1Department of Pharmacology and 2Department of Physiology and Biophysics, Georgetown University School of Medicine, Washington, DC 20007
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ABSTRACT |
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Rumbaugh, Gavin, Kate Prybylowski, Jian Feng Wang, and Stefano Vicini. Exon 5 and Spermine Regulate Deactivation of NMDA Receptor Subtypes. J. Neurophysiol. 83: 1300-1306, 2000. Deactivation of N-methyl-D-aspartate (NMDA) channels after brief agonist exposure determines the duration of their synaptic activation during excitatory neurotransmission. We performed patch-clamp recordings of L-glutamate responses from human embryonic kidney tumoral cells (HEK293) expressing NR1 subunit variants lacking exon 5 together with the NR2B subunit. These responses had deactivation components that lasted several seconds. The presence of exon 5 or spermine greatly accelerated deactivation of L-glutamate responses through alterations in desensitization. These effects were also observed at positive holding potentials and in the presence of physiological Mg2+. Thus NR1 splicing and polyamines may have profound effects on the kinetics of NMDA receptor-mediated synaptic transmission.
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INTRODUCTION |
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N-methyl-D-aspartate (NMDA)
receptors have unique properties that distinguish them from other
ligand-gated ion channels. They are blocked by magnesium at negative
potentials, modulated by polyamines and hydrogen ions, and require
glycine as co-agonist (Dingledine et al. 1999;
McBain and Mayer 1994
). NMDA receptors are composed of
the NR1 subunit and at least one copy of either NR2A, NR2B, NR2C, or
NR2D. The type of NR2 subunit determines agonist affinity, magnesium
sensitivity, deactivation kinetics, modulation by polyamines, and
channel conductance (Dingledine et al. 1999
;
McBain and Mayer 1994
). Alternative splicing in the NR1
subunit gene has been reported for an N terminal cassette, exon 5, and
two C terminal cassettes, exon 21 and exon 22 (Sugihara et al.
1992
). Combinations of these cassettes can produce eight different isoforms. Exon 5 splicing results in receptors with distinct
sensitivity to hydrogen ions, zinc, and polyamines, whereas the
presence of C terminal cassettes affect the regulation by protein
kinase C (Zukin and Bennet 1995
). In this study, we
present the first evidence that NR1 splicing of the N terminal cassette can affect deactivation of NMDA receptor channels.
Using ultrarapid agonist applications by means of a piezoelectric
translator, one can study the deactivation properties of channels in
excised patches with agonist concentration and time course relevant to
those occurring during synaptic transmission (Clements et al.
1992). Electrophysiological studies of recombinant NMDA
receptors expressed in mammalian cells indicated that the NR2 subunit
strongly influences deactivation (Monyer et al. 1994
; Vicini et al. 1998
). On the other hand, we have
previously shown that deactivation kinetics did not change when NR1
isoforms were differentially expressed with the NR2A subunit
(Vicini et al. 1998
). Here we extend the study to
recombinant receptors comprising NR1 subunit splice variants with the
NR2B subunit, which has been recently demonstrated to enhance learning
and memory in mice (Tang et al. 1999
) and is widely
expressed in the mammalian brain (Monyer et al. 1994
;
Zukin and Bennet 1995
).
Polyamines such as spermine and spermidine increase peak current,
decrease single-channel conductance, slow NMDA channel desensitization, and decrease affinity for glutamate (Benveniste and Mayer
1993; Lerma 1992
; Rock and MacDonald
1992
; Williams 1994a
). Yet the action of
spermine on deactivation kinetics, relevant for excitatory synaptic
transmission, has not been investigated. Given the structural similarities between of spermine and exon 5 (Traynelis et al. 1995
), we also compared the effect of spermine on deactivation of NMDA responses from distinct recombinant receptors.
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METHODS |
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NMDA receptor expression vector and transfection
Transfection and subcloning of NMDA receptor subunit cDNAs were
performed as described in greater detail in Vicini et al. (1998). Throughout our work we use for simplicity the
terminology of Sugihara et al. (1992)
in defining the
spliced forms of the NR1 subunits (NR1a, NR1b, NR1e, and NR1g).
