Presentation on theme: "The novel role of Q/R-editing in AMPA receptor trafficking"— Presentation transcript:
1 The novel role of Q/R-editing in AMPA receptor trafficking Ingo GregerMRC Laboratory of Molecular BiologyCambridge, England
2 Glutamate receptors metabotropic (G-protein coupled) pre-post-Kainate RsNMDA-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 compositionGluR1-4 (A-D):- Ca2+ permeability (Ca2+ impermeable)- Rectification (linear I/V)- Conductance (low)GluR2Q/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. MalinowPDZ232211PDZ
5 Receptor assembly takes place in the ER PlasmamembraneEPTGNSecretory pathwayGolgiQCER
6 Receptor assembly takes place in the ER PlasmamembraneEPTGNEndoH resistant(mature)Secretory pathwayGolgithis 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 sensitiveEndoH sensitive(immature)QCERChannel composition and channel density are determined in the ER
7 Specifically GluR2 resides in intracellular compartments 1.Subcellular rat brain fractions*GluR2GluR1NMDAR1Calnexin
8 Specifically GluR2 resides in intracellular compartments 1.GluR1GluR23.Subcellular rat brain fractionsGluR2GluR1NMDAR1Calnexin2.PNS (EndoH+):GluR1GluR2matureimmature% immature:3880
9 GluR2 exits the ER inefficiently chase t (hrs):246912matureimmatureGluR1
10 GluR2 exits the ER inefficiently chase t (hrs):246912matureimmatureGluR1matureimmatureGluR2GluR2 forms a stable ER pool
11 Identifying the GluR2 ER-retention motif 607PGluR1, 3, 4:GluR2:F G I F N S S L W F S L G A F M Q Q G CR - - -Q/RP. SeeburgC
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 permeabilityb. rectificationc. channel conductanceQ/R-editing is very efficient, >99% ofGluR2 in adult brain is edited at this site.- Reduction of Q/R editing in transgenicmice 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 +EndoHmatureimmatureMyc-GluR2:RQ% mature Myc-R210203040*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.+EndoHmatureimmatureMyc-GluR2:RQ% mature Myc-R210203040*n=6; p< 0.02259Myc-GluR2(R)chase t (hrs):matureimmatureMyc-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 isER-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 NDimerization(via N-termini)DimerTetramer2. Dimerization of dimersY. Stern-Bach, E. Gouaux..
18 Q/R-editing affects AMPAR sedimentation % of Total10203040864121416Fraction#P1P21 hr5 hr13 hrGluR2-QGluR2(R) does not assemble into P2.sedimentationP1P2Chase t:Q5 hrRimmaturemature
19 P1 consists of assembly intermediates, P2 of AMPAR tetramersBlue-Native PAGE:RQ11+ SDSFraction#:5671156711TDMP1P2P1P2GluR2(R) is blocked at the step of tetramerisation4 R-subunits are disfavored in tetramers
20 Endogenous GluR2 is largely unassembled - contrasting with GluR1Cultured neurons1sedimentationP1P256711F#:GluR2GluR1MDTD:M100.30.51-2P1P2GluR1GluR2567101112P240GluR1P1GluR230% of Total2010468101214Fraction #Arg607 limits GluR2 numbers in tetramers
21 Arg607 is located at subunit interfaces pore helixTM3GluRKcsA180˚
23 Arg607 is located at subunit interfaces pore helixTM3pore helixselectivityfilterQ607D611Q608W599G603.P-loopKRQNED607matureimmatureThe Q/R site acts like a molecular switch, which restricts GluR2 numbersin 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, affecttraffic/assembly, suggesting that the overall conformation of the P-loopis critical.
25 Ca2+ Edited to Arg Not edited to Arg Arg607 Synapse ER QCERChannel stoichiometry - microscopic propertiesSynaptic channel abundance - macroscopic currentsArg607