title: Reconstitution of homomeric GluA2(flop) receptors in supported lipid membranes: functional and structural properties.
abstract: AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2(flop) receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.
authors: Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom. Electronic address: baranovic@fmp-berlin.de.
authors: Jelena
authors: J
authors: Baranovic
authors: Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom.
authors: Chandra S
authors: CS
authors: Ramanujan
authors: NTT Basic Research Laboratories, Atsugi, Kanagawa 243-0198, Japan.
authors: Nahoko
authors: N
authors: Kasai
authors: Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755-1404.
authors: Charles R
authors: CR
authors: Midgett
authors: Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755-1404.
authors: Dean R
authors: DR
authors: Madden
authors: NTT Basic Research Laboratories, Atsugi, Kanagawa 243-0198, Japan.
authors: Keiichi
authors: K
authors: Torimitsu
authors: Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom.
authors: John F
authors: JF
authors: Ryan
mesh_terms: D000818:Animals; D001581:Benzothiadiazines / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry* / Q000648:ultrastructure; D008564:Membrane Potentials; D018625:Microscopy, Atomic Force; D011487:Protein Conformation; D011810:Quinoxalines / Q000737:chemistry; D051381:Rats; D018091:Receptors, AMPA / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D001581:Benzothiadiazines; D008051:Lipid Bilayers; D008055:Lipids; D008081:Liposomes; D011810:Quinoxalines; D018091:Receptors, AMPA; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; D018698:Glutamic Acid; C104722:glutamate receptor ionotropic, AMPA 2; C004639:cyclothiazide
references: EMBO Rep. 2007 Jan;8(1):51-5
references: 17124511
references: Neurosci Lett. 1993 Feb 5;150(1):80-4
references: 7682309
references: Nature. 2005 Feb 3;433(7025):545-9
references: 15690046
references: Biochim Biophys Acta. 2010 Jul;1800(7):655-61
references: 20307628
references: J Neurosci. 1996 Mar 15;16(6):1982-9
references: 8604042
references: Science. 1991 May 10;252(5007):851-3
references: 1709304
references: Nature. 2000 Oct 26;407(6807):971-7
references: 11069170
references: J Neurosci. 2000 Mar 15;20(6):2073-85
references: 10704481
references: Chem Phys Lipids. 2010 Feb;163(2):117-26
references: 19800876
references: Trends Neurosci. 1995 Apr;18(4):161-8
references: 7539962
references: Neuron. 1993 Jul;11(1):41-52
references: 8338667
references: Eur J Biochem. 2000 Mar;267(5):1382-9
references: 10691975
references: Biophys J. 1983 Aug;43(2):231-6
references: 6193818
references: J Neurosci. 2009 Sep 23;29(38):11912-23
references: 19776277
references: J Neurosci. 2007 Feb 21;27(8):2135-44
references: 17314308
references: Trends Neurosci. 2004 Jun;27(6):321-8
references: 15165736
references: J Neurosci. 2008 Jan 23;28(4):932-43
references: 18216201
references: Nat Protoc. 2007;2(9):2191-7
references: 17853875
references: J Neurosci. 2010 Mar 24;30(12):4449-59
references: 20335481
references: CNS Neurol Disord Drug Targets. 2008 Apr;7(2):129-43
references: 18537642
references: Nat Rev Neurosci. 2007 Feb;8(2):101-13
references: 17237803
references: Methods Enzymol. 2003;372:65-86
references: 14610807
references: J Mol Biol. 1995 Jun 2;249(2):239-43
references: 7783190
references: Front Mol Neurosci. 2012 Jan 02;4:56
references: 22232575
references: Curr Opin Neurobiol. 2006 Jun;16(3):288-97
references: 16713244
references: Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20802-7
references: 22135457
references: J Mol Biol. 2004 Nov 19;344(2):435-42
references: 15522296
references: Neuron. 1994 Dec;13(6):1345-57
references: 7527641
references: Nat Neurosci. 2003 Aug;6(8):803-10
references: 12872125
references: Eur J Biochem. 1997 Feb 1;243(3):798-804
references: 9057848
references: J Mol Biol. 2008 Oct 10;382(3):578-84
references: 18656486
references: Trends Neurosci. 2000 Feb;23(2):75-80
references: 10652548
references: Biochim Biophys Acta. 2005 Jun 30;1712(2):109-27
references: 15919049
references: EMBO J. 2011 Mar 2;30(5):972-82
references: 21317871
references: Science. 1998 Jun 5;280(5369):1596-9
references: 9616121
references: Methods Mol Biol. 2007;363:39-57
references: 17272836
references: Biol Chem. 2006 Feb;387(2):179-87
references: 16497150
references: J Neurochem. 1992 Nov;59(5):1979-82
references: 1383430
references: PLoS Biol. 2009 May;7(5):e1000103
references: 19419241
references: J Struct Biol. 1997 Jul;119(2):149-57
references: 9245755
references: J Neurosci. 1997 Jan 1;17(1):58-69
references: 8987736
references: Nature. 2009 Dec 10;462(7274):745-56
references: 19946266
references: Cell Biochem Biophys. 2005;42(1):75-85
references: 15673930
references: Neurosci Lett. 2006 Jan 2;391(3):102-7
references: 16198054
references: J Physiol. 2003 May 15;549(Pt 1):75-92
references: 12665613
references: Biophys Chem. 1996 Jan 16;58(1-2):87-96
references: 8679921
references: J Mol Biol. 2007 Nov 23;374(2):500-5
references: 17936299