A mutation that alters magnesium block of N-methyl-Daspartate

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1 Proc. Natl. Acad. Sci. USA Vol. 93, pp , August 1996 Neurobiology A mutation that alters magnesium block of N-methyl-Daspartate receptor channels (glutamate/cloned receptor/calcium block/structure/synapse) GEETA SHARMAtt AND CHARLES F. STEVENSt tneuroscience Department, University of California at San Diego, La Jolla, CA 92093; and tmolecular Neurobiology Laboratory and IHoward Hughes Medical Institute, Salk Institute, North Torrey Pines Road, La Jolla, CA Contributed by Charles F. Stevens, May 15, 1996 ABSTRACT N-Methyl-D-aspartate (NMDA) receptors are blocked at hyperpolarizing potentials by extracellular Mg ions. Here we present a detailed kinetic analysis of the Mg block in recombinant wild-type and mutant NMDA receptors. We find that the Mg binding site is the same in the wild-type and native hippocampal NMDA receptor channels. In the mutant channels, however, Mg ions bind with a 10-fold lower affinity. On the basis of these results, we propose that the energy well at the Mg binding site in the mutants is shallow and the binding is unstable because of an increase in the rate of dissociation. We postulate that the dipole formed by the amide group of asparagine 614 of the el subunit contributes to the structure of the binding site but predict that additional ligands will be involved in coordinating Mg ions. N-Methyl-D-aspartate (NMDA) receptors are cation selective ion channels that play a key role in excitatory synaptic transmission of the vertebrate central nervous system (1). These receptors are unique among ligand-gated ion channels in that their conductance is jointly controlled by their natural ligand, glutamate, and by the neuron's membrane potential. The voltage dependence of NMDA receptor activation stems from a block of the channel by physiological concentrations of extracellular Mg ions (2, 3). This block has been characterized quantitatively in native hippocampal NMDA receptor channels where Mg ions bind with fairly high affinity to a site about 80% into the electrostatic field through the pore (4-6). The NMDA receptor is normally a heteromer (7, 8) of two kinds of subunits that are classified into the ; and e families according to amino acid sequence homology (9, 10). Some heteromeric combinations of ; and s subunits expressed in oocytes have been shown to behave like in vivo channels (11-14). Mutation of a single asparagine residue in the region of the protein originally termed "TM2" markedly alters the Mg block (15-17): replacement of a specific asparagine (at the "N/Q/R site") with a glutamine strongly reduces Mg block and does not affect calcium permeability (18, 19). Although an essential channel function is dramatically modified by mutations at the N/Q/R site, the precise nature of the altered functions relative to that of the wild-type channel has not yet been elucidated. We have used the Jahr and Stevens (5) model to show that the heteromeric C1/e1 channels, transiently expressed in a mammalian cell line, behave quantitatively like the native hippocampal receptors and exhibit similar affinity and voltage dependence of Mg binding. In addition, we have extended this analysis to 1/s1(N614Q) mutants. Our data indicate that the mutation reduces the affinity of Mg for its binding site and decreases the voltage dependence slightly. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. MATERIALS AND METHODS Plasmid Construction and Mutagenesis. Mouse NMDA receptor clones C1 and el (7) were subcloned into mammalian expression vector pcdna1/amp (Invitrogen). Site-directed mutagenesis was performed using PCR overlap-extension method (20). The cassette between theaflli site at 1391 bp and HindIll site at 2687 bp was used to mutate the asparagine at position 614 of sl unit. Mutation was confirmed by sequencing the entire cassette. Cell Culture and Transfections. HEK-293 cells were grown on glass coverslips in DMEM and 10% fetal calf serum. Coverslips were coated with 50,ug/ml of poly-d-lysine in 130 mm borate buffer (ph 8.5). Transfections were performed for hr in 3% CO2 using the calcium phosphate precipitation method (21). Mouse subunits of the NMDA receptor were