Abstract
A large number of structurally diverse compounds act as open-channel blockers of NMDA receptors. They may share discrete or overlapping binding sites within the channel. In this study, the effects of mutations in and around the membrane-spanning and pore-forming regions of NMDA receptor subunits were studied with three blockers, MK-801, memantine, and TB-3-4, using recombinant NMDA receptors expressed inXenopus laevis oocytes. Mutations at the critical asparagine residues in the M2 loop of NR1 and NR2B and at a tryptophan residue in M2 of NR2B reduced block by MK-801, memantine, and TB-3-4. Mutations at residues in the pre-M1, M1, M3, post-M3, and post-M4 regions had differential effects on the three blockers. Many mutations in these regions reduced block by MK-801 and TB-3-4 but had no effect on block by memantine. The differential effects on block by memantine and MK-801 are unlikely to be caused by differences in the size of these blockers. Benzyl rings in MK-801 and TB-3-4 may make hydrophobic interactions with aromatic and hydrophobic amino acid residues in the pore. Some mutations in the pre-M1 and M3 regions generated constitutively open channels, characterized by large holding currents. The effects of the various mutants are discussed in the context of models based on the known structure of the pore of the KcsA potassium channel and on previous studies dealing with solvent accessible residues in NMDA receptor subunits as determined by modification after cysteine mutagenesis.
N-Methyl-d-aspartate (NMDA) receptors are ligandgated ion channels that have structural similarities with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors (Dingledine et al., 1999). NMDA receptors have several characteristics that distinguish them from the other glutamate receptors. These include the requirement for two different agonists (glutamate and glycine) to activate the receptor, a high permeability of the channel to Ca2+, and a voltage-dependent block of the channel by Mg2+ (Dingledine et al., 1999). NMDA channels are also blocked by a large number of structurally dissimilar organic blockers including ketamine, MK-801, memantine, and various spider toxins and polyamine derivatives (Huettner and Bean, 1988; Collingridge and Lester, 1989; Chen et al., 1992; Igarashi et al., 1997).
With the cloning of cDNAs encoding subunits of glutamate receptors, it has been possible to begin to delineate the structural features that may contribute to various binding sites and/or functional domains of these receptors. The glutamate binding site is formed by two domains (S1 and S2) in the amino terminus and M3–M4 loop of the NR2 subunit, whereas homologous domains in NR1 form the glycine binding site (Kuryatov et al., 1994; Laube et al., 1997; Anson et al., 1998). The amino-terminal domain preceding S1 seems to be a regulatory domain that may contain binding sites for modulators and antagonists such as spermine, protons, ifenprodil, and Zn2+ (Masuko et al., 1999; Low et al., 2000; Paoletti et al., 2000). The M2 loop region is a critical determinant of divalent cation permeability and Mg2+ block. In particular, asparagine residues in this region form part of a Mg2+ binding site and contribute to the selectivity filter of the channel (Dingledine et al., 1999). These asparagine residues, which are in positions analogous to the Q/R sites that control divalent cation permeability of AMPA and kainate channels, have also been found to influence block by organic channel blockers such as MK-801 (Dingledine et al., 1999). There is little information about how other residues in the membrane-spanning and pore-forming regions of NMDA receptors affect block by compounds such as MK-801, memantine, and other organic channel blockers.
In this article, we have studied mutations in the M2 pore-forming loop and in the M1, M3, and M4 membrane-spanning regions and in some adjacent regions. We determined the effects of these mutations on block by three structurally distinct channel blockers: MK-801, memantine, andN 1-N 4-N 8-tribenzyl-spermidine (TB-3-4). MK-801 is a prototypical, high-affinity NMDA channel blocker with a very slow onset and recovery of block (Wong et al., 1986;Huettner and Bean, 1988), whereas memantine is a low-affinity blocker with much faster rates of block and unblock (Chen et al., 1992;Blanpied et al., 1997). Both molecules have quite rigid structures. TB-3-4 is a potent and selective NMDA blocker and is a much larger and more flexible molecule than either MK-801 or memantine (Igarashi et al., 1997). Our results show that block by these three compounds is affected not only by residues in the M2 loop but also by residues toward the extracellular ends of M1, M3, and M4. Notably, mutations in these regions have differential effects on the different blockers. Surprisingly, many mutants that influence block by TB-3-4 also affect MK-801 but have no effect on block by memantine.
