The role of conserved tryptophan and acidic residues in the human dopamine transporter as characterized by site-directed mutagenesis.
The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na(+)/Cl(-)-dependent transporters. We have explored the roles of these residues using non-conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10-24-fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [(3)H]DA uptake, but caused undetectable [(3)H]CFT binding. Upon anti-C-terminal-hDAT immunoblotting, D345N appeared as broad bands of 66-97 kDa, but this band could not be photoaffinity labeled with cocaine analog [(125)I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid ([(125)I]RTI-82). Unexpectedly, in this mutant, cocaine-like drugs remained potent inhibitors of [(3)H]DA uptake. CFT solely raised the K(m) of [(3)H]DA uptake in wild-type hDAT, but increased K(m) and decreased V(max) in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.
Chen
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Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine, Peoria, Illinois, USA nhc@uic.edu
Vaughan
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Reith
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003042:Cocaine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D017124:Conserved Sequence; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D007700:Kinetics; D008454:Mazindol / Q000493:pharmacokinetics; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014316:Tritium; D014364:Tryptophan
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D014316:Tritium; D001224:Aspartic Acid; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D014364:Tryptophan; D008454:Mazindol; D003042:Cocaine; D004298:Dopamine