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Structure and Function of Neurotransmitter Transporters

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.

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Structure and Function of Neurotransmitter Transporters

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  1. Structure and Function of Neurotransmitter Transporters Erice 2011

  2. 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.

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

  4. Role of neurotransmitter transporters

  5. Pre-synaptic neuron 2K+ Neurotransmitter ATP ADP 3Na+ K+ Na+ Glial cell Cl- 3Na+, H+ G-Protein Ions SIGNAL TRANSDUCTION Post-synaptic neuron

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

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

  8. Electrophysiology as a tool to analyze Transporter Function Most neurotransmitter transporters are electrogenic cotransporters using multiple sodium ions as well as chloride (NSS) or potassium (glutamate transporters)

  9. time Resistive currents: Electrogenic transport current voltage

  10. Example of a common experimental protocol 0 Substrate-induced inward currents Current (nA) + 50 Protocol of Voltage jumps: the holding voltage is -25 mV 8 voltage jumps with 25 mV intervals -25 Voltage (mV) -150 time

  11. current voltage time Capacitative currents: a consequence of Sodium binding/unbinding

  12. NSS transporters: structure, function and chloride site. Eukaryotic NSS transporters mediate cotransport of the neurotransmitter sodium and chloride. For istance the GABA transporter GAT-1: 2Na+out +1Cl-out + GABAout → 2Na+in +1Cl-in + GABAin

  13. GABA Transporter GAT-1 R69 G63 Y140 2Na+:Cl-:GABA NH2 COOH

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

  15. Lithium Interactions In GAT-1, Asp-395 participates in the Na2 site

  16. Loss of Lithium stimulation in D395 mutants

  17. Li stimulation in WT depends on [GABA]

  18. D395 mutants have lost the Li leak currents A. GABA B. Lithium

  19. Where is the chloride binding site ?

  20. Rationale Coordination of Cl- in ClC Channels/antiporters by main chain NH and side chain hydroxyls from serine and tyrosine residues Look 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

  21. Amino acid sequence alignment of a segment of TM VII Between eukaryotic and prokaryotic members of the NSS family Chloride Dependent Chloride Independent

  22. uptake in absence / uptake in presence of Chloride in WT and S331 mutants Only replacements with acidic amino acids render uptake chloride independent

  23. Transport cycle in WT and S331E WT S331E substrate uptake is Chloride-dependent substrate uptake is Chloride-Independent return of unloaded T accelerated by protonation

  24. Uptake of [3H]GABA into reconstituted liposomes inlaid with WT or S331E transporters No uptake in the absence of chloride • Uptake becomes independent on • chloride • 2) Lowering internal pH dramatically • increases uptake

  25. Symmetry in NSS transporters • A clue to understanding alternating access

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

  27. 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

  28. Controversy on Substrate Binding Stoichiometry in LeuT The 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

  29. Glutamate Transporters are different

  30. Cl- Cl- Glutamate transport and currents

  31. T T K+ K+ K+ K+ T T T T T 3Na+,H+ AAA- AAA- nNa+,H+ AAA--X Na+3,H+ 3Na+,H+ Na+3,H+ AAA- AAA- Na+n,H+ AAA--X

  32. Control of inside Form liposomes in: KPi Na,glu Na,glu KPi NaCl + glu* NaCl + glu* Net flux Exchange

  33. Glutamate transporters GltPh: an archeal homologue of brain glutamate transporters Yernool et. al. (2004) Nature 431, 811-818 The 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.

  34. Two Tl+ binding sites in GltPh Boudker et. al. (2007) Nature 445, 387-393

  35. 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?

  36. T T K+ K+ K+ K+ T T T T T 3Na+,H+ AAA- AAA- nNa+,H+ AAA--X Na+3,H+ 3Na+,H+ Na+3,H+ AAA- AAA- Na+n,H+ AAA--X

  37. L-[3H]-Asp D-[3H]-Asp L-[3H]-Glu

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