Transcript 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.
• Glutamate transporters are different.
Forrest, L.R. and Rudnick, G.(2009 Physiology 24, 377-386
Role of neurotransmitter
transporters
Pre-synaptic neuron
2K+
Neurotransmitter
ATP
3Na+
ADP
K+
Na+
Glial cell
Cl3Na+, H+
G-Protein
Ions
SIGNAL TRANSDUCTION
Post-synaptic neuron
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 cotransporters
using multiple sodium ions as well as chloride (NSS) or potassium
(glutamate transporters)
voltage
current
Resistive currents: Electrogenic transport
time
Voltage (mV)
Current (nA)
Example of a common experimental protocol
0
Substrate-induced inward currents
+ 50
Protocol of Voltage jumps:
the holding voltage is -25 mV
8 voltage jumps with 25 mV intervals
-25
-150
time
voltage
current
Capacitative currents: a consequence of Sodium
binding/unbinding
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
GABA Transporter GAT-1
R69
G63
Y140
2Na+:Cl-:GABA
NH2
COOH
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]
D395 mutants have lost the Li leak currents
A. GABA
B. Lithium
Voltage (mV)
Voltage (mV)
-150
-100
-50
50
-150
-100
-50
50
-0.2
-0.2
-0.4
-0.4
-0.6
I(normalized)
-0.6
-0.8
-0.8
-1.0
-1.0
I(normalized)
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
Amino acid sequence alignment of a segment of TM VII
Between eukaryotic and prokaryotic members of the NSS family
Chloride
Dependent
Chloride
Independent
100
3
-
[ H]-GABA uptake (-/+ Cl , %)
uptake in absence / uptake in presence of Chloride
in WT and S331 mutants
80
60
40
20
0
WT 331A 331D 331E
S
S
S
Only replacements with acidic amino acids
render uptake chloride independent
Transport cycle in WT and S331E
WT
substrate uptake is
Chloride-dependent
S331E
substrate uptake is
Chloride-Independent
return of unloaded T
accelerated by protonation
pmol [ H]GABA / mg protein
WT
30
Cl pH 7.4
Cl pH 6.0
Gluc pH 7.4
Gluc pH 6.0
25
20
15
S331E
10
Cl pH 7.4
Cl pH 6.0
Gluc pH 7.4
Gluc pH 6.0
8
6
4
3
10
3
pmol [ H]GABA / mg protein
Uptake of [3H]GABA into reconstituted liposomes inlaid with
WT or S331E transporters
5
0
2
0
0
2
4
6
time (min)
8
10
No uptake in the absence of chloride
0
2
4
6
time (min)
8
10
1) 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, 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
OUT
T
K
K

T


Na , glu
1 glu


T


Na , glu

T

O
K
K
T

IN

Na , glu
 3 Na

O
T

 1H

O

Na , glu
 1K
Cl-



I
 1 glu

I
 3 Na
Cl-

I
 1H

I
 1K

O
TK+
TK+
K+
K+
T
T
3Na+,H+
AAA-
3Na+,H+
AAAnNa+,H+
AAA--X
TNa+3,H+
AAA-
TNa+n,H+
AAA--X
TNa+3,H+
AAA-
Control of inside
Form 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?
60
40
20
3
D-[ H]-Asp uptake (% of WT)
80
0
5C 485N 485S 485A 485E
8
4
D
D
D
D
D
TK+
TK+
K+
K+
T
T
3Na+,H+
AAA-
3Na+,H+
AAAnNa+,H+
AAA--X
TNa+3,H+
AAA-
TNa+n,H+
AAA--X
TNa+3,H+
AAA-
80
60
40
20
3
D-[ H]-Asp uptake (% of WT)
100
0
8
D4
5C
8
D4
5N
8
D4
5S
8
D4
5E
5
D4
5C
5
D4
5N
5
D4
5S
uptake (Li/Na ratio, %)
40
L-[3H]-Asp
D-[3H]-Asp
L-[3H]-Glu
30
20
10
0
W T455C455N455S W T455C455N455S W T455C455N455S
D
D
D
D
D
D
D
D
D
TK+
TK+
K+
K+
T
T
3Na+,H+
AAA-
3Na+,H+
AAAnNa+,H+
AAA--X
TNa+3,H+
AAA-
TNa+n,H+
AAA--X
TNa+3,H+
AAA-
Schematic transport mechanism.
N Reyes et al. Nature 000, 1-6 (2009) doi:10.1038/nature08616
GAT-1 and other NSS
Hebrew Univ.
Columbia Univ.
Annie Bendahan
Elia Zomot
Assaf Ben-Yona
Matthias Quick
Yongfang Zhao
Jonathan Javitch
Glutamate Transporters
Hebrew Univ
MPI Frankfurt
Shlomit Teichman
Shaogang Qu
Noa Rosental
Lucy Forrest
Thomas Crisman