Materials and Methods
NMDA Clones and Site-Directed Mutagenesis.
The NR1 clone used in these studies is the NR1A variant (Moriyoshi et al., 1991) which lacks the 21-amino acid insert encoded by exon-5. This clone, and some of the NR1 mutants in the M2 and M1–M2 linker region (Sakurada et al., 1993) were gifts from Dr. S. Nakanishi (Institute for Immunology, Kyoto University Faculty of Medicine, Kyoto, Japan). The rat and mouse NR2B clones (Kutsuwada et al., 1992; Monyer et al., 1992) were gifts from Drs. P. H. Seeburg (Center for Molecular Biology, University of Heidelberg, Germany) and M. Mishina (University of Tokyo, Tokyo, Japan).
Site-directed mutagenesis was carried out by the method of Sayers et al. (Sayers et al., 1992) or by the method of Ho et al. (1989) using the polymerase chain reaction. For mutations in NR2B we used the rat NR2B clone or, in some cases, the mouse (ε2) NR2B clone containing a 1.7-kilobase HindIII-SphI fragment of the rat NR2B clone with the mutation of interest (Williams et al., 1998). Mutations were confirmed by DNA sequencing using a Seq 4 × 4 personal sequencing system (Amersham Biosciences) over approximately 300 nucleotides containing the mutation. A list of oligonucleotide primers used for mutagenesis has not been included but is available from the authors upon request.
Numbering of Residues.
In NR1 and NR2 subunits, amino acids are numbered from the initiator methionine as in the original paper reporting the sequence of NR1 (Moriyoshi et al., 1991). This differs from the numbering system used in some other laboratories, in which residues are numbered from the start of the mature peptide (Kuner et al., 1996; Beck et al., 1999). In the case of NR1, there is an 18 amino acid signal peptide so, for example, residue NR1(N616) described in this study corresponds to residue NR1(N598) using the alternative numbering scheme (Kuner et al., 1996).
Expression in Oocytes and Voltage-Clamp Recording.
The preparation of capped cRNAs and the preparation, injection, and maintenance of oocytes were carried out as described previously (Williams et al., 1993). Oocytes were injected with NR1 plus NR2 cRNAs in a ratio of 1:5 (0.1–4 ng of NR1 plus 0.5–20 ng of NR2). Macroscopic currents were recorded with a two-electrode voltage-clamp using a GeneClamp 500 amplifier (Axon Instruments, Union City, CA) as described previously (Williams, 1993). Electrodes were filled with 3 M KCl and had resistances of 0.4 to 4 MΩ. Oocytes were continuously superfused with a saline solution (100 mM NaCl, 2 mM KCl, 1.8 mM BaCl2, and 10 mM HEPES, pH 7.5) that contained BaCl2 rather than CaCl2 to minimize Ca2+-activated Cl− currents, and in most experiments oocytes were injected with K+-BAPTA (100 nl of 40 mM, pH 7.0–7.4) on the day of recording.
Concentration-inhibition curves for antagonists were constructed by using six to nine different concentrations of antagonist for each mutation. Each concentration was tested on at least four oocytes. In many experiments with TB-3-4 and memantine, it was possible to construct an entire concentration-inhibition curve on each oocyte. In those experiments, we calculated the IC50 for each individual oocyte, and the reported data are the mean from four or more oocytes. In other experiments, particularly those with MK-801, data were pooled from different oocytes (with a total of four or more oocytes for each concentration of antagonist) to calculate the IC50. Block by MK-801 was very slow, taking several minutes to reach steady state, especially with low concentrations of MK-801, and we did not attempt to measure recovery from block by MK-801 in most oocytes.
