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Mutagenesis rescues spermine and Zn2+ potentiation of recombinant NMDA receptors
Neuron,
vol.
12, 811-818,
April,
1994, Copyright
0 1994 by Cell Press
Mutagenesis Rescues Spermine and Zn*+ Potentiation of Recombinant NMDA Receptors Xin Zheng, Ling Zhang, Cuylaine M. Durand, Michael V. 1. Bennett, and R. Suzanne Zukin Department of Neuroscience Albert Einstein College of Medicine Bronx, New York 10461
Summary Alternative splicing generates distinct forms of the NMDA receptor subunit NRl. NRl subunits with an N-terminal insert (termed Nl) form receptors in Xenopus oocytes with greatly reduced potentiation by spermine and Zn2+. Oocytes expressing NRl receptors with Nl exhibited larger NMDA currents than oocytes expressing corresponding receptors without N 1. In the present study, we used mutational analysis to investigate struttural featuresof the Nl insert that control current amplitudeand spermineandZn*+ potentiation. Neutralization of positive charges in Nl rescued spermine and Znz+ potentiation. Positive charges in N 1 did not affect spermine or Zn*+ affinity. Neutralization of positive charges in Nl diminished the responses to the level of NRl recep tors lacking N 1. The positively charged N 1 may increase NMDA currents by causing a conformational change similar to that produced by spermine and Zn2+ in NRl receptors lacking N 1. Introduction Molecularlydiverseformsof the N-methyl-o-aspartate (NMDA) receptor subunit NRI arise by alternative RNA splicing. Differential splicing of three exons can generate eight NRI splice variants, seven of which have been identified in rodent cDNA libraries (Anantharam et al., 1992; Durand et al., 1992; Nakanishi et al., 1992; Sugihara et al., 1992; Yamazaki et al., 1992; Hollmann et al., 1993). The alternatively spliced exons encode a 21 amino acid sequence in the N-terminal domain (termed Nl)and adjacent sequencesof 37and 38 amino acids in the C-terminal domain (Cl and C2). Splicing out of the exon segment encoding the C2 insert removes the first stop codon, resulting in a new open reading frame that encodes an unrelated sequence of 22 amino acids before a second stop codon is reached. We indicate specific NRI receptor splice variants with subscripts denoting the presence or absence of the alternatively spliced exons from 5’ to 3 (Durand et al., 1993). Thus, the NRl,m subunit has the Nl insert, but lacks Cl and C2; the NRlo17 subunit lacks Nl, but has both Cl and C2. NRI receptor subunits expressed in Xenopus laevis oocytes assemble to form functional homomeric channels. These channels share many of the electrophysiological and pharmacological characteristics of native NMDA receptors, including activation by coagonists glutamate (or NMDA) and glycine, competi-
tive block by D-3-amino, Iphosphonovalerate, voltage-dependent block by Mg2+, voltage-independent block byZnZ+ (>I0 PM), and Ca2+ permeability (Durand et al., 1992; Sugiharaet al., 1992). However, differential utilization of the N-terminal coding exon results in receptor variants that differ in certain physiological and pharmacological properties (Durand et al., 1993; Hollmann et al., 1993). NRl subunits lacking the 21 amino acid Nl insert form receptorsmthat exhibit relatively small currents, higher affinity for agonists, and marked potentiation byspermineand submicromolar Zn2+. NRl subunits containing the Nl sequence form receptors that exhibit relatively large currents, lower affinity for agonists, and less potentiation by spermine (at saturating glycine) or by Zn2+. Receptor binding and electrophysiological studies suggest the presence of distinct recognition sites on the NMDA receptor for polyamines. Spermine and spermidine increase binding of the open channel blocker MK-801 to NMDA receptors, suggesting that polyamines enhance NMDA receptor activity (Ransom and Stec, 1988; Williams et al., 1989; Reynolds and Miller, 1989). This effect is observed in the presence of maximally stimulating concentrations of glutamate and glycine, indicating action at an additional and distinct site on the NMDA receptor. Spermine potentiates NMDA-induced currents in Xenopus oocytes injected with rat brain mRNA (Brackely et al., 1990; McGurk et al., 1990) or with synthetic mRNAs encoding NMDA receptor subunits (Moriyoshi et al., 1991; Durand et al., 1992, 1993), as well as in cultured neurons from striatum (Sprosen and Woodruff, 19!IO), cortex(Rockand Macdonald 1992a, 1992b), hippocampus (Araneda et al., 1993; Benveniste and Mayer, 1993), and spinal cord (lerma, 1992). Potentiation occurs by an increase in maximum response amplitude in the presence of saturating concentrations of NMDA and glycine and, at lower concentrations of glycine, by increasing glycine affinity (McGurk et al., 1990; Benveniste and Mayer, 1993). These observations suggest that there may be two sites for spermine potentiation. Recent studies involving whole-cell (Rock and Macdonald, 1992a, 1992b; Araneda et al., 11993; Benveniste and Mayer, 1993) and single-channel recording (Rock and Macdonald 1992a, 1992b; Araneda et al., 1993) showthat spermine has inhibitoryaswell as potentiating actions on NMDA receptors. Spermine potentiates whole-cell currents and increases the frequency of NMDA channel opening in cortical and hippocampal neurons. At relatively negative membrane potentials, spermine decreases whole-cell currents and apparent singlechannel conductance in a voltagedependent manner, indicative of fast channel block. Both potentiation and inhibition by spermine are also observed for recombinant NMDA NRI receptors lacking the Nl insert (Durand et al., 1992,1993). However, NRI receptors with the Nl insert exhibit spermine
Neuron
812
inhibition and spermine potentiation at low glycine, but little potentiation at saturatingglycine.Thesefindings, together with studies showing the presence of high concentrations of polyamines in the brain (Shaw and Pateman, 1973; Seiler and Schmidt-Glenewinkel, 1975), suggest that polyamines may act at as many as three distinct sites to modulate NMDA receptors physiologically. Like spermine, Zn 2+ has potentiating and inhibitory actions at NMDA receptors. At high concentrations (>lO~M),Zn~+inhibitsNMDAresponsesin hippocampal neurons (Westbrook and Mayer, 1987; Christine and Choi, 1990) and oocytes expressing NRI splice variants (Durand et al., 1992; Hollmann et al., 1993). This block is voltage independent, suggesting that Zn*+ and spermine inhibit at different sites. At submicromolar concentrations, Zn*+ potentiates NMDAinduced responses of recombinant NRI receptors lacking the Nl insert. NRI receptors containing Nl exhibit inhibition at high Zn2+ concentrations, but little or no potentiation at low Zn2+ concentrations (Hollmann et al., 1993). In the present study, we used mutational analysis to investigate the structural features of the Nl insert that alter current amplitudes and control spermine and Zn2’ potentiation of NMDA responses. Because spermine, Zn2+, and the Nl insert are positively charged, we replaced positively charged residues in Nl. Introduction of alanine residues for arginines and lysines in Nl rescued spermine and Zn*+ potentiation and reduced current amplitude. The mutations did not appear to affect spermine or Zn2+ affinity. These findings suggest that the Nl insert itself may potentiate NMDA channel opening and that,‘in NRI receptors lacking the Nl insert, a similar potentiation is produced by spermine and Zn2+. Furthermore, potentiation by the Nl insert requires its positive charges. Results Identification of Amino Acid Residues in the Nl Insert That Reduce Spermine Potentiation at Saturating Glycine Electrophysiological studies indicate that the presence of the Nl insert greatly reduces spermine potentiation at saturating glycine (Durand et al., 1992,1993). To test the importance of the positively charged residues on spermine potentiation, mutant receptor NRlloo subunits were constructed with reduced numbers of positive charges in the N-terminal insert. In mutant 1 (K192A, K193A, R194A), alanine residues were introduced in place of lysine and arginine residues at the N-terminal end of the insert. In mutant 2 (R207A, R208A, K211A), alanines replaced the three positively charged residuesat the C-terminal end of the insert. In mutant 3, alanines replaced all six positively charged residues in the insert. The N-terminal inserts of wildtype and mutant receptors are shown in Figure 1.
1~
N-terminal
Wildtype: Mutant 1:
SKKRNYENLDQLSYDNRRGPK-
Mutant 2:
SKKRNYENLDQLSYDNAAGPA-
Mutant3:
SAAANYENLDQLSYDNAAGPA-
insert
1
SAAANYENLDQLSYDNRRGPK-
Figure 1. The Amino Acid Sequences Type and Mutant NRllm Receptors
of the Nl
The alternatively spliced N-terminal insert charged amino acid residues (bold faced). We tance of these residues on spermine and ZrP the following mutant NRllm receptors: mutant R194A), mutant 2 (R207A, R208A, K211A), and nine in all six positions.
