The novel role of Q/R-editing in AMPA receptor trafficking
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Transcript of The novel role of Q/R-editing in AMPA receptor trafficking
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The novel role of Q/R-editing in AMPA receptor trafficking
Ingo GregerMRC Laboratory of Molecular Biology
Cambridge, England
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Glutamate receptors
• metabotropic (G-protein coupled)
• ionotropic (Ion channels)
pre-
post-Kainate Rs
NMDA-Rs (Ca2+ permeable, voltage dependent)
AMPA-Rs (mostly Ca2+ impermeable, voltage independent)
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Functional properties of AMPAR-heteromers depend on the subunit composition
GluR1-4 (A-D):
- Ca2+ permeability (Ca2+ impermeable)- Rectification (linear I/V)- Conductance (low)
GluR2 Q/R
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Trafficking modes of AMPARs are determined by subunit compositions
regulated trafficking(long C-tails - GluR1)
PDZ
112 2
constitutive trafficking(short C-tails)
R. Malinow
PDZ
2 23
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ER
Golgi
TGN
Plasma membrane
QC
Secretory pathw
ay
EP
Receptor assembly takes place in the ER
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Receptor assembly takes place in the ER
EndoH sensitive(immature)
EndoH resistant(mature)
ER
Golgi
TGN
Plasma membrane
QC
Secretory pathw
ay
EP
Channel composition and channel density are determined in the ER
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GluR1
GluR2
Calnexin
NMDAR1
*
Subcellular rat brain fractions1.
Specifically GluR2 resides in intracellular compartments
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GluR1
GluR2
3.
Specifically GluR2 resides in intracellular compartments
PNS (EndoH+): GluR1 GluR2
38 80% immature:
matureimmature
2.
GluR1
GluR2
Calnexin
NMDAR1
Subcellular rat brain fractions1.
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0 2 4 6 9 12chase t (hrs):
matureimmature GluR1
GluR2 exits the ER inefficiently
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0 2 4 6 9 12chase t (hrs):
matureimmature GluR1
matureimmature GluR2
GluR2 exits the ER inefficiently
GluR2 forms a stable ER pool
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N
C
Identifying the GluR2 ER-retention motif
607P
GluR1, 3, 4:GluR2:
F G I F N S S L W F S L G A F M Q Q G C- - - - - - - - - - - - - - - - R - - -
Q/R
P. Seeburg
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RNA editingenzymatic modification of ribonucleotides (in metazoa)
• Cytidine to Uridine (C/U)
• Adenine to Inosine (A/I)
ADAR
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The Q/R site is a key regulator of ion flux
From Zukin et al., TINS 1997
- The presence of GluR2 in AMPAR changes major functional properties:a. di-valent ion permeabilityb. rectificationc. channel conductance
- Reduction of Q/R editing in transgenic mice results in seizures and early death. (Brusa et al., Science 1995; Feldmeyer et al., Nat. Neurosci. 1999)
P. Seeburg
- Q/R-editing is very efficient, >99% of GluR2 in adult brain is edited at this site.
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The Q/R site determines GluR2 ER-retention
+EndoH
matureimmature
Myc-GluR2: R Q
% m
atur
e M
yc-R
20
10
20
30
40 *
QRn=6; p< 0.02
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0 2 5 9
Myc-GluR2(R)
chase t (hrs):
matureimmature
matureimmature Myc-GluR2(Q)
The Q/R site determines GluR2 ER-retention
Myc-GluR2(Q)Myc-GluR2(R)
surface Myc (green)/ total Myc (red)
…. and thereby channel density at synapses.
+EndoH
matureimmature
Myc-GluR2: R Q
% m
atur
e M
yc-R
20
10
20
30
40 *
QRn=6; p< 0.02
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Summary:
• GluR2 is stably retained in the ER where it forms an intracellular pool. GluR1 exits from the ER within 9-12 hr.
• GluR2 is largely intracellular (ER), GluR1 is predominantly post-ER (cell surface).
• GluR2 ER-exit is controlled by the Q/R site. The edited R-form is ER-retained, the unedited Q-form exits the ER efficiently.
• Unedited GluR2(Q) accumulates at the cell surface/synapses.
The Q/R site thereby determines AMPAR macroscopic currents.
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AMPAR assembly occurs in 2-steps
Dimer
1. Dimerization (via N-termini)
Tetramer
2. Dimerization of dimers
Y. Stern-Bach, E. Gouaux..
N N
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Q/R-editing affects AMPAR sedimentation
P1 P2
Q
R5 hr
Chase t:
sedimentation
immature mature%
of T
otal
0
10
20
30
40
10864 12 14 16
Fraction#
P1
P2
1 hr5 hr
13 hr
GluR2-Q
GluR2(R) does not assemble into P2.
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P1 consists of assembly intermediates, P2 of AMPAR tetramers
6 6 77 11Fraction#:
R Q
M
D
T
5115 11+
SD
S
Blue-Native PAGE:
P1 P1P2 P2
GluR2(R) is blocked at the step of tetramerisation4 R-subunits are disfavored in tetramers
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% o
f Tot
al
GluR1GluR2
6 75 10
GluR1GluR2
11 12
P1 P2
Endogenous GluR2 is largely unassembled - contrasting with GluR1
1
0
10
20
30
40
4 8 126 1410
Fraction #
P1
P2
56 67 7 1111F#:
GluR2GluR1
M
D
T
D:M 10 0.3 0.5 1 -- -
2
P1 P1 P2P2
Cultured neuronssedimentation
Arg607 limits GluR2 numbers in tetramers
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pore helix
TM3
Arg607 is located at subunit interfaces
GluR KcsA
180˚
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QuickTime™ and aVideo decompressor
are needed to see this picture.
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pore helix
TM3
pore helix
selectivity filter
Q607
D611
Q608
W599
G603.
P-loop
K R Q N E D
607
matureimmature
Arg607 is located at subunit interfaces
The Q/R site acts like a molecular switch, which restricts GluR2 numbers in AMPAR tetramers.
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Summary:
• Arg607 restricts GluR2 ER-exit at the level of channel assembly.
• GluR2(R) forms dimers, assembly is blocked at the step of tetramerization.
• GluR2(Q) tetramerizes efficiently.
• Endogenous GluR2 is largely unassembled, GluR1 is mostly tetrameric.
• Other alterations in the pore loop, apart from editing to Arg607, affect traffic/assembly, suggesting that the overall conformation of the P-loop is critical.
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Edited to Arg Not edited to Arg
ER
Synapse
1. Channel stoichiometry - microscopic properties2. Synaptic channel abundance - macroscopic currents
Arg607
Ca2+ Ca2+
QC
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Acknowledgements
Ed ZIFF
Latika KHATRI
Xiangpeng KONG
HHMI
Yimi Amarillo