Impact of disruption of secondary binding site S₂ on dopamine transporter function

Abstract

The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S2) for drugs and substrate in the extracellular vestibule towards the membrane exterior in relation to the primary substrate recognition site (S1). The present experiments are aimed at disrupting S2 by mutating Asp476 and Ile159 to Ala. Both mutants displayed a profound decrease in [3H]DA uptake compared with wild-type associated with a reduced turnover rate kcat. This was not caused by a conformational bias as the mutants responded to Zn2+ (10 µM) similarly as WT. The dopamine transporters with either the D476A or I159A mutation both displayed a higher Ki for DA for the inhibition of [3H]CFT binding than did the WT transporter, in accordance with an allosteric interaction between the S1 and S2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to DAT and possibly other monoamine transporters.

Tamoxifen Directly Interacts with the Dopamine Transporter

“…This distal "S2" site is hypothesized to facilitate binding of substrate to the primary substrate site (Nyola et al, 2010) and possibly even drive the translocation of the dopamine molecule across the membrane (Shan et al, 2011). A potential interaction with the S2 site was assessed with recently described mutants containing disruptions in the S2 site by mutation of Asp476 and Ile159 to Ala (D476A and I159A, respectively) (Zhen and Reith, 2016). Previously, we reported that the K d for binding of [ 3 H]WIN 35,428 to the DAT in WT and the two S2 mutants was the same, whereas the ability of dopamine in competing for [ 3 H]WIN 35,428 binding was reduced in the mutants (Zhen and Reith, 2016).…”

“…A potential interaction with the S2 site was assessed with recently described mutants containing disruptions in the S2 site by mutation of Asp476 and Ile159 to Ala (D476A and I159A, respectively) (Zhen and Reith, 2016). Previously, we reported that the K d for binding of [ 3 H]WIN 35,428 to the DAT in WT and the two S2 mutants was the same, whereas the ability of dopamine in competing for [ 3 H]WIN 35,428 binding was reduced in the mutants (Zhen and Reith, 2016). This is consonant with an allosteric effect of S2 site disruption on S1 site functionality.…”

“…We find that tamoxifen noncompetitively inhibits dopamine uptake and blocks amphetamine-stimulated dopamine efflux. We use cysteine accessibility assays and newly characterized "S2-defective" DAT mutants (Zhen and Reith, 2016) to demonstrate that tamoxifen stabilizes the outwardfacing conformation of the DAT, potentially through an interaction with the S2 domain. This hypothesis is supported by computational docking experiments.…”

Direct and Systemic Administration of a CNS-Permeant Tamoxifen Analog Reduces Amphetamine-Induced Dopamine Release and Reinforcing Effects

“…It is possible that the diphenyl structural motif in 6c could partially account for its blockade of dopamine uptake because this motif also exists in atypical DAT blockers such as modafinil and benztropine, which appear to bind preferentially to the inward-facing conformation of DAT (Reith et al, 2015;Schmitt et al, 2013). In addition to the well-characterized substrate site (S1), crystal structures of the bacterial leucine transporter (Zhen and Reith, 2016), the drosophila DAT, and the serotonin transporter show the presence of secondary allosteric sites (S2 and S3), where compounds can bind and modify the conformation and function of DAT (Reith et al, 2015;Schmitt et al, 2013). It is possible that 6c could occupy one of these allosteric sites.…”

Molecular dynamics of conformation-specific dopamine transporter-inhibitor complexes

“…Attempts to explain this paradox are often pharmacokinetic in nature: of the three drugs, only cocaine reaches the brain fast enough at high concentration to trigger the phasic firing of accumbal dopamine cells needed to elicit euphoria. 15–18 From cysteine accessibility studies, an alternative explanation arose from the finding that cocaine and benztropine bind to different DAT conformations. 19–21 Because cocaine recognizes the OF conformation and benztropine recognizes the IF conformation, it was postulated that behaviorally “typical” (abusable, locomotor psychostimulant) inhibitors such as cocaine stabilize the OF DAT and “atypical” (little or no abuse potential) inhibitors favor IF DAT.…”