Data analysis and curve fitting were carried out using Axograph (Axon Instruments, Foster City, CA) or SigmaPlot (Jandel Scientific, San Rafael, CA) on Macintosh computers. To obtain values for the IC50 of antagonists, concentration-inhibition curves were fit to eq. 1:
To study the voltage-dependence of block, voltage-ramps were constructed by ramping the command signal from −150 mV to +60 or +80 mV over 3 to 4 s. Leak currents, measured in the absence of agonist and blockers, were digitally subtracted. We chose concentrations of blockers that gave a 50 to 80% inhibition at −70 mV at a particular mutant. For analysis of the voltage-dependence of block by TB-3-4 or memantine, data were analyzed using the model of Woodhull (1973) by fitting the data to eq. 2:
To measure Ba2+ permeability, voltage ramps were used to determine current-voltage profiles in extracellular Na+-saline (saline solution; composition as above) and Ba2+-saline (64 mM BaCl2, 2 mM KCl, 10 mM HEPES, pH 7.5) as described previously (Williams et al., 1998). Values for the reversal potentials (Vrev) were calculated by linear regression of data 5 mV either side of an estimated reversal potential. Leak currents were subtracted, and the values of Vrev were corrected for small liquid junction potentials (+3 to +8 mV) measured in Ba2+-saline versus Na+-saline.
Results
Screening Mutations.
We initially made a series of individual point mutations in and around the membrane-spanning and pore-forming loop regions of the NR1 subunit. The mutants were constructed in most cases to alter the functional side chain, for example by neutralizing the charge (E-to-Q, D-to-N), removing an aromatic ring (W-to-L, Y-to-L) or removing a hydroxyl group (S-to-A, T-to-A). The regions that were studied are shown schematically in Fig.1A. We screened each mutation by measuring block using a single concentration of TB-3-4, memantine, and MK-801 at NR1/NR2B receptors (Fig. 1). In these experiments, we also studied the effects of mutations on block by 100 μM Mg2+.
As expected, an NR1(N616Q) mutation in the M2 loop reduced block by all four compounds (Fig. 1C). We found that a number of other mutations, particularly in the pre-M1, M1, M3, post-M3, and pre-M4 regions, reduced inhibition by the organic channel blockers. However, some of these mutants had differential effects on the different blockers. For example, mutations W563L in the M1 region, N650A in the M3 region, and T807C in the pre-M4 region reduced the effects of TB-3-4 and MK-801 but had no effect on block by memantine (Fig. 1C). We subsequently studied in detail mutations at residues that affected block by one or more of the three organic channel blockers. The results from those studies are described below. Only mutations at N616, S617, and L655 had pronounced effects on block by Mg2+ (Fig. 1C). The effects of mutations at N616 and S617 have been studied in detail by other investigators (Kuner et al., 1996; Wollmuth et al., 1998a,b), and we did not carry out further studies of Mg2+ block. Nonetheless, the profile for Mg2+ shown in Fig.1C is clearly a useful comparison with the three other blockers that were studied; there are many more mutations that influence block by TB-3-4, MK-801, and memantine than by Mg2+. In addition to the mutants listed on Fig. 1, we made two mutants at position T648, but these mutants, when coexpressed with NR2B, generated large leak currents in the oocytes, presumably because of formation of constitutively open channels. This is discussed below.
In the following sections, we describe the effects of the various key NR1 mutants identified in Fig. 1. For each of the interesting mutants, we constructed concentration-inhibition curves for TB-3-4, memantine, and MK-801 to quantify the effect of each mutation on each blocker. For TB-3-4 and memantine we also constructed voltage ramps and analyzed them using the Woodhull model of voltage-dependent block to obtain values for the K d(0) and the apparent valence (zδ). In some cases, we made additional mutations at particular positions in NR1 and/or studied mutations at equivalent positions in the NR2B subunit. We have divided the presentation of these results into two sections. One section deals with the M2 loop region (which has previously been studied in most detail with respect to channel blockers), and the other section deals with M1, M3, M4, and adjacent regions.
The M2 Loop/Pore-Forming Region.
