Studies on the structure and functions

of the regulatory domain of the

N-methyl-D-aspartate receptor

(NMDA 受容体の調節領域の構造と機能の解析)

2009. 3

Han Xia

1

CONTENTS

INTRODUCTION………………………………………………………………..2

CHAPER 1

Merterials and Methods…………………………………………………..11

CHAPER 2

Binding of spermine and ifenprodil to a purified, soluble regulatory domain of the NMDA receptor

Introduction……………………………………………………...............20

Results…………………………………………………………………...21

Discussion……………………………………………………….............40

CHAPER 3

Modeling of NR1A and NR2B regulatory domain of the NMDA receptor

Introduction……………………………………………………………...45

Results…………………………………………………………………...46

Discussion…………………………………………………….................56

REFERENCES…………………………………………………………………...57

LIST OF PUBLICAIONS…………………………………………………….70

ACKNOWLEDGEMENTS………………………………………………….71

THESIS COMMITTEE……………………………………………………….72

2

INTRODUCTION The polyamines putrescine, spermidine and spermine are present in almost all

cells and have important roles in protein synthesis, cell division and cell growth (Fig. 1).

Putrescine is synthesized from L-ornithine in a reaction catalyzed by ornithine

decarboxylase, the rate-limiting enzyme in polyamine biosynthesis. Putrescine and

decarboxylated S-adenosyl methionine are substrates for the synthesis of spermidine,

which is a precursor of spermine. Polyamines are organic polycations that are

protonated at physiological pH and can potentially interact with a variety of cellular

targets including nucleic and proteins [1-4].

NH

NH

NHH2N

NNH2H2NN 2

NNH

NH2H2NN NH2

Putrescine

Spermidine

Spermine 2

Fig. 1 Structure of polyamines

The endogenous polyamines putrescine, spermidine and in particular,

spermine modulate a number of ion channel [5, 6]. Spermine and spermidine are present

in high concentration in the nervous system, and uptake and depolarization-induced

release of polyamines from brain slices has been reported [7, 8]. Extracellular spermine

3

has multiple effects at the NMDA subtype of glutamate receptor, including stimulation

that increases the size of NMDA receptor currents and voltage-dependent block [9-15].

Activation of NMDA receptors is associated with the induction of various

forms of synaptic plasticity, including some forms of long-term potentiation and

long-term depression, processes that may underlie learning and memory [16-18].

NMDA receptors are subtype of ligand-gated ionotropic glutamate receptors including

subtypes of AMPA and kainite receptors [19]. Although they share common structural

features, NMDA receptors have several characteristics different from the other two

subtypes of glutamate receptors; 1) Not only glutamate but also glycine are necessary

for activation of NMDA receptors; 2) NMDA receptors show high permeability to Ca2＋;

and 3) NMDA receptor activity is blocked by Mg2＋ at resting membrane potential and

the block is relieved by depolarization[19].

At native NMDA receptors expressed on cultured neurons, there is

considerable variability in the degree of stimulation by spermine of individual neurons

[11-14]. This may be due to the expression of multiple forms of the NMDA receptor

composed of different combinations of subunits. The NR1 family consists of eight

splice variants of the NR1 gene [20-23], eight of which were originally termed

NR1A-NR1H [24]. The NR2 family consists of NR2A, NR2B, NR2C and NR2D cDNA

isolated from rat brain [25-28]. High active NMDA receptor channel is formed in vitro

by co-expression of NR1 and NR2 subunits [25, 26, 29, 30], although the NR1 subunit

alone exhibits a very small response in the Xenopus oocyte expression system [21, 31].

In accord with this, most brain regions express both NR1 and NR2 subunits. The NR1

4

subunit mRNA is found ubiquitously in the brain during development. Mutant mice

defective in the NR1 subunit died shortly after birth and failed to develop

whisker-related neuronal patterns in the trigeminal nuclei [32, 33]. At the embryonic

stages, the NR2B subunit mRNA is expressed in the entire brain, and NR2D subunit

mRNA is expressed in the diencephalons and brainstem [34]. After birth the NR2A

subunit mRNA appears in the entire brain, and NR2C subunit mRNA mainly in the

cerebellum. After birth, expression of NR2B subunit mRNA becomes restricted to the

forebrain, and that of the NR2D subunit mRNA is strongly reduced. The NR2B subunit

knockout mice died within 1 day after birth [35]; disruption of NR2A subunit of the

mouse does not appreciably affect the growth and mating of the mice, but results in

reduction of hippocampal LTP and spatial learning [36]; the NR2D subunit mice grow

normally, but exhibit reduced spontaneous behavioral activity [37]; the deficiency of

either the NR2A or NR2C subunit causes no remarkable behavioral abnormality, but the

disruption of both NR2A and NR2C results in impairment of motor coordination[38].

Thus, multiple NR2 subunits are major determinants of the NMDA receptor channel

diversity, and the molecular compositions and functional properties of NMDA receptor

channel are different depending on the brain regions and developmental stages [39-41].

Each subunit consists of extracellular N-terminal regulatory (R) domain,

agonist binding (S) domain, channel forming domain and intracellular domain (Fig. 2).

There are four membrane (M1-M4) regions in the channel forming domain, and second

membrane segment (M2) is thought to form a re-entrant loop and contribute to the

narrowest constriction of the channel (Fig. 2)[19]. The NMDA receptors are probably

tetramers composed of two molecules each of NR1 and NR2 subunits [19, 42, 43 ].

5

Na+Ca2+

H2NNH2

NR2

Extracellular

Intracellular

HOOC

NR1

HOOC

3 214 32 1 4

2

2 2

2

NR1

NR2

NR1

NR2

Gly Glu

R-domainR-domain SPM

IFEN

SPM

IFEN

N

OH

OH

SPM

IFEN

NH2NH

NH

NH2

(NR1A-H) (NR2A-NR2D)

S-domainS-domain

Fig. 2 Structure of NMDA receptor.

