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1.  Mutational Analysis of the High-Affinity Zinc Binding Site Validates a Refined Human Dopamine Transporter Homology Model 
PLoS Computational Biology  2013;9(2):e1002909.
The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.
Author Summary
The dopamine transporter (DAT) regulates dopaminergic neurotransmission in the brain and is implicated in numerous human disease states. DAT is unique among the monoamine neurotransmitter transporter family because its substrate transport is inhibited by extracellular zinc. DAT homology models rely upon the crystal structure of LeuT solved in 2005. LeuT and DAT share a relatively low overall sequence identity of 22%. In addition, the length of the second extracellular loop of DAT exceeds that of LeuT by 21 residues. The zinc binding site cannot be directly modeled from the LeuT template alone because of these differences. Current available homology models of DAT focused on substrate or inhibitor binding rather than on the second extracellular loop. We exploited the specificity of the zinc binding site to build and calibrate a DAT homology model of the complete transmembrane domain. Our model predicted that the zinc binding site in DAT consists of four zinc co-ordinating residues rather than three that had been previously identified. We verified this hypothesis by site-directed mutagenesis and uptake inhibition studies.
PMCID: PMC3578762  PMID: 23436987
3.  Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter 
The Journal of Biological Chemistry  2012;287(35):29627-29635.
Background: αCaMKII modulates amphetamine-induced dopamine transporter-mediated substrate efflux.
Results: Mice with ablated or blunted αCaMKII function show decreased amphetamine-triggered efflux.
Conclusion: Dopamine transporter function is impaired in mice with targeted αCaMKII mutations and in a mouse model of the Angelman syndrome.
Significance: Such new insights into dopamine transporter function may further illuminate the complex pathophysiology of the Angelman syndrome.
The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP+) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKIIT305D), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP+ efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.
PMCID: PMC3436163  PMID: 22778257
Calcium Calmodulin-dependent Protein Kinase (CaMK); Dopamine; Dopamine Transporters; Neurotransmitter Release; Transport; Amphetamine; Angelman Syndrome

Results 1-6 (6)