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The Journal of Neuroscience, August 15, 1998, 18(16):6163-6175
Control of Voltage-Independent Zinc Inhibition of NMDA Receptors
by the NR1 Subunit
Stephen F.
Traynelis1,
Michele F.
Burgess2,
Fang
Zheng1,
Polina
Lyuboslavsky1, and
Jennifer L.
Powers3
1 Department of Pharmacology, Emory University, Rollins
Research Center, Altanta, Georgia 30322, 2 Georgia
Department of Natural Resources, Environmental Protection Division,
Atlanta, Georgia 30334, and 3 Department of Chemistry,
Kennesaw State University, Kennesaw, Georgia 30144
 |
ABSTRACT |
Zinc inhibits NMDA receptor function through both
voltage-dependent and voltage-independent mechanisms. In this report we have investigated the role that the NR1 subunit plays in
voltage-independent Zn2+ inhibition. Our data show
that inclusion of exon 5 into the NR1 subunit increases the
IC50 for voltage-independent Zn2+
inhibition from 3-fold to 10-fold when full length exon 22 is also
spliced into the mature NR1 transcript and the NMDA receptor complex
contains the NR2A or NR2B subunits; exon 5 has little effect on
Zn2+ inhibition of receptors that contain NR2C and
NR2D. Mutagenesis within exon 5 indicates that the same residues that
control proton inhibition, including Lys211,
also control the effects of exon 5 on Zn2+
inhibition. Amino acid exchanges within the NR1 subunit but outside exon 5 (E181Q, E339Q, E342Q, N616R, N616Q, D669N, D669E, C744A, and
C798A) that are known to decrease the pH sensitivity also decrease the
Zn2+ sensitivity, and concentrations of spermine
that relieve tonic proton inhibition also relieve
Zn2+ inhibition. In summary, our results define the
subunit composition of Zn2+-sensitive NMDA receptors
and provide evidence for structural convergence of three allosteric
regulators of receptor function: protons, polyamines, and
Zn2+.
Key words:
NMDA receptors; zinc; protons; RNA splicing; polyamines; site-directed mutagenesis
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INTRODUCTION |
The group IIB transition metal ion
Zn2+ is widely and variably distributed throughout
neurons in the CNS. Zn2+ is released from
nerve fibers in a Ca2+-dependent manner and
sequestered into neurons via uptake systems with
Km values between 10 and 400 µM
(for review, see Smart et al., 1994
). Although the
Zn2+ concentration within the narrow synaptic cleft
is dynamic and difficult to quantify, estimates of the concentration of
interstitial synaptically released Zn2+ range from 5 to 500 µM (Assaf and Chung, 1984; Howell et al., 1984;
Charlton et al., 1985; Aniksztejn et al., 1987). Of the numerous roles
that have been proposed for Zn2+ in the CNS, perhaps
the best known are its actions on a host of voltage- and ligand-gated
ion channels, including glutamate receptors (Smart et al., 1994; Laube
et al., 1995).
Glutamate receptors can be subdivided on the basis of both structure
and pharmacology into three classes: NMDA, kainate, and AMPA
receptors. The potential involvement of NMDA receptors in synaptic
plasticity and neuronal development (McBain and Mayer, 1994; Scheetz
and Constantine-Paton, 1994; Asztely and Gustafsson, 1996) as well as
neurological disorders such as epilepsy, ischemic cell death, and
neurodegeneration (Choi, 1992; Bradford, 1995; Whetsell, 1996) have
fueled intensive study of all aspects of this receptor, including its
sensitivity to extracellular Zn2+ (Peters et al.,
1987; Westbrook and Mayer, 1987; Choi and Koh, 1998). Inhibition of
NMDA receptor function by extracellular Zn2+ within
the predicted concentration range in the CNS raises the possibility
that the modulatory effects of Zn2+ may be an
important component of some of the processes and disorders that NMDA
receptors are thought to participate in (Peters et al., 1987; Koh et
al., 1996).
In cultured neurons, tens of micromolar Zn2+ can
inhibit NMDA receptors through a voltage-dependent channel block that
appears to occur within the ion channel pore and may involve residues (e.g., Asn616 in M2 region of NR1) that are known to
influence other NMDA channel-blocking ions such as
Mg2+ (Mayer and Vyklicky, 1989; Christine and Choi,
1990; Legendre and Westbrook, 1990; Mori et al., 1992; Sakurada et al.,
1993; Kawajiri and Dingledine, 1993). Lower concentrations of
Zn2+ (<5 µM) can act in a
voltage-independent manner to reduce the frequency of channel opening
as well as the duration of neuronal NMDA receptor-mediated bursts
(Christine and Choi, 1990; Legendre and Westbrook, 1990).
Five cDNAs have been identified that encode NMDA receptor subunits
(NR1, NR2A, NR2B, NR2C, and NR2D; McBain and Mayer, 1994), and zinc
inhibits recombinant receptors comprised of NR1 + NR2 receptors in a
dual voltage-dependent and -independent manner that appears to be
controlled by the NR2 subunit (Mori et al., 1992; Kawajiri and
Dingledine, 1993; Sakurada et al., 1993; Williams, 1996; Chen et al.,
1997; Paoletti et al., 1997). In contrast, very little information
exists about the role of alternative splicing of the NR1 subunit RNA on
Zn2+ inhibition of heteromeric NMDA receptors
(Paoletti et al., 1997), although the eight possible NR1 splice
variants expand structural heterogeneity of the NMDA receptor class
considerably. NR1 splicing occurs in a region- and
developmental-specific manner (Laurie and Seeburg, 1994; Nash et al.,
1997; Paupard et al., 1997), suggesting it may be used by the CNS to
fine tune NMDA receptor responsiveness or influence receptor
localization. We have studied the effects of Zn2+ on
recombinant NMDA receptors containing different NR1 splice variants to
define structural determinants of Zn2+ inhibition.
In this report, we describe striking effects of the N-terminal
alternative exon 5 on voltage-independent Zn2+
inhibition, some of which are reminiscent of the effects of exon 5 on
proton inhibition of NMDA receptors (Zheng et al., 1994; Traynelis et
al., 1995). The similar effects of exon 5 on Zn2+
and proton inhibition lead us to explore whether the modulatory sites
for these endogenous ions share similar structural determinants.
Some of these results have appeared in preliminary form (Burgess et
al., 1996; Burgess and Traynelis, 1997).
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MATERIALS AND METHODS |
NMDA receptor subunit cDNAs. cDNAs encoding the NR1
subunit splice variants (GenBank accession number L08228) as well as NR2A (GenBank numbers AF001423 and CD13211), NR2B (GenBank number
U11419), NR2C (GenBank numbers U08259 and M91563), and NR2D (GenBank
number L31611) subunits were generously provided by Drs. J. Boulter
(University of California at Los Angeles, Los Angeles, CA), S. Heinemann (Salk Institute, La Jolla, CA), S. Nakanishi (Kyoto
University, Kyoto, Japan), and P. Seeburg (Max Planck Institute for
Medical Research, Heidelberg, Germany). NR1(N616R) and NR1(N616Q) were
generously provided by Drs. R. Dingledine (Emory University, Atlanta,
GA) and K. Moriyoshi (Kyoto University, Kyoto, Japan), respectively.
NR1-1a(E181Q), NR1-1a(E339Q), NR1-1a(E342Q), NR1-1b(E363Q),
NR1-1a(D669N), and NR1-1a(D669E) were generously provided by K. Williams (University of Pennsylvania, Philadelphia, PA); the wild-type
sequence for NR1 subunit for these mutants can be accessed by GenBank
number X63255. Other NMDA receptor NR1 subunit mutations (see Fig. 7)
have been previously described (Sullivan et al., 1994; Traynelis et
al., 1995). All experiments were performed with NR1 subunits that
contained exon 21 and exon 22 except those shown in Figures 1 and 2.
