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

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

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

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

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

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

Specifically GluR2 resides in intracellular compartments 1. Subcellular rat brain fractions * GluR2 GluR1 NMDAR1 Calnexin

Specifically GluR2 resides in intracellular compartments 1. GluR1 GluR2 3. Subcellular rat brain fractions GluR2 GluR1 NMDAR1 Calnexin 2. PNS (EndoH+): GluR1 GluR2 mature immature % immature: 38 80

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

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

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

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

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

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

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)

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.

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

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

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

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

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

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.

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.

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

Acknowledgements Ed ZIFF Latika KHATRI Xiangpeng KONG HHMI Yimi Amarillo