Each NMDA receptor subunit contains two large extracellular regulatory domains

(R-domain) and the agonist binding domains (S-domain). The transmembrane domain is

comprised of M1-M3, and a re-entrant loop (M2). There is a intracellular C-terminal

domain of each subunit. The receptor is gated by the co-agonist glutamate (Glu) and

glycine (Gly). The NMDA receptor are probably heterotetramaers composed of two

molecules each of NR1 and NR2 subunits. It indicates that some compounds bound to

the R-domain of NMDA receptors.

6

The multiple effects of spermine on NMDA receptors are summarized in Fig. 3.

spermine potentiates NMDA currents in the presence of saturating concentrations of

glycine (glycine-independent stimulation; mechanism 1 in Fig.3), an effect that involves

an increase in the frequency of channel opening and a decrease in the desensitization of

NMDA receptors [12-14]. A second involves an increase in the desensitization of

NMDA receptors for glycine (glycine-dependent stimulation; mechanism 2 in Fig. 3)

[11-44].

In addition to stimulation, voltage-dependent inhibition by extracellular

spermine was seen at native NMDA receptors (in the absence of extracellular Mg2＋)

possibly representing a direct block of the ion channel by sperimine (mechanism 3 in

Fig. 3) [11-13, 45, 46]. The strong voltage-dependent inhibition may be due to binding

of spermine within the ion channel pore at a site close to, or overlapping with, the

bingidng site for extracellular Mg2＋. There is no evidence to suggest that intracellularly,

produces an incomplete block that probably reflects permeation of spermine through

NMDA channels (Fig. 3) [11, 47].

Stimulation by spermine is dependent on the type of NR1 splice variant and the

type of NR2 subunit present in NMDA receptors [10, 45, 48-51]. Only receptors

expressed from splice variants such as NR1A, that lack a 21 amino acid insert encoded

by exon 5 show glycine-independent stimulation by spremine [48, 49] Furthermore the

various effect of spermine are differentially manifested in heteromeric NR1A/NR2

receptors containing different NR2 subuints (Fig. 3)[10, 45, 51]. NMDA receptors are

inhibited by protons, with a tonic inhibition of about 50% at physiological pH (7.3-7.5)

7

[52, 53]. Sensitivity to protons, like sensitivity to stimulation by spermine, is influenced

by the presence of exon 5 in NR1. Variants that contain the insert (such as NR1B) are

less sensitive to protons than are variats that lack the insert [54]. The

glycine-independent form of spermine stimulation is influenced by extracellular pH.

Ifenprodil is a novel NMDA receptor antagonist that selectively inhibits

NR1/NR2 receptors containing the NR2B subunit [55, 56]. Ifenprodil acts as a

noncompetitive, partial, and voltage-independent antagonist [55, 57, 58]. Its potency

strongly depends on the extracellular pH and is only weakly affected by the insertion of

the NR1 exon 5 [59, 60]. Finally, ifenprodil displays use dependence such that binding

of glutamate increase biding of ifenprodil and vice versa [61, 62].

There are complex interaction between spermine, proton and ifenprodil at

NMDA receptors. Spermine stimulation may involve relief of protein inhibition [54],

whereas ifenprodil inhibition may involve an increase in proton inhibition [60]. Using

site-directed mutagenesis, identified that a cluster of acidic residues located neat the

exon 5 splice site influence senxitivity to spermine and pH, and also identified a several

redidues in the proximal part of the amino terminus of NR1A that appear to form part of

the ifenprodil binding site. This region, containing the biding sites for spermine and

ifenprodil, may influence the downstream agonist binding domain S1 and S2 that

constitue the glycine binding pocket.

8

4. Relative affinity for ifenprodil highlow low low

Effect of spermine1. Glycine-independent stimulation

3. Voltage-dependent inhibition

no

yes

yes

yes

no

no

no

no2. Glycine-dependent stimulation yes yes no no

Na+

in

Ca2+

out Spermine IfenprodilGly Glu

Spermine

NR1A/NR2A NR1A/NR2B NR1A/NR2C NR1A/NR2D

2

1 4

3

Fig.3 Modulation of NMDA receptors.

Diagram of NMDA receptor is showing effect of spermine ,and ifenprodil .Numbers

indicfate various mavroscopic effects of these modulators . Spermine has 3 macroscopic

effects that are differentially controlled by NR2 subunits.

Indeed, the structures of the agonist binding domains of several glutamate

receptor subunits, including GluR0, GluR2, GluR5, NR1 and NR2A have been

determined by X-ray crystallography, and these domains do indeed form bi-lobed

“clamshell” structures similar to bacterial periplasmic binding proteins [68, 84, 72, 43,

83] . The region of the amino terminus preceding S1 also has sequence similarity and a

predicted structure similar to bacterial periplasmic binding proteins, in particular the

leucine/isoleucine/valine binding protein (LIVBP) [64]. We have termed this region

9

the regulatory (R) domain. Mutations at a number of residues in this region of the

NR1 subunit affect spermine stimulation[64, 94]. This region has also been referred to

as the “amino terminal domain” (ATD or NTD) and the “LIVBP-like domain” [73, 88,

95]. We have suggested that a spermine binding site is located in or near the cleft of the

R domain of the NR1 subunit [64].

Ifenprodil is an atypical NMDA antagonist that binds outside the ion channel

pore and selectively inhibits NMDA receptors containing the NR2B subunit [55, 93]. .

Inhibition by ifenprodil is pH-dependent and, mechanistically, may involve an increase

in tonic proton inhibition [59, 60]. Although it was initially suggested that ifenprodil

is an antagonist at the stimulatory polyamine site, this does not seem to be the case.

There is evidence that spermine and ifenprodil act at discrete sites with an allosteric

interaction [77, 64]. Mutations at several residues in the R domain of NR1 greatly

influenced ifenprodil inhibition, and we have suggested that this region may form part

of the ifenprodil binding site [64]. It has also been reported that mutations at residues

in the R domain of NR2B strongly affect ifenprodil inhibition [89], and it was proposed

that the R domain of NR2B forms the ifenprodil binding site whereas the equivalent

region of NR2A forms a high-affinity Zn2+ binding site, involving several amino acid

residues equivalent to those found at the proposed ifenprodil site in NR2B [88, 89].