Inserts
of Wild-
has six positively tested the imporpotentiation with 1 (K192A, Kl93A, mutant 3, with ala-
Mutant and wild-type NRI channels differed in their degree of spermine potentiation at saturating glycine when expressed in Xenopus oocytes. At -60 mV, spermine (250 PM) potentiated NMDA responses of wild-type NRlolr receptors to 176% f 11% of the controlvalue(mean f SEM; n = 9; Figure2A;Figure3A).At the same potential, spermineslightly inhibited NMDA responses of wild-type NRl,m receptors (to 95% f 4% of the control response in the absence of spermine; n = 10). Spermine poientiation was partially or fully rescued in the mutant receptors. At -60 mV, spermine (250 PM) produced an intermediate potentiation of mutant 1 and mutant 2 receptors (to 123% f 3% [n = II] and 147% f 5% [n = 111 of control, respectively). Spermine potentiated mutant 3 receptors to about the same degree that it potentiated NRI~I, receptors (to 174% f 5% of control; n = 7). At -60 mV, the degree of potentiation varied as follows: NRla, = mutant 3 > mutant 2 > mutant 1 > NRltoo receptors (Figure 3A). Pairwise comparison of thedegreeof spermine potentiation indicated that mutant 3 did not differ significantly from NRlor,; all other pairwise comparisons reached significance (p < 0.05; ANOVA analysis followed by Bonferroni’s post-hoc test). These findings suggest that the reduction in spermine potentiation exhibited by NRI receptors with the Nl insert is due entirely to the positive charges in Nl and not to the 21 amino acid “spacer.” The two wild-type receptors did not differ significantly in their affinity for glycine (Durand et al., 1992), and increase in glycine to 20 PM had no further effect on spermine potentiation of mutant 3. In these experiments, NRloII rather than NRlooo receptors were used as a measure of wild-type potentiation, because NRIOII receptors are the predominant receptor variant expressed in forebrain and the best characterized of the NRI receptors. In addition, NRlolI receptors were expressed more consistently than were NRlm receptors. In the present study, spermine potentiated NRlooa responses to the same degree that it potentiated NRloll responses (n = 2; the two receptors were also shown to be potentiated to the same degree by Durand et al. [1993]).
Rescue of ZnW and Spermine 013
Actions
Mutant 1
on NRl Receptors
Mutant 2
Mutant 3
NRlo,,
Figure 2. Neutralization in the Nl Insert Rescues Potentiation
of Positive Charges Spermineand ZnZt
Xenopus oocytes were injected with NRI,~, mutant I, mutant 2, mutant 3, and NRI~,, RNA (10-50 ng per cell). Two to thirteen days after RNA injection, whole-cell recording from oocytes was carried out under two microelectrode voltage clamps at -60 mV. Currents wereelicited by bath ap plication of NMDA (N) Wl PM with 10 PM glycine) in the absence and presence of spermine 6) (250 pM) or Zn*+ (Zn) (1 rM). Potentiation was observed as the increase of NMDA-induced current in the presence of spermine or Zn*+ compared with that in its absence. (A) Potentiation by spermine was partially rescued in mutants 1 and 2 and completely rescued in mutant 3. (B) Potentiation by Znz+ was little changed in mutant 1 but fully rescued in mutants 2 and 3.
The inhibitory action of spermine on native and recombinant NMDA receptor channels is greatly reduced at more depolarized potentials (Durand et al., 1992,1993;Araneda et al., 1993, Benveniste and Mayer, 1993). To evaluate the voltage dependence of spermine actions on mutant and wild-type NRl,m receptors, oocytes were clamped at holding potentials of -40, -60, -80, and -100 mV (Figure 38). At -40 mV, spermine markedly potentiated NMDA responses of NRIOII and mutant 3 receptors, but only slightly potentiated NMDA responses of NRIIm receptors. Responses of mutant 1 and mutant 2 receptors were potentiated to an intermediate degree. When the cells were hyperpolarized, the (net) potentiation by spermine was reduced. At -100 mV, spermine inhibited responses of mutant 1 and NRllm channels. At -60 and -80 mV, spermine inhibited NMDA responses of NRI~oo channels. These observations indicate that the effect of introducing neutral amino acid residues in place of lysines and arginines in the Nl insert is essentially independent of voltage. Positively charged residues within the Nl insert could affect either spermine binding or a subsequent step in the signal transduction mechanism. Spermine concentration-response curves were determined for wild-type and mutant receptors. To minimize spermine inhibition, recordings were made at -40 mV (Figure 4). For four of the receptors, the spermine response curve was biphasic; reduction of potentiation at higher concentrations was presumably due to the onset of inhibition. Although the complex nature of the concentration-response curves makes quantitative analysis difficult, inspection of Figure 4 shows that neutralization of charges in Nl had little effect on spermine potency. The concentration-response curves for mutant 3 and NRloll receptors were nearly
superimposable; half-maximal potentiation by spermi ne was at - 500 FM. Mutant 1 and mutant 2 receptors exhibited potencies for spermine similar to that of NRlol, receptors, but a reduced degtee of potentiation. These findings indicate that the Nl sequence is not inserted in the spermine-binding Site and suggest that it does not affect spermine binding indirectly through a conformational change in the receptor protein. Identification of Nl Residues That Reduce Zn*+ Potentiation At a low concentration (-1 PM), Zn** markedly potentiated NMDA-induced currents in oocytes expressing NRloll and NRlooo, but only modeitly potentiated NRIIa, receptors. At a higher concentlation (100 PM), ZnZ+ inhibited all NRI receptors (Hollmann et al., 1993; see also Durand et al., 1992). Both the inhibitory and potentiating effects were voltage independent. To test the relevance of the charged residues in the Nl insert toZn*+ potentiation, weevaluated this process in wildtypeand mutant NRI receptors. At 1 FC.MZn2+, mutants 2 and 3 showed virtually complete resceeof the potentiation compared with NRIo,, and NRlm receptors; mutant 1 showed no rescue (Figure 28; Figure 5). At 1 PM Zn*+, the degree of potentiation was as follows: NRlloo = mutant 1 < mutant 2 = mutant 3 = NRIo,, = NRlm receptors. The degree of Zn” potentiation observed for NRl,a, and mutant 1 receptors differed significantly from that of mutant 2, mutant 3, NRIoII, and NRlooo receptors (p < 0.05). Differerlces within the two groups were not significant. To test the effect of positively charged residues on Zn*+ affinity, concentration-response curves were constructed for wild-type and mutant NRI receptors. Thetwowild-type and two mutant receptors exhibited
NeUrOn 814
A.
i
zoo
8-j NRloil 8 MS v Y2
u
l
Ml
n
NRlioo
.d
B.
i
rE
60 10-l
Figure4. Neutralization of Does Not Change Spermine
Figure 3. Voltage Type and Mutant
Dependence NRI Receptors
of
Spermine
Action
on
Wild-
Potentiation was measured as the ratio of NMDA-induced current in thepresenceof sperminetothat in itsabsence. Responses were elicited by application of NMDA (300 f1h4 with 10 uM glytine) in the absence and presence of spermine (250 PM) at each holding potential. (A) Spermine potentiation recorded at -60 mV was partially rescued in mutants 1 and 2 and was completely rescued in mutant 3. Standard error bars and numbers of oocytes are indicated. Pairwise comparison of the degree of spermine potentiation indicated that mutant 3 did not differ significantly from NRlon; all other pairwise comparisons reached significance (p < 0.05; ANOVA analysis followed by Bonferroni’s post-hoc test). (B) Spermine potentiation at a range of holding potentials f-40, -60, -80, and -100 mV). Net potentiation by spermine was reduced at more hyperpolarized potentials, presumably because of enhanced inhibition by spermine. In oocytes expressing NRllm or mutant 1 receptors, spermine inhibited NMDA responses at -100 mV. The degree of rescue, measured as the ratio of spermine potentiation of mutant receptor responses to that of NRlo,, receptor responses, varied little with holding potential over the range of potentials examined.
little difference in Zn2+ potencies (Figure 6). Interestingly, at lower concentrations, Zn*+ potentiation of mutant 3 was greater than that of NRloI,. These observations suggest that positively charged residues in Nl do not affect the initial binding of Znz+ to the NMDA receptor. Inspection of the dose-response curves at lower concentrations suggests that the degree of potentiation differs as follows: mutant 3 > NRI~v > NRIIw > mutant 1 receptors. Thus, the two “clusters” of positively charged residues within Nl appeared to affect
Positive Potency
Charges
in the
Nl
Insert
Concentration-response curves were constructed for spermine potentiation of NMDA responses for NRllm (wild-type), mutant 1, mutant 2, mutant 3, and NRlmr receptors. Responses were elicited by application of NMDA (300 PM with 10 hM glycine) in the absenceand presenceof spermine at indicated concentrations at a holding potential of -40 mV. The spermine concentration response curve for mutant 3 was superimposable on that for NRlm, receptors. Potentiation of mutant 2 and mutant 1 re sponses was reduced without a decrease in apparent spermine potency, relative to NRlnl responses. Data points are the steadystate current amplitudes expressed as a percentage of the mean of control responses recorded before and after each test response and represent means + SEM of responses of five to seven oocytes.
Zn2+ potentiation differently. The cluster of positively charged residues at the N-terminal end of Nl had little effect on Zn2+ potentiation; the cluster at the C-terminal end of Nl appeared to account for the entire difference between NRl,w and NRlo,, receptors. The presence of the 21 amino acid spacer in mutant 3 may actually have increased Zn2+ potentiation.
Additivity of Spermine and Zn2+ Effects To evaluate whether spermine and Zn2+ act at the same or different sites, NMDA currents were measured in the presence of the two ligands separately and together (Figure 7). The potentiating effects of spermine (250 PM) and Zn2+ (1 PM) summated in a nearly linear fashion. Thus, the ratio between Pzn + Pspermineand Pzn + Spermine was 1.01 * 0.04 (n = 5), in which Pzn is the potentiation by Zn2+ (response in the presence of Zn2+ minus control response), PSpermineis the potentiation by spermine, and Pzn + spermineis the potentiation by Zn2+ and spermine applied together. The finding of additivity of spermine and Zn2+ potentiation suggests that binding occurs at two independent sites.
