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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.
Forrest, L.R. and Rudnick, G.(2009 Physiology 24, 377-386
Role of neurotransmitter transporters
Post-synaptic neuron
Glial cell
Pre-synaptic neuron
Neurotransmitter
Ions
G-Protein
Na+
Cl-
3Na+, H+
K+
SIGNAL TRANSDUCTION
2K+
3Na+
ATP
ADP
Giros et.al. (1996) Nature 397, 606-612
Giros et.al. (1996) Nature 397, 606-612
Electrophysiology as a tool to analyze Transporter Function
Most neurotransmitter transporters are electrogenic cotransportersusing multiple sodium ions as well as chloride (NSS) or potassium(glutamate transporters)
Resistive currents: Electrogenic transportcu
rren
tvo
ltage
time
Example of a common experimental protocol
-25
Cur
rent
(nA
)V
olta
ge (
mV
)
-150
+ 50
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
curr
ent
volta
ge
time
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
NH2
R69
G63 Y140
COOH
2Na+:Cl-:GABA
GABA Transporter GAT-1
Yamashita et. al. (2005) Nature 437, 215-223
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]
-150 -100 -50 50
-1.0
-0.8
-0.6
-0.4
-0.2
I(normalized)
Voltage (mV)
A. GABA B. Lithium
-150 -100 -50 50
-1.0
-0.8
-0.6
-0.4
-0.2
Voltage (mV)
I(normalized)
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
ChlorideDependent
ChlorideIndependent
Amino acid sequence alignment of a segment of TM VII Between eukaryotic and prokaryotic members of the NSS family
WTS331A
S331DS331E
0
20
40
60
80
100
[3 H]-
GA
BA
upt
ake
(-/+
Cl- , %
)
Only replacements with acidic amino acids render uptake chloride independent
uptake in absence / uptake in presence of Chloridein WT and S331 mutants
WT S331E
substrate uptake is Chloride-dependent
substrate uptake is Chloride-Independent
return of unloaded T accelerated by protonation
Transport cycle in WT and S331E
0 2 4 6 8 10
0
5
10
15
20
25
30WT
pmol
[3 H]G
AB
A /
mg
prot
ein
time (min)
Cl pH 7.4 Cl pH 6.0 Gluc pH 7.4 Gluc pH 6.0
0 2 4 6 8 100
2
4
6
8
10S331E
pmol
[3 H]G
AB
A /
mg
prot
ein
time (min)
Cl pH 7.4 Cl pH 6.0 Gluc pH 7.4 Gluc pH 6.0
Uptake of [3H]GABA into reconstituted liposomes inlaid with WT or S331E transporters
No uptake in the absence of chloride 1) Uptake becomes independent on chloride2) Lowering internal pH dramatically increases uptake
Symmetry in NSS transporters
• A 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 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
Glutamate Transporters are different
Glutamate transport and currents
T
OUT IN
K
gluNa , gluNa ,
K
T
T K T K
T gluNa , T gluNa ,
OIIIIOOO KHNagluKHNaglu 11311131
Cl- Cl-
3Na+,H+3Na+,H+
TK+
K+
TT
TK+
K+
AAA-
TTnNa+,H+
TNa+n,H+
AAA--X
AAA--X
AAA-
AAA-Na+
3,H+
AAA-Na+
3,H+
Control of insideForm liposomes in:KPi Na,glu
KPi Na,glu
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 aspartate
participates in Tl+ site 1
Does this aspartate participate in a cation binding site in the brain glutamate transporters?
0
20
40
60
80
D-[
3 H]-
Asp
upt
ake
(% o
f WT
)
3Na+,H+3Na+,H+
TK+
K+
TT
TK+
K+
AAA-
TTnNa+,H+
TNa+n,H+
AAA--X
AAA--X
AAA-
AAA-Na+
3,H+
AAA-Na+
3,H+
0
20
40
60
80
100
D-[3 H
]-A
sp u
pta
ke (
% o
f W
T)
D-[3H]-AspL-[3H]-Asp L-[3H]-Glu
0
10
20
30
40
upta
ke (
Li/N
a r
atio, %
)
3Na+,H+3Na+,H+
TK+
K+
TT
TK+
K+
AAA-
TTnNa+,H+
TNa+n,H+
AAA--X
AAA--X
AAA-
AAA-Na+
3,H+
AAA-Na+
3,H+
N Reyes et al. Nature 000, 1-6 (2009) doi:10.1038/nature08616
Schematic transport mechanism.
GAT-1 and other NSS
Hebrew Univ. Columbia Univ.
Annie Bendahan Matthias QuickElia Zomot Yongfang ZhaoAssaf Ben-Yona Jonathan Javitch
Glutamate Transporters
Hebrew Univ MPI Frankfurt
Shlomit Teichman Lucy ForrestShaogang Qu Thomas CrismanNoa Rosental