In this paper, we have studied binding of radiolabeled spermine and ifenprodil

to purified soluble R domains of NR1, NR2A, and NR2B, to determine whether these

ligands can bind to the isolated R domains and to determine the relationship between

binding to the R domains and the effects at intact, functional receptors. We found that

10

spermine binds to all three domains, but with the highest affinity at NR1-R. Ifenprodil

binds with high affinity to NR1-R and NR2B-R but not to NR2A-R. We also study the

spermine and ifenprodil binding site in NMDA receptor through homology modeling of

NR1A-R and NR2B-R. Through the simulation we found that there are two binding

sites of spermine in NR1A-R and one is into the cleft of the 3D model of NR1A-R the

other on the surface. But in the 3D model of NR2B-R there is only one spermine

binding site. Ifenprodil bound into the cleft of the 3D model of NR2B-R and bound to

surface of the3D model of NR 1A-R. The results suggest that the ligand which bound to

the cleft of the NR-R maybe regulate the NMDA receptor more than to the surface.

11

CHAPTER 1

Materials and methods

12

Plasmids

For production of COOH-terminal histidine-tagged R domain proteins, DNA

fragments encoding NR1(19-380), NR2A(23-389) and NR2B(27-390) (numbering is

from the initiator methionine, thus these constructs lack the signal peptide) were

amplified by polymerase chain reaction (PCR) using primer pairs of

(5’-TATACATATGCGCGCCGCCTGCCGACCC-3’ and

5’-GGTGCTCGAGGATGATCTTCCTGTCAT-3’) for NR1, (5’-TATACATATGCA-

GAACGCGGCGGCG-3’ and 5’-GGTGCTCGAGGATGATCTTCCTGTCAT-3’) for

NR2A and (5’-TAT-ACATATGCGTTCCCAAAAGAGC-3’ and 5’-CTCGAGTGC-

GGCCGCCACATAATACTTCATCTGCAG-3’) for NR2B. The rat NR1A clone,

which lacks the exon-5 insert [21], and rat NR2A and 2B clones [26] were used as

templates. The DNA fragments obtained by PCR were digested with NdeI and XhoI

for NR1 and NR2A, and with NdeI and NotI for NR2B, and inserted into the same

restriction sites of pET29b (Novagen) to construct plasmids pET29b-NR1(19-380),

pET29b-NR2A(23-389) and pET29b-NR2B(27-390). The PCR fragments were

sequenced over the entire length of each fragment, and the sequences correspond to

those in the NR subunit cDNAs.

Purification of R domain proteins

Escherichia coli BL21(DE3) (Novagen) cells carrying pET29b-NR1(19-380),

pET29b-NR1A(19-380, E181Q), pET29b-NR2A(23-389) or pET29b-NR2B(27-390)

were grown in 2 L of modified Luria-Bertani medium (10 g tryptone, 10 g yeast extract

and 5 g NaCl per liter) containing 50 µg/ml kanamycin at 37 ˚C until A600 reached 0.3.

Protein production was induced by 1 mM isopropyl-β-D-thiogalactopyranoside for 4 h.

13

Inclusion bodies were prepared using methods similar to those described by Chen and

Gouaux (1997). Cells were collected by centrifugation, washed once with 10 mM

Tris-HCl, pH 8.0, containing 1 mM MgCl2, and suspended in 100 ml of 20% sucrose

containing 30 mM Tris-HCl, pH 7.5, 10 mM EDTA and 0.1 mg/ml lysozyme. Cells

were kept on ice for 20 min, and spheroplasts were collected by centrifugation at 30,000

g for 30 min. Spheroplasts were resuspended in 200 ml of 50 mM Tris-HCl, pH 7.5,

0.01% Triton X-100, 1 mM dithiothreitol (DTT) and 100 mM KCl, and sonicated for 2

min using an Ultrasonic Disruptor UD201 (TOMY, Tokyo). After treatment of the cell

lysate with 20 µg/ml DNase I plus 10 mM MgCl2 (30 min on ice), inclusion bodies were

collected by centrifugation at 30,000 g for 15 min. Inclusion bodies were resuspended

in 50 ml of buffer (20 mM Tris-HCl, pH 7.5, 6 M guanidine-HCl, 500 mM NaCl, and 5

mM imidazole), kept on ice for 1 h, and centrifuged at 30,000 g for 15 min. The

supernatant contained about 70 mg protein, half of which (about 35 mg protein) was

applied to each of two 5 ml of Ni-NTA columns (Qiagen). After washing with buffer

(20 mM Tris-HCl, pH 7.5, 6 M guanidine-HCl, 500 mM NaCl, and 20 mM imidazole),

R domain proteins were eluted with an elution buffer (20 mM Tris-HCl, pH 7.5, 6 M

guanidine-HCl, 500 mM NaCl, and 100 mM imidazole).

Proteins (100 μg/ml) were refolded through gradual decrease in the

concentration of guanidine-HCl by stepwise dialysis against Buffer 1 [25 mM

Hepes-KOH, pH 7.5, 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10 µM E-64-C,

a thiol protease inhibitor, 10 µM FUT-175, a serine protease inhibitor, 0.5 mM

phenylmethylsulfonyl fluoride (PMSF), 10% glycerol and 0.01% Brij-35] containing 1

M guanidine-HCl, Buffer 1 containing 0.5 M guanidine-HCl, Buffer 1 containing 0.2 M

14

guanidine-HCl, and Buffer 1 at 4 ˚C for at least 4 h each. R domain proteins were

concentrated to approximately 1 mg/ml by ultrafiltration using PM10 filter (Amicon),

and precipitates were removed by centrifugation. Protein concentration was

determined by the method of Lowry et al. (1951). About 20 mg of R domain protein

was obtained from a 2 liter culture. Monomers of the three R domain proteins (41.6

kDa of NR1-R, 42.3 kDa of NR2A-R and 42.7 kDa of NR2B-R) were isolated by gel

filtration using TSK gel G3000SW (2.15 x 67.5 cm) (TOSOH) and Buffer 2 containing

20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM dithiothreitol, and 0.01% Brij-35.