Rescue
of ZrP
and Spermine
Actions
on NRl
Receptors
815
-,
.I----NRlioo Figure 5. Neutralization Potentiation
Ml
YR
of Positive
Y3 Charges
NlZlo~l
NRlooo
in Nl Rescues
[Zinc]
Zn2+
Potentiation of responses to NMDA (300 )IM with 10 PM glycine) by Zn2+ (1 bM) was measured at -60 mV, as for Figure 2. NRlm, and NRlmo receptors exhibited greater potentiation than did NRlrm receptors. Neutralization of positive charges in mutant 1 did not increase Znz+ potentiation. Mutant 2 and mutant 3 exhibited Zn’+ potentiation comparable with that of NRloll and NRlmo recep tors. Steady-state current amplitudes recorded at -60 mV were normalized to the mean of control responses recorded before and after each test response. At 1 PM, the degreeof Znn potentiation observed for NRllm and mutant 1 receptors differed significantly from that of mutant 2, mutant 3, NRloII, and NRlmo receptors (p < 0.05). Differences within the two groups were not significant. Data are the means + SEM of responses from three tosixoocytes(numberofexperimentsindicatedaboveeach bar).
Response Amplitudes of Wild-Type and Mutant NRl Receptors The effect of neutralization of positive charges in the Nl insert on the amplitude of NMDA-induced responses was also examined (Figure 8). To reduce variability between preparations, responses were compared within a given batch of oocytes, each injected with the same amount of RNA (20 ng per oocyte) and recorded under the same conditions. Oocytes expressing NRllao receptors exhibited responses to NMDA (300 PM with 10 PM glycine) that were about 3-fold greater than those for oocytes expressing NRlolr, in confirmation of Holimann et al. (1993). (Their paper also showed that oocytes expressing NRIw and NRlmo receptors gave responses of similar amplitude.) Changeof IysineandarginineresiduesintheNl insert to alanines reduced current amplitude to that of NRl,-,,, receptors (Figure 8). Current amplitude for NRlloo receptors was significantly greater than those for NRloll and for all three mutant receptors (p < 0.001). Current amplitudes of NRlo,,, mutant 2, and mutant 3 receptors did not differ significantly. It is as yet unknown whether the larger currents observed for receptors with the Nl insert is a result of greater activity at the single-channel level or of more efficient translation, assembly, or insertion of channels. Never-
Figure 6. Neutralization Change Znz+ Potency
of
Positive
10’
10s
108
(n&t) Charges
in Nl
Does
Not
Concentration-response curves were constructed for Znz+ potentiation of NMDA response of NRllm (wild-type), mutant 1, mutant 3, and NRlml receptors at -60 mV. Responses were elicited by application of NMDA (3UO pM with 10 (rM glycine) in the presence of ZnZf at indicated concentrations. Data points are the steady-state current amplitudes expressed as a percentage of the mean of control responses recorded before and after each test response and represent means f SEM of responses from five to seven oocytes. ZnZf potency did notl appear to change. The degree of potentiation varied as follows: mutant 3 > NRloll > NRllm > mutant 1.
theless, these observations suggest the interesting possibility that the Nl insert itself propagates a conformational change to the ion channel to increase NMDA responses. Neutralization of positive charges in Nl byexchangeofaminoacidspreventsthisconformational change, thereby reducing response amplitudes to the level observed for NRI receptors lacking the Nl insert. In NRI receptors lacking Nl, added spermine or Zn*+ may potentiate agonist-evoked re-
Figure
7. Spermine
and Zn2+ Potentiation
In oocytes expressing NRlmI receptors, the control response to NMDA (N) (300 flM with IO PM glycine) was potentiated by spermine (S) (250 vM) and Zn2+ (Zn) (I @vt) at -60 mV. When the two ligands were applied together, the increase in response was thesumoftheincreaseselicited byspermineand byZ#applied separately.
Neuron 816
Nl is unlikely to be due to an electrostatic interaction with spermine or Zn2+ at their binding sites. This finding also indicates that the Nl sequence is unlikely to be inserted in the spermineor Zn2+-binding site. Reducing the net positive charge on Nl by changing lysines and arginines to uncharged alanines also diminishes the response amplitude. Neutralization of positive charges in all three mutants reduces the response amplitude to that observed for NRI receptors lacking the Nl insert. In each case, spermine or Zn2+ potentiates the response toward the level observed for Nl-containing receptors. These findings indicate that the Nl insert with its six positively charged residues may induce a conformational change in the re-
1.6 2
1.6
2 E
1.4
2
1.0
y e Gi I i
0.6
;
0.6
1.e
0.6 0.4
ceptor 0.0 NBloll
MS
Y2
Ml
NRlloo
Figure 8. Neutralization of Positive Charges in the Nl Insert Decreases Response Amplitude Responses of NRl,m receptors were about Sfold larger than those of NRlolr receptors. For all three mutants of the NRltm receptor, responses were reduced to a level comparable with that of NRlmt receptors. Responses of NRl,m receptors were significantly greater than those of NRlmr, mutant 1, mutant 2, and mutant 3 receptors (p < 0.001 for each pairing); responses of NRlm,, mutant 1, mutant 2, and mutant 3 receptors did not differ significantly. The data shown represent the means f SEM of responses to 300 PM NMDA with 10 RM glycine (number of oocytes indicated above the bars). All oocytes used for NRlar, mutant 2, and mutant 3 receptors and six’of nine oocytes used for NRllm receptors were from a single batch from one female, injected with the same amount of RNA (20 ng) and compared under constant conditions. In a separate batch of oocytes (+), the current amplitude for mutant 1 was compared with that for NRlra, (391 f 42 nA; n = 3 oocytes). The data plotted for mutant 1 were resealed by the ratio of NRl,m responses in the two batches of oocytes.
sponses by inducing a conformational change to that induced by the wild-type Nl insert.
similar
Our results indicate that potentjatjon of NRI receptors by spermine and by ZnZ+ is critically affected by the six positively charged amino acid residues of the Nl insert. The presence of the Nl insert in NRI subunits greatly reduces potentiation by Znz+ and by spermine at saturating glycine (Durand et al., 1992, 1993; Hollmann et al., 1993). Neutralization of positive charges at the N-terminal end of the insert (mutant 1) partially rescues spermine but not Znz+ potentiation. Neutralization of the three positive charges at the C-terminal end (mutant 2) partially rescues spermine potentiation and fully rescues Zn2+ potentiation. Replacing all six positively charged residues with alanines (mutant 3) fully rescues spermine and Zn2+ potentiation. Potentiation by spermine and ZnZ+ is essentially additive, indicating possible action at separate sites. Neutralization of positive charges in Nl does not appear to affect spermine or Zn2+ potency. Thus, reduced potentiation in receptors containing
that
is transmitted
to the
ion
channel,
thereby
increasing agonist-evoked currents. Moreover, this effect may be similar to that induced by binding of spermine or Zn~ in receptors lacking Nl. The amplitude of maximally potentiated NRloll responses observed upon application of spermine and Zn2+ together approached that of unpotentiated NRIIW responses, consistentwith the suggestion thatthe insert and potentiators may act through a common mechanism. Apparently, spermine and Zn2+ still bind to Nlcontaining receptors, but with little further potentiation of current amplitude. An important issue is whether polyamines and Zn2+ are endogenous regulators of NMDA receptors. In rat brain, polyamine concentration is in the micromolar range (Shaw and Pateman, 1973). Highest concentrations are in the hippocampus, cerebellum, and corpus callosum. In the hippocampus, spermine and other polyamines are released from and taken up into nerve terminals (Seiler and Dezeure, 1990). Polyamine biosynthesis probably occurs in nerve endings, as suggested by active uptake of ornithine into synaptosomes and relatively high activities of ornithine decarboxylase and S-adenosyl-methionine decarboxylase (Seiler and Dezeure, 1990). Zn2+ is also present in the brain (Donaldson et al., 1973; Dreosti, 1984). In the hippocampus, Zn2+ is stored in presynaptic vesicles and is co-released with glutamate in an activity-dependent manner (Assaf and Chung, 1984; Howell et al., 1984; Charton et al., 1985; Aniksztejn et al., 1987). These findings support the concept that spermine and ZnZ+ act as endogenous physiological modulators of glutamate neurotransmission. Another important issue is whether recombinant homomericchannelssharefunctional propertieswith native NMDA receptors in neurons. A second family of NMDA receptor subunits, NRZA-D, do not themselves form functional channels, but appear to coassemble with NRI subunits to form channels with greatly enhanced activity flkeda et al., 1992; Kutsuwada et al., 1992; Meguro et al., 1992; Monyer et al., 1992; lshii et al., 1993). The finding of genetically diverse NMDA receptor RNAs, many of which are expressed throughout the brain, and the observation that current amplitudes obtained with homomeric NRI channels are relatively low suggest that at least
Rescue of Zn” 817
and Spermine
Actions
on NRI
Receptors
some NMDA receptors are heteromeric channels in vivo. Nevertheless, as noted above, homomeric recep tors share many of the electrophysiological and pharmacological properties of native receptors. Moreover, in preliminary experiments, we observed that NRlorI/ NWB heteromeric receptors exhibit spermine potentiation at saturating glycine. Our whole-cell recording studies indicate that NMDA-induced currents in most hippocampal neurons exhibit spermine potentiation at saturating glytine (Araneda et al., 1993). Our results suggest that at least someof the receptors mediatingthese responses lack the N-terminal insert Nl. In excised outside-out patches of hippocampal neurons, spermine produced potentiation of NMDA responses in only 4 of 15 patches (Araneda et al., 1993). A possible explanation of tfre infrequency of patches showing potentiation by spermine is the relative predominance of Nlcontaining NRI receptors on the cell somata from which the patcheswereobtained. Other studies using whole-cell recording report variable potentiation by spermine (Rock and Macdonald, 1992a; Benveniste and Mayer, 1993). In situ hybridization studies show that NMDA splice variants with the Nl exon predominate in the subthalamic nucleus and in the CA3 of the hippocampus (Standaert et al., 1993). In conclusion, the present study demonstrates that the six positively charged amino acid residues in the Nl insert of NRI receptor splice variants critically affect spermine and ZrP potentiation. Neutralization of the charges by exchange to neutral residues fully rescues potentiation by both ligands. The neutralization of charged residues in Nl also increases response amplitudes. These observations suggest that the Nl insert induces a conformational change in the receptor which increases agonist-evoked currents and occludes the potentiating effects of spermine and Zn*+. The positive charges in Nl are required for these actions; increasing the length of the N-terminal domain by the twenty-one amino acid sequence (without its positive charges) has little effect. The unmodified insert does not appear to affect spermine or ZnZ+ binding. Single-channel measurements will be required to establish the mechanisms of potentiation by Nl and byZn 2+. S p ermine potentiation in hippocampal neurons is due to increased channel open probability; at more negative potentials, spermine also decreases apparent single-channel conductance (Araneda et al., 1993). Whatever the mechanism by which the Nl insert affects response amplitude, alternative splicing of the insert may act as a switch controlling spermine and Zn2+ potentiation of NMDA receptors. For receptors lacking Nl, spermine and ZrP are likely to act as important endogenous modulators of NMDA receptor activity.