According to the terminology of Hollmann et al. (1993)
,
these subunits are designated as NR1-1a, NR1-1b, NR1-4a, and
NR1-4b. The NRla cDNA was subcloned in the pRc/CMV vector (Invitrogen,
Carlsbad, CA), whereas all other cDNAs were into pcDNA I/Amp
(Invitrogen). The parent NR1a plasmid was a gift of Dr. Shigetada
Nakanishi, Kyoto University Faculty of Medicine, Kyoto; the NR2B vector
was a gift of Dr. Richard Huganir (Johns Hopkins University); and the
NR1b, NR1e, and NR1g plasmids were gifts of Dr. Jim Boulter (University
of California, Los Angeles).
Human embryonic kidney 293 cells (HEK293, American Type Culture Collection, Rockville, MD, ATCC No. CRL1573) were grown in minimal essential medium (GIBCO BRL, Gaithersburg, MD), supplemented with 10% fetal bovine serum. Exponentially growing cells were plated on 12-mm glass cover slips (Fisher Scientific, Pittsburgh, PA) and transfected with rat NMDA receptor subunit cDNAs using calcium phosphate precipitation. Studies on the recombinantly expressed receptors were performed within 2-3 days after transfection, and data were obtained for a given subunit combination transfected at least three different times. Cotransfection with pGreenLantern-1 (GIBCO BRL) allowed ready recognition of transfected cells.
Electrophysiology and rapid agonist applications
Electrophysiology and rapid agonist applications were also
performed as described (Vicini et al. 1998). Briefly,
transfected HEK293 cells were studied at room temperature (20-22°C).
Bath solution contained (in mM) 145 NaCl, 5 KCl, 2 CaCl2, and 5 HEPES-NaOH (pH 7.4). Whole cell and
outside-out patch recordings were performed with a patch-clamp
amplifier (Axopatch 200B, Axon Instruments, Foster City, CA) after
capacitance and series resistance compensation. Intracellular (patch
pipette) solutions contained (in mM) 145 Kgluconate, 5 MgCl2, 5 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA), 5 ATP, and 10 HEPES, pH 7.2 with KOH. L-Glutamate and L-cysteate (Sigma, St. Louis, MO) stock solutions in
water at pH 7.4 were diluted to their final concentrations in bath
solution. For fast application of agonists, we used a piezoelectric
translator (PZ 150 M Burleigh Instrument, Fishers, NY). After each
patch recording, ON and OFF rates, as well as
pulse durations, were measured by "blowing out" the patch and
recording currents generated by the liquid junction potential due to a
50:1 dilution of the agonist containing solution (Lester and
Jahr 1992
). As described (Vicini et al. 1998
),
responses in small lifted cells were comparable with those obtained in
excised patches (see also Table 1).
Spermine tetrahydrochloride (Sigma, St. Louis, MO) was added to both
control and L-glutamate-containing solutions and the pH
readjusted. Currents were filtered at 3 kHz with an 8-pole low-pass
Bessel filter (Frequency Devices, Haverhill, MA) and digitized at 10 kHz using an IBM-compatible microcomputer equipped with a Digidata 1200 data acquisition board (Axon Instruments) and PClamp 8 software (Axon
Instruments). Off-line data analysis, curve fitting, and figure
preparation were performed with Origin (MicroCal Software, Northampton,
MA) and PClamp 8 software. Fitting of decay times of the averaged
L-glutamate-activated currents was performed using a
simplex algorithm based on a least-squares exponential fitting routine.
Double exponential equations of the form
I(t) = If *
exp(
t/
f) + Is *
exp(
t/
s), where
If and Is are
the amplitudes of the fast and slow decay components, and
f and
s are their
respective decay time constants used to fit the data. A comparison of
the summed square deviation was used to estimate the quality of single versus double exponential fits. To compare decay time between different
subunit combinations, we used a weighted mean decay time constant
w = [If/(If + Is)] * tf + [Is/(If + Is)] * ts.