Monomers of R domains were eluted at an elution volume of 132 to 152 ml. The

protein was concentrated to approximately 2 mg/ml by ultrafiltration using PM10 filter

(Amicon), and used for the experiments. The percentage of monomer and dimer of

NR1-R, NR2A-R and NR2B-R during the assay was confirmed by gel filtration using

TSK gel G3000SW (0.75 x 30 cm) (TOSOH) and Buffer 2. As a molecular marker,

phosphorylase B (94 kDa), ovalbumin (45 kDa) and lysozyme (14.5 kDa) were used.

Binding of spermine and ifenprodil to R domain proteins

Binding assays were carried out as described previously [66]. The reaction

mixture (0.5 ml) containing 50 to 100 µg R-domain proteins, 10 mM Tris-HCl, pH 7.5,

and 50 mM KCl was preincubated at 30 ˚C for 10 min, and [14C]spermine (463

MBq/mmol, Amersham) or [3H]ifenprodil (15 GBq/mmol, PerkinElmer) was added at 5

to 250 µM for spermine and 0.05 to 0.8 µM for ifenprodil. After incubation at 30 ˚C

for 20 min, the reaction was stopped by addition of 2 ml cold Buffer (10 mM Tris-HCl,

pH 7.5, and 50 mM KCl) and the protein was collected on HAWP02400 filter (0.45 µm,

Millipore). After the filter was washed 2 times with 2 ml of cold Buffer, radioactivity

15

on the filter was measured by a liquid scintillation spectrometer. Blank value (the

level of [14C]spermine or [3H]ifenprodil bound to NR1-R, NR2A-R and NR2B-R in the

reaction mixture kept at 0 ºC) was subtracted from total radioactivity to obtain specific

binding. The dissociation constant (Kd) and maximum binding activity (Bmax) were

calculated from Scatchard plots as described previously [76]. The k1 and k-1 values were

calculated with GraphPad Prism V. 4 (GraphPad software).

Determination of the Kd values of spermine and ifenprodil for NR1-R, NR2A-R

and NR2B-R by CD (circular dichroism) measurements

The CD measurements were performed at 25 ºC using a J-820

spectropolarimeter (Jasco International Co) equipped with a 1.0-mm path-length cuvette.

The samples were scanned from 190 to 250 nm and accumulated 10 times at a

resolution of 0.1 nm with a scanning speed of 50 nm/min and sensitivity of 200 mdeg.

All of the CD data are expressed as molar ellipticity. The concentration of NR1-R,

NR2A-R and NR2B-R used was 0.3-, 0.5- and 0.5 mg/ml, respectively. The buffer

used was the same as that used for binding assay of spermine and ifenprodil.

Increasing amounts of spermine or ifenprodil were added in small aliquots from stock

solutions to 300 μl of protein solution and were allowed to bind for 5 min at room

temperature. The CD spectra of the protein in the absence and presence of increasing

concentrations of spermine (5 - 360 μM) or ifenprodil (0.05 - 2 μM) were recorded. In

this experiment, ifenprodil hemitartrate was used instead of ifenprodil to avoid the

precipitation of proteins. The final change of assay volume was < 3%. The Kd

values were determined according to the double-reciprocal equation plot shown in the

figure.

16

NMDA conloes and site-directed mutagenesis

The NR1 clone used in these studies is the NR1A variant [21], which lacks the

21-amino acid insert encoded by exon 5. This clone was gifts from Dr. S. Nakanishi

(Osaka Bioscience Institute, Osaka, Japan). The wild type mouse and rat NR2B

clones[26,29] were gifts from Drs. M. Mishina (Graduate School of medicine,

University of Tokyo, Tokyo, Japan) and P. H. Seeburg (Center for molecular biology,

University of Heidelberg, Heidelberg, Germany). The preparation of most other NR1

and NR2B mutats has been described previously [64, 65, 67,]. Site-directed mutagenesis

to construct new NR1 and NR2B mutant was carried out by the method of Ho et

al.(1987) using the polymerase chain reaction, with Pfu polymerase purchased from

Stratagene. Amino acids are numbered from the initiator methione in each subunit.

Mutations were confirmed by DNA sequending using a Seq 4´4 personal sequencing

system (Amersham Biosciences) over approximately 300 nuculeotides cotaining the

mutation. Amino acids are numbered from the initiator methione in NR1 and NR2B

clones.

Preparation of oocytes and voltage clamp recording

Adult female Xenopus laevis (Saitama Experimental Animals Supply Co. Ltd.,

Saitama, Japan) were chilled on ice, and the preparation and maintenance of oocytes

were carried out using the methods similar to those described previously [55]. Capped

cRNAs were prepared from linearized cDNA templates using mMessage mMachine kits

(Ambion). Oocytes were injected with NR1 and NR2 cRNAs in a ratio of 1:5 (0.2 - 4

ng of NR1 plus 1 - 20 ng of NR2). Macroscopic currents were recorded with a

17

two-electrode voltage-clamp using Dual Electrode Voltage Clamp Amplifier CEZ-1250

(Nihon Koden, Tokyo). Electrodes were filled with 3 M KCl. Oocytes were

continuously superfused (ca. 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). This solution contained BaCl2

rather than CaCl2, and in most experiments oocytes were injected with

K+-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (100 nl; 40

mM, pH 7.0) on the day of recording to eliminate Ca2+-activated Cl- currents [55].

Oocytes were voltage-clamped at a holding potential of –20 mV. Data were recorded

by using a Digidata 1322A interface with software pCLAMP8 for Windows (Axon

Instruments, USA). Receptors were activated by superfusion of glutamate and glycine

(10 mM).

Data analysis and cureve fitting were carried out using Axongraph (Molecular

Devices) or SigmaPlot (SPSS Inc., Chicago, IL).

Identification of heterodimer formation of R domain by immunoprecipitation and

Western blot analysis

The reaction mixture (0.3 ml) containing either 45 μg NR1-R, 30 μg NR2A-R,

30 μg NR2B-R, 45 μg NR1-R plus 30 μg NR2A-R or 45 μg NR1-R plus 30 μg NR2B-R,

10 mM Tris-HCl, pH 7.5, 50 mM KCl, and 2 μg of anti-NR1 (sc-9058, Santa Cruz) was

kept at 4 °C for 2 h. Then, antibody-coupled protein was precipitated by addition of 2

μl PANSORBIN® Cells (CALBIOCHEM) and incubation at 4 °C for 2 h. The

precipitate was collected by centrifugation at 30,000 g for 5 min, washed 3 times with

Buffer 3 [0.5 M Na-phosphate buffer, pH 7.5, 100 mM NaCl, 1% Triton-X100 and 0.1%

sodium dodecyl sulfate (SDS)], and boiled for 5 min in SDS-PAGE sample buffer.

