Binding of the [125I]3?-(Iodophenyl)tropan-2?-carboxylic acid isopropyl ester to the dopamine transporter at a physiologically relevant temperature: Mutually exclusive binding and different ionic requirements for various uptake blockers and substrates

Abstract

The present study describes the binding of cocaine analog [125I]3 beta-(iodophenyl)tropan-2 beta-carboxylic acid isopropyl ester ([125I]RTI-121), a highly selective ligand for the dopamine transporter (DAT), to rat striatal synaptosomal membranes at 37 degrees C. Saturation analysis of [125I]RTI-121 binding revealed a single binding site with similar affinity for RTI-121 at both 50 and 134 mM NaCl. However, the density of binding sites was reduced at 134 mM NaCl. Various uptake blockers and substrates of the DAT monophasically inhibited the specific binding of [125I]RTI-121. Increasing the NaCl concentration from 50 mM to 134 mM enhanced the affinity of the substrate dopamine and amphetamine for the DAT, without affecting that of the uptake blockers. At 134 mM NaCl, the copresence of GBR12935, BTCP, cocaine, amphetamine, or dopamine decreased the affinity of RTI-121 to the extent predicted by a model in which the binding of all compounds is mutually exclusive. This, along with a different NaCl sensitivity for blockers and substrates, suggests that the two categories of compounds recognize nonidentical but overlapping binding domains on the DAT. In contrast, the mutually exclusive binding with similar NaCl sensitivity for RTI-121 and the other uptake blockers tested here suggests the involvement of common binding domains in the recognition of these blockers.

Modeling of the Interaction of Na+ and K+ with theBinding of Dopamine and [3H]WIN 35,428 to the Human Dopamine Transporter

“…Ion tonicity also presumably played no role in the present results for DA binding, which again was determined primarily by the [K + ]/[Na + ] ratio. In keeping with previously reported effects of Cl ‐ on substrate binding to the DAT (Amejdki‐Chab et al, 1992 b ; Wall et al, 1993 ; Chen et al, 1997 b ), the K i values for DA at given [K + ]/[Na + ] ratios were shifted downward (indicating higher affinity of DA) in the presence of Cl ‐ , emphasizing the importance of keeping the [Cl ‐ ] constant in DA binding measurements.…”

“…In our two previous studies on 3β‐(4‐[ 125 I]iodophenyl)‐tropane‐2β‐carboxylic acid isopropyl ester ([ 125 I]RTI‐121) binding to rat striatal membranes (Chen et al, 1997 a , b ), we advanced preliminary evidence for a differential sensitivity of DA, on the one hand, and 1‐[2‐(diphenylmethoxy)ethyl]‐4‐(3‐phenyl‐2‐propyl)piperazine (GBR 12935)‐ or cocaine‐like blockers, on the other hand, to ambient Na + and K + . The present study addresses the effects of Na + and K + on DA binding in greater detail and takes into account potential effects of Cl ‐ .…”

“…In our present and previous (Chen et al, 1997 a , b ) modeling studies on the effect of varying [Na + ] on blocker binding to the DAT, varying [NaCl] is used because Cl ‐ is generally found to be inert in this respect (Schweri, 1987 ; Amejdki‐Chab et al, 1992 b ; Reith and Coffey, 1993). However, Cl ‐ may stimulate DA binding to the DAT (Amejdki‐Chab et al, 1992 a , b ; Wall et al, 1993 ; Gu et al, 1994), and therefore a counterion other than Cl ‐ is needed when studying DA binding at varying [Na + ].…”

Is Na + required for the binding of dopamine, amphetamine, tyramine, and octopamine to the human dopamine transporter?

“…Although this negates an absolute requirement for Na + in DA binding, a stimulatory role for Na + is still a possibility. Thus, cocaine also blocked leak currents in the absence of Na + (Sonders et al 1997), even though a stimulatory role for Na + in cocaine analog binding to DAT has been shown repeatedly (Reith and Coffey 1993;Chen et al 1997aChen et al , 1997bLi and Reith 1999;Corera et al 2000). In biochemical studies on the role of Na + in DA binding to DAT, the measuring tool is the DA-induced inhibition of binding of a radiolabeled blocker to the DAT because the dissociation of the DA-DAT complex is too rapid, ruling out successful application of commonly used techniques for separating bound from free ligand (see Li and Reith 1999).…”

Binding of 3H-WIN-35,428 and 125I-RTI-121 to Human Platelet Membranes

“…Therefore, as a study continuation, we considered more opportune to use a new radioligand which was claimed to show a greater specific activity than 3 [27,28,31], in order to set a reliable assay, easily reproducible and potentially applicable to comparison studies between healthy subjects and patients with different neuropsychiatric disorders involving the dopamine system.…”