1. The novel role of Q/R-editing in AMPA receptor trafficking
Ingo Greger
MRC Laboratory of Molecular Biology
Cambridge, England

2. Glutamate receptors metabotropic (G-protein coupled)
pre-
post-
Kainate Rs
NMDA-Rs (Ca2+ permeable, voltage dependent)
AMPA-Rs (mostly Ca2+ impermeable, voltage independent)
metabotropic (G-protein coupled)
ionotropic (Ion channels)
Let me say a few general words about GRs to set the stage

3. Q/R 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

4. 1 1 Trafficking modes of AMPARs are determined by subunit compositions
regulated trafficking
(long C-tails - GluR1)
constitutive trafficking
(short C-tails)
R. Malinow
PDZ 2 3 2 2 1 1
PDZ

5. Receptor assembly takes place in the ER
Plasma
membrane EP
TGN
Secretory pathway
Golgi QC ER

6. Receptor assembly takes place in the ER
Plasma
membrane EP
TGN
EndoH resistant
(mature)
Secretory pathway
Golgi
this is also regulated by the ec pathway which together with the sp regulates density.
The location of proteins passing through the sp can be determined by its glycosl status. Proteins with immat.glycans reside in the early part and are sensitive to eh, maturely glycosylat pr have reached later compartments beyond the med. Golgi and are Eh sensitive
EndoH sensitive
(immature) QC ER
Channel composition and channel density are determined in the ER

7. Specifically GluR2 resides in intracellular compartments1.
Subcellular rat brain fractions *
GluR2
GluR1
NMDAR1
Calnexin

8. Specifically GluR2 resides in intracellular compartments1.
GluR1
GluR2 3.
Subcellular rat brain fractions
GluR2
GluR1
NMDAR1
Calnexin 2.
PNS (EndoH+):
GluR1
GluR2
mature
immature
% immature: 38 80

9. GluR2 exits the ER inefficiently
chase t (hrs): 2 4 6 9 12
mature
immature
GluR1

10. GluR2 exits the ER inefficiently
chase t (hrs): 2 4 6 9 12
mature
immature
GluR1
mature
immature
GluR2
GluR2 forms a stable ER pool

11. Identifying the GluR2 ER-retention motif
607 P
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 C

12. RNA editing Cytidine to Uridine (C/U) Adenine to Inosine (A/I)
enzymatic modification of ribonucleotides (in metazoa)
Cytidine to Uridine (C/U)
Adenine to Inosine (A/I)
ADAR

13. 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 permeability
b. rectification
c. channel conductance
Q/R-editing is very efficient, >99% of
GluR2 in adult brain is edited at this site.
- 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

14. The Q/R site determines GluR2 ER-retention
+EndoH
mature
immature
Myc-GluR2: R Q
% mature Myc-R2 10 20 30 40 *
n=6; p< 0.02

15. 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
mature
immature
Myc-GluR2: R Q
% mature Myc-R2 10 20 30 40 *
n=6; p< 0.02 2 5 9
Myc-GluR2(R)
chase t (hrs):
mature
immature
Myc-GluR2(Q)

16. Summary:
GluR2 is largely intracellular (ER), GluR1 is predominantly post-ER
(cell surface).
GluR2 is stably retained in the ER where it forms an intracellular pool.
GluR1 exits from the ER within 9-12 hr.
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.

17. AMPAR assembly occurs in 2-steps
N N
Dimerization
(via N-termini)
Dimer
Tetramer
2. Dimerization of dimers
Y. Stern-Bach, E. Gouaux..

18. Q/R-editing affects AMPAR sedimentation
% of Total 10 20 30 40 8 6 4 12 14 16
Fraction# P1 P2
1 hr
5 hr
13 hr
GluR2-Q
GluR2(R) does not assemble into P2.
sedimentation P1 P2
Chase t: Q
5 hr R
immature
mature

19. P1 consists of assembly intermediates,
P2 of AMPAR tetramers
Blue-Native PAGE: R Q
11+ SDS
Fraction#: 5 6 7 11 5 6 7 11 T D M P1 P2 P1 P2
GluR2(R) is blocked at the step of tetramerisation
4 R-subunits are disfavored in tetramers

20. Endogenous GluR2 is largely unassembled -
contrasting with GluR1
Cultured neurons 1
sedimentation P1 P2 5 6 7 11
F#:
GluR2
GluR1 M D T
D:M 10
0.3
0.5 1 - 2 P1 P2
GluR1
GluR2 5 6 7 10 11 12 P2 40
GluR1 P1
GluR2 30
% of Total 20 10 4 6 8 10 12 14
Fraction #
Arg607 limits GluR2 numbers in tetramers

21. Arg607 is located at subunit interfaces
pore helix
TM3
GluR
KcsA
180˚

23. Arg607 is located at subunit interfaces
pore helix
TM3
pore helix
selectivity
filter
Q607
D611
Q608
W599
G603 .
P-loop K R Q N E D
607
mature
immature
The Q/R site acts like a molecular switch, which restricts GluR2 numbers
in AMPAR tetramers.

24. 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.

25. Ca2+ Edited to Arg Not edited to Arg Arg607 Synapse ERQC ER
Channel stoichiometry - microscopic properties
Synaptic channel abundance - macroscopic currents
Arg607

27. Acknowledgements Ed ZIFF Latika KHATRI Xiangpeng KONG HHMI
Yimi Amarillo