Interaction of two sulfhydryl reagents with a cation recognition site on the neuronal dopamine carrier evidences small differences between [3H]GBR 12783 and [3H]cocaine binding sites

Abstract

We have compared the effect of treating rat striatal cell membranes with ionic hydrophilic sulfhydryl reagents on the specific bindings of [3H]cocaine and of [3H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-[1-3H]propenyl)-piperaz ine) to the neuronal transporter of dopamine. Treatment with 1 mmol/l 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in similar time- and concentration-dependent reductions of the specific binding of both radioligands. None of the uptake blockers tested afforded any protection against 1 mmol/l DTNB. Addition of (sub)millimolar concentrations of CaCl2 or MgCl2, or 250 mmol/l KCl to a treatment medium containing 10 mmol/l Na+ significantly increased the DTNB-induced reduction of the specific binding of both radioligands. Cations were likely to be responsible for this effect since ions in combination with DTNB induced similar reductions in binding when either 1 mmol/l CaCl2 or 50-250 mmol/NaCl were added. Effects of cations on the DTNB-induced inhibition of binding were generally more marked on [3H]GBR 12783 than on [3H]cocaine binding. When added to a medium containing 10 mmol/l Na+ 1 mmol/l DTNB induced a reduction in the Bmax of the specific binding of both radioligands. Addition of 1 mmol/l Ca2+ maintained or increased this Bmax reduction and elicited a decrease in affinity which was significant for [3H]GBR 12783 binding. Treatment of membranes with the sodium salt of p-hydroxymercurybenzenesulfonate (pHMBS) induced time- and concentration-dependent decreases in [3H]GBR 12783 binding which were significantly greater than decreases in [3H]cocaine binding. However, 50 mumol/l pHMBS produced a similar decrease in the Bmax of the specific binding of both radioligands. The pHMBS-induced reduction of [3H]GBR 12783 binding was not reversed by drugs whose action is purely that of uptake inhibition or by substrates of the dopamine carrier. Some of these drugs (100 mumol/l dopamine, 1 mumol/l mazindol or 100 mumol/l cocaine) protected the specific binding of [3H]cocaine against the effects of pHMBS, whereas 1 mmol/l p-tyramine, 10 mumol/l nomifensine and 10 nmol/l GBR 12783 were ineffective. Addition of 120 mmol/l Na+, 1 mmol/l Ca2+ or 10 mmol/l Mg2+ to a treatment medium containing 10 mmol/l Na+ significantly reduced the effects of pHMBS on the specific binding of both radioligands. When striatal cell membranes were treated in a medium containing 130 mmol/l Na+, there was a general decrease in the effects of ions on the reductions of specific binding produced by DTNB or pHMBS.(ABSTRACT TRUNCATED AT 400 WORDS)