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The Journal of Neuroscience, January 15, 1999, 19(2):630-636
Differential Binding of Tropane-Based Photoaffinity Ligands on
the Dopamine Transporter
Roxanne A.
Vaughan1, 3,
Gregory E.
Agoston2,
John
R.
Lever4, and
Amy Hauck
Newman2
1 Molecular Neurobiology Branch and
2 Psychobiology Section, National Institute on Drug
Abuse-Intramural Research Program, National Institutes of Health,
Baltimore, Maryland 21224, and 3 Neuroscience Division,
Yerkes Regional Primate Center, and 4 Department of
Environmental Health Sciences, The Johns Hopkins University School of
Public Health, Baltimore, Maryland 21205
Benztropine and its analogs are tropane ring-containing dopamine
uptake inhibitors that produce behavioral effects markedly different
from cocaine and other dopamine transporter blockers. We investigated
the benztropine binding site on dopamine transporters by covalently
attaching a benztropine-based photoaffinity ligand, [125I]N-[n-butyl-4-(4 -azido-3-iodophenyl)]-4',4"-difluoro-3 -(diphenylmethoxy)tropane ([125I]GA II 34), to the protein, followed by
proteolytic and immunological peptide mapping. The maps were compared
with those obtained for dopamine transporters photoaffinity labeled
with a GBR 12935 analog, [125I]1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)ethyl]piperazine ([125I]DEEP), and a cocaine analog,
[125I]3 -(p-chlorophenyl)tropane-2-carboxylic
acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI
82), which have been shown previously to interact with different regions of the primary sequence of the protein.
[125I]GA II 34 became incorporated in a
membrane-bound, 14 kDa fragment predicted to contain transmembrane
domains 1 and 2. This is the same region of the protein that binds
[125I]DEEP, whereas the binding site for
[125I]RTI 82 occurs closer to the C terminal in a
domain containing transmembrane helices 4-7. Thus, although
benztropine and cocaine both contain tropane rings, their binding sites
are distinct, suggesting that dopamine transport inhibition may occur
by different mechanisms. These results support previously derived
structure-activity relationships suggesting that benztropine and
cocaine analogs bind to different domains on the dopamine transporter.
These differing molecular interactions may lead to the distinctive
behavioral profiles of these compounds in animal models of drug abuse
and indicate promise for the development of benztropine-based molecules for cocaine substitution therapies.
Key words:
cocaine; benztropine; dopamine transporter; photoaffinity
label; dopamine uptake; proteolytic peptide mapping; immunoprecipitation
Copyright © 1999 Society for Neuroscience 0270-6474/99/192630-07$05.00/0
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