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Department of Pharmacology, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania (J.C., K.W.),
Groupe de Recherche
en Thérapeutique Anticancéreuse,
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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The effects of several N-sulfonyl-polyamines, including
N1-dansyl-spermine (N1-DnsSpm) and
N1-(n-octanesulfonyl)-spermine
(N1-OsSpm), were studied at recombinant
N-methyl-D-aspartate (NMDA) receptors expressed
in Xenopus laevis oocytes. N1-DnsSpm and
N1-OsSpm inhibited NMDA receptors and were ~1000-fold
more potent than spermine in oocytes voltage-clamped at 
70 mV. Block
by N1-DnsSpm and N1-OsSpm was strongly voltage
dependent, being more pronounced at hyperpolarized membrane potentials.
With the Woodhull model of voltage-dependent channel block, the values
of Kd(0) were 779 µM, 882 µM, and 7.4 mM and those of z
were 2.58, 2.57, and 1.07 for
N1-DnsSpm, N1-OsSpm, and spermine,
respectively. This suggests that an increase in the voltage dependence
of block together with an increase in affinity contributes to the
increased potencies of N1-DnsSpm and N1-OsSpm
compared with spermine. Sensitivity to N1-DnsSpm was
reduced by mutation NR1(N616Q) and was increased by mutations
NR1(N616G) and NR2A(N615G). The NR1(N616G) and NR2A(N615G) mutations
decreased the Kd(0) value of
N1-DnsSpm without affecting z, whereas the NR1(N616Q)
mutation reduced z. These mutations may alter the accessibility of
part of the polyamine binding site within the channel pore or directly alter the properties of that site. Block by N1-DnsSpm (0.3 µM) was almost complete at 100 mV, and there was no relief of block at extreme negative membrane potentials (100 to
200 mV) at wild-type NR1/NR2A channels. In contrast, block by
N1-DnsSpm was partially relieved at extreme negative
potentials at receptors containing NR1(N616G) or NR2A(N615G),
suggesting that N1-DnsSpm can permeate these mutant
channels but not wild-type NR1/NR2A channels. This is hypothesized to
be due to an increase in the pore size of channels containing
NR1(N616G) or NR2A(N615G), which allows passage of the bulky head group
of N1-DnsSpm. In contrast to N1-DnsSpm,
N1-OsSpm could easily permeate wild-type NR1/NR2A channels,
presumably because the head group of N1-OsSpm can pass
through the narrowest part of the channel pore. N-Sulfonyl-polyamines such as N1-DnsSpm and
N1-OsSpm represent a new class of polyamine antagonists
with which to study glutamate receptor ion channels.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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The endogenous polyamine spermine has a variety of effects on NMDA and non-NMDA glutamate receptors (1, 2). At NMDA receptors, spermine has both stimulatory and inhibitory effects when applied extracellularly (3-7). Inhibition of NMDA receptors by spermine is strongly voltage dependent and may be caused by an open-channel block and/or screening of surface charges around the mouth or vestibule of the ion channel (3, 5, 8). Intracellular spermine can block the ion channel of some subtypes of AMPA and kainate receptors, an effect that is responsible for inward rectification of these receptors (9-12) and may be mechanistically similar to the block of inward-rectifier K+ channels by polyamines (13, 14).
When applied extracellularly, spermine is a relatively weak antagonist
at NMDA receptors and at polyamine-sensitive AMPA and kainate
receptors, blocking these receptors at high micromolar to millimolar
concentrations (3, 4, 10, 15). A number of polyamine-conjugated spider
and wasp toxins are more potent antagonists than spermine at glutamate
receptors (16). These toxins, which include the philanthotoxins,
argiotoxins, and 
-agatoxins, are characterized structurally by the
presence of an aromatic amino acid head group linked through a
carbonamide bond to a polyamine tail such as spermine or a pentamine or
hexamine (17-21). Because of their potencies and specificities,
polyamine-conjugated toxins are potentially valuable tools for studying
the pharmacological and structural properties of glutamate receptor ion
channels and as tools to discriminate subtypes of native glutamate
receptors. However, it is often difficult to obtain these toxins
because they have to be purified from spider venom, requiring access to the appropriate spiders, or to be synthesized in the laboratory. The
syntheses of argiotoxins and -agatoxins, which are potent NMDA
channel blockers, are far from straightforward (22), and commercially
available toxins are often prohibitively expensive.