The M2 loop of NR1 contains an asparagine (N616) that has previously been shown to influence divalent cation permeability and block by Mg2+, MK-801, and other channel blockers (Dingledine et al., 1999). An N-to-Q mutation at this position reduced block by TB-3-4, memantine, and MK-801. The NR2B subunit contains two asparagines (N615 and N616) at positions equivalent to N616 and S617 in NR1, and we studied mutations at both N residues in NR2B. In the M2 region of NR1, mutations at two tryptophan residues (W608 and W611) had small effects on block by memantine (Fig. 1C). We have previously found that mutations at NR1(W608) had little or no effect on Mg2+ block, whereas mutations at NR2B(W607), a position equivalent to NR1(W608), had dramatic effects on Mg2+ block (Williams et al., 1998). In light of this, we studied the effects of NR2B(W607) mutations on block by TB-3-4, memantine, and MK-801. Thus, the studies of the M2 region are focused on NR1(N616), NR2B(N615), NR2B(N616), and NR2B(W607).
N-to-Q mutations at any of the three critical N residues in NR1 and NR2B reduced the potencies of TB-3-4, memantine, and MK-801 (Fig.2, Table 1). The largest effects of the N-to-Q mutations were on block by MK-801, with only modest effects on TB-3-4. An NR2B(W607N) mutation, in the M2 region of NR2B, also greatly reduced the potencies of all three blockers (Fig. 2, Table 1). We studied a number of different mutations at this position and found that substitutions of W-to-N, W-to-L, or W-to-A all greatly reduced the potencies of the blockers whereas W-to-Y or W-to-F mutants had little or no effect (Table 1). Thus, an aromatic residue at position NR2B(W607) seems to be important for block by the organic channel blockers, as has previously been seen with Mg2+ (Williams et al., 1998).
We carried out experiments to determine whether the effects of mutations at NR1(N616) were additive with those of mutations at NR2B(N615) and NR2B(N616). If the effects of any two mutants are not additive, this would suggest that the residues together form a binding site that is ablated by mutation of either residue. The results for MK-801 are shown in Fig. 3A and B, and similar results were seen with TB-3-4 (Table 1). In the case of NR2B(N615Q), a much larger increase in IC50 was seen with NR1(N616Q)/NR2B(N615Q) than with either individual mutation (Fig. 3A, Table 1). In the case of NR2B(N616Q), the combined effects of the NR1(N616Q)/NR2B(N616Q) mutations were not greater than those of the individual mutations (Fig. 3B, Table 1). This suggests that the NR1(N616) and NR2B(N616) residues make equal, nonadditive contributions to the binding site for these blockers whereas the NR2B(N615) residue makes an additional contribution.
Experiments were also carried out to determine whether the effects of mutations at NR2B(W607) were additive with those of mutations at NR1(N616) and at NR2B(N616). The profile for the various antagonists at receptors expressed from combinations of NR1(N616) mutants and NR2B(W607) mutants was complex, but a notable finding was that the effects of combining mutants at NR1(N616) and NR2B(W607) were generally greater than effects seen with each individual mutant (Table 1). We studied a double mutant, NR2B(W607N/N616Q), and found that the effect of this mutant was not greater than that of either of the single mutants NR2B(W607) and NR2B(N616) (Fig. 3C, Table 1). Thus, the effects of mutations at W607 and N616 within the same subunit (NR2B) are not additive.
We studied the effects of mutations on the voltage-dependence of block by TB-3-4 and memantine using voltage ramps analyzed with the Woodhull model of voltage-dependent block. With wild-type receptors and with many of the mutants that we studied, TB-3-4 and memantine produced a complete or near complete block at very hyperpolarized potentials. Examples are shown for TB-3-4 at wild-type receptors and at NR1(L655A)/NR2B receptors in Fig. 4, A and B. Data from these experiments were fit to the Woodhull model (Fig.4, A and B, bottom) to obtain values for theK d(0) and Zδ for each blocker. However, some mutations in the M2 loop region had dramatic effects on the current-voltage profile, with block becoming more pronounced as the cell was hyperpolarized, but with a reversal of the slope conductance and an apparent relief of block at very hyperpolarized potentials. An example is shown in Fig. 4C for TB-3-4 at NR2B(W607N). This type of profile was seen with some mutants at NR1(N616), NR2B(W607), and with combinations of those mutants. We did not attempt to fit data from those mutants to the Woodhull model. The relief of block at very negative membrane potentials presumably reflects permeation of TB-3-4 and memantine through the channels in these mutant receptors. The effects are reminiscent of the effects of some N616 and W607 mutants on block by N 1-dansyl-spermine and Mg2+, in which the mutants increased permeation of the blockers (Chao et al., 1997; Williams et al., 1998). This may have been caused by an increase in the size of the narrow constriction of the channel pore, allowing the normally impermeant or very poorly permeable blockers to permeate when the driving force is sufficiently high. For mutants at which we could determine values for theK d(0) and zδ of the blockers, the mutants increased the K d(0) but had little or no effect on zδ (Table 1).