We thank Alice Wang for technical assistance. We thank Drs. JiaBei Wang and George R. Uhl for helplul advice on sitedirected mutagenesis. We thank Dr. John A. Kessler for his help ful comments on the manuscript. We are gratqful to Dr. S. Nakanishi for providing the NRlmr cDNA. This work was supported by National Institutes of Health grants NS 20752 to R. S. Z., and NS 07412 and HD 04248 to M. V. L. B.; M. V. L. $. is the Sylvia and Robert 5. Olnick Professor of Neuroscience. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordanm with 18 USC Section 1734 solely to indicate this fact.
Experimental
Received
Procedures
Site-Directed Mutagenesis NRlrm was subcloned into tagene) and used to transform
Single-stranded DNA template was rescued with M13K07 helper phage, and sitedirected mutations were made with oligonucleotidedirected in vitro mutagenesis system version 2 (Amersham). Each oligonucleotide was designed to engineer three codon substitutions into cloned NMDA receptor DNAs to generate subunits carrying three amino acid replacemehts in the N-terminal insert. The following oligonucleotides were used for introduction of changes into coding sequences1 EGTCCACCFfTCATAG~CCCGGCCGCACGA~C~CG-3 for mutant 1 (K192A, K193A, R194A); 5-CAGCACCTrCtCTGCCGCGGGTCCGGCCGCGTrGTCATAGGACAGTTG-3 for mutant 2 (R207A, R208A, K2llA). Mutant 3, in which all six positively charged residues were exchanged to alanines, was generated from mutant 1 with the oligonucleotide for mutant 2. Codon substitutions were confirmed by DNA sequencing across the altered region with Sequenase version 2.1 (USB). cRNA Synthesis NRlo,, cDNA was a gift of Dr. S. Nakanishi (Kyoto, Japan); NRl,m and NRlm cDNAs were cloned in this laboratory (Durand et al., 1992). Mutant cDNAs were constructed as described above. To generate templates for transcription, circular plasmid cDNAs were linearized with Not1 (NRl&, BamHl (&Id-type and mutant NRl,,), or Clal (NRl,). Transcription reactions were performed with T7 or T3 polymerase (Ambion T7 or T3 MEGAscript RNA polymerase transcription kit; Ambion) (4 hr at 37°C). Electrophysiolo&al Experiments in Xenopus Qocytes Adult female Xenopus (Xenopus I, Ann Arbor, MI) were anesthetized by immersion in 0.15% aminobenzoic acid ethyl ester, and individual oocytes were isolated as descritied (Kushner et al., 1988,1989). Briefly, oocytes were incubated for 1 hr in Ca2+-free ND-% medium (96 mM NaCI, 2 mM KCI, 1 mM MgCIz, 5 mM HEPES [pH 7.51) containing collagenase type D (2 mg/ml; Boehringer Mannheim), penicillin (10 U/ml), and streptomycin (0.01 mg/ml), after which the follicular layer was t’emoved by manual dissection. Selected stage V and VI oocytes were injected with in vitro transcribed RNA (IO-M ng per cell)‘and maintained at 18OC in ND-% containing Ca2+ (2 mM). Two to thirteen days after injection, oocytes were recorded in MgP+-free frog Ringer’s solution (116 mM NaCI, 2.0 mM KCI, 1.0 mM CarI&, 10 mM HEPES [pH 7.21) under two microelectrode voltage olamp with a Dagan amplifier (Dagan, Inc.). Voltage and currefit electrodes were filled with 1 M KCI, 10 mM HEPES, and 5 mM EDTA and had a resistance of -1 MP. NMDA (300 pM), with or without other drugs, was bath applied to oocytes to elicit dgonist-evoked currents. Glycine (IO PM) was present in all solutions. To cpmpare responses, steady-state currents were normalized to the mean of control responses to NMDA (300 PM) ahd glycine (10 PM) recorded before and after each test response. One-way analysis of variance (ANOVA) was carried out using the lnstat program (GraphPad software, version 1.14, 1990). Acknowledgments
October
27, 1993; revised
January
21,1994.
References the plasmid Bluescript SK- (Strathe Escherichia coli strain DHSaF’.
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Benveniste, M., and Mayer, M. L. (1993). Multiple effects of spermine on N-methyl-o-aspartic acid receptor responses of rat cultured hippocampal neurones. J. Physiol. 464, 131-163. Brackely, P., Goodnow, R. j., Nakanishi, K., Sudan, H. L., and Usherwood, P. N. R. (1990). Spermine and philanthotoxin potentiate excitatory amino acid responses in oocytes injected with rat and chick brain RNA. Neurosci. Lett. 774, 51-56. Charton, G., Rovira, C., BenAri, Y., and Leviel, V. (1985). Spontaneous and evoked release of endogenous Zn* in the hippocampal mossy fiber zone of the rat in situ. Exp. Brain Res. 58, 202205. Christine, C. W., and Choi, receptor-mediated channel rosci. 70, 108-116.
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Nakanishi, N., Axel, R., and Schneider, N. A. (1992). Alternative splicing generates functionally distinct N-methyl-o-aspartate receptors. Proc. Natl. Acad. Sci. USA 89, 8552-8556. Ransom, R. W., and Stec, N. L. (1988). Cooperative modulation of PH]MK801 binding to the N-methyl-o-aspartate receptor-ion channel complex by L-glutamate, glycine, and polyamines. Neurochem. 57,830-836.
Donaldson, J., Pierre,T. S., Minnich, J. L., and Barbeau, A. (1973). Determination of Na+, K+, Mg*‘, Cuz+, Zn2+, and Mn2+ in rat brain regions. Can. J. Biochem. 57, 87-92.
Reynolds, I. J., and Miller, R. J. (1989). lfenprodil of N-methyl-o-aspartate receptor antagonist: polyamines. Mol. Pharmacol. 36, 758-765.
Dreosti, I. E. (1984). ing interaction. In try, Anatomy, and and E. S. Karsakis,
nervous system: theemergZinc, Part A: PhysiochemisFrederickson, C. A. Howell, Alan R. Liss, Inc.), pp. l-26.
Rock, M. D., and Macdonald, R. L. (1992a). The polyamine spermine has multiple actions on N-methyl-aaspartate receptor single channel currents in cultured cortical neurons. Mol. Pharmacol. 47, 83-88.
Durand, G. M., Gregor, P., Zheng, X., Bennett, M. V. L., Uhl, G. R.,andZukin, R.S.(1992).Cloningofanapparentsplicevariant of the rat N-methyl-o-aspartate receptor NMDARI with altered sensitivity to polyamines and activators of protein kinase C. Proc. Natl. Acad. Sci. USA 89, 9359-9363.
Rock, M. D., and Macdonald, R. L. (1992b). Spermine and related polyamines produceavoltagedependent reductionof N-methyl-oaspartate receptor single-channel conductance. Mol. Pharmacol. 42, 157-164.
Zinc in thecentral Neurobiology of Techniques, C. J. eds. (New York:
Durand, G. M., Bennett, M. V. L., and Zukin, R. S. (1993). Splice variants of the N-methyl-n-aspartate receptor NRI identify domains involved in regulation by polyamines and protein kinase C. Proc. Natl. Acad. Sci. USA 90, 6731-6735. Hollmann, M., Boulter, J., Maron, C., Beasley, L., Sullivan, J., Pecht, G., and Heinemann, S. (1993). Zinc potentiates agonistinduced currents at certain splice variants of the NMDA recep tor. Neuron 70, 943-954.