used in a 10:1 ratio (1/s1) for all experiments. A total of 20,g of DNA was applied per 10-cm tissue culture dish. All DNA used was cleaned by banding twice on cesium gradients. Since NMDA receptor expression proved to be highly toxic, 1 mm kynurinic acid and 10 mm MgCl2 were present in the medium continuously after removal of calcium phosphate precipitate. This antagonist cocktail completely prevented cell death. Electrophysiology. Transfected cells were studied hr after transfection. Cells were transferred to a chamber on an inverted microscope and were continuously superfused with Ringer's solution containing 145 mm NaCl/5 mm KCl/1 mm CaCl2/10 mm Hepes/25 mm glucose (ph 7.4) at room temperature. Whole-cell and single-channel recordings (22) were made in a low calcium external solution containing 145 mm NaCl, 5 mm KCl, 0.1 mm CaCl2, 10 mm Hepes, and 25 mm glucose. Pipette solution contained 80 mm CsF, 80 mm CsCl, 5 mm Hepes, and 5 mm EGTA (ph 7.2) in all cases. For solutions with different Mg concentrations, NaCl was substituted with MgCl2, and diffusion potentials were minimized by using an agar bridge. L-Glutamate (100,tM) and glycine (10 AM) were used as agonists in whole-cell experiments. Single channels were activated by the application of 1,uM glutamate and 10,uM glycine. Test solutions containing the agonist were pressure applied for 400 msec. Cells were extensively washed with normal Ringer's solution before the start of the experiment and between applications of test solutions. All chemicals used were of puratronic grade to avoid contamination with heavy metals. -Patch-clamp pipettes were fabricated from borosilicate glass (WPI, Amherst, MA) with tip diameter of 1.5-2,um. Recordings were made using Axopatch 1-B amplifier (Axon Instruments, Burlingame, CA). Data Collection and Analysis. Standard techniques were used to obtain whole-cell and outside-out recordings from single cells (22). Glutamate was applied at a rate not higher than 2 Hz and varied from cell to cell. No appreciable Abbreviation: NMDA, N-methyl-D-aspartate. Present address: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom. 9259

2 9260 Neurobiology: Sharma and Stevens desensitization from response to response was observed. Only cells that showed comparable amplitudes to control zero Mg solution before and after obtaining current-voltage relation were used in analysis to exclude any effects due to desensitization. Data were acquired on line using programs written in AXOBASIC (Axon Instruments). We sampled at 0.5 khz during whole-cell experiments and 10 khz during single channel experiments. Records were filtered at 2 khz. All analysis was done using programs written in AXOBASIC and MATHCAD. Unless otherwise indicated, all quantities are expressed as the mean ± SEM. ORIGIN (Microcal, Amherst, MA) was used to generate final figures. RESULTS AND DISCUSSION A drawing of the receptor (adapted from Hollmann et al. 23) in Fig. 1 illustrates the site of mutation. In all cases, wild-type refers to an asparagine (N) at position 614 in the epsilon subunit and mutant to a glutamine (Q) at this position. State Diagram. According to the analysis by Jahr and Stevens (5) the simplest possible description of the NMDA receptor channel consists of three states: C, the sum of all closed states; 0, the open state; and B, the Mg-blocked state. Transitions to the blocked state (O to B) occur at a rate that ref. depends exponentially on voltage and linearly on extracellular Mg concentration. The B-0 transition occurs at a rate that depends weakly on voltage but is independent of Mg concentration. Transitions occur from both 0 and B to the closed state C and the rates of these transitions are found experimentally not to depend on membrane potential. Following Jahr and Stevens (6), we have used a gating function, f(v), that specifies the fraction of the time the channel is free of Mg block and is described by the equation 1 f(v) = 1 + e(kv)(c/co) [la] where V is the membrane potential (mv) and C is the concentration (mm) of extracellular Mg ions. Jahr and Stevens estimated the values of the constants Co and k to be 3.57 mm and mv-1, respectively, from rate constants extracted from single channel data. Conductance-Voltage Relationships