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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.
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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. • Glutamate transporters are different.
Pre-synaptic neuron 2K+ Neurotransmitter ATP ADP 3Na+ K+ Na+ Glial cell Cl- 3Na+, H+ G-Protein Ions SIGNAL TRANSDUCTION Post-synaptic neuron
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)
time Resistive currents: Electrogenic transport current voltage
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
current voltage time Capacitative currents: a consequence of Sodium binding/unbinding
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
GABA Transporter GAT-1 R69 G63 Y140 2Na+:Cl-:GABA NH2 COOH
Lithium Interactions In GAT-1, Asp-395 participates in the Na2 site
D395 mutants have lost the Li leak currents A. GABA B. Lithium
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
Amino acid sequence alignment of a segment of TM VII Between eukaryotic and prokaryotic members of the NSS family Chloride Dependent Chloride Independent
uptake in absence / uptake in presence of Chloride in WT and S331 mutants Only replacements with acidic amino acids render uptake chloride independent
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
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
Symmetry in NSS transporters • A clue to understanding alternating access
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 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
Cl- Cl- Glutamate transport and currents
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
Control of inside Form liposomes in: KPi Na,glu Na,glu KPi NaCl + glu* NaCl + glu* Net flux Exchange
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.
Two Tl+ binding sites in GltPh Boudker 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?
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
L-[3H]-Asp D-[3H]-Asp L-[3H]-Glu