Evidence for distinct sodium-, dopamine-, and cocaine-dependent conformational changes in transmembrane segments 7 and 8 of the dopamine transporter.
Previously we obtained evidence based on engineering of Zn2+ binding sites that the extracellular parts of transmembrane segment 7 (TM7) and TM8 in the human dopamine transporter are important for transporter function. To further evaluate the role of this domain, we have employed the substituted cysteine accessibility method and performed 10 single cysteine substitutions at the extracellular ends of TM7 and TM8. The mutants were made in background mutants of the human dopamine transporter with either two (E2C) or five endogenous cysteines substituted (X5C) that render the transporter largely insensitive to cysteine modification. In two mutants (M371C and A399C), treatment with the sulfhydryl-reactive reagent [2-(trimethylammonium)-ethyl]methanethiosulfonate (MTSET) led to a substantial inhibition of [3H]dopamine uptake. In M371C this inactivation was enhanced by Na+ and blocked by dopamine. Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A399C. The present findings add new functionality to the TM7/8 region by providing evidence for the occurrence of distinct Na+-, substrate-, and perhaps inhibitor-induced conformational changes critical for the proper function of the transporter.
Norregaard
Lene
L
Molecular Neuropharmacology Group, Department of Pharmacology, Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Loland
Claus Juul
CJ
Gether
Ulrik
U
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004298:Dopamine / Q000493:pharmacokinetics*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005110:Extracellular Space; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011869:Radioligand Assay; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology*; D014316:Tritium; D015032:Zinc / Q000378:metabolism
D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D009419:Nerve Tissue Proteins; D014316:Tritium; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D012964:Sodium; D003042:Cocaine; D015032:Zinc; D004298:Dopamine