structure and function of neurotransmitter transporters

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Structure and Function of Neurotransmitter Transporters. Erice 2011. Sodium-Coupled Neurotransmitter Transporters. Role of neurotransmitter transporters (NSS and glutamate). Electrophysiology as a tool to analyze transporter function. NSS transporters: structure, function and chloride site. - PowerPoint PPT Presentation

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  • Structure and Function of Neurotransmitter TransportersErice 2011

  • Sodium-Coupled Neurotransmitter Transporters Role of neurotransmitter transporters (NSS and glutamate).Electrophysiology as a tool to analyze transporter function.NSS transporters: structure, function and chloride site.Glutamate transporters are different.

  • Forrest, L.R. and Rudnick, G.(2009 Physiology 24, 377-386

  • Role of neurotransmitter transporters

  • Giros et.al. (1996) Nature 397, 606-612

  • Giros et.al. (1996) Nature 397, 606-612

  • Electrophysiology as a tool to analyze Transporter FunctionMost neurotransmitter transporters are electrogenic cotransportersusing multiple sodium ions as well as chloride (NSS) or potassium(glutamate transporters)

  • Resistive currents: Electrogenic transportcurrentvoltage

  • Example of a common experimental protocol -25Current (nA)Voltage (mV)-150+ 50time0Protocol of Voltage jumps:the holding voltage is -25 mV8 voltage jumps with 25 mV intervalsSubstrate-induced inward currents

  • Capacitative currents: a consequence of Sodium binding/unbinding

  • NSS transporters: structure, function and chloride site.

    Eukaryotic NSS transporters mediate cotransport of the neurotransmitter sodiumand chloride.For istance the GABA transporter GAT-1:

    2Na+out +1Cl-out + GABAout 2Na+in +1Cl-in + GABAin

  • NH2R69G63Y140COOH2Na+:Cl-:GABAGABA Transporter GAT-1

  • Yamashita et. al. (2005) Nature 437, 215-223

  • Lithium InteractionsIn GAT-1, Asp-395 participates in the Na2 site

  • Loss of Lithium stimulation in D395 mutants

  • Li stimulation in WT depends on [GABA]

  • D395 mutants have lost the Li leak currentsA. GABAB. Lithium

  • Where is the chloride binding site ?

  • RationaleCoordination of Cl- in ClC Channels/antiporters by main chain NH and side chain hydroxyls from serine and tyrosine residuesLook for serine, threonine and tyrosine residues, located in the transmembrane domains conserved in the Cl- dependent neurotransmitter transporters, but not necessarily in their Cl- independent bacterial counterparts

  • ChlorideDependentChlorideIndependentAmino acid sequence alignment of a segment of TM VII Between eukaryotic and prokaryotic members of the NSS family

  • Only replacements with acidic amino acids render uptake chloride independent uptake in absence / uptake in presence of Chloridein WT and S331 mutants

  • WTS331Esubstrate uptake is Chloride-dependentsubstrate uptake is Chloride-Independent

    return of unloaded T accelerated by protonationTransport cycle in WT and S331E

  • Uptake of [3H]GABA into reconstituted liposomes inlaid with WT or S331E transporters No uptake in the absence of chloride

    Uptake becomes independent on chloride2) Lowering internal pH dramatically increases uptake

  • Symmetry in NSS transportersA clue to understanding alternating access

  • Forrest et.al.(2008) PNAS 105, 10338-10343

  • Transmembrane domain 8 of the {gamma}-aminobutyric acid transporter GAT-1 lines a cytoplasmic accessibility pathway into its binding pocket.Ben-Yona A, Kanner BI.J Biol Chem. 2009 Apr 10;284(15):9727-32. Epub 2009 Feb 6

  • Controversy on Substrate Binding Stoichiometry in LeuTThe mechanism of a neurotransmitter:sodium symporter--inward release of Na+ and Substrate is triggered by substrate in a second binding site.Shi L, Quick M, Zhao Y, Weinstein H, Javitch JA.Mol Cell. 2008 Jun 20;30(6):667-77.Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site.Piscitelli CL, Krishnamurthy H, Gouaux E.Nature. 2010 Dec 23;468(7327):1129-32

  • Glutamate Transporters are different

  • Glutamate transport and currents

  • Glutamate transportersGltPh: an archeal homologue of brain glutamate transportersYernool et. al. (2004) Nature 431, 811-818The structure is in excellent agreement with functional data on site-directed mutants from the mammalian glutamate transporters, including the inferred proximity of the tips of HP1 and HP2.

  • Two Tl+ binding sites in GltPhBoudker et. al. (2007) Nature 445, 387-393

  • The side-chain of a conserved aspartateparticipates in Tl+ site 1 Does this aspartate participate in a cation binding site in the brain glutamate transporters?

  • N Reyes et al. Nature 000, 1-6 (2009) doi:10.1038/nature08616Schematic transport mechanism.

  • GAT-1 and other NSS

    Hebrew Univ. Columbia Univ.

    Annie Bendahan Matthias QuickElia Zomot Yongfang ZhaoAssaf Ben-Yona Jonathan JavitchGlutamate Transporters

    Hebrew Univ MPI Frankfurt

    Shlomit Teichman Lucy ForrestShaogang Qu Thomas CrismanNoa Rosental