Data values are expressed as means ± SE unless otherwise indicated.
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RESULTS |
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In contrast to receptors comprising NR1 splice variants with the
NR2A subunit (Vicini et al. 1998), cotransfection of the NR2B subunit with NR1 splice variants with and without exon 5 resulted
in significant differences in deactivation. As shown in Fig.
1A, the deactivation of
current produced by brief application of glutamate was described by a
double exponential function. The weighted deactivation time constant
(
w) in small lifted cells expressing
NR1a/NR2B (
exon5) subunits was four times larger than that in cells
expressing NR1b/NR2B (+exon5) subunits (Table 1). Faster deactivation
in the presence of exon 5 was also observed with NR1 splice variants
that did not contain C-terminus cassettes. Similar results were
obtained from excised outside-out patches (Table 1). The
w in cells expressing NR1e/NR2B subunits was
also larger than in cells expressing NR1g/NR2B subunits (Table 1). The
persistent channel openings during L-glutamate responses in
NR1a/2B transfections, as illustrated in Fig. 1B, produced a
slow deactivation lasting several seconds.
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The similar action of spermine and exon 5 on proton sensitivity of NMDA
receptor has prompted the discovery of structural similarities between
polyamines and the surface loop of exon 5 (Traynelis et al.
1995). We therefore investigated the effect of spermine on
deactivation of NMDA responses from distinct recombinant receptors. As
illustrated in Fig. 2, spermine
significantly accelerated the decay of responses from cells transfected
with NR1a/NR2B cDNAs, whereas it was ineffective on cells transfected
with the NR1b variant (containing exon 5).
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A decreased entry and a faster recovery from desensitization, as well as decreased affinity, may explain why the presence of both exon 5 and spermine produced faster deactivating responses. Therefore we compared the entry into and recovery from desensitization between responses from cells transfected with NR1a/NR2B (n = 12) and NR1b/NR2B (n = 8) with and without spermine. As illustrated in Fig. 3, A and B, the rate of entry into desensitization was significantly slower for NR1b/NR2B receptors than NR1a/NR2B receptors. The rate of recovery was also slower for NR1a/NR2B receptors (Fig. 3, C and D). Time constants of double exponential curves used for fitting the recovery time course were 22 and 1,230 ms (40% contribution of the slower component to peak amplitude) for NR1a/NR2B responses and 35 and 950 ms (17%) for NR1b/NR2B responses. Spermine was effective in decreasing the entry into desensitization with NR1a/NR2B but not with NR1b/NR2B responses (Fig. 3B), and it accelerated the recovery from desensitization of NR1a/NR2B responses (Fig. 3D). Rate constants for recovery of NR1a/NR2B responses in the presence of spermine were 17 and 1,980 ms (20%). These results taken together indicate that the major effect exon 5 and spermine have on the NR1/NR2B NMDA receptor subtype is to accelerate deactivation by decreasing entry into desensitization and increasing recovery. This is also confirmed by the much greater contribution of the fast deactivation component to the peak response with both spermine (Fig. 2C) and exon 5 (Fig. 1 legend).
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The presence of exon 5 (Durand et al. 1992) or spermine
(Williams 1994a
) decrease agonist affinity. Because
agonist affinity affects deactivation (Lester and Jahr
1992
), we also investigated the action of spermine on the much
faster currents produced by rapid application of the low-affinity
agonist, L-cysteate. As originally reported (Lester
and Jahr 1992
), the fast deactivation for these low-affinity
agonists is caused by increased unbinding rates that result in reduced
entry into desensitization. Responses to brief applications of
L-cysteate were slower for NR1a/NR2B than NR1b/NR2B
receptors. Deactivation of currents produced in transfected cells by
rapid application of L-cysteate (20 mM, 8 ms) were
characterized by single exponential curves. The time constant in eight
cells expressing NR1a/NR2B subunits was 43 ± 5 ms and in 10 cells
expressing NR1b/NR2B subunits was 29 ± 3 ms (P < 0.05, independent t-test).