for Wild-Type Channels. As will be seen later, the kinetic properties of Mg block in mutants cannot be determined from single channel currents. For this reason, and because we wish to examine receptor properties over a wider range of Mg concentrations than is permitted by analysis of single channel currents (6), we initially NH2 Proc. Natl. Acad. Sci. USA 93 (1996) focus on macroscopic currents. Current-voltage relations were obtained from wild-type cells in the presence of extracellular Mg concentrations ranging from 10,tM to 10 mm. Typical data collected from a wild-type cell in the presence of two different Mg concentrations are illustrated in Fig. 2A. For wild-type channels, current i(v) at a given voltage V is related to the conductance g(v) in the presence of Mg by the equation i(v) = g(v)(v- Vr), [2a] where Vr is the reversal potential. Because g(v) is constant in the absence of extracellular Mg ions, the conductance function, g(v), normalized with respect to conductance at positive potentials, gives an estimate off(v): g(v) f(v) = [2b] gmax' where gmax is the value of g(v) in the absence of Mg block at positive values of V (either +40 mv or +60 mv). Such estimates off(v) were plotted against voltage for data from 30 cells in six Mg concentrations. A normalized conductancevoltage plot for the cell of Fig. 2A is exhibited in Fig. 2C. As the extracellular Mg concentration is increased, the normalized conductance curves shift to the right along the voltage axis as greater depolarization is required to overcome the Mg block. The smooth curve in Fig. 2C is a fit of the gating function, f(v), given by Eq. la, with values of the constants k and Co as described below. The three-state theory (smooth curve in Fig. 2C) from Eq. la provides an accurate description for the behavior of wild-type channels exposed to a wide range of external Mg concentrations. The average value of k in Eq. la was found to be mv-1, an estimate that agrees very well with the mv-1 calculated for NMDA receptors from hippocampal receptors (5, 6); Co was 3.57 mm for both. The good agreement between the values of k and Co in wild-type and native channels demonstrates that the wild-type recombinant receptors are quantitatively like the hippocampal receptors studied by Jahr and Stevens (5, 6) with respect to the position and affinity of the Mg binding site. Conductance-Voltage Relationships for Mutant Channels. Since the gating function in Eq. la provides a good fit to the data from wild-type cells, we have extended this analysis to mutant channels. Typical whole-cell current-voltage relations obtained in two different Mg concentrations from one mutant cell are presented in Fig. 2B. Unlike in wild-type channels, the current-voltage relations obtained from mutant cells were not out membrane in ligand binding sites GLUR2 NEFGIFNSLWFSLGAFMRQGCDIS ~1 DALTLSSAMWFSWGVLLNSGIGEG el1 GPSFTIGKAIWLLWGLVFNNSVPVQ WildType: ~1/E1 COOH Mutant: 51/E1(N614--Q) FIG. 1. Sequence comparison for the putative TM2 region of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluR 2 and NMDA receptor subunits C1 and el. The highlighted amino acids are at the site that is conserved throughout the glutamate receptor channels and is also the site of mutation in the el subunit. For simplicity, an old terminology is used to represent the transmembrane regions.

3 Neurobiology: Sharma and Stevens Proc. Natl. Acad. Sci. USA 93 (1996) 9261 A. Wild-Type B. Mutant a) ] / luojam Ca MWm mv mm Mg 300 IM Mg </-1000 pa D -8o ,oM Mg C. Conductance - Voltage Relation FIG. 2. Current-voltage and conductance plots for wild-type and mutant channels. (A) Typical whole-cell current (evoked by glutamate application) plotted against voltage for wild-type cells in the presence of 300,u M and 3 mm extracellular Mg. (Upper inset) The linear current-voltage relation in the absence of Mg. (Lower inset) Sample traces. (B) Current-voltage plots from a mutant cell in 10,uM and 3 mm Mg. (Upper inset) The nonlinear current-voltage relationship in the absence of external Mg ions. (Lower inset) Sample records. All experiments were performed in 0.1 mm external calcium. Sample traces from both wild-type and mutant cells are displayed for holding potentials ranging from -80 mv to +40 mv