Covalent and noncovalent chemical modifications of arginine residues decrease dopamine transporter activity

Abstract

Rotating disk electrode voltammetry was used to measure dopamine (DA) transport in rat striatum and in human embryonic kidney cells expressing the rat dopamine transporter (DAT). The goals of this study were to determine 1) if arginine (Arg) selective agents could alter DA transport, and 2) if DA analogs and DAT inhibitors could attenuate the effects of these agents on the DAT. Phenylglyoxal (PG), Hill coefficient 2.5, and other Arg selective agents decreased DA transport velocities. DA, Hill coefficient 1.0, and its analogs 3-hydroxyphenethylamine and 4-hydroxyphenethylamine attenuated the effects of PG on the DAT while phenethylamine did not. The tropane-based DAT inhibitors cocaine, WIN 35065-2, and WIN 35428 also attenuated the effects of PG. Benztropine, GBR 12935, and GBR 12909 did not. Thus, Arg residues are important for DAT activity and the results suggest that DA and cocaine both interact with Arg residues. Structure-activity studies suggest that DA interacts with Arg through its catechol hydroxyl groups and cocaine through the ester linkage attached to carbon 2 of the tropane ring. The results that 1). DA and cocaine may interact with the same functionally important Arg residue at the DAT, and 2). some members of the tropane and 1,4-dialkylpiperazine classes of DAT inhibitors may interact differently with DAT-derived Arg residue(s) furthers the notion that DAT activity sparing antagonists of cocaine can be designed.

Rotating disk electrode voltammetric measurements of serotonin transporter kinetics in synaptosomes

“…RDEV measurements of 5-HT uptake were made using modifications of previously published procedures used to measure dopamine uptake in rat striatal suspensions (Bjorklund et al, 2007; Earles and Schenk, 1998; Robinson et al, 2005; Volz et al, 2004; Volz and Schenk, 2004; Wayment et al, 2001) and rat vesicular preparations (Volz et al, 2006b). SERT activity was determined by subtracting the initial velocities of 5-HT clearance in the presence of 1 μM paroxetine from initial velocities measured in the presence of vehicle (physiologic buffer).…”

Nonclassical Pharmacology of the Dopamine Transporter: Atypical Inhibitors, Allosteric Modulators, and Partial Substrates

“…Conformationspecific DAT interaction was first suggested by the finding that cocaine and benztropine differentially affect the vulnerability of extracellular-facing DAT cysteine residues toward reaction with membrane impermeant sulfhydryl-reducing reagents, indicating that these inhibitors stabilize different transporter conformations . Similarly, binding of cocaine-like compounds was shown to protect DAT transmembrane arginine residues from covalent reaction with phenylglyoxal, whereas benztropine-like compounds failed to impact phenylglyoxal reactivity (Volz et al, 2004). Whole-cell binding studies performed in the presence of Zn 21 (a DAT modulator that loosely binds the extracellular face of the transporter, stabilizing an outward-facing conformation) or in the absence of extracellular Na 1 (which increases the relative number of inward-facing DATs) further hint at specific conformational effects that vary depending on the structure of the bound inhibitor (Loland et al, 2002;Schmitt and Reith, 2011).…”

Age‐dependent differences in dopamine transporter and vesicular monoamine transporter‐2 function and their implications for methamphetamine neurotoxicity

“…Rotating disk electrode voltammetry was used to measure the initial velocities of inwardly directed DA transport into, and outwardly directed METH-induced DA efflux from, rat striatal suspensions as well as the initial velocities of inwardly directed vesicular DA transport into membrane-associated vesicles purified from rat striata as described previously (Volz et al, 2007a; Volz et al, 2006a; Volz et al, 2006b; Volz et al, 2004; Volz and Schenk, 2004). Briefly, untreated and unanesthetized male Sprague-Dawley rats from Charles River Laboratories (Raleigh, NC) were sacrificed by decapitation and striata (but not the nucleus accumbens) were removed.…”