Seiler, malian
N., and Dezeure, F. (1990). Polyamine cells. Int. J. Biochem. 22, 211-218.
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Seiler, N., and Schmidt-Glenewinkel, T. (1975). Regional distribution of putrescine, spermidine and spermine in relation to the distribution of RNAand DNA in the rat nervous system. J. Neurothem. 24, 791-795. Shaw, G. C., and Pateman, A. J. (1973). The regional distribution of the polyamines spermidine and spermine in brain. J. Neurothem. 20, 1225-1230.
Howell,C. A.,Welch, M. G.,and Frederickson, C. F. (1984). Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308, 736-738.
Sprosen, T. S., and Woodruff, G. N. (1990). Polyamines potentiate NMDA induced whole-cell currents in cultured striatal neurons. Eur. J. Pharmacol. 779, 477-478.
Ikeda, K., Nagasawa, M., Mori, H., Araki, K., Sakimura, K., Watanabe, M., Inoue, Y., and Mishima, M. (1992). Cloning and expression of the E4 subunit of the NMDA receptor channel. FEBS Lett. 373, 34-38.
Standaert, D. G., Testa, C. M., Penney, I. B., Jr, and Young, A. B. (1993). Alternatively spliced isoforms of the NMDARI glutamate receptor subunit: differential expression in the basal ganglia of the rat. Neurosci. Lett. 752, 161-164.
Ishii, T., Moriyoshi, K., Sugihara, H., Sakurada, K., Kadotani, H., Yokoi, M., Akazawa, C., Shigemoto, R., Mizumo, N., Masu, M., and Nakanishi, S. (1993). Molecular characterization of the family of the N-methyl-o-aspartate receptor subunits. J. Biol. Chem.268, 2836-2843.
Sugihara, H., Moriyoshi, K., Ishii,T., Masu, M., and Nakanishi, S. (1992). Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing. Biochem. Biophys. Res. Commun. 785, 826-832.
Kushner, L., Lerma, J., Zukin, R. S., and Bennett, M. V. L. (1988). Coexpression of N-methyl-o-aspartate and phencyclidine recep tors in Xenopus oocytes injected with rat brain mRNA. Proc. Natl. Acad. Sci. USA 85, 3250-3254. Kushner, L., Lerma, J., Bennett, M. V. L., and Zukin, R. S. (1989). Using the Xenopos oocyte system for expression and cloning of neuroreceptors and channels. In Methods of Neuroscience, P. M. Conn, ed. (Orlando, Florida: Academic Press), pp. 3-28. Kutsuwada, T., Kashiwabuchi, N., Mori, H., Sakimura, hiya, E., Araki, K., Meguro, H., Masaki, H., Kumanishi, kawa, M., and Mishina, M. (1992). Molecular diversity
K., KusT., Araof the
Westbrook, C. L., and Mayer, M. L. (1987). Micromolar concentrations of Zn++ antagonize NMDA and CABA responses in hippo campal neurones. Nature 328, 640-643. Williams, K., Romano, C., and Molinoff, M. R. (1989). Develop mental switch in the expression of NMDA receptors occurs in viva and in vitro. Mol. Pharmacol. 36, 575-581. Yamazaki, M., Mori, H., Araki, K., Mori, K. J., and Mishina, M. (1992). Cloning, expression and modulation of a mouse NMDA receptor subunit. FEBS Lett. 300, 39-45.
vol.
12, 811-818,
April,
1994, Copyright
0 1994 by Cell Press
Mutagenesis Rescues Spermine and Zn*+ Potentiation of Recombinant NMDA Receptors Xin Zheng, Ling Zhang, Cuylaine M. Durand, Michael V. 1. Bennett, and R. Suzanne Zukin Department of Neuroscience Albert Einstein College of Medicine Bronx, New York 10461
Summary Alternative splicing generates distinct forms of the NMDA receptor subunit NRl. NRl subunits with an N-terminal insert (termed Nl) form receptors in Xenopus oocytes with greatly reduced potentiation by spermine and Zn2+. Oocytes expressing NRl receptors with Nl exhibited larger NMDA currents than oocytes expressing corresponding receptors without N 1. In the present study, we used mutational analysis to investigate struttural featuresof the Nl insert that control current amplitudeand spermineandZn*+ potentiation. Neutralization of positive charges in Nl rescued spermine and Znz+ potentiation. Positive charges in N 1 did not affect spermine or Zn*+ affinity. Neutralization of positive charges in Nl diminished the responses to the level of NRl recep tors lacking N 1. The positively charged N 1 may increase NMDA currents by causing a conformational change similar to that produced by spermine and Zn2+ in NRl receptors lacking N 1. Introduction Molecularlydiverseformsof the N-methyl-o-aspartate (NMDA) receptor subunit NRI arise by alternative RNA splicing. Differential splicing of three exons can generate eight NRI splice variants, seven of which have been identified in rodent cDNA libraries (Anantharam et al., 1992; Durand et al., 1992; Nakanishi et al., 1992; Sugihara et al., 1992; Yamazaki et al., 1992; Hollmann et al., 1993). The alternatively spliced exons encode a 21 amino acid sequence in the N-terminal domain (termed Nl)and adjacent sequencesof 37and 38 amino acids in the C-terminal domain (Cl and C2). Splicing out of the exon segment encoding the C2 insert removes the first stop codon, resulting in a new open reading frame that encodes an unrelated sequence of 22 amino acids before a second stop codon is reached. We indicate specific NRI receptor splice variants with subscripts denoting the presence or absence of the alternatively spliced exons from 5’ to 3 (Durand et al., 1993). Thus, the NRl,m subunit has the Nl insert, but lacks Cl and C2; the NRlo17 subunit lacks Nl, but has both Cl and C2. NRI receptor subunits expressed in Xenopus laevis oocytes assemble to form functional homomeric channels. These channels share many of the electrophysiological and pharmacological characteristics of native NMDA receptors, including activation by coagonists glutamate (or NMDA) and glycine, competi-
tive block by D-3-amino, Iphosphonovalerate, voltage-dependent block by Mg2+, voltage-independent block byZnZ+ (>I0 PM), and Ca2+ permeability (Durand et al., 1992; Sugiharaet al., 1992). However, differential utilization of the N-terminal coding exon results in receptor variants that differ in certain physiological and pharmacological properties (Durand et al., 1993; Hollmann et al., 1993). NRl subunits lacking the 21 amino acid Nl insert form receptorsmthat exhibit relatively small currents, higher affinity for agonists, and marked potentiation byspermineand submicromolar Zn2+. NRl subunits containing the Nl sequence form receptors that exhibit relatively large currents, lower affinity for agonists, and less potentiation by spermine (at saturating glycine) or by Zn2+. Receptor binding and electrophysiological studies suggest the presence of distinct recognition sites on the NMDA receptor for polyamines. Spermine and spermidine increase binding of the open channel blocker MK-801 to NMDA receptors, suggesting that polyamines enhance NMDA receptor activity (Ransom and Stec, 1988; Williams et al., 1989; Reynolds and Miller, 1989). This effect is observed in the presence of maximally stimulating concentrations of glutamate and glycine, indicating action at an additional and distinct site on the NMDA receptor. Spermine potentiates NMDA-induced currents in Xenopus oocytes injected with rat brain mRNA (Brackely et al., 1990; McGurk et al., 1990) or with synthetic mRNAs encoding NMDA receptor subunits (Moriyoshi et al., 1991; Durand et al., 1992, 1993), as well as in cultured neurons from striatum (Sprosen and Woodruff, 19!IO), cortex(Rockand Macdonald 1992a, 1992b), hippocampus (Araneda et al., 1993; Benveniste and Mayer, 1993), and spinal cord (lerma, 1992). Potentiation occurs by an increase in maximum response amplitude in the presence of saturating concentrations of NMDA and glycine and, at lower concentrations of glycine, by increasing glycine affinity (McGurk et al., 1990; Benveniste and Mayer, 1993). These observations suggest that there may be two sites for spermine potentiation. Recent studies involving whole-cell (Rock and Macdonald, 1992a, 1992b; Araneda et al., 11993; Benveniste and Mayer, 1993) and single-channel recording (Rock and Macdonald 1992a, 1992b; Araneda et al., 1993) showthat spermine has inhibitoryaswell as potentiating actions on NMDA receptors. Spermine potentiates whole-cell currents and increases the frequency of NMDA channel opening in cortical and hippocampal neurons. At relatively negative membrane potentials, spermine decreases whole-cell currents and apparent singlechannel conductance in a voltagedependent manner, indicative of fast channel block. Both potentiation and inhibition by spermine are also observed for recombinant NMDA NRI receptors lacking the Nl insert (Durand et al., 1992,1993). However, NRI receptors with the Nl insert exhibit spermine
Neuron
812
inhibition and spermine potentiation at low glycine, but little potentiation at saturatingglycine.Thesefindings, together with studies showing the presence of high concentrations of polyamines in the brain (Shaw and Pateman, 1973; Seiler and Schmidt-Glenewinkel, 1975), suggest that polyamines may act at as many as three distinct sites to modulate NMDA receptors physiologically. Like spermine, Zn 2+ has potentiating and inhibitory actions at NMDA receptors. At high concentrations (>lO~M),Zn~+inhibitsNMDAresponsesin hippocampal neurons (Westbrook and Mayer, 1987; Christine and Choi, 1990) and oocytes expressing NRI splice variants (Durand et al., 1992; Hollmann et al., 1993). This block is voltage independent, suggesting that Zn*+ and spermine inhibit at different sites. At submicromolar concentrations, Zn*+ potentiates NMDAinduced responses of recombinant NRI receptors lacking the Nl insert. NRI receptors containing Nl exhibit inhibition at high Zn2+ concentrations, but little or no potentiation at low Zn2+ concentrations (Hollmann et al., 1993). In the present study, we used mutational analysis to investigate the structural features of the Nl insert that alter current amplitudes and control spermine and Zn2’ potentiation of NMDA responses. Because spermine, Zn2+, and the Nl insert are positively charged, we replaced positively charged residues in Nl. Introduction of alanine residues for arginines and lysines in Nl rescued spermine and Zn*+ potentiation and reduced current amplitude. The mutations did not appear to affect spermine or Zn2+ affinity. These findings suggest that the Nl insert itself may potentiate NMDA channel opening and that,‘in NRI receptors lacking the Nl insert, a similar potentiation is produced by spermine and Zn2+. Furthermore, potentiation by the Nl insert requires its positive charges. Results Identification of Amino Acid Residues in the Nl Insert That Reduce Spermine Potentiation at Saturating Glycine Electrophysiological studies indicate that the presence of the Nl insert greatly reduces spermine potentiation at saturating glycine (Durand et al., 1992,1993). To test the importance of the positively charged residues on spermine potentiation, mutant receptor NRlloo subunits were constructed with reduced numbers of positive charges in the N-terminal insert. In mutant 1 (K192A, K193A, R194A), alanine residues were introduced in place of lysine and arginine residues at the N-terminal end of the insert. In mutant 2 (R207A, R208A, K211A), alanines replaced the three positively charged residuesat the C-terminal end of the insert. In mutant 3, alanines replaced all six positively charged residues in the insert. The N-terminal inserts of wildtype and mutant receptors are shown in Figure 1.