Extracellular Mg2+ produce a glycine-independent
and subunit-specific potentiation of NMDA responses, and this
potentiation has been proposed to occur at the same site as spermine
(Paoletti et al. 1995). This raises the possibility that
the action of both spermine and exon 5 may not have physiological
relevance. We therefore investigated NMDA responses in cells
transfected with NR1a/NR2B subunit cDNA in the presence of a
physiological concentration of Mg2+ (Fig.
4). For these experiments, recordings
were performed at positive holding potentials to remove the
voltage-dependent Mg2+ blockade (McBain
and Mayer 1994
). As illustrated in Fig. 4, A and
B, 2 mM Mg2+ failed to alter the
deactivation of NMDA currents. In addition, when spermine was added to
Mg2+, the weighted time constants of the
responses were significantly decreased. However, when 10 mM
Mg2+ was used, a small but significant shortening
of the decay time of NMDA responses was observed
(
w 650 ± 62 ms in control and 430 ± 35 ms with 10 mM, Mg2+
n = 4). The difference in
w
between NR1a/NR2B and NR1b/NR2B receptor was maintained at positive
potentials (not shown).
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DISCUSSION |
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We demonstrate for the first time that NMDA currents produced by
receptors comprising distinct NR1 splice variants differ in
deactivation. For cells expressing NR1a/NR2B receptors, persistent channel openings lasting several seconds were observed. Similar prolonged deactivation components have been observed in patch responses
and in NMDA-mediated excitatory postsynaptic currents (NMDA-EPSCs) from
hippocampal pyramidal neurons (Kirson and Yaari 1996; Spruston et al. 1995
) implying a
substantial contribution of NR1a/2B channels to native NMDA receptors.
We also observed that effect of exon 5 on deactivation of NMDA
responses from NR1a/NR2B transfected cells is mimicked by spermine. Our
report that spermine accelerates deactivation kinetics is relevant
for excitatory synaptic transmission. In fact, NMDA-EPSCs at
autaptic synapses in hippocampal neurons in culture are accelerated by
histamine acting on the spermine site (Bekkers 1994).
However, these findings could not be repeated for NMDA responses in
hippocampal slices (Bekkers et al. 1996
), possibly due
to the variability of action of histamine and spermine on distinct
receptor subtypes. Indeed, the presence of the NR1 subunit with exon 5 as well as the presence of the NR2A subunit prevents the action of
spermine on the NMDA receptor (Durand 1992
;
Williams et al. 1994
). Similar observations were
reported with histamine (Williams 1994b
).
Slow deactivation of NMDA receptors is determined by entry and
exit from desensitized states (Lester and Jahr 1992).
Given the reported decreased entry into desensitization with polyamines such as spermine and spermidine (Benveniste and Mayer
1993
; Lerma 1992
; Williams
1994a
), one would predict deactivation to be accelerated. We
confirmed this prediction and propose that a decreased entry and a
faster recovery from desensitization may explain in part why the
presence of both exon 5 and spermine produced faster deactivating responses. The fast component of deactivation is mainly related to
unbinding and is determined by agonist affinity (Lester and Jahr
1992
). A contribution to faster deactivation could also be expected from the reported decrease in agonist affinity with exon 5 (Durand et al. 1992
) or spermine (Williams
1994a
). Indeed, we observed that the fast decay components of
L-glutamate and L-cysteate responses were
shorter with exon 5. Additionally, the fast decay component of
L-glutamate responses in NR1a/NR2B cells also became shorter with spermine.
