Kinetic Analysis of the Chloride Dependence of the Neuronal Uptake of Dopamine and Effect of Anions on the Ability of Substrates to Compete with the Binding of the Dopamine Uptake Inhibitor GBR 12783

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Abstract
The specific binding of [3H]l‐[2‐(diphenyl‐methoxy)ethyl]‐4‐(3‐phenyl‐2‐propenyl)piperazine([3H]GBR 12783) to the dopamine (DA) neuronal carrier present in membranes prepared from rat striatum was not affected when Cl– was replaced by either Br– or NO3–. In media containing Cl–, Br–, or NO3–, d‐amphetamine and DA competed with the radioligand in a monophasic manner with Hill coefficients of close to 1 (0.94–1.12). Replacement of CT by Br– impaired the ability of some substrates (d‐amphetamine, DA, p‐hydroxyamphetamine, and m‐tyramine) to compete with [3H]GBR 12783. The potency of Br– to decrease the affinity of substrates for the specific binding site was significantly correlated (t = 7.07, p < 0.001) with their affinity for this binding site. These results suggest that the various substrates tested could bind to recognition sites in which Cl– is differently involved; as a consequence, substrates could bind to the neuronal carrier by means of partly different links. In experiments dealing with the specific uptake of [3H]DA, F–, NO3–, is‐ethionate–, or acetate– was unable to substitute for Cl–, whereas Br– was quite a total substitute. Replacement of Cl– by equimolar concentrations of either NO3 or isethionate– resulted in inhibition curves of DA specific uptake with Hill coefficients of close to 1 (0.77 and 1.04, respectively); this indicates that both NO3– and isethionate” are devoid of inhibitory effects on neuronal uptake and are quite ineffective substitutes for Cl–. Kinetic analyses performed in different experimental conditions provide consistent results revealing that decreasing the CT concentration induced a raise in apparent Km of DA without modification of the Fmax. On the other hand, the apparent Km values of Cl– for the stimulation of the neuronal uptake of DA decreased when the DA concentration increased. These data are consistent with a co‐transport of Cl– and DA by the neuronal uptake system, with CT being the driving force.

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