at 20 mv intervals and have a duration of 400 msec. (C) Conductance as a function of voltage for wild-type and mutant cells derived as described in the text and fitted with Eq. la. linear in the absence of Mg (Fig. 2B). The current i(v) in the presence of Mg can then be viewed as a combination of a conductance function in 0 Mg, Go(V), and the gating function, f(v), given by the equation i(v)- Go(V)f(V)( V- Vr), [2c] where V is the membrane potential and Vr is the reversal potential estimated from the current-voltage relation. Go(V) is found from current-voltage relations in the absence of Mg [for this case f(v) = 1] by means of defining the equation i*(v) = Go(V)(V- Vr*), [2d] where i* is the current and Vr* is the reversal potential in the absence of Mg. This Go(V) was then used to determine f(v) in the presence of Mg with the relation f(v) ( Vr)(1 V* r). [2e] ()(V- Vr) i*(v) )[e Data were collected from 20 mutant cells at four Mg concentrations. Fig. 2C presents f(v), determined for the cell of Fig. 2B, as a function of voltage. Eq. la, with values of constants k and Co selected for an optimal fit, was used to generate the smooth curve that presents the theoreticalf(v) plotted against voltage. Thus, the gating function of the form given in Eq. la provides a satisfactory fit to the data obtained from both mutant and wild-type cells, but with modified values of k and C0 for the mutant receptors. The gating function, as described for wild-type receptors by Eq. la, can also be written as f(v) = 1 + e-k(v-vo) [lb] Vo, the voltage at which the gating function is half-maximal, is given by the relation 1 /C\ Vo= kln() [ic] where Co is the dissociation constant for Mg at the blocking site. Eq. lb is well approximated by linear relationship near Vo and the deviation (V Vo) of voltage from the midvoltage Vo - is given by 1 k f(v) = + 4(V- Vo). [1d] The gating function then has two important features. (i) From Eq. ld, the slope of the gating function curves near their midvoltage is specified by k and is, therefore, a measure of the position of the binding site the field through the ion conduction pathway. (ii) The term Co measures the affinity (Kd) of Mg for its site and, therefore, permits the depth of the energy well at the Mg binding site to be calculated. We have used the physical significance of k and C0 to examine the changes in both the affinity and position of the Mg binding site of the mutant receptors. Voltage Dependence of the Mg Block. Results from individual cells were best fitted with slightly different values of k. The average k for the mutant gating function curves was calculated to be mv-1 for the mutants as compared with mv-1 for the wild-type channels, and the difference is statistically significant (P < , Kolmogorov- Smirnov test). In the wild-type channels, the Mg site is about 80% the way into the electrostatic field through the pore, as compared with about 67% for the mutant channels. This 17% shift in the value of k was found to be independent of Mg concentration. Affinity of Mg for Its Binding Site. To quantify the amount of block at each Mg concentration, we used the parameter VO defined in Eq. lc. Fig. 3 illustrates the relationship between VO and Mg concentration C for both wild-type and mutant receptors. The data points from wild-type receptors in low Mg concentration presented in Fig. 3 deviate systematically from the straight line predicted by a three-state model (Eq. lc, a deviation that is expected because Eq. 1 was derived as a limiting case for physiological concentrations of Mg; see Jahr and Stevens, ref. 5). At lower concentrations, it becomes necessary to use a fourth state (B' that can be reached only from the open 0 state), based on the presence of a Mg-independent but voltagedependent blocking mechanism observed in single-channel recordings (5), for a satisfactory description of the data. The smooth curve for wild-type receptors in Fig. 3 is the four-state equation with no free parameters. We used the numerical values of all the rate constants previously estimated (see Table 1 in ref. 6). We find that the affinity of Mg for its binding site in the recombinant 1/El channels is quantitatively the same as that of the native hippocampal receptors studied earlier.

