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

The novel role of Q/R-editing in AMPA receptor trafficking. Ingo Greger MRC Laboratory of Molecular Biology Cambridge, England. pre-. Kainate Rs NMDA-Rs (Ca 2+ permeable, voltage dependent) AMPA-Rs (mostly Ca 2+ impermeable, voltage independent). post-. Glutamate receptors.

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

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

  2. pre- • Kainate Rs • NMDA-Rs (Ca2+ permeable, voltage dependent) • AMPA-Rs (mostly Ca2+ impermeable, voltage independent) post- Glutamate receptors • metabotropic (G-protein coupled) • ionotropic (Ion channels)

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

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

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

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

  8. 3. GluR1 GluR2 Specifically GluR2 resides in intracellular compartments 1. 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): 0 2 4 6 9 12 mature GluR1 immature

  10. mature GluR2 immature GluR2 forms a stable ER pool GluR2 exits the ER inefficiently chase t (hrs): 0 2 4 6 9 12 mature GluR1 immature

  11. Q/R 607 P F G I F N S S L W F S L G A F M QQ G C - - - - - - - - - - - - - - - - R - - - GluR1, 3, 4: GluR2: P. Seeburg Identifying the GluR2 ER-retention motif N C

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

  13. - 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) From Zukin et al., TINS 1997 The Q/R site is a key regulator of ion flux P. Seeburg

  14. R Q Myc-GluR2: * 40 30 mature % mature Myc-R2 20 immature 10 Q R 0 n=6; p< 0.02 +EndoH The Q/R site determines GluR2 ER-retention

  15. 0 2 5 9 chase t (hrs): R Q Myc-GluR2: surfaceMyc (green)/ total Myc (red) Myc-GluR2(R) * 40 30 mature % mature Myc-R2 20 immature Myc-GluR2(Q) 10 Q R 0 n=6; p< 0.02 +EndoH Myc-GluR2(R) Myc-GluR2(Q) mature mature immature immature …. and thereby channel density at synapses. The Q/R site determines GluR2 ER-retention

  16. The Q/R site thereby determines AMPAR macroscopic currents. 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.

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

  18. GluR2-Q 1 hr 5 hr 40 P2 13 hr 30 P1 % of Total 20 10 0 4 6 8 10 12 14 16 Fraction# mature immature GluR2(R) does not assemble into P2. Q/R-editing affects AMPAR sedimentation sedimentation P1 P2 Chase t: Q 5 hr R

  19. 4 R-subunits are disfavored in tetramers 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

  20. 2 GluR1 GluR2 F#: 6 7 11 5 6 7 11 T D M P2 P2 P1 P1 - - - D:M 10 0.3 0.5 1 Arg607 limits GluR2 numbers in tetramers Endogenous GluR2 is largely unassembled - contrasting with GluR1 Cultured neurons 1 sedimentation P1 P2 GluR1 GluR2 5 6 7 10 11 12 P2 40 GluR1 P1 GluR2 30 % of Total 20 10 0 4 8 10 12 14 6 Fraction #

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

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

  23. 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.

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

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

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