NR1 splice variants that contain the two C-terminal alternate exons but
lack exon 5 are referred to in the literature as NR1-1a (Hollmann et
al., 1993), NR1011 (Durand et al., 1992), and NR1A
(Sugihara et al., 1992). NR1-1b, NR1111, and NR1B
describe the corresponding cDNA that contains exon 5. We use the
nomenclature of Hollmann et al. (1993), and for simplicity, refer
whenever possible to the presence or absence of NR1 exon 5.
Expression of NMDA receptors in Xenopus oocytes.
cRNA was synthesized from linearized template cDNA according to
manufacturer specifications (Ambion). Quality of synthesized cRNA was
assessed by gel electrophoresis, and quantity was estimated by
spectroscopy and gel electrophoresis. Stage V and VI oocytes were
surgically removed from the ovaries of Xenopus laevis
anesthetized with 3-amino-benzoic acid ethylester (1 gm/l). Clusters of
~30 oocytes were incubated with 292 U/ml Worthington (Freehold, NJ)
type IV collagenase or 1.3 mg/ml collagenase (Life Technologies,
Gaithersburg, MD; 17018-029) for 2 hr in Ca2+-free
solution comprised of (in mM) 115 NaCl, 2.5 KCl, and 10 HEPES, pH 7.5, with slow agitation to remove the follicular cell layer.
Oocytes were then washed extensively in the same solution supplemented
with 1.8 mM CaCl2 and maintained in Barth's
solution comprised of (in mM): 88 NaCl, 1 KCl, 24 NaHCO3, 10 HEPES, 0.82 MgSO4,
0.33 Ca(NO3)2, and 0.91 CaCl2 and supplemented with 100 µg/ml gentamycin, 40 µg/ml streptomycin, and 50 µg/ml penicillin. Oocytes were injected
within 24 hr of isolation with 5 ng of NR1 subunit and 5-10 ng of NR2
subunit in a 50-100 nl volume and incubated in Barth's solution at
18°C for 3-18 d; some oocytes were stored at 4°C after 3-5 d.
Voltage-clamp recordings from Xenopus oocytes.
Two electrode voltage-clamp recordings were made 3-18 d postinjection.
Oocytes were placed in a dual-track recording chamber with a single
perfusion line that split to perfuse two oocytes, which increased the
efficiency of data collection and allowed side-by-side comparisons to
be made on oocytes injected with different subunit combinations. Dual
recordings were made using Warner model OC725B two-electrode voltage clamps as recommended by the manufacturer. The bath clamps communicated across silver chloride wires placed into each side of the
recording chamber, both of which were assumed to be at a reference
potential of 0 mV. Oocytes were perfused with a solution comprised of
(in mM) 90 NaCl, 1 KCl, 10 HEPES, and 1.0 BaCl2, pH 7.3, and held at 
20 to 40 mV.
ZnCl2 solutions (10 mM) were made fresh every
10 hr and added directly to the recording solution to obtain the
desired nominal Zn2+ concentration for all
experiments except those with NR2B- and NR2A-expressing receptors. For
these receptors, we used tricine to buffer Zn2+
concentrations <3 µM. We used a stability constant
(KS) for Zn-tricine of
10
5 M (Vieles et al., 1972; Tripathi
et al., 1987; Paoletti et al., 1997) to calculate the nominal free
Zn2+ concentration according to:
![[<UP>Zn</UP><SUP>2<UP>+</UP></SUP>]<SUB><UP>FREE</UP></SUB>=&agr; K<SUB><UP>S</UP></SUB>[<UP>Zn</UP><SUP>2<UP>+</UP></SUP>]<SUB><UP>TOTAL</UP></SUB>/[<IT>Tricine</IT>]](/content/vol18/issue16/fulltext/6163/img001.gif)
|
(1)
|
in which we assume that the [Tricine] is unchanged
by addition of Zn2+ to the solution (i.e.,
[Tricine] is much greater than
[Zn2+]TOTAL). 
is defined
by:
![&agr;=1+[<UP>H</UP><SUP><UP>+</UP></SUP>]/K<SUB><UP>A</UP></SUB>](/content/vol18/issue16/fulltext/6163/img002.gif)
|
(2)
|
where the KA for tricine is
108.15. Similar free Zn2+
concentrations were obtained with the use of programs that calculate
free ion concentrations (WINMAXC or BAD; Zheng et al., 1998). HEPES does not contribute to Zn2+ buffering (Benitez et
al., 1991). We estimate a free Zn2+ at pH 7.4 that
is ~80% of that reported at pH 7.3 by Paoletti et al. (1997). Our
contaminant Zn2+ (Paoletti et al., 1997) is ~300
nM (Zheng et al., 1998) and arises predominantly from the
NaCl. We assume that only free (or fully hydrated)
Zn2+ acts at NMDA receptors, and calculated free
Zn2+ concentrations at pH 8.0 using the log
stability constants (KSn) for
Zn(OH)n+2n of 5.0 (n = 1), 11.1 (n = 2), 13.6 (n = 3), and
14.8 (n = 4; Martell and Smith, 1974) according to:
|
(3)
|
Secure impalements were usually obtained in solution containing
1 mM CaCl2 before switching to the
BaCl2-containing solution. Voltage and current electrodes
had resistances of 2-10 M
and were filled with 300 mM
KCl. NMDA receptor currents were evoked with 20 µM
glutamate plus 7-10 µM glycine superfused for 1-2 min; washout time was 3-5 min between drug applications. For
NR2A-containing receptors, all experiments were performed in 10 µM glycine, which is 10× our measured EC50
value for glycine (1 µM; n = 8; data not
shown). Solution exchange was computer-controlled through an
eight-valve manifold. The flow rate varied minimally between various
valves and throughout the experiment.
Data analysis. Only current responses to glutamate plus
glycine that were >50 nA at pH 7.3 (40 mV) and were not potentiated by 1 µM Zn2+ (Hollmann et al., 1993)
were analyzed to minimize the contribution to our responses of
previously described NR1 homomeric receptors, which are probably
heteromeric receptors that incorporate Xenopus glutamate
receptor subunits (Soloviev et al., 1996). By this criteria, ~6% of
recordings were discarded; response amplitude was typically several
hundreds of nanoamperes at 20 mV. Inhibition curves were analyzed as
shown in Figure 3A, Zn2+ concentrations
were corrected for 0.3 µM contaminant
Zn2+ for NR2B-containing receptors. For proton
inhibition curves, the oocytes were prewashed with the test pH before
and after agonist application. Experimental manipulations were
expressed as a percent of pre-event and post-event control responses,
and the data were pooled together. Zn2+ inhibition
data were fitted (least squares criterion) to the equation:
|
(4)
|
where n is the Hill slope, IC50 is the
nominal concentration of Zn2+ that produces 50%
inhibition, and minimum a residual current response. For all
receptors, minimum was fixed to 0 if the fitting algorithm
returned a value <5.6%, our estimated limit of detection for the
maximal Zn2+ concentration tested (mean SEM for
responses at 100 µM Zn2+ was 2.55%).
For mutant receptors and NR2C- and NR2D-containing receptors,
minimum was fixed to 0. Hill slopes ranged from 0.5 to 1.6;
IC50 values for wild-type NR1/NR2B,C,D receptors will be
slight underestimates of true IC50 for voltage-independent effects if voltage-dependent inhibition by high concentrations (30-100
µM) of Zn2+ occurs at holding
potentials between 20 and 40 mV; Hill slopes will be slightly
overestimated for the same reason. Throughout the text the
IC50 values that are reported reflect the nominal Zn2+ concentration; effective
Zn2+ activity will be approximately half the nominal
concentration given the activity coefficient for
Zn2+ of 0.5 (Lobo and Quaresma, 1989; Lide, 1997).