18

Western blotting was performed by the method of Nielsen et al. (1982) using 3 μg

protein and ECL Western blotting reagents (GE Healthcare). NR1-R, NR2A-R and

NR2B-R were detected with anti-NR1, anti-NR2A (sc-9056, Santa Cruz), and

anti-NR2B (sc-9057, Santa Cruz). These commercially available antibodies were

raised against epitopes in the first 70 to 300 amino acids of the amino terminus (i.e., the

R domain) of each subunit.

Homology modeling of NR1 and NR2B regulatory domain of NMDA receptor and

docking of spermine and ifenprodil

Three dimensional structures of NR1A-R and NR2B-R were constructed by

homology modeling with the Amber99 force field in MOE (version 2007.09, CCG Inc.,

Montreal, Canada) based on the Brookhaven Protein Databank 1EWK. Furthermore, the

structure of the docking complex between NR1A-R and spermine, NR1A-R and

ifenprodil, NR2B-R and spermine, and NR2B-R and ifenprodil were predicted with

ASEDock of MOE. The resulting three-dimensional structure of each complex was

displayed using MolFeat (Ver. 3.6, FiatLux, Tokyo, Japan).

19

CHAPTER 2

Binding of spermine and ifenprodil to a purified,

soluble regulatory domain of the NMDA receptor

20

Introduction The binding of spermine and ifenprodil to the amino terminal regulatory (R)

domain of the N-methyl-D-aspartate receptor was studied using purified regulatory

domains of the NR1, NR2A and NR2B subunits, termed NR1-R, NR2A-R and NR2B-R.

The R domains were overexpressed in Escherichia coli and purified to near

homogeneity. The Kd values for binding of [14C]spermine to NR1-R, NR2A-R and

NR2B-R were 19, 140 and 33 µM, respectively. [3H]Ifenprodil bound to NR1-R (Kd,

0.18 µM) and NR2B-R (Kd, 0.21 µM), but not to NR2A-R at the concentrations tested

(i.e. 0.1 to 0.8 μM). These Kd values were confirmed by circular dichroism

measurements. The Kd values reflected their effective concentrations at intact

NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine

and ifenprodil on NMDA receptors occur through their binding to the regulatory

domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or

ifenprodil to a mixture of NR1-R and NR2A-R or NR1-R and NR2B-R was additive

with that of each individual R domain. Binding of spermine to NR1-R and NR2B-R

was not inhibited by ifenprodil and vice versa, indicating that the binding sites for

spermine and ifenprodil on NR1-R and NR2B-R are distinct.

21

Results Purification of R domain proteins

The regulatory (R) domains of native NMDA receptor subunits are in the

extracellular environment, as is the S1-S2 agonist-binding domain (Fig. 4a), and we

reasoned that the R domains could be expressed and purified as soluble proteins, as has

been reported for S1-S2 fusion proteins from several glutamate receptors [69, 68, 84, 72,

43, 83]. The R domains of NR1, NR2A, and NR2B, comprising the first 360 to 370

amino acids after the signal peptide (Fig. 4b) were subcloned into pET29b with a

histidine tag at their COOH-terminus. These R domain proteins were expressed

separately in E. coli, purified from inclusion bodies using Ni-NTA column

chromatography, and refolded by dialysis. As shown in Fig. 4c, the soluble NR1-R,

NR2A-R and NR2B-R proteins were purified to near homogeneity. The molecular

mass of these proteins on SDS-PAGE is similar to that predicted based on their amino

acid sequences (41.6 kDa of NR1-R, 42.3 kDa of NR2A-R and 42.7 kDa of NR2B-R),

although the mobility of NR2A-R was slightly greater than that of NR1-R and NR2B-R.

In Fig. 4d, the molecular size of these purified R domains in solution is shown.

NR2A-R and NR2B-R mainly consisted of monomer and dimer forms, but NR1-R

contained a polymerized form together with monomers and dimers. The percentage of

the monomer form of NR1-R, NR2A-R and NR2B-R was 37, 73 and 71%, respectively.

The elution profile of NR1-R, NR2A-R and NR2B-R was not influenced by adding 0.1

mM spermine to the elution buffer, suggesting that spermine is not involved in

polymerization or dimerization of each regulatory domain (data not shown). These

preparations were used for subsequent binding assays.

22

kDa

94 -

68 -

45 -

32 -

18 -

14821 2７

S1 S2NR2B-RSP M1 M2 M3 M4

390

14641 2３

S1 S2NR2A-RSP M1 M2 M3 M4

389

S19381 1９

SPS2NR1-R

M1 M2 M3 M4

380

(a)

(c) (d)

Elution Volume (ml) 4 5 6 7 8 9 10

A280

0

0.1

0.2

0.3

Elution Volume (ml) 4 5 6 7 8 9 10

A280

0

0.1

0.2

0.3

Elution Volume (ml) 4 5 6 7 8 9 10

NR2A-R

NR1-R

NR2B-R

A280

0

0.1

0.2

0.3

94k 45k 14.5k

(b)

M2

M3M4 M1

S1 S2agonist

N

C

R-domain NR1

NR2B

NR2A

Fig. 4 Structure and purity of R domain proteins. (a) Schematic of an NMDA receptor

subunit showing the extracellular R and S1-S2 domains, three membrane-spanning domains (M1,

M3, M4), a re-entrant loop (M2) and intracellular C terminus. (b) Schematics of NR1, NR2A, and

NR2B subunits showing the positions of the signal peptides (sp), the R domains used in this study,

the S1 and S2 domains, and the membrane spanning and re-entrant loops (M1-M4). Amino acids

are numbered (above each schematic) from the initiator methionine. (c) Sodium dodecyl

sulfate-polyacrylamide gel electrophoresis of purified R domains (NR1-R, NR2A-R and NR2B-R).