4 9262 Neurobiology: Sharma and Stevens Proc. Natl. Acad. Sci. USA 93 (1996) A. Wild-Type Mutant B. Wild-Type 10 tlm Mg 50 PiM Mg C. Mutant 100MMa C. Mutant 10 AM Mg 1 mm Mg Magnesium Conc.(lM) FIG. 3. E1(N614Q) mutation alters the affinity of Mg for its binding site. Voltage at which whole-cell conductance is half maximal is plotted against Mg concentration. Diamonds represent data from wild-type cells (4-11 per point) and are fitted with a smooth curve obtained from the four state gating function (see text). Triangles are data from mutant channels (three to six cells per point). Data were fitted by Eq. Ic. The 1e (N614Q) channels, however, have a lower Mg affinity. This lower affinity of Mg for its binding site in the mutant channels appears as a shift of the curve to the right along the concentration axis in Fig. 3. The fit of data from mutant cells was obtained by changing the Mg dissociation rate 10-fold [exp(0.017v + 3.2) msec-1 rather than exp(0.017v 0.96) msec-1], but we could also have obtained the same fit by + adjusting association rates. Because mutant channels do not show any significant block at Mg concentrations lower than 100,uM, we could not study the gating function at low levels of extracellular Mg for these channels and, therefore, have used the three-state theory in Eq. la for the mutant channels. In summary, the wild-type channels behave quantitatively like native hippocampal channels that have been described before. Mutant channels are, however, modified; the affinity of Mg for its binding site is reduced 10-fold, and the voltage dependence is reduced by about 17%. The analysis presented so far uses equilibrium measurements, and only ratios of rate constants appear in the gating function Eq. la. In the following, we provide single-channel data indicating that the stability of Mg at its binding site (the dissociation rate), rather than the association rate, is altered by the mutation. Mg Block at the Single-Channel Level.Wild-type channels expressed in HEK-293 cells behave like native channels both in the presence and absence of Mg ions; the number of "flickers" (that is, interruptions due to Mg block) increases with Mg concentration, but the interruption time remains essentially constant (0.4 msec at -80 mv, 10 cells). Representative data without Mg present and at two different Mg concentrations are shown in Fig. 4 A and B. Mutant channels, on the other hand, behave differently. Five patches showed the main conductance level of mutant channels is similar to that of wild-type channels, but the mutant channels also exhibit an obvious subconductance level that is not evident in records of wild-type channels. In the presence of Mg, no interruptions are visible for mutant channels, but the single-channel conductance decreases with increasing concentrations of extracellular Mg. In the presence of 0.1 mm extracellular Mg, single-channel conductance was found to be ps, whereas in the presence of 1 mm external Mg, it was reduced to 24.5 ± 0.88 ps. Single-channel records presented in Fig. 4C illustrate this behavior. FIG. 4. l1/el(n614q) mutation leads to shorter interruption times and reduces single-channel conductance in the presence of Mg. (A) Single-channel records from wild-type and mutant outside-out patches in the absence of Mg. (B) Single-channel openings from two wild-type channels in the presence of 10 jum and 50 ALM Mg (both records from the same patch). The number of interruptions increases with Mg concentration but the conductance remains unaltered. (C) Singlechannel openings from mutant channels in 10/uM and 1 mm Mg (two different patches). The decrease in conductance and the absence of interruptions is apparent from the records. The observed decrease in single-channel conductance in the presence of high Mg concentration leads us to believe that rate at which Mg ions dissociate from their blocking site is considerably increased in the mutants. If the rate of association had decreased, we would have seen fewer interruptions without any change in single-channel conductance. If the rate of dissociation were the only difference between mutant and wild-type receptors, the mean block time would decrease 10-fold to about 40,usec (because the dissociation constant Co is increased 10-fold), an event much too brief for us to resolve, and the measured single-channel conductance would decrease in high concentrations of Mg, as observed in our experiments. Unfortunately, because individual interruptions cannot be resolved, direct estimation of association and dissociation rates is no longer possible for the mutant channels. Our results show that the behavior of Mg block in heteromeric ~1/E1 recombinant wild-type channels is quantitatively the same as that of native channels present in primary cultures of hippocampal neurons (6) and in pyramidal cells in hippocampal slices (24). Extension of this analysis to the 1l/s1(N614Q) mutant channels shows that the three-state model (Eq. la) is an adequate approximation for the Mg block in mutants as well but that the affinity of Mg for its binding site within the channel is decreased by about an order of magnitude. In addition to the change in affinity, the voltage dependence of Mg block is reduced by 17%. This change in voltage dependence could be explained in a number of ways. (i) The energy minimum of the binding site might move so that the fraction of the electrostatic field that a Mg ion moves across is now 0.67 instead of 0.8. If almost all the voltage drop occurs across 10 A along the pore (25) and the energy minimum of binding site moves by 17%, translation of the binding site would be 1.7 A toward the outer vestibule of the channel. (ii) Instead of moving the binding site, the mutation might distort the electrostatic field through the pore. (iii) As described in a model proposed by Zarei and Dani (25), a hydrated Mg ion occupying the blocking site could exhibit a large voltage dependence for the block from outside because of coupling to the movement of the permeant ion off of its binding site within the pore. It is conceivable that this mutation has an allosteric