Composite proton inhibition curves were constructed using pH 7.6 as a
control and fitted using the same equation, except with proton activity
substituted for [Zn2+]. Proton IC50
values were determined from the pH-derived activities using an activity
coefficient of 0.8 (Lide, 1997).
Current-voltage (I-V) relationships were
recorded in response to a 4 sec voltage ramp (80 to +40 mV) before,
during, and after drug application. The NMDA receptor I-V
curve was determined from the difference of current responses in the
presence and absence of agonist, and the reversal potential was
determined from a fitted polynomial function (fifth to ninth order;
least squares criterion) or interpolation.
Statistical significance for all tests was set at p < 0.05, and minimum detectable differences were calculated for a power of
0.7. Error bars in all figures are SEM. Paired and unpaired t tests and ANOVA were performed to evaluate the difference
between means; for t tests we used Welch's approximate
t statistic when the underlying distributions had different
variances.
Voltage-dependent channel block of wild-type and mutant NMDA
receptors. Because both Zn2+ and polyamines can
cause a voltage-dependent block of the NMDA receptor channel, it is
important to determine whether this effect complicates any of the
experiments we performed. Although we observe a weak voltage-dependent
channel block of wild-type NR1-1/NR2B receptors by 10 µM
Zn2+ at hyperpolarized potentials (e.g., 70 mV;
n = 9; p < 0.05), we can detect no
voltage-dependent block by extracellular Zn2+ at our
typical recording potential, as measured by the ratio of current
recorded 30 mV negative to the reversal potential to that recorded 30 mV positive to the reversal potential
(IVREV30/IVREV+30), determined from current-voltage curves (control
IVREV30/IVREV+30 = 1.0 ± 0.02, mean ± SEM; 10 µM
Zn2+
IVREV30/IVREV+30 = 1.0 ± 0.06; p > 0.4; n = 9;
minimum detectable difference, 15%). We also find no significant
effects of exon 5 on channel block induced by addition of 10 µM Zn2+ (n = 9 oocytes; data not shown) or 0.5 mM Mg2+
(n = 28 oocytes; data not shown), and no effects of
exon 5 on the current-voltage curve recorded when all external
Na+ was replaced by 61.5 mM
Ba2+ (n = 28 oocytes; data not
shown). These data suggest the effects of exon 5 on voltage-independent
Zn2+ inhibition that we describe here are unlikely
to involve ion permeation or block, except perhaps at very high
concentrations of Zn2+. To evaluate the ability of
spermine to relieve Zn2+ inhibition (see Fig.
7), we have used an NR1 subunit that contains a mutation in the M2
region of the receptor that has previously been shown to relieve
voltage-dependent channel block by both Zn2+ and
spermine (Mori et al., 1992; Kawajiri and Dingledine, 1993; Sakurada et
al., 1993; Kashiwagi et al., 1997). To confirm that under our recording
conditions these M2 mutations relieved voltage-dependent Zn2+ inhibition of NR1-1/NR2B receptors, we
constructed current-voltage curves in the presence and absence of
extracellular Zn2+ and spermine for wild-type and
mutant receptors. We found receptors that contained an arginine residue
in place of the M2 Asn616 in NR1-1a and M2
Asn637 in NR1-1b (mutant numbering differs because
of inclusion of 21 amino acids encoded by exon 5) showed no detectable
channel block by Zn2+ at 30 mV (pooled control
IVREV30/IVREV+30 = 1.1 ± 0.04; 30 µM Zn2+
IVREV30/IVREV+30 = 1.0 ± 0.1; p > 0.1; n = 11;
minimum detectable difference, 13%) or at 70 mV (data not shown;
n = 23), consistent with the findings of other
laboratories (Mori et al., 1992; Kawajiri and Dingledine, 1993;
Sakurada et al., 1993). Similarly, voltage-dependent channel block by 1 mM spermine (see Fig.
5A,B) was also abolished by the M2
pore mutation in NR1-1a(N616R)/NR2B and NR1-1b(N637R)/NR2B receptors
(pooled
IVREV30/IVREV+30 = 1.2 ± 0.03; n = 11) compared with wild-type
NR1-1/NR2B receptors (IVREV30/IVREV+30 = 0.6 ± 0.05; n = 16; p < 0.05 by unpaired t test). Similar results were found at more
hyperpolarized potentials and with 100 µM spermine
(n = 27; Kashiwagi et al., 1997).
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RESULTS |
Zn2+ sensitivity of alternatively spliced
NR1 subunits
Extracellular Zn2+ is known to inhibit NMDA
receptors by both voltage-independent and voltage-dependent mechanisms.
Voltage-dependent inhibition appears to involve weak channel block
(Mayer et al., 1989; Christine and Choi, 1990; Legendre and Westbrook,
1990) and is controlled by some of the same amino acid residues that control channel block by Mg2+ (Mori et al., 1992;
Kawajiri and Dingledine, 1993; Sakurada et al., 1993; see Materials and
Methods). In this study we have examined the ability of NR1 to control
voltage-independent inhibition of NMDA receptors by extracellular
Zn2+. To do this, we first evaluated the
IC50 values for Zn2+ inhibition of
responses to maximal glutamate and glycine of all NR1 splice variants
expressed in combination with the NR2B subunit in Xenopus
oocytes. Current responses were recorded from oocytes expressing NMDA
receptor subunits that were voltage clamped at depolarized holding
potentials (20 to 40 mV) at which voltage-dependent channel block
by Zn2+ is minimal (see Materials and Methods).
Figure1 summarizes the effects of
extracellular Zn2+ on the function of NMDA receptors
comprised of different NR1 splice variants and the NR2B subunit.
Inclusion of the N-terminal alternative exon 5 reduced the sensitivity
to extracellular Zn2+ by more than fourfold for
receptors that had spliced in exon 22 starting at the alternative
acceptor site 2647 bp [numbered as in GenBank number X63255; see
Hollmann et al. (1993), their Fig. 1]. By contrast, exon 5 had
variable effects on the Zn2+ sensitivity of
recombinant receptors that had the original stop codon deleted by use
of an internal acceptor splice site at 3003 bp within exon 22 [i.e.,
NR1-3 and NR1-4 by the nomenclature of Hollmann et al. (1993)]. In two
of four batches of oocytes, exon 5 reduced Zn2+
sensitivity of NR1-3/NR2B and NR1-4/NR2B receptors by less than twofold
when the alternative acceptor splice site within exon 22 was used; in
the other batches of oocytes expressing NR1-3/NR2B and NR1-4/NR2B
receptor, exon 5 had much larger effects. This exon 22-linked
variability might be related to interactions between proteins
endogenous to the oocyte and the PDZ2 site (Kornau et al., 1995)
encoded in the new reading frame that results from the deletion of the
stop codon (data not shown). Interestingly, the effects of exon 5 on
Zn2+ inhibition were largely unaffected by the
presence or absence of alternative exon 21 (Fig. 1).
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Figure 1.
Control of Zn2+ and proton
inhibition by RNA splicing of the NR1 subunit. A, Coding
regions are shown schematically for eight NR1 splice variants
(nomenclature according to Hollmann et al., 1993; the suffix
a or b denotes absence or presence of
exon 5) that arise by alternative use of exons, alternative use of
acceptor splice sites, or both. The N terminal is shown to the
left and the C terminal is at the right.