1~
N-terminal
Wildtype: Mutant 1:
SKKRNYENLDQLSYDNRRGPK-
Mutant 2:
SKKRNYENLDQLSYDNAAGPA-
Mutant3:
SAAANYENLDQLSYDNAAGPA-
insert
1
SAAANYENLDQLSYDNRRGPK-
Figure 1. The Amino Acid Sequences Type and Mutant NRllm Receptors
of the Nl
The alternatively spliced N-terminal insert charged amino acid residues (bold faced). We tance of these residues on spermine and ZrP the following mutant NRllm receptors: mutant R194A), mutant 2 (R207A, R208A, K211A), and nine in all six positions.
Inserts
of Wild-
has six positively tested the imporpotentiation with 1 (K192A, Kl93A, mutant 3, with ala-
Mutant and wild-type NRI channels differed in their degree of spermine potentiation at saturating glycine when expressed in Xenopus oocytes. At -60 mV, spermine (250 PM) potentiated NMDA responses of wild-type NRlolr receptors to 176% f 11% of the controlvalue(mean f SEM; n = 9; Figure2A;Figure3A).At the same potential, spermineslightly inhibited NMDA responses of wild-type NRl,m receptors (to 95% f 4% of the control response in the absence of spermine; n = 10). Spermine poientiation was partially or fully rescued in the mutant receptors. At -60 mV, spermine (250 PM) produced an intermediate potentiation of mutant 1 and mutant 2 receptors (to 123% f 3% [n = II] and 147% f 5% [n = 111 of control, respectively). Spermine potentiated mutant 3 receptors to about the same degree that it potentiated NRI~I, receptors (to 174% f 5% of control; n = 7). At -60 mV, the degree of potentiation varied as follows: NRla, = mutant 3 > mutant 2 > mutant 1 > NRltoo receptors (Figure 3A). Pairwise comparison of thedegreeof spermine potentiation indicated that mutant 3 did not differ significantly from NRlor,; all other pairwise comparisons reached significance (p < 0.05; ANOVA analysis followed by Bonferroni’s post-hoc test). These findings suggest that the reduction in spermine potentiation exhibited by NRI receptors with the Nl insert is due entirely to the positive charges in Nl and not to the 21 amino acid “spacer.” The two wild-type receptors did not differ significantly in their affinity for glycine (Durand et al., 1992), and increase in glycine to 20 PM had no further effect on spermine potentiation of mutant 3. In these experiments, NRloII rather than NRlooo receptors were used as a measure of wild-type potentiation, because NRIOII receptors are the predominant receptor variant expressed in forebrain and the best characterized of the NRI receptors. In addition, NRlolI receptors were expressed more consistently than were NRlm receptors. In the present study, spermine potentiated NRlooa responses to the same degree that it potentiated NRloll responses (n = 2; the two receptors were also shown to be potentiated to the same degree by Durand et al. [1993]).
Rescue of ZnW and Spermine 013
Actions
Mutant 1
on NRl Receptors
Mutant 2
Mutant 3
NRlo,,
Figure 2. Neutralization in the Nl Insert Rescues Potentiation
of Positive Charges Spermineand ZnZt
Xenopus oocytes were injected with NRI,~, mutant I, mutant 2, mutant 3, and NRI~,, RNA (10-50 ng per cell). Two to thirteen days after RNA injection, whole-cell recording from oocytes was carried out under two microelectrode voltage clamps at -60 mV. Currents wereelicited by bath ap plication of NMDA (N) Wl PM with 10 PM glycine) in the absence and presence of spermine 6) (250 pM) or Zn*+ (Zn) (1 rM). Potentiation was observed as the increase of NMDA-induced current in the presence of spermine or Zn*+ compared with that in its absence. (A) Potentiation by spermine was partially rescued in mutants 1 and 2 and completely rescued in mutant 3. (B) Potentiation by Znz+ was little changed in mutant 1 but fully rescued in mutants 2 and 3.
The inhibitory action of spermine on native and recombinant NMDA receptor channels is greatly reduced at more depolarized potentials (Durand et al., 1992,1993;Araneda et al., 1993, Benveniste and Mayer, 1993). To evaluate the voltage dependence of spermine actions on mutant and wild-type NRl,m receptors, oocytes were clamped at holding potentials of -40, -60, -80, and -100 mV (Figure 38). At -40 mV, spermine markedly potentiated NMDA responses of NRIOII and mutant 3 receptors, but only slightly potentiated NMDA responses of NRIIm receptors. Responses of mutant 1 and mutant 2 receptors were potentiated to an intermediate degree. When the cells were hyperpolarized, the (net) potentiation by spermine was reduced. At -100 mV, spermine inhibited responses of mutant 1 and NRllm channels. At -60 and -80 mV, spermine inhibited NMDA responses of NRI~oo channels. These observations indicate that the effect of introducing neutral amino acid residues in place of lysines and arginines in the Nl insert is essentially independent of voltage. Positively charged residues within the Nl insert could affect either spermine binding or a subsequent step in the signal transduction mechanism. Spermine concentration-response curves were determined for wild-type and mutant receptors. To minimize spermine inhibition, recordings were made at -40 mV (Figure 4). For four of the receptors, the spermine response curve was biphasic; reduction of potentiation at higher concentrations was presumably due to the onset of inhibition. Although the complex nature of the concentration-response curves makes quantitative analysis difficult, inspection of Figure 4 shows that neutralization of charges in Nl had little effect on spermine potency. The concentration-response curves for mutant 3 and NRloll receptors were nearly
superimposable; half-maximal potentiation by spermi ne was at - 500 FM. Mutant 1 and mutant 2 receptors exhibited potencies for spermine similar to that of NRlol, receptors, but a reduced degtee of potentiation. These findings indicate that the Nl sequence is not inserted in the spermine-binding Site and suggest that it does not affect spermine binding indirectly through a conformational change in the receptor protein. Identification of Nl Residues That Reduce Zn*+ Potentiation At a low concentration (-1 PM), Zn** markedly potentiated NMDA-induced currents in oocytes expressing NRloll and NRlooo, but only modeitly potentiated NRIIa, receptors. At a higher concentlation (100 PM), ZnZ+ inhibited all NRI receptors (Hollmann et al., 1993; see also Durand et al., 1992). Both the inhibitory and potentiating effects were voltage independent. To test the relevance of the charged residues in the Nl insert toZn*+ potentiation, weevaluated this process in wildtypeand mutant NRI receptors. At 1 FC.MZn2+, mutants 2 and 3 showed virtually complete resceeof the potentiation compared with NRIo,, and NRlm receptors; mutant 1 showed no rescue (Figure 28; Figure 5). At 1 PM Zn*+, the degree of potentiation was as follows: NRlloo = mutant 1 < mutant 2 = mutant 3 = NRIo,, = NRlm receptors. The degree of Zn” potentiation observed for NRl,a, and mutant 1 receptors differed significantly from that of mutant 2, mutant 3, NRIoII, and NRlooo receptors (p < 0.05). Differerlces within the two groups were not significant. To test the effect of positively charged residues on Zn*+ affinity, concentration-response curves were constructed for wild-type and mutant NRI receptors. Thetwowild-type and two mutant receptors exhibited
NeUrOn 814
A.
i
zoo
8-j NRloil 8 MS v Y2
u
l
Ml
n
NRlioo
.d
B.