To look for polyamine derivatives that have activities similar to those of the polyamine-conjugated spider toxins, we have studied the properties of several N1-substituted polyamines. Derivatives of spermine and spermidine, such as N1-DnsSpm and N1-OsSpm (Fig. 1), that have an alkyl- or aryl-sulfonyl group attached to one of the terminal amino groups, were recently found to be potent inhibitors of calmodulin-activated phosphodiesterase activity and of polyamine uptake.1 These compounds are stable and are relatively easy to synthesize. In the current study, N1-DnsSpm and N1-OsSpm were found to be potent voltage-dependent blockers that could differentially block and/or permeate recombinant NMDA receptors. N1-Sulfonyl-polyamines are useful new tools to study polyamine block of glutamate receptors and channel structure of those receptors.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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Expression in oocytes and voltage-clamp recording.
The
preparation of cRNAs and the preparation, injection, and maintenance of
oocytes were carried out as described previously (7, 23, 24). Oocytes
were injected with NR1 plus NR2 cRNAs in a ratio of 1:5 (0.5-4 ng of
NR1 plus 2.5-20 ng of NR2). For experiments with NR2C and NR2D, the
mouse cDNA clones 
3 and 4 were used (25, 26).
. Oocytes were continuously superfused
(~5 ml/min) with a Mg2+-free saline solution (96 mM NaCl, 2 mM KCl, 1.8 mM
BaCl2, 10 mM HEPES, pH 7.5), which contained
BaCl2 rather than CaCl2 to minimize
Ca2+-activated Cl
currents (27).
In most experiments, oocytes were injected with K+-BAPTA
(50-100 nl of 40 mM, pH 7.4) on the day of recording to eliminate a slowly activating Cl current that is seen
even in the presence of extracellular Ba2+ (23).
I-V curves were measured by using linear voltage ramps over 6-24 sec
as described in Results. In some experiments, control ramps with
glutamate were measured before and after ramps with polyamines and the
control ramps were averaged. In other experiments, control ramps were
measured before but not after ramps with polyamines. In all
experiments, leak currents were measured with ramps before and after
the test ramps, and leak currents were digitally subtracted.
Site-directed mutagenesis. The NR1 mutants were prepared by using a 2.6-kb SphI/SalI fragment of plasmid pN60 (28) inserted into the same sites of M13mp18 (29). Similarly, the NR2 mutants were prepared using a 2.2-kb BamHI/XmaI fragment of pBSNR2A and a 2.1-kb BamHI/SphI fragment of PBSNR2B inserted into the same sites of M13mp18 and M13mp19, respectively. Mutagenesis was carried out according to the method of Kunkel et al. (30) or Sayers et al. (31) with the Sculptor in vitro mutagenesis system (Amersham International, Buckinghamshire, UK). The oligonucleotides for preparation of mutants were CAA TGC CGG AGC CGA GCA GGA CGC (antisense) for NR1(N616G), GCC TGG TCT TCC AGA ATT CTG TGC C (sense) for NR2A(N614Q), TGG TCT TCA ACC AGT CTG TGC CTG T (sense) for NR2A(N615Q), TGG TCT TCA ACG GTT CTG TGC CTG (sense) for NR2A(N615G), GTC TGG TGT TTC AGA ACT CCG TAC C (sense) for NR2B(N615Q), and TGG TGT TTA ACC AGT CCG TAC CTG T (sense) for NR2B(N616Q) (mutated nucleotides are underlined). Mutated DNA fragments were isolated from the replicative form of M13 and religated into the corresponding sites of pN60, pBSNR2A, and pBSNR2B. Mutations were confirmed by DNA sequencing (32). The NR1(N616Q) mutant (33) was provided by Dr. S. Nakanishi (Institute for Immunology, Kyoto University Faculty of Medicine, Kyoto, Japan). The NR1(N616R) mutant (34) was provided by Dr. R. J. Dingledine (Department of Pharmacology, Emory University, Atlanta, GA). Amino acids are numbered from the initiator methionine in NR1 and NR2 clones (28, 35). This numbering system differs from the system used in some laboratories in which amino acids are numbered from the first residue in the mature peptide (36). Thus, residues NR1(N616) and NR2A(N615) correspond to NR1(N598) and NR2A(N596), respectively, in the article by Wollmuth et al. (36).