The position of exons 5 and 21 are shown as a broken
box; two different boxes surround exon 22, each indicating the
use of a different acceptor splice site. Membrane-associated regions
are shown as boxes (open, transmembrane;
closed, membrane-associated). NR1-3 and NR1-4 contain a
new 66 base pair reading frame terminated by a previously out of frame
stop codon as a result of the use of an alternative splice-acceptor
site within exon 22, which deletes the original stop codon.
B, Composite Zn2+ inhibition curves
are shown for NR1-1 splice variants ± exon 5 coexpressed with
NR2B. All error bars are SEM. Low Zn2+
concentrations (0.03-0.3 µM) were achieved using tricine
buffer (see Materials and Methods); all other concentrations were
corrected for 0.3 µM contaminant Zn2+.
C, Composite proton inhibition curves are shown for
NR1-1 splice variants ± exon 5 coexpressed with NR2B reexpressed
as a percent of the fitted maximum. D, Mean-fitted
IC50 values for Zn2+ inhibition of
wild-type NR1-a/NR2B receptors (open bars) are
significantly lower than NR1-b/NR2B (filled bars;
p < 0.05 for all). E,
IC50 values for proton inhibition of wild-type NR1-a/NR2B
receptors (open bars) are also increased by exon 5 (filled bars). The number of oocytes recorded for
each experiment is shown in parentheses.
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Inclusion of exon 5 into the NR1 subunit has previously been shown to
reduce the proton sensitivity of NR1-1/NR2 receptors (Traynelis et al.,
1995). Because of the similar effects of exon 5 on proton and
Zn2+ inhibition of receptors that contain the full
length NR1 subunit, we tested whether C-terminal splicing of NR1
controlled the effects of exon 5 on proton inhibition of heteromeric
receptors in a parallel manner to Zn2+ inhibition.
Figure 1, D and E, compares the effects of NR1
RNA splicing on proton and Zn2+ inhibition of
NR1/NR2B receptors and shows that exon 5 relieves proton inhibition
irrespective of C-terminal splicing of NR1 subunit. Similar results for
proton inhibition have previously been reported for NR1 splice variants
expressed in Xenopus oocytes in the absence of NR2 subunits
[Traynelis et al. (1995), their footnote 13]. Figure
2A compares more
directly the effects of exon 5 on proton and Zn2+
inhibition of various C-terminal splice variants of the NMDA receptor.
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Figure 2.
Comparison of the effects of exon 5 on proton and
Zn2+ inhibition. A, Ratio of the
fitted IC50 values for proton (closed bars)
and Zn2+ (open bars) inhibition of
NR1 splice variants coexpressed with NR2B. B, Ratio of
the fitted IC50 values for proton and
Zn2+ inhibition of NR1-1 splice variants coexpressed
with different NR2 subunits. Data describing proton inhibition for NR2A
and C are from Traynelis et al. (1995). Data for proton inhibition of
NR2B are from Figure 1; IC50 values for proton inhibition
of NR1-1a/NR2D and NR1-1b/NR2D were 71 (n = 5) and
154 nM (n = 10), respectively.
C, Correlation between the effectiveness of exon 5 to
relieve proton and Zn2+ inhibition for different NR1
splice variants coexpressed with different NR2 subunits. NR1 variants
that use the alternate acceptor splice site within exon 22 are not
shown because of the oocyte-dependent variability in the effects of
exon 5.
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The NR2 subunit controls the relief of voltage-independent
Zn2+ inhibition of exon 5
The NR2 subunit is known to control allosteric regulation of NMDA
receptors. For example, both extracellular Mg2+ and
polyamines can relieve tonic proton inhibition of NMDA receptors that
contain the NR2B subunit, but not other NR2 subunits (Williams et al.,
1994; Zhang et al., 1994; Paoletti et al., 1995; Traynelis et al.,
1995; Williams, 1995). To test whether the NR2 subunit influences the
ability of exon 5 to decrease voltage-independent Zn2+ inhibition, we constructed
Zn2+ inhibition curves for receptors comprised
of NR1-1 ± exon 5 and each of the NR2 subunits. Table
1 summarizes these data, which show that
exon 5 can reduce the sensitivity to extracellular
Zn2+ of receptors that contain NR1-1 and NR2A or
NR2B by 4-fold to 10-fold, although the IC50 for
voltage-independent Zn2+ inhibition differs by more
than an order of magnitude between NR2A- and NR2B-containing receptors
(Paoletti et al., 1997). By contrast, not only did we find that
receptors comprised of NR1-1 plus NR2C (Williams, 1996; Chen et al.,
1997) or NR2D subunits were much less sensitive to extracellular
Zn2+, but exon 5 was also less effective in reducing
the sensitivity of these receptors to Zn2+ (Table
1). Because exon 5 exerts similar effects on both the proton and
Zn2+ sensitivity of NR1 splice variants (Fig.
2A), we also compared the effects of different NR2
subunits on proton and Zn2+ sensitivity of NMDA
receptors. As shown in Figure 2, B and C, we
found a correlation between the effects of exon 5 on proton and
Zn2+ sensitivity for various heteromeric receptors.
This finding, together with similar results from NR1 splice variants,
raises the possibility that inhibition by Zn2+ and
protons might share common structural or functional determinants.
Although we can detect no channel block by 10 µM
Zn2+ at approximately 30 mV (see Materials and
Methods), it is still possible that some of the higher
Zn2+ concentrations required to inhibit exon
5-containing receptors might complicate our evaluation of
voltage-independent inhibition by causing some channel block. To test
whether exon 5 acts only on voltage-independent Zn2+
inhibition, we examined Zn2+ inhibition of receptors
that carried NR1 mutations, NR1-1a(N616R) and NR1-1b(N637R), that have
been reported to relieve voltage-dependent Zn2+
inhibition (Mori et al., 1992; Kawajiri and Dingledine, 1993; Sakurada
et al., 1993). Evaluation of the Zn2+ effects of
these mutant NR1 subunits showed that although they are somewhat less
sensitive to voltage-independent inhibition of Zn2+
(see Fig. 4B), exon 5 still increases the
IC50 value for Zn2+ inhibition to a
similar extent as in wild-type receptors (Table 1). This result
supports the idea that the actions of exon 5 cannot be explained by
effects on voltage-dependent block at high concentrations of
Zn2+.
Common structural determinants of proton and
Zn2+ inhibition
The ability of exon 5 to reduce Zn2+ inhibition
suggests that voltage-independent Zn2+ inhibition
may share structural features with proton inhibition, further
supporting an emerging trend of convergence among allosteric modulatory
systems (see Discussion). To evaluate whether a structural argument can
be made in support of the idea that Zn2+ and protons
act through convergent mechanisms to inhibit NMDA receptors, we tested
whether mutations in exon 5 that alter its ability to relieve proton
inhibition similarly alter its ability to relieve
Zn2+ inhibition. Figure
3 summarizes these experiments and shows
that residues including and near Lys211 control the
effects of exon 5 on Zn2+ inhibition. Figure
4A shows that a strong
correlation exists between the ability of these various exon 5 mutations to relieve proton and Zn2+ inhibition,
consistent with the idea that similar structural components within exon
5 influence both proton and Zn2+ inhibition. Does
the correlation between NR1-1b mutations within exon 5 that influence
Zn2+ and proton inhibition extend to other portions
of the receptor? We (Burgess et al., 1996) and others (Kashiwagi et
al., 1997) have observed that mutations at Asn616 in
the M2 pore-forming region of the molecule can reduce the sensitivity
of the receptor to protons. Figure 4B shows that this decrease in pH sensitivity is also correlated with a decrease in
Zn2+ sensitivity (Table 1). In addition, we have
also now extended the results of Sullivan et al. (1994) to show that
two extracellular cysteine residues (Cys744 and
Cys798 in NR1-1a) that control redox modulation,
proton inhibition, polyamine relief of proton inhibition, and
Zn2+ potentiation of NR1-1a expressed in
Xenopus oocytes in the absence of NR2 subunits (J. M. Sullivan and S. F. Traynelis, unpublished data) also control
Zn2+ inhibition of heteromeric receptors. We also
report that several acidic residues that have previously been shown to
alter proton inhibition of NMDA receptor function (Williams et al.,
1995; Kashiwagi et al., 1996; K. Williams, unpublished observations)
cause similar changes in the Zn2+ sensitivity (Fig.
