Localization of Cocaine Analog [125I]RTI 82 Irreversible Binding to Transmembrane Domain 6 of the Dopamine Transporter

Abstract

The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [125 I]3␤-(p-chlorophenyl)tropane-2␤-carboxylic acid, 4-azido-3-iodophenylethyl ester ([ 125 I]RTI 82). The incorporation site of this compound was mapped to transmembrane domains (TMs) 4 -6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (CNBr), which hydrolyzes proteins on the C-terminal side of methionine residues. CNBr hydrolysis of The dopamine transporter (DAT)2 is a neuronal protein that clears dopamine (DA) from the synapse via Na ϩ /Cl Ϫ -dependent active transport. DA reuptake is necessary for appropriate control of dopaminergic function and may be dysregulated in disorders such as Parkinson disease and drug abuse (1). Cocaine and many other compounds bind to DAT and inhibit DA reuptake, resulting in increased transmitter levels believed to mediate drug reinforcement (2). Despite extensive pharmacological investigation of transport inhibitors, the molecular mechanisms by which they are recognized by DAT and exert their effects remain largely unknown.DAT and the related norepinephrine and serotonin transporters (NET and SERT) are integral membrane proteins that consist of 12 transmembrane domains (TMs), connecting intracellular (IL) and extracellular (EL) loops, and intracellular N and C termini (3). Substrate translocation is thought to occur by an alternating access mechanism that sequentially exposes extracellular and intracellular portions of the substrate permeation pathway to opposite sides of the membrane (4). The three-dimensional structures of these proteins are unknown, although they are recently proposed to be similar to that of the homologous Aquifex aeolicus leucine transporter, LeuT Aa (5). The substrate binding site on LeuT Aa is composed of residues from TMs 1, 3, 6, and 8, and some analogous sites have been identified in DAT, NET, and SERT (6, 7).LeuT Aa , however, is not cocaine-sensitive, and its structure does not indicate how cocaine or other uptake blockers might interact at monoamine transporters. Cocaine binding to DAT is reduced by mutagenesis of many amino acids throughout the primary sequence (6, 7), but in most cases it is not clear if these effects are due to disruption of specific ligand interaction sites or to conformational alterations that indirectly impact binding. Thus the structure of the inhibitor binding site on DAT and the identities of the residues that dock cocaine and other blockers

The binding sites for cocaine and dopamine in the dopamine transporter overlap

“…Recently, it was shown that the photoreactive cocaine analog [ 125 I]RTI 82 incorporated into TMD 6 (ref. 17), which is an important part of the cocaine-binding site in the models presented here. Together with these previous findings, our results provide strong evidence that cocaine and its analogs are true competitive inhibitors.…”

Transmembrane Domain 6 of the Human Serotonin Transporter Contributes to an Aqueously Accessible Binding Pocket for Serotonin and the Psychostimulant 3,4-Methylene Dioxymethamphetamine

“…Modeling and mutagenesis studies of cocaine and cocaine analog binding in hDAT predict that cocaine would interact significantly with hSERT TM6 residues (16,57,58). Specifically, hDAT residues Phe-320, Ser-321, and Leu-322, which correspond by homology to hSERT Phe-335, Ser-336, and Leu-337, are predicted to interact with the N-methylamine of (Ϫ)-2␤-carbomethoxy-3␤-(4-fluorophenyl)tropane (CFT), a cocaine analog, and thus, we predicted that the homologous hSERT residues would be protected by cocaine.…”

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain

“…Several studies have reported the existence of multiple binding sites for different inhibitors on DAT, and studies with irreversible ligands may be important in mapping these respective sites [24][25][26][27]. Both photoaffinity ligands [28][29][30] and alkylating reagents [31][32][33][34], including the tropane analog [ 125 I]RTI-82 [35], have been used to identify the ligand binding sites of cocaine and related tropane analogs on DAT. In addition, alkylating agents including isothiocyanate and bromoacetamide tagged to cocaine, rimcazole and benztropine analogs have been successful as irreversible DAT analogs [31,32,36].…”