5 Neurobiology: Sharma and Stevens effect on the permeant ion site and distorts it in a fashion that reduces the coupling between Mg and the permeant ion. This would also appear to reduce the voltage dependence of Mg binding. Premkumar and Auerbach (26) have recently published a study of NMDA receptor currents in wild-type and channels with a mutation similar to the one we have studied here. At the qualitative level, the results of the two studies coincide. Premkumar and Auerbach find that the mutant channels have two distinct conductance levels and that Mg, at physiological concentrations, decreases the single-channel conductance. The quantitative results of the Premkumar and Auerbach study differ in many essential ways, however, from what we have reported. Unfortunately, identifying the sources of these differences seems impossible. Premkumar and Auerbach studied channels bearing mutations in both subunit classes (instead of just one) and used expression in oocytes; furthermore, they did not compare their results with those on native channels as reported by Jahr and Stevens (5, 6). We, on the other hand, have studied channels expressed in mammalian cells and have used a quantitative formulation that permits us to compare behavior of our channels their native counterparts. In addition, because Premkumar and Auerbach limited themselves to the study of single-channel properties, they were restricted to a narrower range of Mg concentrations. Nevertheless, the qualitative agreement between the two laboratories strengthens our conclusions. Structure-Function Relation of NMDA Channel. Mg binding sites have been characterized in several metalloproteins and enzymes and are envisioned to be small pockets in the protein where each Mg ion usually forms six coordinate bonds with the surrounding ligands. Mg is bound preferentially by nitrogen and oxygen atoms and sites showing strong selectivity for this cation probably contain a nitrogen base and at least one phosphate or carboxylate group (27). Phosphoenzymes of known structure often exhibit a Mg binding motif termed the carboxylate cluster site. Each carboxylate cluster consists of three to four side-chain carboxylates grouped in a solvent exposed cleft. For example, crystal structure of bacterial protein CheY shows that bound Mg is held by three protein oxygen atoms provided by three surrounding aspartate residues and three oxygen atoms provided by the solvent (28). The side chain of glutamine is -2 A longer than that of asparagine. Therefore, the conservative substitution of an asparagine with a glutamine presumably changes only the position of the dipole formed by the amide group. Since the three-dimensional structure of the channel is unknown, we cannot predict the direction in which the extended dipole swings. There are two consequences of this mutation. First, it reduces the depth of the energy well at the binding site, thereby increasing the rate of dissociation of Mg ions. Using Eyring rate theory, we estimate a change in energy at the binding site to be about 1.4 kcal/mol (1 kcal = 4.18 kj) for a 10-fold change in the dissociation rate. Second, if the observed change in voltage dependence is real, the mutation shifts the energy minimum of the site with respect to the electrostatic field. Considering that the Mg ion requires six coordination sites, only one of which might be provided by the dipole formed by the amide group, it is conceivable that the other coordination sites for Mg lie higher up in the electrostatic field. The loss of the amide link would then result in the observed upward shift in the energy minimum reflected in k. It is thus plausible that the e1(n614q) mutation is at the Mg binding site and perturbs ion-ligand interaction by simply Proc. Natl. Acad. Sci. USA 93 (1996) 9263 changing the size of the binding cavity. Of course, a movement of the dipole is also expected to produce steric hindrance and subtly change the symmetry of the channel changing not only Mg binding but also some other properties of the channel. Thus, the dipole formed by the amide group of asparagine could well be at a position deep in the electrostatic field and contribute to the binding of Mg ions, but it is unlikely that the binding site will be formed exclusively by a ring of amides contributed by several subunits of the receptor. We propose that additional amino acids, probably oxyanionic in nature, are involved in binding Mg ions in the pore of the NMDA receptor. We thank Dr. M. Mishina for the gift of ;1 and el DNA and Gerlinde Pecht for help with sequencing. This work was supported by National Institutes of Health Grant NS (C.F.S.) and the Howard Hughes Medical Institute (C.F.S.). 1. Collingridge, G. L. & Lester, R. A. J. (1989) Pharmacol. Rev. 41, Mayer, L. 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