4B). The tight correlation between proton and Zn2+ IC50 values across these various
NMDA receptor mutants provide strong evidence that some elements of the
mechanism that control proton and Zn2+ inhibition
must be similar and that common structural determinants of proton and
Zn2+ inhibition are spread throughout the NR1
subunit.
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Figure 3.
Structural determinants within exon 5 that control
Zn2+ inhibition. A, Typical recording
of mutant NMDA receptor NR1-1b(K211R)/NR2B current responses from which
Zn2+ inhibition curves were determined. The
thick bars (top) show application of
solutions nominally containing 0, 100, 30, 10, 3, or 1 µM
Zn2+ plus maximal glutamate and glycine. Before
recording glutamate responses in the presence 30 and 100 µM Zn2+, the oocyte was prewashed with
30 and 100 µM Zn2+ to control for any
shift in the preresponse leak current. Measurements of the preresponse
baseline (open circles) and the peak response
(open squares) were made from the same position relative
to the response waveform. B, Broken lines
show the Zn2+ inhibition curves for wild-type
NR1-1a/NR2B and NR1-1b/NR2B receptors. Symbols show
inhibition of NR1-1b/NR2B receptors with amino acid substitutions at
amino acid positions 207, 208, and 211 in the NR1-1b subunit.
C, IC50 values were determined for
Zn2+ inhibition of NR1-1b/NR2B receptors containing
the mutations indicated (open bars). The filled
bar shows the IC50 values for receptors comprised
of wild-type NR1-1a/NR2B for comparison. The broken line
shows the IC50 of wild-type NR1-1b/NR2B. The amino acid
sequence of exon 5 is written above (capitals letters are amino acids
encoded by exon 5). Full inhibition curves for mutant receptors labeled
in bold are shown in B. Numbers in parentheses show the
number of oocytes used to construct each inhibition curve. m207-211
describes an NR1-1b mutant subunit that contains three amino acid
substitutions (K207G, R208G, and K211G); m192-194 contains NR1-1b
mutations K192G, K193G, and R194G, and m197-205 contains NR1-1b
mutations E197A, D200A, and D205A.
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Figure 4.
Common structural determinants of pH and
Zn2+ inhibition. A, There is a strong
correlation between the pH and Zn2+ sensitivity of
the NMDA receptor across a variety of different NR1 exon 5 mutations.
All Zn2+ inhibition curves were determined from
heteromeric receptors consisting of NR1-1/NR2B subunits;
IC50 values describing pH inhibition are data from NR1
subunits expressed in Xenopus oocytes in the
absence of NR2 (Traynelis et al., 1995), except for wild-type NR1-1b,
NR1-1b(m207-211), NR1-1b(K214G), and NR1-1b(K211G), which were
determined from heteromeric receptors containing NR2B. The amino acids
encoded by exon 5 are shown above, with the appropriate symbol denoting
the residues that were altered in the triple mutants to G (m192-194,
m207-211) or A (m197-205). B, There is a strong
correlation between pH and Zn2+ sensitivity for
NR1-1 mutations outside exon 5 that reduce pH sensitivity. All
Zn2+ and proton IC50 values were
determined from responses of heteromeric NR1-1/NR2B receptors
recorded at five or more different proton or Zn2+
concentrations. The position of residues with marked effects on proton
and Zn2+ sensitivity are shown on a linear
representation of the NR1 subunit; M1-4 denote membrane-associated
regions. For all experiments, the number of oocytes recorded for each
proton or Zn2+ inhibition curve ranged between 7 and
40. Broken lines show linear regressions.
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Interactions between exon 5 and NR1 mutations
To evaluate the interactions between exon 5 and NR1 mutations that
alter proton and Zn2+ inhibition, we used
thermodynamic cycles (Carter et al., 1984) to calculate the coupling
energy between the effects of exon 5 and various NR1 mutations on
proton inhibition. The coupling energy is the difference in free energy
with respect to proton inhibition between the NR1-1 exon 5-containing
mutant receptor and receptors containing wild-type NR1 + exon 5 and
mutant NR1 exon 5. For our calculations we assume that the
IC50 values we measure approximate the
KD for proton binding to a single
site. We did not examine the coupling energy with respect to
Zn2+ because its IC50 is decreased
by the mutations to a point at which further reduction by exon 5 might
not be fully detectable because of voltage-dependent channel block by
high Zn2+ concentrations. Figure
5 shows the independent effects on proton inhibition of exon 5 and NR1-1 mutations (coexpressed with NR2B) within
a pore-forming domain, at a potential site of disulfide linkage, and at
an acidic residue. As a control for the reliability of this approach,
we examined the mutations at two conserved extracellular cysteine
residues (Cys744 and Cys798 in
NR1-1a) that control a variety of important features of NMDA receptor
function and may combine to form a disulfide bridge (Sullivan et al.,
1994). As expected, the coupling coefficient was less than that
predicted for independent effects of amino acid substitutions at the
two cysteine residues, consistent with the idea that the hypothesized
disulfide linkage mediates the effects of these residues on the proton
sensor. The predicted independence of the effects of exon 5 and
mutations that alter the pH inhibition suggest that these perturbations
in proton sensitivity are additive and thus occur by different
mechanisms.
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Figure 5.
Thermodynamic cycle analysis of mutant NR1
subunits. The mutations in exon 5 containing NR1-1b subunits are
labeled with the numbering of NR1-1a subunits for clarity. However, the
position of these mutations should be increased by 21 residues because
of insertion of exon 5. The actual numbering of the mutant pairs is (± exon 5) E342Q/E363Q, N616R/N637R, C744A/C765A, and C798A/C819A. The
coupling coefficient was calculated as indicated assuming our
experimental IC50 values approximate the
KD values for protonation. The coupling energies
( G) in kilocalories per mole for these various mutations and
splice variants are 0.23 for E342/E363Q ± exon 5, 0.05 for
N616/N637R ± exon 5, 0.12 for C745/C765A ± exon 5, 0.17 for C798/C819A ± exon 5, and 0.8 for C745A and C798A. Proton
IC50 values not included in Figures 3 or 4 are (in
nM): 1071 NR1-1b(E363Q)/NR2B, 446 NR1-1b(N637R)/NR2B, 558 NR1-1b(C765A), 688 NR1-1b(C819A), and 135 NR1-1a(C744A, C798A)/NR2B.
All inhibition curves were constructed for wild-type and mutant NR1-1a
and NR1-1b subunits coexpressed with NR2B except for NR1-1b(C765A) and
NR1-1b(C819A), which were expressed in Xenopus oocytes
in the absence of NR2 (Sullivan et al., 1994).