The M1, M3, and M4 Domains.
The effects of mutations in these regions are listed in Table 2 and are summarized (together with the effects on mutations in the M2 region) in Fig. 5. Mutations that produced a 3-fold change in IC50 (shaded areas on Fig. 5) were considered to be significant. In the pre-M1 and M1 regions of NR1, an F558L mutation had a modest effect on block by TB-3-4 and MK-801 without affecting memantine (Table 2, Fig. 5), whereas mutations at W563 had larger effects on block by TB-3-4 and MK-801, again with no effect on block by memantine (Fig. 6B, top, and Table 2).
In the M3 and post-M3 regions, mutations at residues Y647, A652, A653, V656, E662, and D669 in NR1 had modest effects. More pronounced effects were seen with mutations at A645, N650, and L655 in NR1 (Fig. 6, Table2). In the M4 and post-M4 regions, mutations at T807 in NR1 had large effects on block by TB-3-4 and MK-801 but little or no effect on block by memantine (Fig. 6B, Table 2). This was similar to the effects of mutants at W563 in M1 and N650 in M3 (Fig. 6B).
We studied a number of different mutations (W-to-L, -A, -Y, and -F) at NR1(W563). The results are shown in Fig.7A and Table 2. The W-to-L or W-to-A mutations had large effects on block by TB-3-4 and MK-801, increasing the IC50 andK d(0) by 8- to 49-fold, whereas the W-to-Y and W-to-F mutations had no effect. None of the mutations altered block by memantine (Table 2). We also studied mutations at the equivalent position, W559, in the M1 region of the NR2B subunit. Similar to the NR1(W563L) mutation, an NR2B(W559L) mutation greatly reduced inhibition by TB-3-4 and MK-801 (increasing the values of IC50 and K d(0) by 59- to 330-fold) but had no effect on block by memantine (Fig. 5, Table 2). A W-to-A mutation at NR2B(W559) was nonfunctional when coexpressed with NR1. Mutations of W559 to Y or F had smaller effects than the W-to-L mutation on block by TB-3-4 and MK-801. In all cases, the magnitude of the change in K d(0) for TB-3-4 was similar to that for the change in IC50. Taken together, these data suggest residues NR1(W563) and NR2B(W559) influence binding of TB-3-4 and MK-801, and that the aromatic ring of the tryptophan (also found in Y and F but not in L or A residues) is an important determinant of block in wild-type channels.
At position NR1 (N650), an N-to-A mutation increased the IC50 and K d(0) values of TB-3-4 by 21- to 29-fold, whereas an N-to-D mutation had the opposite effect, reducing the values by 10-fold. An N-to-Q mutation at NR1(N650) had little effect (Table 2; Fig. 7B). Mutations at this position had smaller effects on block by MK-801 (2- to 7-fold) than on block by TB-3-4 and had no effect on memantine. If the amino groups of MK-801 and memantine (which have only one amino group each) and one of the three amino groups of TB-3-4 bind at the N-site residues in M2, it is conceivable that the side chain of N650 in M3 interacts with another amino group on TB-3-4. This could explain the decrease in potency seen with the N-to-A mutation and the increase in potency seen with the N-to-D mutation at NR1(N650). A mutation at the equivalent position in NR2B, NR2B(N649), also reduced block by TB-3-4.
A large decrease in affinity was seen with a T-to-C mutation but not with T-to-S or T-to-V mutations at NR1(T807) (Fig. 7C). The lack of effect of the S and V mutations suggests that neither the size nor the hydrophobicity of the side chain at this position is the key determinant. A T-to-A mutation at this position gave rise to receptors that generated only very small currents (1–5 nA).
Mutations That Generate Constitutively Open Channels.
