title: The N-terminus of the norepinephrine transporter regulates the magnitude and selectivity of the transporter-associated leak current.
abstract: The norepinephrine (NE) transporter (NET) mediates the removal of NE from synaptic spaces and is a major target for antidepressants, amphetamine and cocaine. Previously, we have shown that syntaxin 1A (SYN 1A) supports human NET (hNET) cell surface expression, that hNET/SYN 1A interactions are direct and mediated by the hNET N-terminus, and that the hNET/SYN 1A association limits substrate-induced hNET-associated currents [Sung, U., Apparsundaram, S., Galli, A., Kahlig, K.M., Savchenko, V., Schroeter, S., Quick, M.W., Blakely, R.D., 2003. A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity. J. Neurosci. 23, 1697-1709]. These data raise the possibility that the hNET N-terminus, and potentially its interaction with SYN 1A, might regulate other hNET conductance states, including the hNET-mediated leak current. Importantly for monoamine transporters, the leak conductance has been shown to play a critical role in regulating cell membrane potential and possibly neuronal excitability [Quick, M.W., 2003. Regulating the conducting states of a mammalian serotonin transporter. Neuron 40, 537-549]. Here we demonstrate that deletion of the binding domain for SYN 1A in the NET N-terminus robustly enhances the NET-mediated leak current as well as its selectivity for Cl- permeation under particular intracellular ionic compositions. In addition, we show that the NET N-terminus coordinates the ability of intracellular Na+ and Cl- to regulate the leak conductance. These data suggest that the NET N-terminus regulates and defines the ionic specificity of the NET-mediated leak current.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, 465 21st Ave. South, Nashville, TN 37232-8548, USA.
authors.forename: Francesca
authors.initials: F
authors.lastname: Binda
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Lute
authors.forename: Concetta
authors.initials: C
authors.lastname: Dipace
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
authors.forename: Aurelio
authors.initials: A
authors.lastname: Galli
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D002712:Chlorides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004558:Electric Stimulation / Q000379:methods; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009638:Norepinephrine / Q000378:metabolism*; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D017434:Protein Structure, Tertiary / Q000502:physiology; D012964:Sodium / Q000494:pharmacology; D050827:Syntaxin 1 / Q000502:physiology; D014162:Transfection / Q000379:methods
chemical_list: D002712:Chlorides; D050484:Norepinephrine Plasma Membrane Transport Proteins; D050827:Syntaxin 1; D012964:Sodium; D009638:Norepinephrine