Cavara NA et al. (2010), Residues at the tip of the pore loop of NR3B-co...

XB-ART-42588

BMC Neurosci 2010 Oct 19;11:133. doi: 10.1186/1471-2202-11-133.

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Residues at the tip of the pore loop of NR3B-containing NMDA receptors determine Ca2+ permeability and Mg2+ block.

Cavara NA , Orth A , Hicking G , Seebohm G , Hollmann M .

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Members of the complex N-methyl-D-aspartate receptor (NMDAR) subfamily of ionotropic glutamate receptors (iGluRs) conventionally assemble from NR1 and NR2 subunits, the composition of which determines receptor properties. Hallmark features of conventional NMDARs include the requirement for a coagonist, voltage-dependent block by Mg2+, and high permeability for Ca2+. Both Mg2+ sensitivity and Ca2+ permeability are critically dependent on the amino acids at the N and N+1 positions of NR1 and NR2. The recently discovered NR3 subunits feature an unprecedented glycine-arginine combination at those critical sites within the pore. Diheteromers assembled from NR1 and NR3 are not blocked by Mg2+ and are not permeable for Ca2+. Employing site-directed mutagenesis of receptor subunits, electrophysiological characterization of mutants in a heterologous expression system, and molecular modeling of the NMDAR pore region, we have investigated the contribution of the unusual NR3 N and N+1 site residues to the unique functional characteristics of receptors containing these subunits. Contrary to previous studies, we provide evidence that both the NR3 N and N+1 site amino acids are critically involved in mediating the unique pore properties. Ca2+ permeability could be rescued by mutating the NR3 N site glycine to the NR1-like asparagine. Voltage-dependent Mg2+ block could be established by providing an Mg2+ coordination site at either the NR3 N or N+1 positions. Conversely, "conventional" receptors assembled from NR1 and NR2 could be made Mg2+ insensitive and Ca2+ impermeable by equipping either subunit with the NR3-like glycine at their N positions, with a stronger contribution of the NR1 subunit. This study sheds light on the structure-function relationship of the least characterized member of the NMDAR subfamily. Contrary to previous reports, we provide evidence for a critical functional involvement of the NR3 N and N+1 site amino acids, and propose them to be the essential determinants for the unique pore properties mediated by this subunit.

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

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	Figure 1. Agonist-induced current responses of coexpressed mutant NMDAR subunits. Mean current responses elicited by application of glycine alone (Gly, 10 μM) or together with 100μM glutamate (Glu) for NR1/NR2 and NR1/NR3 diheteromers assembled from wild type or mutant subunits. White bars represent mean amplitudes after the application of glycine, black bars denote mean responses after the coapplication of glutamate/glycine; all current amplitudes are shown in nA. The right diagram depicts the ratio of glycine- to glutamate/glycine-induced current responses for each combination. Data shown here are mean values ± SEM, n = 11-48 for NR1/NR2 combinations, n = 5-45 for NR1/NR3 diheteromers; p < 0.05; p < 0.01; **p < 0.005 (Student's t-test).
	Figure 2. Ca2+ permeability of coexpressed mutant NMDAR subunits. Overview of the Ca2+ permeabilities of diheteromers featuring N and N+1 site mutant NMDAR subunits. To the right, PCa2+/Pmono ratios for the investigated combinations are given; the graphs represent those values on a relative scale. For NR1/NR2 combinations, measurements of Ca2+ permeability were performed after the application of 100 μM glutamate (Glu) and 10 μM glycine (Gly). NR1/NR3 diheteromers were activated by glycine alone. n = 5-9.
	Figure 3. Mg2+ block of coexpressed mutant NMDAR subunits: IV relations. IV relationships of the Mg2+ block of N and N+1 site mutant NMDAR diheteromers. Shown are measurements between -150 mV and +50 mV after application of 10 μM glycine (Gly) alone or in coapplication with 100 μM glutamate (Glu) in the absence (blue traces) and presence (red bars) of Mg2+. Traces represent averages from 3-4 experiments per subunit combination (normalized to +20 mV).
	Figure 4. Mg2+ block of coexpressed mutant NMDAR subunits: mean values and current traces. A. Amino acid sequence of the pore loop regions of rodent NR1, NR2B, and NR3B. The native amino acids at the N and N+1 positions are boxed. B. Mean values (± SEM) of the Mg2+ block of N and N+1 site mutant NMDAR diheteromers. 0.5 mM Mg2+ was applied after activation of the receptor with 10 μM glycine (Gly) alone or in coapplication with 100 μM glutamate (Glu). Statistical significance was determined relative to the respective wild type combination. n = 5-27, p < 0.05; p < 0.01; **p < 0.005. C. Representative current traces of the Mg2+ block of diheteromers featuring N and N+1 site mutant subunits. Agonists (conc. as in B) were applied for 60 s (long bar); Mg2+ was added for 20 s during the application (denoted by the short bar).
	Figure 5. Coordination of Mg2+ within the pore of wild type and N and N+1 site mutant NMDARs. Conformation of the pore region of an NR1/NR3 diheteromer. A. Side view of the selectivity filter of an NR1/NR3 diheteromer. Note that not the entire transmembrane region is shown. A similar conformation is employed by NR1/NR2 receptors. The N site is located at the tip of the pore loop; the N+1 site is located adjacent to that in the helix (marked by the red asterisk). Below, the same diheteromer is shown in a bottom view. The extensive protein loops belong to the NR1 subunits. To the right, a schematic representation of the selectivity filter of NMDARs is depicted: One Na+ ion (blue ball) and one Mg2+ ion (green ball) are coordinated in the pore with a water molecule (red) serving as a spacer. Bottom right: coordination of six water molecules by an Mg2+ ion. B.-G. Coordination of Mg2+ in NMDAR diheteromers. Mg2+ is depicted as a green ball; the blue ball represents Na+. Below each structure is a schematic view of the coordination by water (red with white protons), side chain oxygens (red dots) and backbone oxygens (red dots with blue rings).

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