Biophysical Characterization of the Cocaine Binding Pocket in the Serotonin Transporter Using a Fluorescent Cocaine Analogue as a Molecular Reporter

Abstract

Cocaine is one of the most widely abused psychostimulants, causing major medical and socioeconomic problems (1). Currently, there is no effective treatment against cocaine addiction available; therefore, clarifying the molecular mechanisms underlying the psychostimulatory effects and addictive properties of cocaine should prove critical for potential development of future therapeutic strategies. Cocaine and related drugs act by inhibiting clearance of released monoamine neurotransmitters from the synaptic cleft (2-4). This clearance of monoamines occurs via three distinct but highly homologous monoamine transporters, the serotonin transporter (SERT), 1 the dopamine transporter (DAT), and the norepinephrine transporter (NET) (2-4). Cocaine binds with high affinity to all three transporters and is generally believed to act as a competitive blocker of substrate translocation (4, 5). Several studies have provided evidence that inhibition of the DAT is the predominant mechanism behind the stimulatory effects and addictive properties of cocaine (6 -8). However, this hypothesis has been challenged by recent studies on mice in which the DAT gene has been deleted (1). Despite the absence of the DAT gene, it was surprisingly observed that these mice self-administered cocaine, indicating a possible important role of also the SERT and NET (9, 10).The SERT belongs together with DAT and NET to a family of Na ϩ /Cl Ϫ -dependent solute carriers that are characterized functionally by their dependence on the presence of Na ϩ and Cl Ϫ in the extracellular fluid (3, 11). All Na ϩ /Cl Ϫ -dependent carriers are believed to share a common topology characterized by the presence of 12 transmembrane segments connected by alternating extracellular and intracellular loops with an intracellular location of the N and C terminus (3, 4, 11). Despite intense efforts, including many mutagenesis studies (12-18) and studies using photoaffinity labeling (19), surprisingly little is known about the binding site for cocaine-like substances in the monoamine transporters. Although cysteine-scanning mutagenesis of transmembrane segment 3 in the SERT has suggested that two residues in the middle of the transmembrane segment could be in close proximity to the cocaine binding site (20), no direct contact sites have been established between cocaine and specific transporter residues.

Comparative Molecular Field Analysis Using Selectivity Fields Reveals Residues in the Third Transmembrane Helix of the Serotonin Transporter Associated with Substrate and Antagonist Recognition

“…The residue homologous to Ile172 in the LeuT Aa structure projects into a hydrophobic binding pocket to accommodate the hydrophobic methyl groups of the substrate (Yamashita et al, 2005). In addition, cocaine analogs are thought to bind in a highly hydrophobic microenvironment (Rasmussen et al, 2001). Altogether, our results support the involvement of Ile172 with cocaine recognition but not in the binding of any of the antidepressants.…”

Substrate-induced Unlocking of the Inner Gate Determines the Catalytic Efficiency of a Neurotransmitter:Sodium Symporter

“…5C) (51). TEMPO strongly quenched the fluorescence from TMR conjugated to E192C, consistent with partial burying of the fluorophore (Fig.…”

“…Here, we covalently coupled TMR-maleimide to a cysteine inserted at position 192 of LeuT (LeuT E192C TMR ) and performed a series of collisional quenching experiments offering dynamic rearrangement data in support of a direct coupling between substrate binding and a conformational rearrangement at the intracellular end of TM5. Thus, leucine elicited a Na ϩ -dependent increase in the accessibility of TMR to the aqueous quencher iodide while decreasing accessibility to the hydrophobic quencher (TEMPO) (51).…”

2002 Medicinal Chemistry Division Award Address: Monoamine Transporters and Opioid Receptors. Targets for Addiction Therapy

“…We synthesized 16 (RTI-233) as the first fluorescent 3-phenyltropane analogue and used the compound for biophysical characterization of the cocaine binding pocket at the 5-HTT. 105 The data obtained using steady-state fluorescence anisotropy and collisional quenching experiments with aqueous and hydrophobic quenchers were consistent with a highly hydrophobic microenvironment in the binding pocket for cocaine-like uptake inhibitors. However, the bound cocaine analogue was still accessible for aqueous quenching and thus partially exposed to solvent.…”