i
rE
60 10-l
Figure4. Neutralization of Does Not Change Spermine
Figure 3. Voltage Type and Mutant
Dependence NRI Receptors
of
Spermine
Action
on
Wild-
Potentiation was measured as the ratio of NMDA-induced current in thepresenceof sperminetothat in itsabsence. Responses were elicited by application of NMDA (300 f1h4 with 10 uM glytine) in the absence and presence of spermine (250 PM) at each holding potential. (A) Spermine potentiation recorded at -60 mV was partially rescued in mutants 1 and 2 and was completely rescued in mutant 3. Standard error bars and numbers of oocytes are indicated. Pairwise comparison of the degree of spermine potentiation indicated that mutant 3 did not differ significantly from NRlon; all other pairwise comparisons reached significance (p < 0.05; ANOVA analysis followed by Bonferroni’s post-hoc test). (B) Spermine potentiation at a range of holding potentials f-40, -60, -80, and -100 mV). Net potentiation by spermine was reduced at more hyperpolarized potentials, presumably because of enhanced inhibition by spermine. In oocytes expressing NRllm or mutant 1 receptors, spermine inhibited NMDA responses at -100 mV. The degree of rescue, measured as the ratio of spermine potentiation of mutant receptor responses to that of NRlo,, receptor responses, varied little with holding potential over the range of potentials examined.
little difference in Zn2+ potencies (Figure 6). Interestingly, at lower concentrations, Zn*+ potentiation of mutant 3 was greater than that of NRloI,. These observations suggest that positively charged residues in Nl do not affect the initial binding of Znz+ to the NMDA receptor. Inspection of the dose-response curves at lower concentrations suggests that the degree of potentiation differs as follows: mutant 3 > NRI~v > NRIIw > mutant 1 receptors. Thus, the two “clusters” of positively charged residues within Nl appeared to affect
Positive Potency
Charges
in the
Nl
Insert
Concentration-response curves were constructed for spermine potentiation of NMDA responses for NRllm (wild-type), mutant 1, mutant 2, mutant 3, and NRlmr receptors. Responses were elicited by application of NMDA (300 PM with 10 hM glycine) in the absenceand presenceof spermine at indicated concentrations at a holding potential of -40 mV. The spermine concentration response curve for mutant 3 was superimposable on that for NRlm, receptors. Potentiation of mutant 2 and mutant 1 re sponses was reduced without a decrease in apparent spermine potency, relative to NRlnl responses. Data points are the steadystate current amplitudes expressed as a percentage of the mean of control responses recorded before and after each test response and represent means + SEM of responses of five to seven oocytes.
Zn2+ potentiation differently. The cluster of positively charged residues at the N-terminal end of Nl had little effect on Zn2+ potentiation; the cluster at the C-terminal end of Nl appeared to account for the entire difference between NRl,w and NRlo,, receptors. The presence of the 21 amino acid spacer in mutant 3 may actually have increased Zn2+ potentiation.
Additivity of Spermine and Zn2+ Effects To evaluate whether spermine and Zn2+ act at the same or different sites, NMDA currents were measured in the presence of the two ligands separately and together (Figure 7). The potentiating effects of spermine (250 PM) and Zn2+ (1 PM) summated in a nearly linear fashion. Thus, the ratio between Pzn + Pspermineand Pzn + Spermine was 1.01 * 0.04 (n = 5), in which Pzn is the potentiation by Zn2+ (response in the presence of Zn2+ minus control response), PSpermineis the potentiation by spermine, and Pzn + spermineis the potentiation by Zn2+ and spermine applied together. The finding of additivity of spermine and Zn2+ potentiation suggests that binding occurs at two independent sites.
Rescue
of ZrP
and Spermine
Actions
on NRl
Receptors
815
-,
.I----NRlioo Figure 5. Neutralization Potentiation
Ml
YR
of Positive
Y3 Charges
NlZlo~l
NRlooo
in Nl Rescues
[Zinc]
Zn2+
Potentiation of responses to NMDA (300 )IM with 10 PM glycine) by Zn2+ (1 bM) was measured at -60 mV, as for Figure 2. NRlm, and NRlmo receptors exhibited greater potentiation than did NRlrm receptors. Neutralization of positive charges in mutant 1 did not increase Znz+ potentiation. Mutant 2 and mutant 3 exhibited Zn’+ potentiation comparable with that of NRloll and NRlmo recep tors. Steady-state current amplitudes recorded at -60 mV were normalized to the mean of control responses recorded before and after each test response. At 1 PM, the degreeof Znn potentiation observed for NRllm and mutant 1 receptors differed significantly from that of mutant 2, mutant 3, NRloII, and NRlmo receptors (p < 0.05). Differences within the two groups were not significant. Data are the means + SEM of responses from three tosixoocytes(numberofexperimentsindicatedaboveeach bar).
Response Amplitudes of Wild-Type and Mutant NRl Receptors The effect of neutralization of positive charges in the Nl insert on the amplitude of NMDA-induced responses was also examined (Figure 8). To reduce variability between preparations, responses were compared within a given batch of oocytes, each injected with the same amount of RNA (20 ng per oocyte) and recorded under the same conditions. Oocytes expressing NRllao receptors exhibited responses to NMDA (300 PM with 10 PM glycine) that were about 3-fold greater than those for oocytes expressing NRlolr, in confirmation of Holimann et al. (1993). (Their paper also showed that oocytes expressing NRIw and NRlmo receptors gave responses of similar amplitude.) Changeof IysineandarginineresiduesintheNl insert to alanines reduced current amplitude to that of NRl,-,,, receptors (Figure 8). Current amplitude for NRlloo receptors was significantly greater than those for NRloll and for all three mutant receptors (p < 0.001). Current amplitudes of NRlo,,, mutant 2, and mutant 3 receptors did not differ significantly. It is as yet unknown whether the larger currents observed for receptors with the Nl insert is a result of greater activity at the single-channel level or of more efficient translation, assembly, or insertion of channels. Never-
Figure 6. Neutralization Change Znz+ Potency
of
Positive
10’
10s
108
(n&t) Charges
in Nl
Does
Not
Concentration-response curves were constructed for Znz+ potentiation of NMDA response of NRllm (wild-type), mutant 1, mutant 3, and NRlml receptors at -60 mV. Responses were elicited by application of NMDA (3UO pM with 10 (rM glycine) in the presence of ZnZf at indicated concentrations. Data points are the steady-state current amplitudes expressed as a percentage of the mean of control responses recorded before and after each test response and represent means f SEM of responses from five to seven oocytes. ZnZf potency did notl appear to change. The degree of potentiation varied as follows: mutant 3 > NRloll > NRllm > mutant 1.
theless, these observations suggest the interesting possibility that the Nl insert itself propagates a conformational change to the ion channel to increase NMDA responses. Neutralization of positive charges in Nl byexchangeofaminoacidspreventsthisconformational change, thereby reducing response amplitudes to the level observed for NRI receptors lacking the Nl insert. In NRI receptors lacking Nl, added spermine or Zn*+ may potentiate agonist-evoked re-
Figure
7. Spermine
and Zn2+ Potentiation
In oocytes expressing NRlmI receptors, the control response to NMDA (N) (300 flM with IO PM glycine) was potentiated by spermine (S) (250 vM) and Zn2+ (Zn) (I @vt) at -60 mV. When the two ligands were applied together, the increase in response was thesumoftheincreaseselicited byspermineand byZ#applied separately.
Neuron 816
Nl is unlikely to be due to an electrostatic interaction with spermine or Zn2+ at their binding sites. This finding also indicates that the Nl sequence is unlikely to be inserted in the spermineor Zn2+-binding site. Reducing the net positive charge on Nl by changing lysines and arginines to uncharged alanines also diminishes the response amplitude. Neutralization of positive charges in all three mutants reduces the response amplitude to that observed for NRI receptors lacking the Nl insert. In each case, spermine or Zn2+ potentiates the response toward the level observed for Nl-containing receptors. These findings indicate that the Nl insert with its six positively charged residues may induce a conformational change in the re-
1.6 2
1.6
2 E
1.4
2
1.0
y e Gi I i
0.6
;
0.6
1.e
0.6 0.4
ceptor 0.0 NBloll
MS
Y2
Ml
NRlloo
Figure 8. Neutralization of Positive Charges in the Nl Insert Decreases Response Amplitude Responses of NRl,m receptors were about Sfold larger than those of NRlolr receptors. For all three mutants of the NRltm receptor, responses were reduced to a level comparable with that of NRlmt receptors. Responses of NRl,m receptors were significantly greater than those of NRlmr, mutant 1, mutant 2, and mutant 3 receptors (p < 0.001 for each pairing); responses of NRlm,, mutant 1, mutant 2, and mutant 3 receptors did not differ significantly. The data shown represent the means f SEM of responses to 300 PM NMDA with 10 RM glycine (number of oocytes indicated above the bars). All oocytes used for NRlar, mutant 2, and mutant 3 receptors and six’of nine oocytes used for NRllm receptors were from a single batch from one female, injected with the same amount of RNA (20 ng) and compared under constant conditions. In a separate batch of oocytes (+), the current amplitude for mutant 1 was compared with that for NRlra, (391 f 42 nA; n = 3 oocytes). The data plotted for mutant 1 were resealed by the ratio of NRl,m responses in the two batches of oocytes.
sponses by inducing a conformational change to that induced by the wild-type Nl insert.