Data analysis. Data analysis and curve fitting were carried out using Axograph (Axon Instruments) or SigmaPlot (Jandel Scientific, San Rafael, CA) on Macintosh computers. To obtain values for the IC50 and Hill slope (nH) of antagonists, concentration-inhibition curves were fit to eq.1:
![I<SUB>glu + PA</SUB> = 100/1 + ([antagonist]/IC<SUB>50</SUB>)<SUP><IT>n</IT><SUB><IT>H</IT></SUB></SUP>](/content/vol51/issue5/fulltext/861/img001.gif)
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(1) |
![I<SUB>glu + PA</SUB>/I<SUB>glu</SUB> = &agr;/[1 + {[PA]/<IT>K</IT><SUB><IT>d</IT></SUB>(<IT>0</IT>)<IT> </IT>exp (z&dgr;FV/RT)}]](/content/vol51/issue5/fulltext/861/img002.gif)
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(2) |
is the fraction of the block that is
voltage-dependent, Kd (0) is the
equilibrium dissociation constant of the polyamine at a transmembrane
potential of 0 mV, z is the charge of the polyamine, is the
fraction of the membrane electric field sensed by the polyamine at its
binding site within that field, F is the Faraday constant, R is the gas
constant, and T is the absolute temperature. We included the function
in eq. 2 because in some oocytes the response to glutamate in the
presence of the polyamine was not fully relieved at positive
potentials, possibly because the polyamines have an additional
voltage-independent component of block or because the I-V curve in the
presence of polyamine was measured after the control I-V curve with
glutamate and the response to glutamate showed a small run-down or
run-up over time.
Materials.
The syntheses of N1-DnsSpm,
N1-OsSpm, N1-DnsSpd, and N8-DnsSpd
(hydrochloride salts) are reported elsewhere.1
L-Glutamate and glycine were purchased from Sigma Chemical
(St. Louis, MO). Spermine tetrahydrochloride was purchased from Aldrich Chemical (Milwaukee, WI) or Calbiochem (San Diego, CA). The NR1 clone
(28) was a gift from Dr. S. Nakanishi (Institute for Immunology, Kyoto
University, Kyoto, Japan). The splice variant of NR1 used in these
studies was NR1A (28, 38). The NR2A and NR2B clones (39) were gifts
from Dr. P.H. Seeburg (Center for Molecular Biology, University of
Heidelberg, Germany). The 3 and 4 (mouse NR2C and NR2D) clones
(25, 26) were gifts from Dr. M. Mishina (Department of Pharmacology,
University of Tokyo, Japan).
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Results |
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Summary
Introduction
Procedures
Results
Discussion
References
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Potencies of polyamine derivatives.
Polyamines and their
N-dansylated derivatives inhibited responses to glutamate
and glycine at NR1/NR2A receptors. The potencies of spermine,
spermidine, and their derivatives were measured in oocytes
voltage-clamped at 70 mV (Fig. 2 and Table
1). N1-DnsSpm was 1700-fold more potent than
the parent compound spermine (Fig. 2 and Table 1). Similarly,
N-dansylation at either terminal amino group of spermidine
produced compounds, N1-DnsSpd and N8-DnsSpd,
that were 200-400-fold more potent than spermidine (Table 1). To
determine whether the dansyl moiety itself was a potent NMDA receptor
antagonist, we measured the effects of dansylamide and of
dansylethylamide at NR1/NR2A receptors in oocytes voltage-clamped at
70 mV. These two compounds, at a concentration of 10 µM, inhibited responses to glutamate by only
5-9% (data not shown). Thus, the potent inhibitory effects of
dansylated polyamines do not lie within the dansyl group itself and
require the polyamine tail in addition to the dansyl head group.
Another derivative of spermine, N1-OsSpm (Fig. 1), which
has an N1-alkyl rather than an N1-aryl
substitution, had a potency similar to that of N1-DnsSpm
(Table 1). The subunit-specificity of N1-DnsSpm was
determined by measuring its potency at NR1/NR2 receptors containing
different NR2 subunits. N1-DnsSpm was ~50-fold more
potent at NR1/NR2A and NR1/NR2B receptors than at NR1/NR2C and NR1/NR2D
receptors (Table 1). The block of NR1/NR2A receptors by
N1-DnsSpm (0.3 µM) was noncompetitive
with respect to glutamate and glycine, with the degree of block being
unaffected by concentrations of glutamate and glycine over a range of
0.3-10 µM (data not shown).