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Polyamine relief of voltage-independent
Zn2+ inhibition
Polyamines such as spermine have been shown to relieve tonic
proton inhibition in a manner analogous to the effects of exon 5, suggesting that some extracellular modulatory systems (protons and
spermine) previously considered to be independent actually work through
convergent pathways (Traynelis et al., 1995). To further examine the
links between proton and Zn2+ inhibition, we tested
whether polyamines can mimic the effects of exon 5 on
Zn2+ inhibition in an manner analogous to the
ability of polyamines to mimic exon 5-induced relief of proton
inhibition. Because both Zn2+ (see above) and
polyamines can block the NMDA receptor pore, we coexpressed
NR1-1a(N616R) and NR1-1b(N637R) with NR2B to minimize complications
attributable to voltage-dependent effects of Zn2+
(Mori et al., 1992; Kawajiri and Dingledine, 1993; Sakurada et al.,
1993) and polyamines (Kashiwagi et al., 1997; see Materials and
Methods). Because polyamines can have both potentiating and inhibiting
actions, we first performed experiments to ensure that these mutations,
which abolished voltage-dependent polyamine block, did not abolish the
ability of polyamines to relieve tonic proton inhibition and potentiate
receptor function. Figure
6A shows the voltage-dependent inhibition by polyamines as well as
voltage-independent relief of proton block (i.e., polyamine
potentiation). Figure 6, B and C, confirms that
replacement of the M2 asparagine with arginine removes
voltage-dependent channel block by spermine (Kashiwagi et al., 1997)
and also shows that potentiation of the current response by polyamines
is reduced. Such a result could occur if the M2 mutation altered the
ability of spermine to interact with the receptor, or if the pore
mutations we used unexpectedly decreased the proton sensitivity. To
distinguish between these two possibilities, we examined the proton
sensitivity of NR1-1a(N616R)/NR2B receptors. The experiments described
in Figure 6D-F confirm that the M2 pore mutations decrease the proton sensitivity of the NMDA receptors and
show that the level of decreased polyamine potentiation can be fully
accounted for from the reduced proton sensitivity (i.e., less tonic
inhibition to relieve means less potentiation by polyamines). These
results suggest that the interaction between polyamines and receptors
containing either NR1-1a or NR1-1a(N616R) subunits in combination with
NR2B are similar. Kashiwagi et al. (1997) reported similar effects of
pore mutations on pH inhibition while these experiments were in
progress.
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Figure 6.
Effects of mutations in the NR1-1 M2 pore-forming
region on proton inhibition and spermine relief of proton inhibition.
A, Potentiation and block of NR1-1a/NR2B receptors by
extracellular spermine (SP) is evident in the
current-voltage relationship. B, Exchange of
Asn616 in NR1 for Arg relieves voltage-dependent
channel block by spermine at hyperpolarized potentials (see Materials
and Methods), but also attenuates spermine potentiation of receptor
responses. C, Ratio of the current response obtained in
the presence of 1 mM spermine to the control response
recorded at fixed holding potentials illustrates more clearly both
potentiation and block of wild-type NR1-1a/NR2B receptors by spermine
(open squares, n = 6-8 oocytes at
each holding potential). Filled squares show relief of
voltage-dependent channel block by spermine and attenuation of spermine
potentiation at pH 7.3 for mutant NR1 subunits (n = 9-11 oocytes at each holding potential). Identical results were found
for 0.1 mM spermine (n = 56, data not
shown). D, Composite proton inhibition curves were
constructed for wild-type NR1-1a/NR2B receptors (63 oocytes) and
NR1-1a(N616R)/NR2B (48 oocytes) receptors by normalizing current
responses recorded at different pH values to those observed at pH 7.6 and expressing the mean response at each pH as a percent of the fitted
maximum (see Materials and Methods). NR1 N616R decreased the amount of
tonic proton inhibition for receptors that both lack and contain exon 5 (data not shown; n = 15 oocytes). E,
Spermine potentiation, defined as the current recorded in the presence
of 300 µM spermine expressed as a percent of the control
current response, is shown as a function of pH for NR1-exon 5/NR2B
receptors (data from 45 oocytes) and NR1-1a(N616R)/NR2B receptors
(n = 37). F, Plotting the degree of
spermine potentiation against the degree of proton inhibition for the
same data show that there is no apparent difference in the effect of
spermine on wild-type and mutant receptors. Differences in spermine
potentiation shown in panels C and E
therefore result from the change in pH sensitivity shown in
D.
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To test whether voltage-independent Zn2+ inhibition,
like proton inhibition, can be relieved by both exon 5 and polyamines, we constructed Zn2+ inhibition curves in the absence
and presence of 300 µM spermine for NR1-1a(N616R)/NR2B
and NR1-1b(N637R)/NR2B receptors. In the absence of exon 5, spermine
caused an increase in the IC50 for Zn2+
inhibition from 4 to 24 µM (Fig.
7A; a sixfold shift). However, for NR1 subunits that contained exon 5, spermine had much more modest
effects, shifting the Zn2+ IC50 value
from 65 to 151 µM (Fig. 7B; a 2.3-fold shift).
These data suggest that spermine can increase the IC50
value for Zn2+ in an exon 5-dependent manner. If we
assume that spermine-Zn2+ binding (discussed below)
only minimally influences free Zn2+ concentration,
then we can evaluate the concentration-effect relationship for
spermine relief of Zn2+ inhibition by examining the
current response in the presence of 10 µM
Zn2+ with or without a variable concentration of
spermine present (Fig. 7C). The result of this experiment
suggests that spermine can relieve Zn2+
inhibition almost completely with an EC50 value of ~90
µM (n = 15) under these conditions.
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Figure 7.
Relief of Zn2+ inhibition by
spermine. A, B, To test whether spermine
also relieves voltage-independent Zn2+ inhibition in
an manner analogous to the effect of exon 5, Zn2+
inhibition curves were constructed for exon 5-lacking
NR1-1a(N616R)/NR2B receptors in the absence and presence of 300 µM spermine. This concentration of spermine induces no
channel block of these mutant receptors (Fig. 5C).
Spermine shifted the IC50 for Zn2+
inhibition of receptor function by sixfold when exon 5 was absent
(A) and by 2.3-fold for receptors that contain
exon 5 (B). C, Top
panel, Zn2+ inhibition of glutamate-evoked
NR1-1a(N616R)/NR2B receptor-mediated currents recorded in either 10 or
100 µM spermine at 30 mV. Calibration: 150 sec, 50 nA.
Bottom panel, Spermine decreases the inhibition of
NR1-1a(N616R)/NR2B receptor responses observed in 10 µM
Zn2+ in a dose-dependent manner with an
IC50 value of 90 µM; spermine can similarly
relieve the small amount of inhibition of exon 5-containing
NR1-b(N637R)/NR2B receptors as well (open circles).
D, To test whether the actions of spermine persist in
the absence of polyamine potentiation, we evaluated the effect of
spermine on Zn2+ inhibition at pH 8.0 at which there
is no spermine potentiation (i.e., because there is no inhibition by
protons). Under these conditions, we still observe relief of
Zn2+ inhibition, consistent with the idea that
spermine, like exon 5, can relieve Zn2+ inhibition.
Numbers in parentheses are the number of oocytes.
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Because spermine potentiates the NMDA receptors, any reduction by
Zn2+ of polyamine potentiation would reduce current
response amplitude and thus, appear as an opposing effect to polyamine
relief of Zn2+ inhibition. That is, the current
recorded in the presence of both spermine and Zn2+
might be less than that recorded in spermine alone in part because of
interference of spermine potentiation by Zn2+. This
could cause an underestimation of the effects of spermine on
Zn2+ inhibition. To evaluate the effects of spermine
on Zn2+ inhibition without this confounding
variable, we tested the effects of spermine on Zn2+
inhibition at pH 8.0, a pH value at which there is almost no tonic
proton inhibition, and thus, no polyamine potentiation (Fig. 7D). Even in the absence of any potentiating actions of
polyamines, we still see a relief of Zn2+ inhibition
by polyamines to approximately the same level reported at pH 7.3. Similar results to those shown in Figure 6, C and
D, were found when the above experiments were repeated with
wild-type NR1-1/NR2B receptors that lack or contain exon 5 (n = 45 oocytes; data not shown).
