Cavara NA et al. (2009), Effects of NR1 splicing on NR1/NR3B-type excita...

XB-ART-39809

BMC Neurosci 2009 Apr 06;10:32. doi: 10.1186/1471-2202-10-32.

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Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors.

Cavara NA , Orth A , Hollmann M .

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BACKGROUND: N-methyl-D-aspartate receptors (NMDARs) are the most complex of ionotropic glutamate receptors (iGluRs). Subunits of this subfamily assemble into heteromers, which - depending on the subunit combination - may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity. RESULTS: Here, we have investigated whether the NR3B subunit is capable of assembly with each of the eight functional NR1 splice variants, and whether the resulting receptors share the unique functional properties described for NR1-1a/NR3. We provide evidence that functional excitatory glycine receptors formed regardless of the NR1 isoform, and their pharmacological profile matched the one reported for NR1-1a/NR3: glycine alone fully activated the receptors, which were insensitive to glutamate and block by Mg2+. Surprisingly, amplitudes of agonist-induced currents showed little dependency on the C-terminally spliced NR1 variants in NR1/NR3B diheteromers. Even more strikingly, NR3B conferred proton sensitivity also to receptors containing NR1b variants - possibly via disturbing the "proton shield" of NR1b splice variants. CONCLUSION: While functional assembly could be demonstrated for all combinations, not all of the specific interactions seen for NR1 isoforms with coexpressed NR2 subunits could be corroborated for NR1 assembly with NR3. Rather, NR3 abates trafficking effects mediated by the NR1 C terminus as well as the N-terminally mediated proton insensitivity. Thus, this study establishes that NR3B overrides important NR1 splice variant-specific receptor properties in NR1/NR3B excitatory glycine receptors.

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Species referenced: Xenopus laevis
Genes referenced: gnl3 grin1 grin2b nodal1 nodal2 nodal3.1

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	Figure 1. Comparison of agonist-induced currents for all NR1 splice variant/NR3B combinations. A. Ratio of current responses elicited by application of glycine (10 μM, Gly) and glycine plus glutamate (300 μM, Glu) for NR1 subunits expressed alone, NR1/NR2B heteromers, and NR1/NR3B heteromers. Data shown here are mean values ± SEM, n = 10–23 from 2–5 independent experiments per combination; p < 0.05; p < 0.01; **p < 0.005 (Student's t-test). B. Representative current traces of diheteromers, shown exemplary for combinations with NR1–3a. Black bars denote agonist application.
	Figure 2. Comparison of glutamate/glycine-induced steady-state current amplitudes for all NR1 isoforms with either NR2B or NR3B. A. Relative current responses for each of the eight NR1 splice variants coexpressed with NR2B (upper panel) or NR3B (lower panel). Amplitudes were normalized to the responses mediated by NR1-1a/NR2B and NR1-1a/NR3B, respectively. All currents were recorded after the application of 300 μM glutamate and 10 μM glycine. Data shown here are mean values ± Gaussian error propagation, n = 6–10 from 2 independent experiments per combination. B. Current potentiation by the N-terminal NR1b splice variants over NR1a variants when coexpressed with either NR2B (light grey) or NR3B (dark grey). Responses of the NR1a variants were independently set to 1 and responses mediated by NR1b variants were normalized to the responses of the respective NR1a variants. Data are mean values ± Gaussian error propagation, n = 6–10 from 2 independent experiments per combination. ns = not significant. C. Relative glutamate/glycine-induced current responses of NR1–3a- and NR1–3b-containing NMDARs depending on the pH value. Current responses were normalized to the values measured at pH 7.2. Data are mean values ± Gaussian error propagation, n = 6. alone = expressed alone; ns = not significant.
	Figure 3. Comparison of the Mg2+ block of NR3B-containing diheteromers with each of the NR1 splice variants. A. Current-voltage (IV) relationships for NR1–3b/NR2B (upper diagram, Glu/Gly-induced currents) and NR1–3b/NR3B (middle and lower diagrams for Glu/Gly- and Gly-induced currents, respectively) recorded between -150 mV and +50 mV. Recordings were performed in the presence of 300 μM glutamate (Glu) and/or 10 μM glycine (Gly) in the presence (dark grey traces) and absence (light grey traces) of 0.5 mM Mg2+. Traces represent averages from 4 experiments per combination (normalized to +20 mV). B. Comparison of the Mg2+ block of agonist-evoked current responses at -70 mV for each NR1 splice variant expressed alone, with NR2B, or with NR3B. Mean values ± SEM, n = 9–18 from 2–5 independent experiments per combination; p < 0.05; p < 0.01; **p < 0.005 (Student's t-test). C. Exemplary potentiation by glutamate of glycine-induced NR1–3b/NR3B receptor currents in oocytes with high expression levels of XenNR2B. Note that only the portion of current induced by additional glutamate is blocked by Mg2+. Current responses are mean values ± SEM normalized to the glycine-induced response (set to 100% and marked with #), n = 6. The inset shows a representative current trace from this batch recorded during sequential applications of agonists and Mg2+.

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Anantharam, Combinatorial RNA splicing alters the surface charge on the NMDA receptor. 1992, Pubmed, Xenbase