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20 mV to minimize
voltage-dependent block. No stimulation by N1-DnsSpm was
seen when experiments were carried out with high (10 µM)
or low (0.1 µM) concentrations of glycine (data not
shown). This suggests that polyamine stimulation does not occur with
concentrations of N1-DnsSpm that produce a profound
voltage-dependent block and that, if N1-DnsSpm does have
stimulatory effects at NMDA receptors, the potency of those effects is
not increased to the same extent as the potency of the
voltage-dependent block. Thus, although it remains possible that
N1-DnsSpm can have stimulatory effects on NMDA receptors,
such effects are not seen when studying macroscopic currents using low
micromolar concentrations of N1-DnsSpm. These observations
are consistent with the hypothesis that the stimulatory effects of
spermine and related polyamines involve binding sites that are outside
the ion channel pore, distinct from the site that mediates
voltage-dependent block (1, 3, 6). Experiments were also carried out to
look for stimulation by high (10-100 µM) concentrations
of N1-DnsSpm at NR1/NR2B receptors (data not shown), but
those concentrations produced a large block of glutamate responses even
at depolarized membrane potentials, which may mask stimulatory effects
of N1-DnsSpm.
Voltage dependence of block and effects of mutations in the M2
regions of NR1 and NR2.
The inhibitory effects of spermine and of
polyamine-derived spider toxins are voltage dependent. Therefore,
experiments were carried out to study the voltage dependence of block
by N1-sulfonyl-polyamines. In some experiments, we measured
steady state currents induced by glutamate or glutamate plus
N1-DnsSpm in oocytes voltage-clamped at different holding
potentials. Inhibition by N1-DnsSpm was strongly voltage
dependent, being more pronounced at hyperpolarized than at depolarized
membrane potentials (data not shown). The voltage dependence of block
by N1-DnsSpm and N1-OsSpm was studied
quantitatively by using voltage ramps analyzed according to the model
of Woodhull (37) (Fig. 3 and Tables 2 and
3). At wild-type NR1/NR2A receptors, the values of
Kd(0) were similar for
N1-DnsSpm (779 µM) and
N1-OsSpm (882 µM), as were the values for
z (2.58 for N1-DnsSpm and 2.57 for N1-OsSpm;
Tables 2 and 3). In these two polyamine derivatives, the sulfonamide
nitrogen is a weak acid and is not charged at pH 7.5, and the
dimethylamino group on the dansyl moiety of N1-DnsSpm is
also uncharged at this pH. Thus, N1-DnsSpm and
N1-OsSpm each have a total charge of +3, due to protonation
of the amino groups in the polyamine tail. Assuming that all three
amino groups enter the transmembrane electric field (i.e., z = 3),
the value of (the average depth of the transmembrane field sensed by the polyamines) is 0.86. We also studied block by spermine (300 µM) at NR1/NR2A receptors by using voltage ramps. The
value of Kd(0) was 7.4 ± 3.5 mM and that of z was 1.07 ± 0.17 (mean ± standard error, 5 oocytes) for block by spermine. These
values are similar to those reported for block by spermine at native NMDA receptors on cultured hippocampal neurons, at which the
Kd(0) value was 27 mM and the z value was 1.17 (3).
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are unchanged (Fig. 3 and Table 2). In contrast, the
NR1(N616Q) mutation had only a small effect on the
Kd(0) of N1-DnsSpm,
but this mutation significantly reduced the value of z for
N1-DnsSpm (Fig. 3 and Table 2).
We also studied the effects of some of the mutations in M2 on block of
NR1/NR2A receptors by N1-OsSpm. IC50 values for
N1-OsSpm were derived from concentration-inhibition curves
using oocytes voltage-clamped at 70 mV. The potency of
N1-OsSpm at NR1/NR2A receptors at 70 mV was reduced by
mutation NR1(N616Q) and increased by mutation NR2A(N615G) (Table 3),
similar to the effects of these mutations on the potency of
N1-DnsSpm. However, mutation NR1(N616G), which increases
the potency of N1-DnsSpm, had no effect on the potency of
N1-OsSpm (Table 3). The voltage dependence of block by
N1-OsSpm was studied at wild-type and mutant receptors by
using voltage ramps and Woodhull modeling. Because N1-OsSpm
showed marked permeation of wild-type and mutant channels (see below),
we used concentrations of N1-OsSpm that were 5-10-fold
higher than their IC50 values measured at 70 mV. Mutation
NR1(N616Q) did not alter the Kd(0)
value of block by N1-OsSpm but significantly reduced
the value of z (Table 3). In contrast, mutation NR2A(N615G) reduced
the Kd(0) of
N1-OsSpm without affecting the z (Table 3). These
results are similar to the effects of NR1(N616Q) and NR2A(N615G) on the
z and Kd(0) values of block by
N1-DnsSpm (Table 2).