The role of spermine-Zn(II) complexation in spermine relief of
Zn2+ inhibition
Polyamines are known to chelate transition metals such as copper
and zinc under certain conditions. For example, two spermine molecules
can complex with Zn2+, and their interaction is
stabilized at alkaline pH and by certain anions (Antonelli et al.,
1984; Wojciechowska et al., 1991). Accordingly, it is important to
consider whether the effects of spermine we observe on
voltage-independent Zn2+ inhibition of NMDA receptor
function simply reflect spermine binding of Zn2+.
Although extensive data exists for Cu(II)-polyamine complexes as a
function of pH, considerably less data exists for Zn(II)-polyamine complexes at different pH values. Although most Cu2+
is complexed with spermine at physiological pH (Palmer and Powell, 1974; Wojciechowska et al., 1991), Zn2+ is known to
form weaker complexes with polyamines (Antonelli et al., 1984;
Wojciechowska et al., 1991). For example, at pH 6, <2% of the
Zn2+ is complexed with spermine compared with 24%
of Cu2+.
Could the relief of Zn2+ inhibition of NMDA receptor
function by spermine that we observe be explained by spermine chelation of Zn2+? Two experimental observations make this
unlikely. First, it is not possible to account for the different
effects of spermine on receptors that lack and contain exon 5 by simply
removing a fraction of the extracellular Zn2+ from
the solution. Second, spermine-transition metal complexes are
pH-sensitive, with metal-spermine complexes being more prevalent at
alkaline pH. However, we observe similar relief by spermine of
Zn2+ inhibition at pH 6.6, 7.3, and 8.0. Zn2+ at 10 µM inhibited NR1-exon
5(N616R)/NR2B responses to 48 ± 5% (pH 6.6; n = 8), 47 ± 3% (pH 7.3; n = 6), and 48 ± 4%
(pH 8.0; n = 11) of control, and this level of
inhibition was reduced by 300 µM spermine to 77 ± 3% (pH 6.6), 76 ± 3% (pH 7.3), and 75 ± 2% (pH 8.0) of
control (p > 0.4 by ANOVA). Thus, if
significant Zn2+-spermine complexation does occur
at pH 7.3, our data suggest that either the complexed ion is
biologically active or the spermine-water exchange rate of the complex
is fast enough to allow Zn2+ to rapidly interact
with a binding site on the receptor protein.
Interactions between protons and Zn2+
Does Zn2+ inhibition involve an enhancement of
the proton sensitivity of the NMDA receptor such that tonic proton
inhibition is enhanced in the presence of Zn2+? Such
a mechanism has been proposed to underlie inhibition of NMDA receptors
by phenylethanolamines such as ifenprodil and its analogs (Mott et al.,
1998). We performed two experiments to evaluate the functional
interaction between proton and Zn2+ inhibition.
First, we measured the Zn2+ sensitivity of NMDA
receptors at different pH values to assess whether alkaline pH relieved
Zn2+ inhibition. We found that that NR1-1a/NR2B
receptors are similarly sensitive to extracellular
Zn2+ at both pH 8.0 (IC50, 2.2 µM; n = 10) and pH 6.6 (IC50, 3.0 µM; n = 10)
pH values, compared with pH 7.3 (IC50, 2.5 µM; Table 1). Modest effects of pH are expected because
the ionization of residues that interact with Zn2+
almost certainly will be affected by this pH change. However, the lack
of a strong monotonic relationship between pH and
Zn2+ sensitivity is inconsistent with the idea that
Zn2+ inhibits receptor function by enhancement of pH
sensitivity. Second, we tested whether extracellular
Zn2+ could alter proton sensitivity. Consistent with
our idea that Zn2+ inhibition does not reflect an
enhancement of proton inhibition, we find no significant difference in
proton inhibition at pH 6.6 or 6.8 in the absence or presence of 1 µM extracellular Zn2+
(n = 19; p > 0.4; minimum detectable
difference 17%). Zn2+ appears to slightly enhance
potentiation of current responses by pH 8.0 (122 ± 6% of
control; p < 0.05). Given the limited solubility of
hydroxide-Zn(II) complexes that form at alkaline pH values (see Eq. 3,
Materials and Methods), we might expect an enhanced current at pH 8.0 versus pH 7.3 if a small portion of the Zn2+
precipitated slowly out of solution at pH 8.0. These data support the
conclusion that Zn2+ does not inhibit receptors by
enhancing the pH sensitivity through a stabilization of the protonated
state. Moreover, the data also show that NMDA receptor responses
partially inhibited by protons are sensitive to
Zn2+, and vice versa.
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DISCUSSION |
The two most important conclusions to emerge from this study are
(1) that alternative splice variants of the NR1 subunit control voltage-independent inhibition by extracellular Zn2+
and (2) that this form of Zn2+ inhibition shares
several prominent structural and functional features with proton
inhibition. The effects of alternative exon splicing on
Zn2+ inhibition have interesting implications for
the role of Zn2+ in information processing.
Postsynaptic neurons that contact presynaptic terminals that release
Zn2+ in a Ca2+-dependent manner
might include NR1 exon 5 in the mature mRNA to either relieve tonic
inhibition of postsynaptic receptor function by ambient levels of
Zn2+ in the extracellular space (Zheng et al., 1998)
or shift NMDA receptor responsiveness to synaptically released
Zn2+. On a structural level, the three parallels
that we have described between protons and Zn2+
inhibition (similar sensitivity to NR1 exon 5, polyamines, and NR1
mutations) suggest these two modulatory sites either contain overlapping structural components or converge on a common downstream mechanism.
Control of Zn2+ inhibition by alternative
exon splicing
NR1 exon 5 has widespread and interesting effects on NMDA receptor
function (Durand et al., 1992, 1993; Sugihara et al., 1992; Hollmann et
al., 1993). Exon 5 is thought to encode an extracellular surface loop
that may act as a tethered ligand that can influence several
extracellular allosteric modulators, including protons and polyamines,
as well as potentiation by Zn2+ of NR1 subunits
expressed without NR2 in Xenopus oocytes (Durand et al.,
1992, 1993; Hollmann et al., 1993, Westbrook, 1994; Traynelis et al.,
1995). Thus, the ability of exon 5 to influence voltage-independent inhibition by extracellular protons and Zn2+ fits
well with the idea that this portion of the protein may control access
of extracellular ions to a complex regulatory site on the extracellular
surface of the receptor. What ramifications does this altered
sensitivity have for neurobiology? Exon 5 is expressed in a
region-specific manner and regulated throughout development of the
nervous system (Laurie and Seeburg, 1994; Nash et al., 1997; Paupard et
al., 1997; Weiss et al., 1998). Unfortunately, no data exist that allow
a direct comparison of the relative abundance of NMDA receptors that
lack and contain NR1 exon 5. However, it seems safe to suggest that in
regions such as hippocampal CA3, in which exon 5 is expressed and
Zn2+ is released, that the presence of exon
5-containing NR1 subunits should decrease the response of the neurons
to both ambient levels of Zn2+ as well as
synaptically released Zn2+.
