The Uptake Inhibitors Cocaine and Benztropine Differentially Alter the Conformation of the Human Dopamine Transporter

Reith, Maarten E. A.; Berfield, Janet L.; Wang, Lijuan C.; Ferrer, Jasmine V.; Javitch, Jonathan A.

doi:10.1074/jbc.m011785200

Cited by 97 publications

(118 citation statements)

References 31 publications

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“…This cysteine was also protected from reaction when cocaine was present, consistent with a model in which cocaine stabilizes an outward facing conformation of DAT (58). This dynamic rearrangement is underscored by our findings that cocaine is not a neutral blocker but rather alters the conformation of extracellular and intracellular cysteines in DAT (59,60). That cocaine binding is greatly enhanced by sodium (61) is also consistent with cocaine binding preferentially to a C2-or C3-like configuration.…”

Section: Table 1 Tyrosine Kinetics Of Tyt1 Variantssupporting

confidence: 72%

State-dependent Conformations of the Translocation Pathway in the Tyrosine Transporter Tyt1, a Novel Neurotransmitter:Sodium Symporter from Fusobacterium nucleatum

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Yano

Goldberg

et al. 2006

Journal of Biological Chemistry

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The gene of a novel prokaryotic member (Tyt1) of the neurotransmitter:sodium symporter (NSS) family has been cloned from Fusobacterium nucleatum. In contrast to eukaryotic and some prokaryotic NSSs, which contain 12 transmembrane domains (TMs), Tyt1 contains only 11 TMs, a characteristic shared by ϳ70% of prokaryotic NSS homologues. Nonetheless upon heterologous expression in an engineered Escherichia coli host, Tyt1 catalyzes robust Na ؉ -dependent, highly selective L-tyrosine transport. Genetic engineering of Tyt1 variants devoid of cysteines or with individually retained endogenous cysteines at positions 18 or 238, at the cytoplasmic ends of TM1 and TM6, respectively, preserved normal transport activity. Whereas cysteine-less Tyt1 was resistant to the inhibitory effect of sulfhydryl-alkylating reagents, N-ethylmaleimide inhibited transport by Tyt1 variants containing either one or both of the endogenous cysteines, and this inhibition was altered by the substrates sodium and tyrosine, consistent with substrate-induced dynamics in the transport pathway. Our findings support a binding model of Tyt1 function in which an ordered sequence of substrate-induced structural changes reflects distinct conformational states of the transporter. This work identifies Tyt1 as the first functional bacterial NSS member putatively consisting of only 11 TMs and shows that Tyt1 is a suitable model for the study of NSS dynamics with relevance to structure/function relationships of human NSSs, including the dopamine, norepinephrine, serotonin, and ␥-aminobutyric acid transporters.

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Section: Table 1 Tyrosine Kinetics Of Tyt1 Variantssupporting

confidence: 72%

State-dependent Conformations of the Translocation Pathway in the Tyrosine Transporter Tyt1, a Novel Neurotransmitter:Sodium Symporter from Fusobacterium nucleatum

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Yano

Goldberg

et al. 2006

Journal of Biological Chemistry

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“…Newman and colleagues have identified dissimilarities between benztropine and cocaine actions at the DAT via distinctive structureactivity relationship profiles (Newman and Kulkarni, 2002). In addition, benztropine altered the accessibility of alkylation agents to wild-type DAT cysteine residues in a pattern distinct from the pattern generated by cocaine, WIN 35,428, and mazindol (Reith et al, 2001). The latter study demonstrated a primary alkylation pattern difference at C90, a DAT extracellular loop cysteine residue that immediately follows TM 1.…”

Section: Discussionmentioning

confidence: 86%

Recognition of Benztropine by the Dopamine Transporter (DAT) Differs from That of the Classical Dopamine Uptake Inhibitors Cocaine, Methylphenidate, and Mazindol as a Function of a DAT Transmembrane 1 Aspartic Acid Residue

