title: Antagonist-induced conformational changes in dopamine transporter extracellular loop two involve residues in a potential salt bridge.
abstract: Ligand-induced changes in the conformation of extracellular loop (EL) 2 in the rat (r) dopamine transporter (DAT) were examined using limited proteolysis with endoproteinase Asp-N and detection of cleavage products by epitope-specific immunoblotting. The principle N-terminal fragment produced by Asp-N was a 19kDa peptide likely derived by proteolysis of EL2 residue D174, which is present just past the extracellular end of TM3. Production of this fragment was significantly decreased by binding of cocaine and other uptake blockers, but was not affected by substrates or Zn(2+), indicating the presence of a conformational change at D174 that may be related to the mechanism of transport inhibition. DA transport activity and cocaine analog binding were decreased by Asp-N treatment, suggesting a requirement for EL2 integrity in these DAT functions. In a previous study we demonstrated that ligand-induced protease resistance also occurred at R218 on the C-terminal side of rDAT EL2. Here using substituted cysteine accessibility analysis of human (h) DAT we confirm cocaine-induced alterations in reactivity of the homologous R219 and identify conformational sensitivity of V221. Focused molecular modeling of D174 and R218 based on currently available Aquifex aeolicus leucine transporter crystal structures places these residues within 2.9Å of one another, suggesting their proximity as a structural basis for their similar conformational sensitivities and indicating their potential to form a salt bridge. These findings extend our understanding of DAT EL2 and its role in transport and binding functions.
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States.
authors.forename: Jon D
authors.initials: JD
authors.lastname: Gaffaney
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States.
authors.forename: Madhur
authors.initials: M
authors.lastname: Shetty
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States.
authors.forename: Bruce
authors.initials: B
authors.lastname: Felts
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States.
authors.forename: Akula-Bala
authors.initials: AB
authors.lastname: Pramod
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States.
authors.forename: James D
authors.initials: JD
authors.lastname: Foster
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States. Electronic address: keith.henry@med.und.edu.
authors.forename: L Keith
authors.initials: LK
authors.lastname: Henry
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States. Electronic address: roxanne.vaughan@med.und.edu.
authors.forename: Roxanne A
authors.initials: RA
authors.lastname: Vaughan
mesh_terms: D000818:Animals; D004298:Dopamine / Q000378:metabolism; D018492:Dopamine Antagonists / Q000737:chemistry / Q000494:pharmacology*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D018492:Dopamine Antagonists; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D004298:Dopamine
keywords: Amphetamine; Cocaine; Methamphetamine; Molecular modeling; SCAM