Labeling of dopamine transporter transmembrane domain 1 with the tropane ligand N-[4-(4-azido-3-[125I]iodophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane implicates proximity of cocaine and substrate active sites.
The novel photoaffinity ligand N-[4-(4-azido-3-(125)I-iodophenyl)-butyl]-2-beta-carbomethoxy-3beta-(4-chlorophenyl) tropane ([(125)I]MFZ 2-24) was used to investigate the site for cocaine binding on the dopamine transporter (DAT). [(125)I]MFZ 2-24 irreversibly labeled both rat striatal and expressed human DAT with high affinity and appropriate pharmacological specificity. Tryptic proteolysis of [(125)I]MFZ 2-24 labeled DAT followed by epitope-specific immunoprecipitation demonstrated that the ligand becomes adducted almost exclusively to transmembrane domains (TMs) 1-2. Further localization of [(125)I]MFZ 2-24 incorporation achieved by proteolyzing labeled wild-type and methionine mutant DATs with cyanogen bromide identified the sequence between residues 68 and 80 in TM1 as the ligand adduction site. This is in marked contrast to the previously identified attachment of the photoaffinity label [(125)I]RTI 82 in TM6. Because [(125)I]MFZ 2-24 and [(125)I]RTI 82 possess identical tropane pharmacophores and differ only in the placement of the reactive azido moieties, their distinct incorporation profiles identify the regions of the protein adjacent to different aspects of the cocaine molecule. These findings thus strongly support the direct interaction of cocaine on DAT with TM1 and TM6, both of which have been implicated by mutagenesis and homology to a bacterial leucine transporter as active sites for substrates. These results directly establish the proximity of TMs 1 and 6 in DAT and suggest that the mechanism of transport inhibition by cocaine involves close interactions with multiple regions of the substrate permeation pathway.
Parnas
M Laura
ML
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, USA.
Gaffaney
Jon D
JD
Zou
Mu Fa
MF
Lever
John R
JR
Newman
Amy Hauck
AH
Vaughan
Roxanne A
RA
D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003042:Cocaine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D003488:Cyanogen Bromide / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D008024:Ligands; D008297:Male; D008715:Methionine; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling; D013379:Substrate Specificity; D014326:Tropanes / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D001386:Azides; D050483:Dopamine Plasma Membrane Transport Proteins; D007457:Iodine Radioisotopes; D008024:Ligands; D050505:Mutant Proteins; C527694:N-(4-(4-azido-3-(125I)iodophenyl)butyl)-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane; D010446:Peptide Fragments; D014326:Tropanes; C076379:RTI 82; D008715:Methionine; D014357:Trypsin; D003042:Cocaine; D003488:Cyanogen Bromide