Each lane of the 12% acrylamide gel contained 2 µg protein, and the gel was stained with Coomassie

Brilliant Blue R250. Numbers on the left represent molecular mass in kDa. (d) Gel filtration of

purified R domain proteins used for radioligand binding assays. The pattern indicates the extent of

dimerization and polymerization after the isolation and concentration of the monomer fraction of R

domain proteins. Peaks corresponding to monomers are indicated by an arrow in each panel.

23

Binding of spermine and ifenprodil to R domain proteins

Binding of [14C]spermine to the soluble R domain proteins was determined.

The binding of spermine to NR1-R, NR2A-R and NR2B-R was saturable (Fig. 5, upper

panels), and the Kd values for spermine at these regulatory domains were 19, 140 and

33 µM, respectively (Fig. 5, lower panels). Thus, although spermine can bind to all

three R domains, it binds with highest affinity to NR1-R. Bmax values, which indicate

proportion of active protein, for NR1-R, NR2A-R, and NR2B-R were 6.3, 6.9 and 7.3

nmol/mg R domain protein, which were equivalent to 0.24, 0.29 and 0.32 mol/mol R

domain, respectively. Judging from the percentage of monomer and dimer (Fig. 4d)

and Bmax values for spermine binding (Fig. 5b), spermine may bind to both monomer

and dimer forms of NR1-R, NR2A-R and NR2B-R. The Hill coefficient of spermine

binding to these three R domains was almost 1.0, suggesting that spermine binding to

each R domain is independent, even though dimers can be formed. As a control,

binding of spermine was studied using an unrelated protein, ovalbumin, and a mutant of

NR1-R, E181Q, which drastically reduces spermine stimulation as studied in functional

assays [64]. There was little binding of spermine to ovalbumin, and binding to the

E181Q mutant was reduced compared to the wild-type NR1-R domain (Fig. 5). The

Kd value of spermine at the E181Q mutant was 74 μM compared with 19 μM for wild

type NR1-R. Such a decrease in spermine binding to the E181Q mutant was nearly

parallel with the decrease in spermine stimulation in functional assays [64]. This is

consistent with the proposal that residue E181 forms part of the spermine binding site

on NR1.

24

The Kd value for binding of spermine to the NR2B R domain was also

calculated by determining the association (k1) and dissociation (k-1) rate constants from

studies of the time course of association and dissociation of [14C]spermine (Fig. 6).

Values of k1 were obtained using spermine concentrations of 12.5–100 μM, and values

of k-1 were obtained from dissociation plots after addition of non-labeled 3 mM

spermine. Under these conditions, the association rate was not fully

concentration-dependent. The Kd value was calculated as the average using values of k1

and k-1 at concentrations of 12.5, 25, and 50 μM [14C]spermine. The values for k1 and k-1

were 8500 M-1 min-1 and 0.22 min-1, respectively. Accordingly, the Kd was determined

to be as 25.9 μM, which is similar to the Kd measured in the stauration binding analysis

shown in Fig. 5.

We then measured the effects of spermine on recombinant NMDA receptors

expressed in oocytes to compare the concentration of spermine required to potentiate

intact, functional NMDA receptors with the effects of spermine seen at isolated R

domains. Since spermine stimulation is seen at both NR1/NR2A and NR1/NR2B

receptors in the presence of 10 μM glutamate and 1 μM glycine at pH 7.5 [9], the EC50

values for spermine were measured under these conditions. To minimize

voltage-dependent block by spermine, oocytes were voltage-clamped at -20 mV. As

shown in Fig. 7, the EC50 values for spermine stimulation at NR1/N2A and NR1/NR2B

receptors were 278 µM and 78 µM, respectively. The ratio of EC50 values at NR2A

versus NR2B correlates well with the Kd values for spermine binding, although EC50

values are higher than Kd values.

25

Binding of [3H]ifenprodil to the purified R domain proteins was also studied.

Ifenprodil bound to NR1-R and NR2B-R, but there was no specific binding of 0.1 to 0.8

µM [3H]ifenprodil to NR2A-R. As shown in Fig. 8, binding of ifenprodil to NR1-R

and NR2B-R was saturable, with Kd values of 0.18 µM and 0.21 µM, respectively.

These values were close to the IC50 (0.34 μM) of ifenprodil at NR1/NR2B [55]. Bmax

values for both NR1-R and NR2B-R were 5.6 and 5.2 nmol/mg R domain protein,

which were equivalent to 0.24 and 0.21 mol/mol R domain protein, respectively.

These values were similar to the binding capacity for spermine at these proteins. Hill

coefficient for ifenprodil binding to NR1-R and NR2B-R was almost 1.0, suggesting

that ifenprodil binding on each R domain is independent, even though homodimers can

be formed, and that there is likely one binding site per R domain.

The binding of spermine and ifenprodil to R domain proteins was also

measured by CD. The CD spectra of R domain proteins were analyzed from 190 to

250 nm with increasing concentrations of spermine or ifenprodil, and the shifts of

magnitude at wavelength 208.6 nm reflecting a-helical structure were plotted. As

shown in Figs. 9 and 10, the Kd values of spermine for NR1-R, NR2A-R and NR2B-R

were 20, 133 and 35 μM, respectively, and those for ifenprodil at NR1-R and NR2B-R

were 0.14 and 0.17 μM, respectively. There was no change in the CD spectra of

NR2A-R by 2 μM ifenprodil. The results are similar to those seen in studies with

radiolabeled spermine and ifenprodil and confirm that their binding to NR1-R, NR2A-R

and NR2B-R is saturable and specific.