Ukairo

Bondi

Newman

et al. 2005

J Pharmacol Exp Ther

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Binding of cocaine to the dopamine transporter (DAT) protein blocks synaptic dopamine clearance, triggering the psychoactive effects associated with the drug; the discrete drug-protein interactions, however, remain poorly understood. A longstanding postulate holds that cocaine inhibits DAT-mediated dopamine transport via competition with dopamine for formation of an ionic bond with the DAT transmembrane aspartic acid residue D79. In the present study, DAT mutations of this residue were generated and assayed for translocation of radiolabeled dopamine and binding of radiolabeled DAT inhibitors under identical conditions. When feasible, dopamine uptake inhibition potency and apparent binding affinity K i values were determined for structurally diverse DAT inhibitors. The glutamic acid substitution mutant (D79E) displayed values indistinguishable from wild-type DAT in both assays for the charge-neutral cocaine analog 8-oxa-norcocaine, a finding not supportive of the D79 "salt bridge" ligand-docking model. In addressing whether the D79 side chain contributes to the DAT binding sites of other portions of the cocaine pharmacophore, only inhibitors with modifications of the tropane ring C-3 substituent, i.e., benztropine and its analogs, displayed a substantially altered dopamine uptake inhibition potency as a function of the D79E mutation. A single conservative amino acid substitution thus differentiated structural requirements for benztropine function relative to those for all other classical DAT inhibitors. Distinguishing the precise mechanism of action of this DAT inhibitor with relatively low abuse liability from that of cocaine may be attainable using DAT mutagenesis and other structure-function studies, opening the door to rational design of therapeutic agents for cocaine abuse.Millions of individuals worldwide are addicted to cocaine, a public health crisis that also carries a substantial burden to society in the form of medical expenses, lost earnings, and increased crime associated with psychostimulant abuse. Although therapeutics including buprenorphine and methadone are clinically accessible to treat opiate abuse and addiction, no such Food and Drug Administration-approved medication is available for the treatment of cocaine addiction. Pharmacological and behavioral studies have established that the euphoric and addictive effects of cocaine are initiated by its binding to the dopamine transporter (DAT) protein, blocking clearance of dopamine from central nervous system synapses and thereby prolonging dopaminergic neurotransmission in brain areas associated with reward (Ritz et al., 1987). The DAT is therefore a logical target for development of medications for cocaine abuse. Elucidating the relationship between the DAT and its substrates and inhibitors is essential, especially regarding cocaine points of contact with the DAT protein relevant to its inhibition of dopamine uptake.Because dopamine and all classical DAT inhibitors possess a protonated nitrogen atom, a longstanding model for cocaine

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“…Furthermore, BZT analogs are less effective than cocaine in maintaining high rates of responding in self-administration procedures (Woolverton et al, 2001). These findings suggest differences between these drugs and cocaine analogs in their interactions with the DA transporter (Vaughan et al, 1999;Reith et al, 2001;Chen et al, 2004), which may be responsible for the diminished cocaine-like pharmacological activity of the BZT analogs.…”

mentioning

confidence: 72%

Relationship between in Vivo Occupancy at the Dopamine Transporter and Behavioral Effects of Cocaine, GBR 12909 [1-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine], and Benztropine Analogs

Desai

Kopajtic²,

French³

et al. 2005

J Pharmacol Exp Ther

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Analogs of benztropine (BZT) bind to the dopamine (DA) transporter and inhibit DA uptake but often have behavioral effects that differ from those of cocaine and other DA-uptake inhibitors. To better understand these differences, we examined the relationship between locomotor-stimulant effects of cocaine 125 I]RTI-121 and effect at short times after injection and less than predicted at longer times after injection. This result suggests that the apparent rate of occupancy of the DA transporter, in addition to percentage of sites occupied, contributes to the behavioral effects of cocaine. The present results suggest that among drugs that act at the DA transporter, the slower apparent rates of occupancy with the DA transporter by the BZT analogs may contribute in an important way to differences in their effectiveness.

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The Uptake Inhibitors Cocaine and Benztropine Differentially Alter the Conformation of the Human Dopamine Transporter

Cited by 97 publications

References 31 publications

State-dependent Conformations of the Translocation Pathway in the Tyrosine Transporter Tyt1, a Novel Neurotransmitter:Sodium Symporter from Fusobacterium nucleatum

State-dependent Conformations of the Translocation Pathway in the Tyrosine Transporter Tyt1, a Novel Neurotransmitter:Sodium Symporter from Fusobacterium nucleatum

Recognition of Benztropine by the Dopamine Transporter (DAT) Differs from That of the Classical Dopamine Uptake Inhibitors Cocaine, Methylphenidate, and Mazindol as a Function of a DAT Transmembrane 1 Aspartic Acid Residue

Relationship between in Vivo Occupancy at the Dopamine Transporter and Behavioral Effects of Cocaine, GBR 12909 [1-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine], and Benztropine Analogs

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