An aspartate residue (D669) in the extracellular loop of NR1, just
distal to the M3 segment, has been found to influence stimulation by
spermine and inhibition by protons (44). This residue also has a small
influence on voltage-dependent block by spermine. Mutations that
neutralize the negative charge at D669 (D669N and D669A) reduced
voltage-dependent block by spermine, whereas a mutation that retains
the negative charge (D669E) did not alter voltage-dependent block by
spermine, although all three mutations decrease sensitivity to pH and
to spermine stimulation (44). It was proposed that screening of the
negative charge at D669 of NMDA receptors may contribute to
voltage-dependent block by spermine (44). It is notable that in the
linear amino acid sequence D669 is in a position analogous to D652 of
the GluR1 subunit of AMPA receptors, a position also occupied by an
aspartate residue in all other GluR subunits. In a study that generated
three-dimensional models of this region of GluR1 and GluR6 subunits,
GluR1(D652) (equivalent to D669 in NR1) was one of two acidic residues
that, after agonist binding, was proposed to move to a position near the mouth of the ion channel (45). This residue could be important for
attracting cations to the channel (45). If D669 in NR1 occupies a
position in the tertiary structure of NR1 analogous to that of D652 in
GluR1 (45), then NR1(D669) could be positioned near the entrance of the
ion channel of NMDA receptors. In this case, the inhibitory effect of
spermine could be due in part to an interaction of an amino group of
spermine with NR1(D669), masking the negative charge at D669, and thus
reducing the attraction of Na+ and Ba2+ and,
consequently, the unitary conductance of the channel. To determine
whether D669 influences block by N1-DnsSpm, we measured the
effects of 1 µM N1-DnsSpm on responses to
glutamate (10 µM; with 10 µM glycine) at
NR1/NR2A receptors containing NR1(D669E) and NR1(D669N) in oocytes
voltage-clamped at 70 mV. N1-DnsSpm inhibited responses
to glutamate by 77 ± 5% (wild-type), 80 ± 2% (D669E), and
58 ± 2% (D669N; p < 0.01, one-way analysis of
variance with Dunnett's test). Thus, mutation D669N but not D669E
reduces block by N1-DnsSpm, similar to effects seen with
spermine (44), although the effect of the NR1(D669N) mutation is much
smaller than the effects of mutations at NR1(N616). This suggests that
charge screening, at least of residue D669, contributes only in small
part to block by N1-DnsSpm. It may be that
N1-DnsSpm has more points of interaction with the channel
pore than does spermine, and thus mutations at one contact point (D669) have a smaller effect on block by N1-DnsSpm than on block
by spermine.
Permeation through wild-type and mutant channels.
A number of
different characteristics have been reported for the voltage-dependent
block of macroscopic NMDA currents by spermine and polyamine analogs.
Spermine produces an incomplete block of NMDA receptors even at
extremely hyperpolarized potentials of 100 to 200 mV (3, 46). The
shallow slope conductance and incomplete block seen with spermine (Fig.
5A) (3, 46) may reflect permeation of spermine through
the ion channel of NMDA receptors (3) or screening of surface charges
rather than fast channel block (8). Some polyamine analogs, such as
1,10-diaminodecane, seem to act as classic channel blockers, producing
a complete block of macroscopic currents (47, 48). Other analogs, in particular, long-chain penta-amines such as BE4444, cause a complete block of NMDA responses, but the block is relieved by 
50% at extreme
negative membrane potentials, presumably reflecting permeation of
BE4444 through the ion channel of NMDA receptors (46). To determine
whether the N1-sulfonyl-polyamines can permeate NMDA
channels, we compared the effects of spermine with those of
N1-DnsSpm and N1-OsSpm at extreme negative
potentials (Fig. 5) by using concentrations of spermine (300 µM), N1-DnsSpm (0.3 µM), and
N1-OsSpm (0.3 µM) that are close to the
IC50 values for these antagonists at 70 mV (see Table 1).
N1-DnsSpm produced a complete block of NR1/NR2A receptors
at membrane potentials of ~100 mV, and little or no recovery of the
response was seen at extreme negative potentials (Fig. 5B). In
contrast, block by N1-OsSpm was incomplete and showed a
partial recovery at extreme negative membrane potentials (Fig. 5C).
These results suggest that N1-OsSpm, but not
N1-DnsSpm, can easily permeate the ion channel of NR1/NR2A
receptors.