In initial experiments in which we screened a large number of mutations in the NR1 subunit, we found that oocytes expressing NR1/NR2B receptors with a T-to-A or T-to-S mutation at T648 (located in M3) had very large holding currents when voltage-clamped at −70 mV. An example is shown in Fig. 8A. Application of glutamate and glycine to these mutants did induce inward currents, but the currents were often irreversible on wash-out of the agonists (data not shown), and we did not attempt to study the effects of TB-3-4, memantine, and MK-801 at the T648 mutants. We reasoned that the large holding currents required to voltage-clamp cells expressing NR1(T648) mutants may be due to the expression of constitutively open channels in receptors containing these mutants. If this were the case, replacing extracellular Na+ (the main charge carrier) with the large impermeant cationN-methyl-d-glucamine (NMDG) should reduce the currents. We also hypothesized that extracellular Mg2+, applied in the absence of agonists, would block these constitutively open channels, provided that the mutation does not disrupt the Mg2+ binding site. To test this, we studied the effects of substituting NMDG for Na+ in the extracellular solution, and we also studied the effects of extracellular Mg2+. Substitution of Na+ by NMDG or the addition of 100 μM Mg2+ greatly reduced the holding currents in cells expressing receptors containing NR1(T648) mutants but had little effect on wild-type receptors (Fig. 8, A and C). In light of this, we also examined the effects of NMDG and Mg2+ at many of the other key mutations that we had characterized and at some adjacent and nearby residues (Fig. 8C). Only mutations at Q556, P557, T648, and L657 in NR1 produced holding currents that were larger than wild-type and were sensitive to NMDG and Mg2+ (Fig. 8, B and C). An NR2B(N649D) mutation had a similar effect. In all cases the increased holding current, and consequent reduction by NMDG or Mg2+, were modest compared with the effects seen with NR1(T648) mutants.
Effects of Mutations on Permeability of Ba2+.
Mutations at the N residues in the M2 loop and at NR2B(W607) have previously been shown to affect permeability of divalent cations including Ca2+ and Ba2+(Williams et al., 1998; Dingledine et al., 1999). We carried out experiments to determine whether the key residues identified in this study affect permeability of Ba2+. Reversal potentials were measured in extracellular solutions that contained Na+ or Ba2+ as the main charge carrier. The shift in reversal potential between Na+ and Ba2+ is an index of the permeability of the channel to Ba2+. We used Ba2+ rather than Ca2+ in these studies to minimize Ca2+-activated Cl− conductances (Leonard and Kelso, 1990). At wild-type NR1/NR2B receptors, the reversal potentials were −1.6 ± 0.3 mV (Na+) and +21.1 ± 0.6 mV (Ba2+). As shown in Fig.9, mutations at NR1(N616) and NR2B(W607) in the M2 loop had characteristic effects on Ba2+permeability (Dingledine et al., 1999). For example, mutations of NR1(N616) to Q, G, or W markedly reduced Ba2+permeability and an N-to-R mutation had an even larger effect. Mutations of NR2B(W607) to L, N, or A, but not to Y or F, reduced Ba2+ permeability, highlighting the importance of an aromatic ring (as found in W, Y, and F) at this position. An NR2B(W559L) mutation had a small effect on Ba2+permeability but two other mutations at this position had no effect. All of the other key mutants that were studied had no effect on Ba2+ permeability (Fig. 9). This highlights the specificity of these mutations and indicates that these residues are determinants of block by TB-3-4 and MK-801 but not of Ba2+ permeability.
Discussion
In this study, we have identified residues in several regions of NMDA receptor subunits that differentially affect block by MK-801, memantine, and TB-3-4. A model that summarizes this work is shown in Fig. 10. The model is based, in part, on previous studies in which solvent-accessible residues were probed with MTS reagents after cysteine substitution (Kuner et al., 1996; Beck et al., 1999). The M2 segment of NR1 was proposed to contain an α-helix terminating at N616 followed by an extended structure or random coil (Kuner et al., 1996). The critical asparagines in the M2 loop of NR1 and NR2B form the binding sites for Mg2+ and contribute to the narrow constriction of the pore (Wollmuth et al., 1996; Wollmuth et al., 1998a,b).
