Apparent Homomeric NR1 Currents Observed in Xenopus Oocytes are Caused by an Endogenous NR2 Subunit Carsten Schmidt and Michael Hollmann⁎ Lehrstuhl für Biochemie I–Rezeptorbiochemie, Ruhr-Universität Bochum, Universitätsstraβe 150, 44801 Bochum, Germany Received 7 September 2007; received in revised form 21 November 2007; accepted 26 November 2007 Available online 8 December 2007 Functional N-methyl-D-aspartate receptors NMDARs are thought to be heteromeric receptor complexes consisting of NR1 and NR2 subunits. However, recombinant NR1 subunits expressed in Xenopus oocytes assemble functional ion channels even without exogenous NR2 subunits and with a different pharmacology, suggesting a homomeric subunit stoichiometry. To explain this phenomenon, we screened oocytes for Xenopus NR2 subunits and found all four subunit-encoding mRNAs (XenNR2A–XenNR2D) to be present endogenously, with those encoding the XenNR2B subunit being particularly abundant. We cloned the full- length XenNR2B cDNA and co-expressed it with NR1 in oocytes. A detailed electrophysiological characterization revealed that the pharmacology of NR1/XenNR2B was identical with that of the presumed homomeric NMDARs expressed from NR1 subunits. By contrast, heteromeric receptors containing the rat NR2B subunit showed significant pharmacological differences compared with NR1/XenNR2B receptors. These results demon- strate that recombinant NR1 subunits expressed in Xenopus oocytes interact with an endogenously expressed NR2B subunit and form hybrid hetero- meric NMDARs. These findings confirm the current views that NMDARs are obligatory heteromeric complexes and that functional homomeric NMDARs do not exist. © 2007 Elsevier Ltd. All rights reserved. Edited by J. Karn Keywords: NMDA receptor; Xenopus laevis; oocyte; homomeric NR1; XenNR2B Introduction Since the expression cloning of the first ionotropic glutamate receptor (iGluR) subunit GluR1, 1 the Xenopus laevis oocyte expression system has been extensively used for electrophysiological analyses of all functional members of the iGluR family. 2,3 Such analyses include the expression cloning of the first N-methyl-D-aspartate (NMDA) receptor NMDAR subunit NR1-1a 4 and the subsequent characteriza- tion of this subunit and its seven functional splice variants. 4–6 Each of these NR1 splice variants assembled into functional and putatively homo- meric receptor complexes that were activated by glutamate or NMDA in the presence of glycine as co- agonist, but not by α-amino-3-hydroxy-5-methyl-4- isoxazole propionate (AMPA) or kainate. 2,7 The currents obtained showed all the hallmark features known from physiological NMDARs, such as a voltage-dependent Mg 2+ block and sensitivity to the competitive antagonist 2-amino-5-phosphonovale- rate (APV) or to the open-channel blocker 5-methyl- 10,11-dihydro-5H-dibenzo[a,b]cyclohept-5,10-imi- nmaleate (MK801). 2,4,6,8 However, native NMDARs *Corresponding author. E-mail address: Michael.Hollmann@rub.de. Abbreviations used: NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; XenNR, Xenopus NMDAR subunit; iGluR, ionotropic glutamate receptor; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate; APV, 2-amino-5-phosphonovalerate; MK801, 5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohept-5, 10-iminmaleate; HEK, human embryonic kidney; rNR, rat NMDAR subunit; RACE, rapid amplification of cDNA ends; UTR, untranslated region; CNQX, 6-cyano-7- nitroquinoxaline-2,3-dione; KYA, kynureic acid; I/V, current–voltage; CPE, cytoplasmic polyadenylation element; GSP, gene-specific primer. doi:10.1016/j.jmb.2007.11.105 J. Mol. Biol. (2008) 376, 658–670 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.