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.

Forrest, L.R. and Rudnick, G.(2009 Physiology 24,

Role of neurotransmitter transporters

Giros et.al. (1996) Nature 397,

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)

Resistive currents: Electrogenic transport current voltage time

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

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

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

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

Yamashita et. al. (2005) Nature 437,

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

Loss of Lithium stimulation in D395 mutants

Li stimulation in WT depends on [GABA]

A. GABAB. Lithium D395 mutants have lost the Li leak currents

Where is the chloride binding site ?

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

Chloride Dependent Chloride Independent Amino 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 Chloride in WT and S331 mutants

WTS331E substrate uptake is Chloride-dependent substrate uptake is Chloride-Independent return of unloaded T accelerated by protonation Transport cycle in WT and S331E

Uptake of [ 3 H]GABA into reconstituted liposomes inlaid with WT or S331E transporters No uptake in the absence of chloride1)Uptake becomes independent on chloride 2) Lowering internal pH dramatically increases uptake

Symmetry in NSS transporters A clue to understanding alternating access

Forrest et.al.(2008) PNAS 105,

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 Apr 10;284(15): 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 Jun 20;30(6): Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site. Piscitelli CL, Krishnamurthy H, Gouaux E. Nature Dec 23;468(7327):

Glutamate Transporters are different

Glutamate transport and currents Cl -

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

Control of inside Form liposomes in: KPiNa,glu KPi Na,glu NaCl + glu* NaCl + glu* Net fluxExchange

Glutamate transporters GltPh: an archeal homologue of brain glutamate transporters Yernool et. al. (2004) Nature 431, 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 Glt Ph Boudker et. al. (2007) Nature 445,

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

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

D-[ 3 H]-AspL-[ 3 H]-Asp L-[ 3 H]-Glu

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

N Reyes et al. Nature 000, 1-6 (2009) doi: /nature08616 Schematic transport mechanism.

GAT-1 and other NSS Hebrew Univ. Columbia Univ. Annie Bendahan Matthias Quick Elia Zomot Yongfang Zhao Assaf Ben-Yona Jonathan Javitch Glutamate Transporters Hebrew Univ MPI Frankfurt Shlomit Teichman Lucy Forrest Shaogang Qu Thomas Crisman Noa Rosental