The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.
"The reduced effect of allosteric modulators on A7-modified receptors is thought to reflect an increase in open probability that occurs after modification of the A7 residue. In MTS-treated A7C receptors, open probability is essentially at its maximum and thus agents that slow channel opening such as protons and zinc have limited opportunity to act (Yuan et al., 2005). A similar effect could explain the reduced effect of ethanol on these channels because Wright et al. (1996) showed that although 200 mM ethanol reduced mean open time and frequency of bursts, it did not introduce new closed times. "
[Show abstract][Hide abstract] ABSTRACT: N-Methyl-D-aspartate (NMDA) receptors gate a slow and calcium-rich component of the postsynaptic glutamate response. Like all ionotropic glutamate receptors, NMDA subunits contain a highly conserved motif (SYTANLAAF) in the transmembrane (TM) 3 domain that is critically involved in channel gating. Mutation of an alanine in this domain (A7; underlined above) results in constitutively open receptors that show reduced sensitivity to several allosteric modulators. In this study, we examined the effects of ethanol, a substance that inhibits NMDA currents via an unknown mechanism, on tonically active NMDA receptors expressed in human embryonic kidney 293 cells. Ethanol (100 mM) inhibited currents from GluN1(A7R)/GluN2A and GluN1(A7R)/GluN2B receptors by approximately 50%, whereas those from GluN1/GluN2B(A7R) receptors were reduced by less than 10%. In cysteine-substituted GluN1 and GluN2 A7 mutants, estimated ethanol IC₅₀ values for agonist-gated currents were 101, 117, 103, and 69 mM for GluN1(A7C)/GluN2A, GluN1(A7C)/GluN2B, GluN1/GluN2A(A7C), and GluN1/GluN2B(A7C) receptors, respectively. After exposure to the thiol-modifying reagent 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), A7C mutants showed robust agonist-independent currents and reduced sensitivity to ethanol (IC₅₀ values of 371, 256, 715, and 958 mM, respectively, as above). In contrast, cysteine modification of the ligand-binding domain resulted in constitutively open receptors that showed robust ethanol inhibition. Ethanol inhibition of MTSET-treated GluN1(A7C) receptors was further reduced by TM3/TM4 mutations previously shown to reduce ethanol sensitivity of agonist-gated receptors. Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits.
Journal of Pharmacology and Experimental Therapeutics 01/2012; 340(1):218-26. DOI:10.1124/jpet.111.187179 · 3.97 Impact Factor
"In addition, a DRPEER motif in NR1 (Watanabe et al., 2002), a tryptophan residue in the M2 regions of NR2 subunits (Williams et al., 1998) and the common SYTANLAAF motif in TM3 (Yuan et al., 2005; Wada et al., 2006) affect the Mg 2+ block. Comparing the sequences of NR1, NR2 and NR3 subunits reveals a remarkable conservation of these regions, although especially within the QRN site and the SYTANLAAF motif several exchanges between NR1, NR2 and NR3 subunits are found. "
[Show abstract][Hide abstract] ABSTRACT: Glycine has diverse functions within the mammalian central nervous system. It inhibits postsynaptic neurons via strychnine-sensitive glycine receptors (GlyRs) and enhances neuronal excitation through co-activation of N-methyl-D-aspartate (NMDA) receptors. Classical Ca(2+)-permeable NMDA receptors are composed of glycine-binding NR1 and glutamate-binding NR2 subunits, and hence require both glutamate and glycine for efficient activation. In contrast, recombinant receptors composed of NR1 and the glycine binding NR3A and/or NR3B subunits lack glutamate binding sites and can be activated by glycine alone. Therefore these receptors are also named "excitatory glycine receptors". Co-application of antagonists of the NR1 glycine-binding site or of the divalent cation Zn(2+) markedly enhances the glycine responses of these receptors. To gain further insight into the properties of these glycine-gated NMDA receptors, we investigated their current-voltage (I-V) dependence. Whole-cell current-voltage relations of glycine currents recorded from NR1/NR3B and NR1/NR3A/NR3B expressing oocytes were found to be linear under our recording conditions. In contrast, NR1/NR3A receptors displayed a strong outwardly rectifying I-V relation. Interestingly, the voltage-dependent inward current block was abolished in the presence of NR1 antagonists, Zn(2+) or a combination of both. Further analysis revealed that Ca(2+) (1.8 mM) present in our recording solutions was responsible for the voltage-dependent inhibition of ion flux through NR1/NR3A receptors. Since physiological concentrations of the divalent cation Mg(2+) did not affect the I-V dependence, our data suggest that relief of the voltage-dependent Ca(2+) block of NR1/NR3A receptors by Zn(2+) may be important for the regulation of excitatory glycinergic transmission, according to the Mg(2+)-block of conventional NR1/NR2 NMDA receptors.
"Previous studies have suggested that the M3 segment functions as a transduction element, coupling ligand binding to channel opening (Jones et al., 2002; Yuan et al., 2005). M3 contains the highly conserved SYTANLAAF motif, and many cysteine mutations in this region exhibit state-dependent accessibility, implying that M3 movement occurs in response to activation. "
[Show abstract][Hide abstract] ABSTRACT: N-Methyl-D-aspartate (NMDA) receptors play a critical role in both development of the central nervous system and adult neuroplasticity. However, although the NMDA receptor presents a valuable therapeutic target, the relationship between its structure and functional properties has yet to be fully elucidated. To further explore the mechanism of receptor activation, we characterized two gain-of-function mutations within the NR1 M3 segment, a transmembrane domain proposed to couple ligand binding and channel opening. Both mutants (A7Q and A7Y) displayed significant glycine-independent currents, indicating that their M3 domains may preferentially adopt a more activated conformation. Substituted cysteine modification experiments revealed that the glycine binding clefts of both A7Q and A7Y are inaccessible to modifying reagents and resistant to competitive antagonism. These data suggest that perturbation of M3 can stabilize the ligand binding domain in a closed cleft conformation, even in the absence of agonist. Both mutants also displayed significant glutamate-independent current and insensitivity to glutamate-site antagonism, indicating partial activation by either glycine or glutamate alone. Furthermore, A7Q and A7Y increased accessibility of the NR2 M3 domain, providing evidence for intersubunit coupling at the transmembrane level and suggesting that these NR1 mutations dominate the properties of the intact heteromeric receptor. The equivalent mutations in NR2 did not exhibit comparable phenotypes, indicating that the NR1 and NR2 M3 domains may play different functional roles. In summary, our data demonstrate that the NR1 M3 segment is functionally coupled to key structural domains in both the NR1 and NR2 subunits.
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