The novel role of Q/R-editing in AMPA receptor trafficking

Ingo GregerMRC Laboratory of Molecular Biology

Cambridge, England

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)

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

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

ER

Golgi

TGN

Plasma membrane

QC

Secretory pathw

ay

EP

Receptor assembly takes place in the ER

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

GluR1

GluR2

Calnexin

NMDAR1

*

Subcellular rat brain fractions1.

Specifically GluR2 resides in intracellular compartments

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.

0 2 4 6 9 12chase t (hrs):

matureimmature GluR1

GluR2 exits the ER inefficiently

0 2 4 6 9 12chase t (hrs):

matureimmature GluR1

matureimmature GluR2

GluR2 exits the ER inefficiently

GluR2 forms a stable ER pool

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

RNA editingenzymatic modification of ribonucleotides (in metazoa)

• Cytidine to Uridine (C/U)

• Adenine to Inosine (A/I)

ADAR

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.

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

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

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.

AMPAR assembly occurs in 2-steps

Dimer

1. Dimerization (via N-termini)

Tetramer

2. Dimerization of dimers

Y. Stern-Bach, E. Gouaux..

N N

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.

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

% 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

pore helix

TM3

Arg607 is located at subunit interfaces

GluR KcsA

180˚

QuickTime™ and aVideo decompressor

are needed to see this picture.

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.

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.

Edited to Arg Not edited to Arg

ER

Synapse

1. Channel stoichiometry - microscopic properties2. Synaptic channel abundance - macroscopic currents

Arg607

Ca2+ Ca2+

QC

Acknowledgements

Ed ZIFF

Latika KHATRI

Xiangpeng KONG

HHMI

Yimi Amarillo