INTERACTION OF 2 SULFHYDRYL-REAGENTS WITH A CATION RECOGNITION SITE ON THE NEURONAL DOPAMINE CARRIER EVIDENCES SMALL DIFFERENCES BETWEEN [H-3] GBR-12783 AND [H-3] COCAINE BINDING-SITES

We have compared the effect of treating rat striatal cell membranes wi th ionic hydrophilic sulfhydryl reagents on the specific bindings of [ H-3]cocaine and of [H-3]GBR 12783 y)ethyl]4-(3-phenyl-2-[1-H-3]propeny l)-piperazine) to the neuronal transporter of dopamine. Treatment with 1 mmol/l 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in simil ar time- and concentration-dependent reductions of the specific bindin g of both radioligands. None of the uptake blockers tested afforded an y protection against 1 mmol/l DTNB. Addition of (sub)millimolar concen trations of CaCl2 or MgCl2, or 250 mmol/l KCl to a treatment medium co ntaining 10 mmol/l Na+ significantly increased the DTNB-induced reduct ion 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/l NaCl were added. Effects of cations on the DTNB-induced in hibition of binding were generally more marked on [H-3]GBR 12783 than on [H-3]cocaine binding. When added to a medium containing 10 mmol/l N a+ 1 mmol/lDTNB induced a reduction in the B-max of the specific bindi ng of both radioligands. Addition of 1 mmol/l Ca2+ maintained or incre ased this B-max reduction and elicited a decrease in affinity which wa s significant for [H-3]GBR 12783 binding. Treatment of membranes with the sodium salt of p-hydroxymercurybenzenesulfonate (pHMBS) induced ti me- and concentration-dependent decreases in [H-3]GBR 12783 binding wh ich were significantly greater than decreases in [H-3]cocaine binding. However, 50 mu mol/l pHMBS produced a similar decrease in the B-max o f the specific binding of both radioligands. The pHMBS-induced reducti on of [H-3]GBR 12783 binding was not reversed by drugs whose action is purely that of uptake inhibition or by substrates of the dopamine car rier. Some of these drugs (100 mu mol/l dopamine, 1 mu mol/l mazindol or 100 mu mol/l cocaine) protected the specific binding of [H-3]cocain e against the effects of pHMBS, whereas 1 mmol/l p-tyramine, 10 mu mol /l nomifensine and 10 nmol/l GBR 12783 were ineffective. Addition of 1 20 mmol/l Na+, 1 mmol/l Ca2+ or 10 mmol/l Mg2+ to a treatment medium c ontaining 10 mmol/l Na+ significantly reduced the effects of pHMBS on the specific binding of both radioligands. When striatal cell membrane s were treated in a medium containing 130 mmol/l Na+, there was a gene ral decrease in the effects of ions on the reductions of specific bind ing produced by DTNB or pHMBS. Cation concentrations which interfered with the actions of DTNB and pHMBS were approximately those which bloc ked the specific binding of [H-3]GBR 12783 when they were present duri ng association of the radioligand (K+, Ca2+, Mg2+), or, in the case of Na+, which are effective in reducing this blockade (Bonnet et al. 198 8). The present data are consistent with the existence of mutually exc lusive binding sites for [H-3]GBR and [H-3]-cocaine on the neuronal do pamine transporter. The hypothesis of a cation recognition site which could gate admission of uptake inhibitors or carrier substrates to the ir binding domain on the transporter is discussed.