To determine whether the binding sites for spermine and ifenprodil on

26

individual NR1 and NR2 R domains are altered through an interaction with the R

domains of the other subunits, the binding of spermine and ifenprodil was compared

using NR1-R only, NR2-R only and a mixture of NR1-R and NR2-R. These

experiments were carried out using 20 µM spermine and 0.2 µM ifenprodil. As shown

in Fig. 11, the binding of spermine and ifenprodil was quantitative, being dependent on

the amount of R domain protein (50 to 100 µg). Furthermore, binding of either ligand

to NR1-R and NR2A-R or NR2B-R was simply additive. We also carried out

experiments to confirm that heterodimers of NR1-R + NR2A-R and NR1-R + NR2B-R

do indeed form in solution under these conditions. This was done by

immunoprecipitation with an anti-NR1-R antibody followed by Western blotting with

anti-NR1 or anti-NR2 antibodies (Fig. 12). Although NR2A-R and NR2B-R were not

immunoprecipitated by anti-NR1, these two R domains were coimmunoprecipitated

with NR1-R by anti-NR1, indicating that heterodimer formation between NR1-R and

NR2A-R or NR2B-R does indeed occur after the individual NR1 and NR2 R domains

are added together in solution. The percentage of heterodimer formation was

estimated to be 20 to 40%, which was similar to the percentage of homodimer formation

(see Fig. 4). The results suggest that spermine and ifenprodil bind independently to

each regulatory domain, even when dimer formation occurs. The results are in

accordance with the data showing Hill coefficients of unity for binding of spermine and

ifenprodil to NR1-R and NR2-R (Figs. 5 and 8).

27

0123456

012

345

Spe

rmin

e bo

und/

NR

2A-R

(nm

ol/m

g pr

otei

n)

Spermine (mM)

Spe

rmin

e bo

und/

NR

2B-R

(nm

ol/m

g pr

otei

n)

Spermine (mM) 0 20 40 60

(a) (b) (c) NR1-R NR2A-R NR2B-R

012

345

Spe

rmin

e bo

und/

NR

1-R

(nm

ol/m

g pr

otei

n)

Spermine (mM) 0 100 200 300 0 40 80 120

Kd = 32.5 mM

Spermine bound/NR1-R(nmol/mg protein)

Spermine bound/NR2A-R(nmol/mg protein)

Spermine bound/NR2B-R(nmol/mg protein)

SP

M b

ound

/NR

1-R

/[SP

M]

(nm

ol/m

g pr

otei

n/mM

)

SP

M b

ound

/NR

2A-R

/[SPM

](n

mol

/mg

prot

ein/mM

)

SP

M b

ound

/NR

2B-R

/[SPM

](n

mol

/mg

prot

ein/mM

)

Hill coefficient =1.005 Hill coefficient =1.003 Hill coefficient =0.998

0

0.1

0.2

0.3

0.4

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 70

0.01

0.02

0.03

0.040.05

0

0.1

0.2

0.3

0 1 2 3 4 5 6 7 8

Ovalbumin

NR1-R,Kd = 18.5 mM

NR2A-R,Kd = 140 mM

NR1-R

NR1-R (E181Q)

NR2A-R

NR2B-R,

Fig. 5 Binding of spermine to R domain proteins.

Saturation isotherms (upper panels) and Scatchard plots (lower panels) of the binding of

[14C]spermine to NR1-R (a), NR2A-R (b), and NR2B-R (c). The Hill coefficient was

calculated from the plot of the logarithmic form of a Hill equation. Binding of

[14C]spermine to NR1-R (E181Q) and that to ovalbumin was also shown in upper

figures of (a) and (b), respectively. Values are means ± S. D. from three experiments.

SPM, spermine.

28

Fig. 6 Time course of [14C]spermine binding to NR2B-R and its dissociation by

nonlabeled 3 mM spermine.

The binding assay was performed using 12.5 (), 25 (), 50 () and 100 () μM

[14C]spermine as substrate, and nonlableled 3 mM spermine was added to the reaction

mixture at 16 min after the onset of incubation. [14C]spermine bound to NR2B-R was

measured as described in Materials and methods. Values are means ± S. D. from three

experiments.

29

1.0

1.4

1.6

1.8

2.0

1.2

2.4

2.2

1 10 100 1000 10000

Stim

ulat

ion

by s

perm

ine

(-fol

d)

(1 µ

M G

ly, 1

0 µM

Glu

)

NR1/NR2BNR1/NR2A

Spermine (µM)

(EC50 = 278 µM) (EC50 = 78 µM)

Fig. 7 Effects of spermine at intact, functional NMDA receptors.

The effects of spermine on currents induced by glutamate and glycine at NR1/NR2A (○)

and NR1/NR2B (●) were measured in oocytes expressing recombinant receptors and

voltage clamped at –20 mV. Values are mean ± S. D. from five experiments. The

Hill coefficients for spermine at NR1/NR2A and NR1/NR2B were 1.17 and 1.05,

respectively.

30

Ifenp

rodi

lbou

nd/N

R2B

-R(n

mol

/mg

prot

ein)

(b)

Ifenprodil (mM)

Ifenp

rodi

l bou

nd/N

R1-

R(n

mol

/mg

prot

ein)

(a)

Ifenprodil (mM)

0

1

2

3

4

5

0 0.2 0.4 0.6 0.80

1

2

3

4

5

0 0.2 0.4 0.6 0.8

NR1-R NR2-RHill coefficient =0.984 Hill coefficient =1.001

Ifenp

rodi

l bou

nd/N

R2B

-R/[I

fenp

rodi

l](n

mol

/mg

prot

ein/mM

)

(nmol/mg protein) Ifenprodil bound/NR2B-R

Ifenp

rodi

l bou

nd/N

R1-

R/[I

fenp

rodi

l](n

mol

/mg

prot

ein/mM

)

(nmol/mg protein) Ifenprodil bound/NR1-R

05

101520253035

0 1 2 3 4 5 60

5

10

15

20

25

0 1 2 3 4 5 6

NR1-R,Kd = 0.18 mM

NR2B-R,Kd = 0.21 mM

NR2A-R

NR2B-R

Fig. 8 Binding of ifenprodil to NR1-R and NR2-R.

Saturation isotherms (upper panels) and Scatchard plots (lower panels) of the binding of

[3H]ifenprodil to NR1-R (a) and NR2B-R (b). The Hill coefficient was calculated

from the plot of the logarithmic form of a Hill equation. Binding of [3H]ifenprodil to

NR2A-R is also shown in upper figure of (b). Values are means ± S. D. from three

experiments.