The action of both spermine and exon 5 in the presence of physiological
Mg2+ confers physiological relevance to our findings. In
support of this observation, Williams et al. (1994)
failed to observed competition between Mg2+ and spermine
with 1 mM Mg2+. It is possible, however, that
Mg2+ and spermine may have similar actions at
nonphysiological Mg2+ concentrations (10 mM) as observed by
Paoletti et al. (1995)
. Our finding that spermine
decreases deactivation at both positive and negative holding voltages
also indicates that the observed effect is not related to the reported
voltage-dependent channel blocking mechanisms (Rock and
MacDonald 1992
). In the presence of Mg2+, the
faster voltage-dependent block at negative holding potential masks the
action of spermine. However, as the membrane depolarizes the action of
spermine should be felt even in physiological Mg2+.
A decrease in NMDA-EPSC decay during development has been proposed to
underlie a reduction in plasticity of excitatory synapses relevant to
the so-called "critical period" (Carmignoto and
Vicini 1992; Crair and Malenka 1995
;
Hestrin 1992
). Evidence is growing that the
developmental increase in the NR2A subunit may play a major role in
these changes (Flint et al. 1997
; Stocca and
Vicini 1998
; Tovar and Westbrook 1999
). Our
results imply that when the NR2B subunit is expressed, NR1 splicing can
also play an important role in developmental changes in NMDA-EPSC
kinetics. Indeed, developmental regulation and cell-specific expression
of NR1 mRNA splice variants has been shown by in situ hybridization
(Laurie and Seeburg 1994
; Paupard et al.
1997
). Specifically, the NR1a spliced forms were widely and
abundantly distributed throughout the brain, whereas the NR1b variants
were located in similar patterns in fewer areas. A clear developmental
increase of NR1b forms occurs in areas such as the thalamus and in
cerebellar granule neurons (Laurie and Seeburg 1994
;
Paupard et al. 1997
). One can speculate that in these
areas a developmental change of NMDA-EPSC kinetics would parallel the
increased expression of exon 5 in cell populations expressing the NR2B
subunit. A rapid turnover of the NR1 subunit has recently been
demonstrated in mammalian neurons (Huh and Wenthold 1999
). One could also speculate that an activity-dependent,
rapid control of kinetics of NMDA responses could be achieved through changes in the expression of NR1 splice variants. In support of this
hypothesis, transformer-2-
, a specific factor that is activity dependent in humans, controls splicing of several proteins including the NR1 subunit (Daoud et al. 1999
).
NMDA receptor channels with long deactivation would be persistently
active in consequence to spontaneous synaptic release. This may have
relevance for the reported trophic activity of NMDA (Sheetz and
Constantine-Paton 1994). At the same time, however, the
capability to remain active for longer periods would enhance calcium
entry, possibly leading to neurotoxic damage in specific pathological
conditions. Indeed, neurotoxic death of retinal ganglion cells has been
associated with enhanced expression of NR1a mRNA and protein after
optic nerve crush (Kreutz et al. 1998
). In contrast, the
presence of exon 5 and spermine may increase excitotoxicity in the
focal injury area during ischemia-induced acidosis by decreasing proton
inhibition of NMDA receptors (Traynelis et al. 1995
). If exon 5 is associated with neurotoxic damage, our finding that splicing
of NR1 subunit determines functional properties of NMDA responses
together with the reported differential distribution of splice forms in
the basal ganglia (Standaert et al. 1993
) may be related
to the pathogenesis of Parkinson's disease and other neurological disorders.
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ACKNOWLEDGMENTS |
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We are grateful to Dr. Barry B. Wolfe, Dr. Wei Jian Zhu, and S. Grossman for critical reading of the manuscript and Dr. Dennis R. Grayson (University of Illinois, Chicago) for subcloning NR1b, e, and g plasmids into eukaryotic expression vectors.
This work was supported by National Institute of Mental Health Grants R01 MH-58946, KO2 MH-01680, and F31-MH-11943.
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FOOTNOTES |
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Address for reprint requests: S. Vicini, Dept. of Physiology and Biophysics, Georgetown University School of Medicine, 3900 Reservoir Rd., NW, Washington, DC 20007.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 September 1999; accepted in final form 15 November 1999.
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REFERENCES |
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