Deletion of the C-Terminal Domain of the NR2B Subunit Alters Channel Properties and Synaptic Targeting of N-Methyl-D-Aspartate Receptors in Nascent Neocortical Synapses Ralf Mohrmann, 1 Georg Ko ¨ hr, 2 Hanns Hatt, 1 Rolf Sprengel, 2 and Kurt Gottmann 1 * 1 Lehrstuhl fu ¨ r Zellphysiologie, Ruhr-Universita ¨t Bochum, Bochum, Germany 2 Department of Molecular Neuroscience, Max-Planck Institute for Medical Research, Heidelberg, Germany Channel properties and synaptic targeting of N-methyl- D-aspartate (NMDA) receptors determine their impor- tance in synaptic transmission, long-term synaptic plas- ticity, and developmental reorganization of synaptic circuits. To investigate the involvement of the C-terminal domain of the NR2B subunit in regulating channel prop- erties and synaptic localization, we analyzed gene- targeted mice expressing C-terminally truncated NR2B subunits (NR2B C/C mice; Sprengel et al. [1998] Cell 92:279 – 89). Because homozygous NR2B C/C mice die perinatally, we studied embryonic neocortical neurons differentiating in culture. At early stages in vitro, neurons predominantly expressed NR1/NR2B receptors, as shown by the NR2B subunit-specific antagonist ifen- prodil. At these nascent synapses, NMDA excitatory postsynaptic currents (EPSCs) in neurons from NR2B C/C mice showed a strong-amplitude reduction to 20% of control, but AMPA EPSCs were unaltered. Analysis of the MK-801 block of NMDA receptor-mediated whole-cell currents revealed a decreased peak open probability of NMDA receptor channels (to about 60%) in neurons from NR2B C/C mice, although their single channel conduc- tance was unchanged. To study effects on synaptic tar- geting, we determined the fraction of synaptically local- ized NMDA receptors relative to the whole-cell NMDA receptor population. In neurons from NR2B C/C mice, the synaptic NMDA receptor fraction was drastically re- duced, suggesting that the C-terminal domain of the NR2B subunit plays a major role in synaptic targeting of NMDA receptors at nascent synapses. With increasing time in culture, the reduction in NMDA EPSCs in neurons from NR2B C/C mice diminished. This is explained by the expression of additional NMDA receptor subtypes containing NR2A subunits at more mature synapses. © 2002 Wiley-Liss, Inc. Key words: NMDA receptors; synaptic targeting; open probability; C-terminal domain; NR2B subunit The functional role of N-methyl-D-aspartate (NMDA) receptors in excitatory synaptic transmission, in long-term plasticity of glutamatergic synapses, and in developmental refinement of synaptic connections is based on the bio- physical properties of synaptic NMDA receptors (Bliss and Collingridge, 1993; Katz and Shatz, 1996). NMDA recep- tors are heteromeric receptor-channel complexes that are composed of NR1 and NR2 subunits, with the type of NR2 subunit largely determining the biophysical and pharmacological properties of NMDA receptor subtypes (Hollmann and Heinemann, 1994; McBain and Mayer, 1994; Zukin and Benett, 1995). During differentiation of cortical neurons, the NR1/NR2B receptor represents the major NMDA receptor subtype in nascent glutamatergic synapses (Kirson and Yaari, 1996; Lindlbauer et al., 1998; Tovar and Westbrook, 1999; Mohrmann et al., 2000). The NR2A subunit expression occurs delayed in an activity-dependent manner (Monyer et al., 1994; Hoff- mann et al., 1997, 2000; Quinlan et al., 1999). The intracellular, C-terminal domains of NR2 sub- units have been shown to interact with putative scaffold- ing proteins of postsynaptic density, such as PSD-95 (Kor- nau et al., 1995, 1997; Kim et al., 1996; Mu ¨ller et al., 1996; Niethammer et al., 1996; Kim and Huganir, 1999). This type of interaction has been suggested to control synaptic targeting and clustering of NMDA receptors. Moreover, the C-terminal domains of the NR2 subunits contain potential phosphorylation sites for tyrosine kinases and other protein kinases that play a central role in mod- ulation of NMDA receptor channel properties (Moon et al., 1994; Wang and Salter, 1994; Ko ¨hr and Seeburg, 1996; Omkumar et al., 1996; Leonard and Hell, 1997; Yu Contract grant sponsor: Deutsche Forschungsgemeinschaft; Contract grant number: Go 710/2-2. *Correspondence to: Dr. Kurt Gottmann, Lehrstuhl fu ¨ r Zellphysiologie, Ruhr-Universita ¨t Bochum, D-44780 Bochum, Germany. E-mail: kurt.gottmann@ruhr-uni-bochum.de Received 29 October 2001; Revised 25 January 2002; Accepted 28 January 2002 Published online 5 April 2002 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.10219 Journal of Neuroscience Research 68:265–275 (2002) © 2002 Wiley-Liss, Inc.