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70 mV (Table 4). These concentrations of
N1-DnsSpm produced a 60-80% block of glutamate responses
at 80 mV in the wild-type and mutant receptors (Fig. 6B). I-V curves were constructed by using linear voltage ramps from 200 or 185 mV
to +40 mV at a rate of 9-10 mV/sec (Fig. 6A). To measure recovery from
block (which is assumed to reflect permeation of
N1-DnsSpm), the ratio of the glutamate-induced current in
the absence and presence of N1-DnsSpm was measured at 80
and 170 mV (Fig. 6B). This ratio will be larger at 170 mV than at
80 mV if there is recovery from block at extreme negative membrane
potentials. At wild-type receptors, no recovery was seen at 170 mV.
However, a pronounced recovery was seen at NR1(N616G)/NR2A,
NR1/NR2A(N615G), and NR1(N616Q)/NR2A(N615G) receptors (Fig. 6). These
data suggest that N1-DnsSpm, which does not easily permeate
wild-type NR1/NR2A receptors, can readily permeate receptors containing
NR1(N616G) or NR2A(N615G). We also studied permeation at channels
containing NR1(N616Q) together with wild-type NR2A and at channels
containing NR2A(N615Q) together with NR1(N616Q) (Fig. 6); permeation by
N1-DnsSpm was not seen at these mutants. Thus, the increase
in permeation of N1-DnsSpm is selective for the NR1(N616G)
and NR2A(N615G) mutations.
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Discussion |
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Summary
Introduction
Procedures
Results
Discussion
References
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One of the goals of this study was to identify polyamine
derivatives that share the potencies of the polyamine-conjugated toxins
such as the argiotoxins and philanthotoxins but are easier to
synthesize than those toxins. The sulfonamide polyamine derivatives described here represent a new class of polyamine antagonists of
glutamate receptors. Because these compounds are stable and their
syntheses are straightforward, N1-DnsSpm,
N1-OsSpm, and related analogs should be useful tools for
studies of glutamate receptor ion channels. The
N1-substituted polyamines that we have studied were
200-1700-fold more potent than the parent compounds, with
N1-DnsSpm and N1-OsSpm being the most potent.
Indeed, N1-DnsSpm (IC50 = 0.3 µM
at 70 mV) was only ~30-fold less potent than
argiotoxin636 and Agel-505 (IC50 = 0.01 µM at 70 mV) (18, 19). The potency of
N1-DnsSpm is similar to that reported for
philanthotoxin-343 (IC50 = 10-56 µM at 60
mV) (20, 21). Although N1-DnsSpm and N1-OsSpm
were ~1000-fold more potent than spermine at 70 mV, the values of
Kd(0) determined for these compounds
(800-900 µM) were only ~10-fold lower than
the Kd(0) value for spermine (7.4 mM). However, N1-DnsSpm and
N1-OsSpm showed a much steeper voltage dependence (z = 2.58) than did spermine (z = 1.07), suggesting that an increase in
voltage dependence, together with a modest increase in the affinity of binding, is responsible for the much greater potency, at 70 mV, of
the N1-sulfonyl polyamines compared with spermine.
The subunit selectivity of N1-DnsSpm was similar to that
reported for a number of structurally diverse channel blockers acting at NMDA receptors. Thus, N1-DnsSpm was less potent at
NR1/NR2C and NR1/NR2D receptors than at NR1/NR2A and NR1/NR2B
receptors, similar to the profile seen with Mg2+, MK-801
(25, 35, 39), spermine (6, 51), and argiotoxin636 (19). The
structural features of NR2 subunits that control sensitivity to many of
these antagonists are unknown, although regions of NR2B and NR2C that
influence Mg2+ block have been described (52). Spermine
itself blocks NR1/NR2A and NR1/NR2B receptors at micromolar
concentrations but has no effect on NR1/NR2C and NR1/NR2D receptors at
concentrations of 
300 µM (6, 51). Because of its
increased potency compared with spermine, N1-DnsSpm may be
a useful probe for studying the subunit-specific properties that
influence sensitivity to polyamines.
At wild-type NR1/NR2A channels, Woodhull (37) analysis of the block
yielded very similar values of z (~2.58) for N1-DnsSpm
and N1-OsSpm. In this analysis, z represents the valence of
the blocker, and represents the depth of the binding site for the
blocker within the membrane field. Thus, if the charge on the polyamine analogs is +3 at pH 7.5, the value of is 0.86, suggesting that the
"binding site" for polyamines lies deep within the channel. Indeed,
the z values for the N1-sulfonyl polyamines (z = 2.57-2.58) were larger than the value for spermine seen at NR1/NR2
receptors (z = 1.07) or at native NMDA receptors (z = 1.17) (3),
suggesting that N1-DnsSpm and N1-OsSpm may bind
much deeper in the channel pore than spermine itself, even though
spermine has four positively charged amino groups at physiologic pH.