The model is also related to the known structure of the KcsA potassium channel (Doyle et al., 1998). There is some amino acid homology between the M2 loops of glutamate receptor subunits and the pore helix of KcsA, although in the case of NMDA receptor subunits, the homology is very limited (Fig. 11). It is possible that the M1 through M3 region of NMDA receptors has a structure similar to KcsA; M1 corresponds to the outer helix and M3 to the inner helix of KcsA. Indeed, secondary structure prediction of these regions of NR1 and NR2B suggests the presence of α helices in M1, M2, and M3 (Fig.11). KcsA channel subunits have a pore-forming loop containing an α helix followed by a random coil; this structure may be similar to the M2 loop region of glutamate receptor subunits (Panchenko et al., 2001). Notably, the secondary structure predicted for NR1 has an M2 helix that ends just before N616, consistent with the model proposed by Kuner et al. (1996). A shorter helix is predicted in the M2 region of NR2B (Fig.11). In this context, it is notable that there is very little amino acid identity between the M2 regions of NR1 and NR2B. It is thus conceivable that the pore loops in NR1 and NR2B are somewhat different in terms of their structure, length, or positioning within the pore. Compared with KcsA, NMDA receptor subunits contain another putative helix, M4, so the overall packing and arrangement of the membrane spanning helices in glutamate receptor subunits may be different from those in KcsA.
Mutations at the critical asparagines in M2 (N616 in NR1, N615 and N616 in NR2B) had marked effects on block by MK-801 and memantine and modest effects on block by TB-3-4. It is likely that these residues interact with the amino group of MK-801 and memantine and, possibly, with one of the amino groups of TB-3-4. The effects of mutations at NR1(N616) and NR2B(N615) were additive, suggesting that they make separate contributions to a binding site for the blocker. Some N-site mutations not only reduced the affinity but also increased the apparent permeation of TB-3-4 and memantine, similar to effects on block byN 1-dansyl-spermine (Chao et al., 1997). Mutations such as NR1(N616G) presumably increase the size of the narrow constriction, allowing the cationic blockers to actually permeate the channel when the driving force is sufficiently large. The values of zδ for TB-3-4 and memantine at wild-type receptors were 1.38 and 0.89, respectively. Assuming that only one molecule of each blocker binds within the transmembrane electrical field, this would correspond to δ values of 0.46 for TB-3-4 and 0.89 for memantine. TB-3-4 is a long and highly flexible molecule. If it bound in the channel in an extended conformation, then the positive charges might be distributed over some distance and it is difficult to strictly interpret the δ values as a depth of field for this molecule. In the case of memantine, it is possible that two molecules of memantine bind simultaneously within the channel (Sobolevsky and Koshelev, 1998). This would yield an average δ value of 0.45 for memantine, consistent with the tip of the helix being located about halfway across the membrane electrical field (Fig. 10).
We have shown previously that mutations at NR2B(W607) alter block and permeation of extracellular Mg2+ (Williams et al., 1998). The effects of the NR2B(W607) mutants seen in the present study were reminiscent of their effects on block and permeation of Mg2+, with an aromatic residue at this position being important for block by the organic blockers. Mutations at the equivalent residue in NR1, W608, have little or no effect. We suggested previously that NR2B(W607) may contribute directly to the narrow constriction and Mg2+ binding site (Williams et al., 1998). In this case, it could also contribute to the binding site for organic channel blockers such as MK-801, TB-3-4, and memantine (Fig. 10). Another possibility is that NR2B(W607) forms part of a structural backbone that is important for control of the pore structure. There is a conserved tryptophan in an equivalent position in KcsA (W67), the side chain of which is not exposed to the lumen of the channel but forms part of a cuff of aromatic residues that constrain the opening of the channel pore (Doyle et al., 1998).