similar
Our results indicate that potentjatjon of NRI receptors by spermine and by ZnZ+ is critically affected by the six positively charged amino acid residues of the Nl insert. The presence of the Nl insert in NRI subunits greatly reduces potentiation by Znz+ and by spermine at saturating glycine (Durand et al., 1992, 1993; Hollmann et al., 1993). Neutralization of positive charges at the N-terminal end of the insert (mutant 1) partially rescues spermine but not Znz+ potentiation. Neutralization of the three positive charges at the C-terminal end (mutant 2) partially rescues spermine potentiation and fully rescues Zn2+ potentiation. Replacing all six positively charged residues with alanines (mutant 3) fully rescues spermine and Zn2+ potentiation. Potentiation by spermine and ZnZ+ is essentially additive, indicating possible action at separate sites. Neutralization of positive charges in Nl does not appear to affect spermine or Zn2+ potency. Thus, reduced potentiation in receptors containing
that
is transmitted
to the
ion
channel,
thereby
increasing agonist-evoked currents. Moreover, this effect may be similar to that induced by binding of spermine or Zn~ in receptors lacking Nl. The amplitude of maximally potentiated NRloll responses observed upon application of spermine and Zn2+ together approached that of unpotentiated NRIIW responses, consistentwith the suggestion thatthe insert and potentiators may act through a common mechanism. Apparently, spermine and Zn2+ still bind to Nlcontaining receptors, but with little further potentiation of current amplitude. An important issue is whether polyamines and Zn2+ are endogenous regulators of NMDA receptors. In rat brain, polyamine concentration is in the micromolar range (Shaw and Pateman, 1973). Highest concentrations are in the hippocampus, cerebellum, and corpus callosum. In the hippocampus, spermine and other polyamines are released from and taken up into nerve terminals (Seiler and Dezeure, 1990). Polyamine biosynthesis probably occurs in nerve endings, as suggested by active uptake of ornithine into synaptosomes and relatively high activities of ornithine decarboxylase and S-adenosyl-methionine decarboxylase (Seiler and Dezeure, 1990). Zn2+ is also present in the brain (Donaldson et al., 1973; Dreosti, 1984). In the hippocampus, Zn2+ is stored in presynaptic vesicles and is co-released with glutamate in an activity-dependent manner (Assaf and Chung, 1984; Howell et al., 1984; Charton et al., 1985; Aniksztejn et al., 1987). These findings support the concept that spermine and ZnZ+ act as endogenous physiological modulators of glutamate neurotransmission. Another important issue is whether recombinant homomericchannelssharefunctional propertieswith native NMDA receptors in neurons. A second family of NMDA receptor subunits, NRZA-D, do not themselves form functional channels, but appear to coassemble with NRI subunits to form channels with greatly enhanced activity flkeda et al., 1992; Kutsuwada et al., 1992; Meguro et al., 1992; Monyer et al., 1992; lshii et al., 1993). The finding of genetically diverse NMDA receptor RNAs, many of which are expressed throughout the brain, and the observation that current amplitudes obtained with homomeric NRI channels are relatively low suggest that at least
Rescue of Zn” 817
and Spermine
Actions
on NRI
Receptors
some NMDA receptors are heteromeric channels in vivo. Nevertheless, as noted above, homomeric recep tors share many of the electrophysiological and pharmacological properties of native receptors. Moreover, in preliminary experiments, we observed that NRlorI/ NWB heteromeric receptors exhibit spermine potentiation at saturating glycine. Our whole-cell recording studies indicate that NMDA-induced currents in most hippocampal neurons exhibit spermine potentiation at saturating glytine (Araneda et al., 1993). Our results suggest that at least someof the receptors mediatingthese responses lack the N-terminal insert Nl. In excised outside-out patches of hippocampal neurons, spermine produced potentiation of NMDA responses in only 4 of 15 patches (Araneda et al., 1993). A possible explanation of tfre infrequency of patches showing potentiation by spermine is the relative predominance of Nlcontaining NRI receptors on the cell somata from which the patcheswereobtained. Other studies using whole-cell recording report variable potentiation by spermine (Rock and Macdonald, 1992a; Benveniste and Mayer, 1993). In situ hybridization studies show that NMDA splice variants with the Nl exon predominate in the subthalamic nucleus and in the CA3 of the hippocampus (Standaert et al., 1993). In conclusion, the present study demonstrates that the six positively charged amino acid residues in the Nl insert of NRI receptor splice variants critically affect spermine and ZrP potentiation. Neutralization of the charges by exchange to neutral residues fully rescues potentiation by both ligands. The neutralization of charged residues in Nl also increases response amplitudes. These observations suggest that the Nl insert induces a conformational change in the receptor which increases agonist-evoked currents and occludes the potentiating effects of spermine and Zn*+. The positive charges in Nl are required for these actions; increasing the length of the N-terminal domain by the twenty-one amino acid sequence (without its positive charges) has little effect. The unmodified insert does not appear to affect spermine or ZnZ+ binding. Single-channel measurements will be required to establish the mechanisms of potentiation by Nl and byZn 2+. S p ermine potentiation in hippocampal neurons is due to increased channel open probability; at more negative potentials, spermine also decreases apparent single-channel conductance (Araneda et al., 1993). Whatever the mechanism by which the Nl insert affects response amplitude, alternative splicing of the insert may act as a switch controlling spermine and Zn2+ potentiation of NMDA receptors. For receptors lacking Nl, spermine and ZrP are likely to act as important endogenous modulators of NMDA receptor activity.
We thank Alice Wang for technical assistance. We thank Drs. JiaBei Wang and George R. Uhl for helplul advice on sitedirected mutagenesis. We thank Dr. John A. Kessler for his help ful comments on the manuscript. We are gratqful to Dr. S. Nakanishi for providing the NRlmr cDNA. This work was supported by National Institutes of Health grants NS 20752 to R. S. Z., and NS 07412 and HD 04248 to M. V. L. B.; M. V. L. $. is the Sylvia and Robert 5. Olnick Professor of Neuroscience. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordanm with 18 USC Section 1734 solely to indicate this fact.
Experimental
Received
Procedures
Site-Directed Mutagenesis NRlrm was subcloned into tagene) and used to transform
Single-stranded DNA template was rescued with M13K07 helper phage, and sitedirected mutations were made with oligonucleotidedirected in vitro mutagenesis system version 2 (Amersham). Each oligonucleotide was designed to engineer three codon substitutions into cloned NMDA receptor DNAs to generate subunits carrying three amino acid replacemehts in the N-terminal insert. The following oligonucleotides were used for introduction of changes into coding sequences1 EGTCCACCFfTCATAG~CCCGGCCGCACGA~C~CG-3 for mutant 1 (K192A, K193A, R194A); 5-CAGCACCTrCtCTGCCGCGGGTCCGGCCGCGTrGTCATAGGACAGTTG-3 for mutant 2 (R207A, R208A, K2llA). Mutant 3, in which all six positively charged residues were exchanged to alanines, was generated from mutant 1 with the oligonucleotide for mutant 2. Codon substitutions were confirmed by DNA sequencing across the altered region with Sequenase version 2.1 (USB). cRNA Synthesis NRlo,, cDNA was a gift of Dr. S. Nakanishi (Kyoto, Japan); NRl,m and NRlm cDNAs were cloned in this laboratory (Durand et al., 1992). Mutant cDNAs were constructed as described above. To generate templates for transcription, circular plasmid cDNAs were linearized with Not1 (NRl&, BamHl (&Id-type and mutant NRl,,), or Clal (NRl,). Transcription reactions were performed with T7 or T3 polymerase (Ambion T7 or T3 MEGAscript RNA polymerase transcription kit; Ambion) (4 hr at 37°C). Electrophysiolo&al Experiments in Xenopus Qocytes Adult female Xenopus (Xenopus I, Ann Arbor, MI) were anesthetized by immersion in 0.15% aminobenzoic acid ethyl ester, and individual oocytes were isolated as descritied (Kushner et al., 1988,1989). Briefly, oocytes were incubated for 1 hr in Ca2+-free ND-% medium (96 mM NaCI, 2 mM KCI, 1 mM MgCIz, 5 mM HEPES [pH 7.51) containing collagenase type D (2 mg/ml; Boehringer Mannheim), penicillin (10 U/ml), and streptomycin (0.01 mg/ml), after which the follicular layer was t’emoved by manual dissection. Selected stage V and VI oocytes were injected with in vitro transcribed RNA (IO-M ng per cell)‘and maintained at 18OC in ND-% containing Ca2+ (2 mM). Two to thirteen days after injection, oocytes were recorded in MgP+-free frog Ringer’s solution (116 mM NaCI, 2.0 mM KCI, 1.0 mM CarI&, 10 mM HEPES [pH 7.21) under two microelectrode voltage olamp with a Dagan amplifier (Dagan, Inc.). Voltage and currefit electrodes were filled with 1 M KCI, 10 mM HEPES, and 5 mM EDTA and had a resistance of -1 MP. NMDA (300 pM), with or without other drugs, was bath applied to oocytes to elicit dgonist-evoked currents. Glycine (IO PM) was present in all solutions. To cpmpare responses, steady-state currents were normalized to the mean of control responses to NMDA (300 PM) ahd glycine (10 PM) recorded before and after each test response. One-way analysis of variance (ANOVA) was carried out using the lnstat program (GraphPad software, version 1.14, 1990). Acknowledgments
October
27, 1993; revised
January
21,1994.
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