However, there are a number of limitations and caveats to using the
Woodhull model to analyze block by polyamines. First, it is not known
whether only one molecule of N1-DnsSpm (or any of the other
polyamines) enters and binds in the channel at a given time or whether
two or more molecules can bind simultaneously. In the case of inward
rectifier K+ channels, for example, it has been proposed
that two molecules of spermine can simultaneously enter and block the
channel in an end-to-end manner (53). Second, it is not known whether
the entire polyamine tail of N1-DnsSpm and
N1-OsSpm enters the pore of the NMDA channel, or if only
part of the polyamine tail, containing, for example, one or two charged amino groups, enters the membrane electric field. However, if only one
molecule of N1-DnsSpm enters the channel, most or all or
the polyamine tail would have to be within the membrane electric field
to yield a z value of 2.58, assuming that z = 3. A third
limitation to analyzing the voltage-dependence of block is that
N1-OsSpm shows marked permeation of wild-type channels and
N1-DnsSpm permeates some of the mutant NMDA channels.
Although we used concentrations of the polyamines that produced a large
block and analyzed data over a voltage range where the block develops, it is not known to what extent permeation of a polyamine molecule influences the block caused by another molecule subsequently entering the channel. It is conceivable that in channels where permeation occurs, two polyamines could be present simultaneously: one blocking at
the polyamine binding site or approaching that site and another unblocking and passing through the channel into the oocyte.
Polyamines have several stable conformations. In the fully extended
all-trans conformation of N1-DnsSpm and
N1-OsSpm, the distances between the amino groups are
~0.52 nm (diaminopropane moieties) and ~0.65 nm (diaminobutane
moiety), with a total distance of ~1.2 nm between the first and third
charged amino groups. These positively charged groups may each interact
with one or more amino acid side chains within the channel pore,
constituting part of the polyamine binding site. Again, this places
limitations on the interpretation of the depth-of-field () value
derived from the apparent valence, z. If the polyamine derivatives
enter the membrane field in an extended conformation, the separation
between the first and third charged amino groups could span 20% of the membrane electrical field, assuming a lipid bilayer thickness of 6 nm.
Mutations at the critical asparagine residues in the M2 regions of NR1
and NR2A had complex effects on the potency (at 70 mV), the
Kd(0) value, and the z value of
block by N1-DnsSpm and N1-OsSpm. Mutations
NR1(N616G) and NR2A(N615G) increased the potency of
N1-DnsSpm, an effect that was probably due to an increase
in the affinity of the binding site for N1-DnsSpm because
these mutations reduced the Kd(0)
value of N1-DnsSpm without affecting z. A possible
explanation for these results is that the asparagine residues present
at position NR1(N616) and NR2A(N615) normally hinder the binding of
N1-DnsSpm and that when these residues are replaced by
glycine residues, in NR1(N616G) and NR2A(N615G), the
N1-DnsSpm has increased access to the interaction points of
its binding site. Mutation of NR1(N616) to glutamine (Q), which has a
bulkier side chain than asparagine, had a different effect on block by
N1-DnsSpm, reducing the potency and the z but having
only a small effect on the Kd(0)
value of N1-DnsSpm. The NR1(N616Q) mutation may reduce
the interaction of one of the charged groups on N1-DnsSpm
with the channel protein or may reduce the depth to which N1-DnsSpm can enter into the channel. Indeed, the
NR1(N616Q) mutation had a similar effect on block by
N1-OsSpm, reducing the potency and the z of that
compound, suggesting that the mutation affects the interaction of the
polyamine tail rather than the head group in these polyamine
derivatives. As was seen with N1-DnsSpm and
N1-OsSpm, block by MK-801 and TCP is influenced by
mutations at NR1(N616) (33, 34, 42). Thus, NR1(N616) may contribute
directly to the binding sites for N1-DnsSpm and MK-801.
Alternatively, mutations at this site could alter block by perturbing
other features of the channel structure or by alterations in pore size.
