, S. Benmansour, J. Costentin, E. M. Parker, L. X. Cubeddu

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60 Scopus citations


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 GBR 12783) or [3H]mazindol) using striatal membrane preparations. Raising the incubation temperature from 0°C to 25°C or 37°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°C < 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°C (from 126 to 406 nM). In contrast, increasing the temperature resulted in a more important increase in the Ki of uptake inhibitors (33 > Ki ratio > 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. These results suggest that: 1) raising the incubation temperature up to 37°C allows discrimination between substrates and competitive inhibitors of the neuronal utake; 2) the binding of substrates is entropy-driven and seems to be hydrophobic; and 3) the binding of carrier blockers is enthalpy-driven and could induce a conformational change in the carrier and/or involve electrostatic bonds with the neuronal carrier of dopamine.

Original languageEnglish (US)
Pages (from-to)1206-1214
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number3
StatePublished - 1990

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology


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