Common functional and structural determinants of proton and
Zn2+ inhibition
The idea that the structural determinants of proton
and Zn2+ binding might overlap was first suggested
on the basis of the similarity between the pKa values of amino acid
residues that frequently coordinate Zn2+ and the
IC50 for proton inhibition of NMDA receptors (Peters et
al., 1987; Westbrook and Mayer, 1987; Traynelis and Cull-Candy, 1990,
1991; Traynelis, 1998). However, it is a particularly thorny issue to
address with functional studies, given the proposed mechanism of action
for protons. Traynelis and Cull-Candy (1991) have argued that a
protonated NMDA receptor is nonfunctional, suggesting that the proton
inhibition curve reflects an increasing fraction at any moment of
receptors that remain only briefly protonated. If this view proves
correct, it explains the additive rather than synergistic effects of
proton and Zn2+ inhibition. This is because a
nonfunctional protonated receptor, regardless of whether it binds
Zn2+, does not contribute to the current response.
Thus the properties of residual currents at acidic pH should mirror
properties of larger currents at alkaline pH because only unprotonated
and thus fully functional receptors contribute to the response.
There are several possible ways to explain our data describing parallel
effects of spermine, exon 5, and selected NR1 residues on proton and
Zn2+ inhibition. Zn2+-binding to
any protein reflects its coordination chemistry, which is flexible for
Zn2+ given its pseudonoble gas configuration
(d10). Zn2+ is known
to form tetrahedral, trigonal-bipyramid, square pyramid, octahedral,
and other complexes in free solution (Huhey, 1983), and additional
geometries could be dictated by the structure and flexibility of the
binding site within the protein. In other
Zn2+-binding proteins, typically at least three
electron pairs are provided by the protein to hybrid
Zn2+ orbitals (Frausto da Silva and Williams, 1991);
water or additional amino acid residues may satisfy the other 1-3
coordination sites, depending on the coordination geometry. If we
assume at least three electron donors exist that define the
Zn2+-binding site on the NMDA receptor (Fig.
8), we then must hypothesize that the
neutralization of the partial or formal negative charge on these
residues caused by Zn2+ binding is sufficient to
shut down receptor function. Alternatively, binding of
Zn2+ might decrease the degrees of freedom of one of
the electron-donating groups and thus prevent the conformational
changes necessary for channel opening.
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Figure 8.
Structural models of proton and
Zn2+ action. A, If the proton sensor
is a single ionizable amino acid residue
(X) rather than carbohydrate or lipid, it
could be physically distinct from the residues that bind
Zn2+ (R1-3). At least three
electron pairs are usually required to coordinate
Zn2+; fourth and/or fifth electron pairs could be
provided by water or other amino acid residue(s) depending on
coordination geometry. In this scenario, the acidic NR1 amino acid
residues (D, E) studied in Figures 3 and
4 could enhance the manner by which both protons or
Zn2+-binding influence channel gating, rather than
altering the binding of either ion. Both exon 5 and polyamines are
shown influencing the local microenvironment (gray
area) of each regulatory site, which could shift the pKa of the
proton sensor and alter the association of Zn2+ with
its binding site. B, Although the proton sensor and
Zn2+-binding site might be physically distinct,
certain acidic residues alternatively could enhance the
electronegativity and partial negative charge of the proton sensor
and electron donors to Zn2+, thereby
decreasing the KD for proton and
Zn2+ binding. Exon 5 and polyamines could
influence the proton- and Zn2+-binding sites
indirectly through association with these acidic residues, although
this idea contradicts the additive effects of exon 5 and mutation E342Q
in NR1. C, If a single amino acid residue constitutes
the proton sensor, it might contribute to the Zn2+
coordination site. This would transmit the effects of acidic residues
that control the pKa of the proton sensor directly to the affinity of
Zn2+ for its binding site. Exon 5 and
polyamines could, by shielding the proton sensor, directly influence
Zn2+ binding. Alternatively, exon 5 and
polyamines might alter the pKa of the proton sensor by interacting with
acidic residues that themselves influence the electronegativity of the
proton sensor (but see Fig. 5).
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At this juncture there are three different models that could explain
our data. First, we could hypothesize that protons bind to a site
separate from any of the electron donors to Zn2+
(Fig. 8A), and that binding of
Zn2+ or protons inhibits receptor function by
controlling in a similar manner movement of the channel gate. This
functional convergence could create the parallel effects we observe of
nonexon 5 NR1 mutations on both proton and Zn2+
effects, provided all of these mutations uncouple both
Zn2+ and proton binding from the downstream
mechanisms. The effects of polyamines and exon 5 that we observe on
proton and Zn2+ inhibition are simplest to explain
if both binding sites are close enough so that their local environments
can be influenced by charged polyamines or residues near
Lys211 in exon 5. Second, the proton and
Zn2+-binding sites could be separate but under the
control of a handful of NR1 anionic residues
(Glu181, Glu342,
Asp669, etc.) that increase the partial negative
charge on the proton sensor and are electron donors to
Zn2+. In this scenario, the positive charges on exon
5 and polyamines could interact with these residues to reduce their
effects on the proton and Zn2+-binding sites (Fig.
8B), although this idea is hard to reconcile with the
additive effects of exon 5 and certain anionic residues (Fig. 5).
Third, the proton sensor could be a part of the
Zn2+-binding site. In this scenario, similar effects
on protein charge distribution and conformation might follow either
Zn2+ binding to its coordination site or protonation
of a critical electron donor to Zn2+ (Fig.
8C). Both spermine and exon 5 could reduce the effectiveness of Zn2+ and protons in this model by shielding the
proton sensor to shift its pKa (Traynelis et al., 1995), while at the
same time altering the ionic microenvironment of the
Zn2+-binding site and/or interfering with
Zn2+ access to the site. Alternatively, exon 5 and
polyamines might interact with a variety of acidic residues that
control the partial charge on the proton sensor (but see Fig. 5). An
important part of this idea is that spermine and exon 5 must not
associate so tightly with the proton sensor-electron donor so as to
neutralize the partially or formally charged amino acid side chains,
because such neutralization would inhibit the receptor (rather than
relieve proton and Zn2+ inhibition). This last model
postulates three unique molecular interactions at the same binding
site: protonation, coordination of a metal ion, and shielding.
Whereas identification of the proton sensor and residues that
coordinate Zn2+ would help in the evaluation of
these models, efforts to find these sites using site-directed
mutagenesis are likely to be fraught with interpretative difficulties.
Removal of a residue that contributes, say, to Zn2+
binding may cause only a shift in its IC50 rather than
abolish Zn2+ binding if other nearby residues can
also donate electron pairs, albeit less willingly. This reduces the
search for Zn2+-binding partners to interpretation
of partial shifts in the Zn2+ sensitivity. Although
construction of multiple mutations might pinpoint additional candidate
residues that interact with Zn2+ (Fig. 5), the
number of mutants to be i
Top
Abstract
Introduction
![1/(1+[<UP>OH</UP><SUP><UP>−</UP></SUP>]K<SUB><UP>S1</UP></SUB>+[<UP>OH</UP><SUP><UP>−</UP></SUP>]<SUP>2</SUP>K<SUB><UP>S2</UP></SUB>+[<UP>OH</UP><SUP><UP>−</UP></SUP>]<SUP>3</SUP>K<SUB><UP>S3</UP></SUB>+[<UP>OH</UP><SUP><UP>−</UP></SUP>]<SUP>4</SUP>K<SUB><UP>S4</UP></SUB>).](/content/vol18/issue16/fulltext/6163/img004.gif)
![(100−<IT>minimum</IT>)/(1+([<UP>Zn</UP><SUP>2<UP>+</UP></SUP>]/<UP>IC</UP><SUB>50</SUB>)<SUP>n</SUP>)+<IT>minimum </IT>](/content/vol18/issue16/fulltext/6163/img006.gif)