The NR2A(N615G) mutation decreased the Kd(0) of N1-OsSpm, similar to its effect on N1-DnsSpm, whereas NR1(N616G), which increases the affinity for N1-DnsSpm, had no effect on block by N1-OsSpm. The NR1(N616G) mutation may have a predominantly volume-specific effect, allowing easier access of the bulky head group of N1-DnsSpm to the channel but having little or no effect on the accessibility and binding of N1-OsSpm. On the other hand, the NR2A(N616G) mutation affects the affinities of both N1-DnsSpm and N1-OsSpm, suggesting that this residue may alter the properties of another part of the polyamine binding site or may have effects on the accessibility of this site that are similar for N1-DnsSpm and N1-OsSpm. These results also indicate a nonequivalent or nonsymmetrical role for the asparagines at the narrowest part of the channel in NR1 and NR2A, as was seen in studies of the permeability of small organic cations (36).
Block by N1-OsSpm was incomplete and was partially relieved
at extreme negative membrane potentials, whereas block by
N1-DnsSpm was complete and showed no relief at extreme
negative potentials. The relief of block by N1-OsSpm at
100 to 200 mV is similar to that seen with linear penta-amines such
as BE4444 (46) and presumably reflects permeation of
N1-OsSpm through the ion channel of NMDA receptors. Thus,
if N1-OsSpm can permeate the ion channel, presumably in a
linear conformation, but N1-DnsSpm cannot, it is likely
that the bulky aromatic head group of N1-DnsSpm (see Fig.
1) is responsible for the impermeant properties of this molecule. In
receptors containing the NR1(N616G) or NR2A(N615G) mutants, permeation
of N1-DnsSpm was seen at extreme negative membrane
potentials. Based on the permeability ratios of K+ and
organic cations such as tetraethylammonium, the NR1(N616G) and
NR2A(N615G) mutants have been estimated to increase the size of the
narrowest region of the NMDA channel from 0.55 nm in NR1/NR2A receptors
to 0.75 nm in NR1(N616G)/NR2A receptors and to 0.67 nm in
NR1/NR2A(N615G) receptors (36). The results of the present work are
consistent with the hypothesis that the bulky head-group of
N1-DnsSpm normally prevents it from easily permeating the
channel, but that increasing the pore size with the N-to-G mutations
allows N1-DnsSpm to easily permeate NR1/NR2A channels. We
estimated the diameter of the naphthalene ring on N1-DnsSpm
to be 0.8-0.85 nm, which is too large to easily permeate wild-type
channels with a pore size of 0.55 nm (36, 49, 50). Because
N1-DnsSpm can easily permeate receptors with NR2A(N615G),
the narrowest constriction in NR1/NR2A(N615G) channels may be > 0.67 nm (36). However, it is not known whether the size of the head
group on N1-DnsSpm, rather than its chemical structure, is
the key determinant of permeation through NMDA channels. Nevertheless,
N1-DnsSpm and other sulfonamide polyamine derivatives
represent new tools that can be used to study block and permeation of
glutamate receptor channels. Studying reversal of block at extreme
negative membrane potentials using modified polyamines such as
N1-DnsSpm provides a means of probing channel pore
structure using extracellular application of N1-DnsSpm and
whole-cell recording, without the need to use the polyamine itself as
the major charge carrier through the channels.
| |
Acknowledgments |
|---|
We are grateful to Drs. S. Nakanishi, P. H. Seeburg, and M. Mishina for providing the wild-type NR1 and NR2 clones; to Drs. S. Nakanishi and R. J. Dingledine (Emory University, Atlanta, GA), for providing some of the NR1 mutants; and to Albert Pahk for technical assistance with some experiments.
| |
Footnotes |
|---|
Received August 27, 1996; Accepted January 17, 1997
1 N. Seiler, F. Douaud, J. Renault, J.-G. Delcros, R. Havouis, P. Uriac, and J.P. Moulinoux. Targeting of tumors via the polyamine uptake system: a model study with N1-dansyl-spermine. I. Effects on enzymes and on cells in culture. Submitted for publication.
This work was supported by United States Public Health Service Grant NS35047 from the National Institute of Neurological Disorders and Stroke, by a Grant-in-Aid from the American Heart Association, and by a grant from the Japan Health Sciences Foundation.
Send reprint requests to: Keith Williams, Ph.D., Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084.
| |
Abbreviations |
|---|
NMDA, N-methyl-D-aspartate;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
N1-DnsSpm, N1-dansyl-spermine;
N1-OsSpm, N1-(n-octanesulfonyl)-spermine;
N1-DnsSpd, N1-dansyl-spermidine;
N8-DnsSpd, N8-dansyl-spermidine;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
TCP, N-[1-(2-thienyl)cyclohexyl]piperidine;
I-V, current-voltage.
| |
References |
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Summary
Introduction
Procedures
Results
Discussion
References
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