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.
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.
Ukairo
Okechukwu T
OT
Division of Pharmaceutical Sciences, Duquesne University, Mellon Hall, Room 453, 600 Forbes Ave., Pittsburgh, PA 15282, USA.
Bondi
Corry D
CD
Newman
Amy Hauck
AH
Kulkarni
Santosh S
SS
Kozikowski
Alan P
AP
Pan
Stephen
S
Surratt
Christopher K
CK
D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000187:drug effects / Q000378:metabolism*; D001590:Benztropine / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism; D006224:Cricetinae; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008024:Ligands; D008454:Mazindol / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008774:Methylphenidate / Q000378:metabolism; D018613:Microscopy, Confocal; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008024:Ligands; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; D001590:Benztropine; D008774:Methylphenidate; D001224:Aspartic Acid; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D008454:Mazindol; D003042:Cocaine; D004298:Dopamine