In addition to M2, the other regions in which we identified residues that influence channel block were the pre-M1, M1, M3, post-M3, and pre-M4 regions. In the model (Fig. 10), the channel is shown as a structure with a pore formed by the helix and random coil in M2 and an outer vestibule formed by the M3 and post-M3 segments. The pre-M1 and M4 regions may also contribute to the vestibule (Beck et al., 1999). The helices are proposed to span a membrane distance of 34 Å with the dimension of the narrow constriction being 5.5 Å (Villarroel et al., 1995; Wollmuth et al., 1996). Thus, in this model, it is possible to show the relative positions of residues along each helix, but the relative positioning of residues between helices is uncertain and the relative positions of residues that lie in random coil structures (e.g., in the pre-M1 and post-M3 regions) is unknown. In addition to residues that affect sensitivity to blockers, we identified several mutations that generate constitutively open NMDA channels. The largest effects were seen with mutations at T648 in M3, but mutations at Q556 and P557 in M1 and L657 in M3 also had effects (Fig. 10, dark blue circles). These mutants presumably affect gating of the channel. Residue T648 is near residue A653, which is equivalent to a residue in the δ2 channel (an “orphan” channel related by sequence homology to glutamate receptors) at which an A-to-T mutation generates constitutively open channels responsible for the Lurchermouse phenotype (Zuo et al., 1997). A-to-T mutations at NR1(A653) or NR2B(A652) do not generate constitutively open channels (K. Williams, unpublished observations), but the effects of the T648 mutants suggest that the M3 region in NMDA receptors is, like the equivalent region in δ2, important for channel gating.
Many, but not all, of the residues identified in the current study that influence channel blockers were previously shown to be exposed in the channel lumen (Kuner et al., 1996; Beck et al., 1999). The side chains of residues that are exposed to the lumen could interact directly with channel blockers, forming part of their binding sites. It is possible that residues N650, Y647, and possibly A653 in NR1, and Y646 and N649 in NR2B interact directly with MK-801 and TB-3-4 (Fig. 10). At least some of these residues are solvent exposed and positioned at a level such that MK-801 and TB-3-4 could interact with their side chains and simultaneously interact with the N residues in M2. In particular, NR1(N650) may bind to an amino group of TB-3-4 because an N-to-A mutation reduces the affinity for TB-3-4, whereas an N-to-D mutation increases affinity. The roles of residues at the top of M1 and M3 and in the pre-M1 and post-M3 regions are more difficult to interpret. Mutations at residues NR1(W563) and NR2B(W559) had pronounced effects on block by MK-801 and TB-3-4, but these residues were reported to not be solvent exposed (Beck et al., 1999). We found that an aromatic residue (W, Y, or F) at these positions was important for block by MK-801 and TB-3-4. The aromatic residue may be important for stabilizing the channel structure and/or stabilizing a binding site within the channel. Also, it seems unlikely that MK-801 (drawn to scale on Fig. 10) could interact directly with residues at the top of the M1 and M3 helices while simultaneously interacting with the M2 asparagines, although a direct interaction of TB-3-4 with these residues is conceivable.
Clearly, there are many residues that affect block by MK-801 but do not affect block by memantine (Fig. 10, green circles). Memantine and MK-801 have a similar overall size and both are influenced by the critical asparagines in M2. It is unlikely that differences in the effects of the M1, M3, and M4 mutants can be explained on the basis of the sizes of MK-801 versus memantine. However, MK-801 contains benzyl rings and is more likely to make hydrophobic interactions with aromatic residues in the pore. It is possible that some residues that influence MK-801 (but not memantine) do so by altering access of the blocker to its binding site or by allosteric disruptions of the binding site. It is also possible that some mutants that influence two or more antagonists do so indirectly or allosterically rather than by directly altering side chains (or the positioning of backbone peptide groups) within the binding site. However, the mutations at these positions do not simply have general nonspecific effects on the pore structure, evidenced by their lack of effect on block by memantine and Mg2+ and their lack of effect on Ba2+ permeability.
Acknowledgment
We are grateful to Dr. A. Shirahata for supplying TB-3-4.
Footnotes
- Received July 26, 2001.
- Accepted December 4, 2001.
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↵1 Present address: College of Pharmacy, Nihon University, Funabashi, Japan.
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Supported by Unites States Public Health Service grant NS35047 and the Hamaguchi Foundation for the Advancement of Biochemistry, Japan.
Abbreviations
- NMDA
- N-methyl-d-aspartate
- AMPA
- α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
- TB-3-4
- N1-N4-N8-tribenzyl-spermidine
- BAPTA
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- NMDG
- N-methyl-d-glucamine
- The American Society for Pharmacology and Experimental Therapeutics