Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. complexes to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating (1/2/1/2) subunit arrangement the GluN1/GluN2A NMDA receptor adopts an adjacent (1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors. AMPA-type kainate-type and NMDA-type) assemble as tetramers of four homologous subunits. The various subunits share a common modular architecture consisting of an extracellular N-terminal domain (NTD) 4 an agonist-binding domain (ABD) a transmembrane domain (TMD) and an intracellular C-terminal domain (CTD; 1 2 AMPA and kainate receptors can function as Vatalanib homomers although they preferentially assemble as heteromers. In contrast NMDA receptors are obligate heteromers usually composed of two GluN1 and two GluN2 subunits. Heteromeric AMPA and kainate receptors appear to have a 2:2 subunit Vatalanib stoichiometry and an alternating subunit arrangement (3 4 However there have been conflicting results regarding the subunit arrangement in NMDA receptors with evidence for either an adjacent (5 6 or an alternating arrangement (7-10). We have developed a method based on AFM imaging for determining the arrangement of subunits within ionotropic receptors (11-13). The method involves engineering specific epitope tags onto each subunit and expressing the receptors in a suitable cell line (tsA 201). Crude membrane fractions from the transfected cells are solubilized in detergent and the receptors are isolated by affinity Vatalanib chromatography. The receptors are incubated with subunit-specific antibodies and the resulting receptor-antibody complexes are imaged by AFM. Receptors with two bound antibodies are identified and the angles between the Vatalanib antibodies are measured. A frequency distribution of these angles then reveals the structure of the receptor. In the present study we have used this method to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating subunit arrangement the GluN1/GluN2A NMDA receptor adopts an adjacent arrangement. We conclude that contrary to the current view the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. EXPERIMENTAL PROCEDURES Constructs The following constructs were used: wild type (WT) rat GluA1 rat GluA2igQ with a His8/Myc tag between residues 22 and 23 (…FGV22HHHHHHHHEQKLISEEDLS23SN … ; tag underlined) WT rat GluN1-1a GluN1 with a hemagglutinin (HA)/His8 tag between residues 416 and 417 in the ABD (…TMS416YPYDVPDYAHHHHHHHHD417GTC … ; tag underlined) GluN1 with a Myc tag between residues 416 and 417 (…TMS416EQKLISEEDLD417GTC … ; tag underlined) WT rat GluN2A GluN2A with a FLAG/His8 epitope tag between residues 851 and 852 that is 15 residues downstream of the TMD (…CFTG851DYKDDDDKHHHHHHHHV852CSD … ; tag Vatalanib underlined) and GluN2A with an HA/His8 tag between residues 425 and 426 in the ABD (…DPL425EQKLISEEDLHHHHHHHHT426ETC … ; tag underlined). All constructs were in the vector pcDNA3.1 except the two AMPA receptor constructs which were in p3αpA (a derivative of pcDNA3). Antibodies The following antibodies were used: mouse monoclonal anti-GluA1 (Millipore; clone RH95 MAB2263 raised against an Vatalanib N-terminal peptide of rat GluA1) mouse monoclonal anti-GluN1 (Abcam; ab134308 S308-48 raised against amino acids 42-361 of GluN1) mouse monoclonal anti-GluN1 (Millipore; clone 54.1 MAB363 raised against amino acids 660-811 of GluN1) rabbit monoclonal anti-GluN2A (Millipore; clone A12W 4 raised against residues 1265-1464 of mouse GluN2A) mouse monoclonal anti-Myc (Invitrogen; R950-25) mouse monoclonal anti-His (Fitzgerald; clone Ntrk2 His-17 10 rabbit polyclonal anti-His (Fitzgerald; 70R-HR005) mouse monoclonal anti-V5 (Invitrogen; R960-25) mouse monoclonal anti-HA (Covance; HA.11 clone 16B12 MMS-101P) mouse monoclonal anti-FLAG (Sigma; clone M2 F3165) mouse monoclonal anti-β-actin (Sigma; clone AC-15 A5441) Cy3-conjugated goat anti-mouse (Sigma; C2181) Cy3-conjugated goat anti-rabbit (Sigma; C2306) and fluorescein isothiocyanate-conjugated goat anti-mouse (Sigma; F8771). The specificity of all primary antibodies used to decorate the various AMPA and NMDA.