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

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Presentation on theme: "The novel role of Q/R-editing in AMPA receptor trafficking Ingo Greger MRC Laboratory of Molecular Biology Cambridge, England."— Presentation transcript:

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) ionotropic (Ion channels) pre- post- Kainate Rs NMDA-Rs (Ca 2+ permeable, voltage dependent) AMPA-Rs (mostly Ca 2+ impermeable, voltage independent)

3 Functional properties of AMPAR-heteromers depend on the subunit composition GluR1-4 (A-D): - Ca 2+ permeability (Ca 2+ impermeable) - Rectification (linear I/V) - Conductance (low) GluR2 Q/R

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

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

6 EndoH sensitive (immature) EndoH resistant (mature) ER Golgi TGN Plasma membrane QC Secretory pathway EP Channel composition and channel density are determined in the ER

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

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

9 chase t (hrs): mature immature GluR1 GluR2 exits the ER inefficiently

10 chase t (hrs): mature immature GluR1 mature immature GluR2 GluR2 exits the ER inefficiently GluR2 forms a stable ER pool

11 N C 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

12 RNA editing 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 The presence of GluR2 in AMPAR changes major functional properties: a. di-valent ion permeability b. rectification c. 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.

14 The Q/R site determines GluR2 ER-retention +EndoH mature immature Myc-GluR2: RQ % mature Myc-R * Q R n=6; p< 0.02

15 0259 Myc-GluR2(R) chase t (hrs): mature immature mature immature 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 mature immature Myc-GluR2: RQ % mature Myc-R * Q R n=6; p< 0.02

16 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.

17 AMPAR assembly occurs in 2-steps Dimer 1.Dimerization (via N-termini) Tetramer 2. Dimerization of dimers Y. Stern-Bach, E. Gouaux.. N

18 Q/R-editing affects AMPAR sedimentation P1P2 Q R 5 hr Chase t: sedimentation immature mature % of Total Fraction# P1 P2 1 hr 5 hr 13 hr GluR2-Q GluR2(R) does not assemble into P2.

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

20 % of Total GluR1 GluR GluR1 GluR P1P2 Endogenous GluR2 is largely unassembled - contrasting with GluR Fraction # P1 P F#: GluR2GluR1 M D T D:M P1 P2 Cultured neurons sedimentation Arg607 limits GluR2 numbers in tetramers

21 pore helix TM3 Arg607 is located at subunit interfaces GluRKcsA 180˚

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23 pore helix TM3 pore helix selectivity filter Q607 D611 Q608 W599 G603. P-loop KRQNED 607 mature immature Arg607 is located at subunit interfaces 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 Edited to ArgNot edited to Arg ER Synapse 1.Channel stoichiometry - microscopic properties 2.Synaptic channel abundance - macroscopic currents Arg607 Ca 2+ QC

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27 Acknowledgements Ed ZIFF Latika KHATRI Xiangpeng KONG HHMI Yimi Amarillo


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