title: Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux.
abstract: The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 microM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.
authors.affiliation: Department of Molecular Physiology and Biophysics, Center for Molecular Neuroscience, Kennedy Centerfor Research on Human Development, Vanderbilt University, 7124 MRBIII, 465 21st Avenue S., Nashville, TN 37232, USA.
authors.forename: Francesca
authors.initials: F
authors.lastname: Binda
authors.forename: Concetta
authors.initials: C
authors.lastname: Dipace
authors.forename: Erica
authors.initials: E
authors.lastname: Bowton
authors.forename: Sabrina D
authors.initials: SD
authors.lastname: Robertson
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Lute
authors.forename: Jacob U
authors.initials: JU
authors.lastname: Fog
authors.forename: Minjia
authors.initials: M
authors.lastname: Zhang
authors.forename: Namita
authors.initials: N
authors.lastname: Sen
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Colbran
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Gnegy
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.forename: Kevin
authors.initials: K
authors.lastname: Erreger
authors.forename: Aurelio
authors.initials: A
authors.lastname: Galli
mesh_terms: D000595:Amino Acid Sequence; D000661:Amphetamine / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000166:cytology; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008636:Mesencephalon / Q000166:cytology; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism; D050827:Syntaxin 1 / Q000378:metabolism*; D014162:Transfection
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D050827:Syntaxin 1; D000661:Amphetamine; D005982:Glutathione Transferase; D004298:Dopamine