31

0

5

10

15

20

25

Θ (x

104

degc

m2 d

mol

e-1)

Spermine (mM) 0 100 200 300 400

Kd = 133 mM

0.01 0.02 0.03 0.04-0.01

(a) NR2A-R(b) NR2B-R

0

10

20

30

40

50

Spermine (mM) 0 100 200 300 400Θ

(x10

4de

gcm

2 dm

ole-1

)

-0.04 0.04 0.08 0.12

Kd = 35.0 mM

NR1-R (c)

[Spermine]-1 (mM)-1 [Spermine]-1 (mM)-1

[Shi

ft in

ellip

ticity

] -1

[x10

-6de

gcm

2 dm

ole-1

]-1

[Shi

ft in

ellip

ticity

] -1

[x10

-6de

gcm

2 dm

ole-1

]-1

0

10

20

30

40

0 20 40 60 80Θ (x

104

degc

m2 d

mol

e-1)

Spermine (mM)

2468

12

-0.1 0.1 0.2 0.3

10

[Shi

ft in

ellip

ticity

] -1

[x10

-6de

gcm

2 dm

ole-1

]-1

Kd = 20.0 mM

[Spermine]-1 (mM)-1

4

8

12

16

20

2

4

6

8

10

Fig. 9 Measurement of the Kd values for spermine at NR1-R, NR2A-R and

NR2B-R by CD.

CD measurements were performed as described in Materials and methods.

Concentration-dependent effects of spermine on shifts of magnitude at 208.6 nm are

shown in the upper panels, and the Kd values of spermine were determined from

double-reciprocal plots as shown in the lower panels. Values are mean ± S. D. from

three experiments.

32

0

4

8

12

16

0.5 1.0 1.5 2.0 2.50

(a) NR1-R

Θ (x

104

degc

m2 d

mol

e-1)

Ifenprodil (mM)

5

10

15

20

25

-10 -5 5 10 15

Kd = 0.14 mM

[Ifenprodil]-1 (mM)-1

[Shi

ft in

ellip

ticity

] -1

[x10

-6de

gcm

2 dm

ole-1

]-1

(b) NR2B-R

Θ (x

104

degc

m2 d

mol

e-1)

Ifenprodil (mM)

0

10

20

30

0.5 1.0 1.5 2.0 2.50

[Ifenprodil]-1 (mM)-1

[Shi

ft in

ellip

ticity

] -1

[x10

-6de

gcm

2 dm

ole-1

]-1

2468

101214

-10 -5 5 10 15 20 25

Kd = 0.17 mM

Fig. 10 Measurement of the Kd values of ifenprodil for NR1-R, and NR2B-R by

CD.

CD measurements were performed as described in Materials and methods.

Concentration-dependent effects of ifenprodil on shifts of magnitude at 208.6 nm are

shown in the upper panels, and the Kd values of ifenprodil were determined from

double-reciprocal plots as shown in the lower panels. Values are mean ± S. D. from

three experiments.

33

[3H

]Ifen

prod

il bo

und

(pm

ol)

NR1-R NR2A-R NR2B-R

50 (mg) 100 50 100 50 100

NR1-R + NR2A-R

NR1-R + NR2B-R

50 + 50 50 + 50

[14 C

]Spe

rmin

e bo

und

(pm

ol)

NR1-R NR2A-R NR2B-R

50 (mg) 100 50 100 50 100

NR1-R + NR2A-R

NR1-R + NR2B-R

50 + 50 50 + 50

(a)

(b)

0

100

200

300

400

0

100

200

300

400

Spermine

Ifenprodil

Fig. 11 Binding of spermine and ifenprodil to various combinations of R domain

proteins.

Binding was measured using 50 to 100 µg of NR1-R NR2A-R or NR2B-R, alone or in

combination. The concentrations of [14C]spermine and [3H]ifenprodil were 20 µM and

0.2 µM, respectively. Values are means ± S. D. from three experiments.

34

NR1 NR2A NR2B NR1+NR2A

NR1+NR2B

Anti-NR1

Anti-NR2A

Anti-NR2B

Immunoprecipitation with anti-NR1 Detection by

Western blotting

Fig.12 Identification of heteromer formation of R domains by

immunoprecipitation followed by Western blot analysis.

The R domains shown in the figure were immunoprecipitated by anti-NR1 as described

in Materials and methods. Immunoprecipitated protein was detected by Western blot

analysis using anti-NR1, anti-NR2A and anti-NR2B. Experiments were repeated twice,

and essentially the same results were obtained.

35

Distinct binding sites for spermine and ifenprodil on NR1-R and NR2B-R

Experiments were carried out to determine if there are interactions between

spermine and ifenprodil on the purified NR1-R protein. As shown in Table 1, binding

of spermine to NR1-R was inhibited by nonlabeled spermine and spermidine, but not by

tribenzylspermidine (TB-34), a polyamine derivative that is a potent NMDA channel

blocker but does not have effects at the stimulatory polyamine site [74]. Binding was

also unaffected by agonists (glycine and glutamate) that bind in the S1-S2 domains, and

by ifenprodil. The binding of ifenprodil to NR1-R was inhibited by nonlabeled

ifenprodil, but not by agonists or by spermine and spermidine (Table 1). Similarly, the

binding of spermine to NR2B-R was inhibited by nonlabeled spermine and spermidine,

but not by agonists and ifenprodil, and that of ifenprodil was inhibited by nonlabeled

ifenprodil, but not by agonists and spermine and spermidine (Table 2). The results

indicate that spermine and ifenprodil bind to distinct sites on NR1-R and NR2B-R.

36

Table 1 Spermine and ifenprodil binding to purified NR1-R

[14C]Spermine bound [3H]Ifenprodil bound Addition

nmol/mg protein % nmol/mg protein %

None

Spermine (300 µM)

Spermidine (1 mM)

Tribenzylspermidine (100 µM)

Ifenprodil (10 µM)

Glycine (100 µM)

Glutamate (100 µM)

3.20

0.08

0.09

3.28

2.83

3.23

3.45

±

±

±

±

±

±

±

0.23

0.01

0.02

0.21

0.25

0.24

0.27

100

3

3

103

88

101

108

2.98

2.63

2.71

0.27

3.15

2.88

±

±

±

±

±

±

0.17

0.14

0.17

0.01

0.16

0.19

100

88

91

n.d.

9

106

97

The concentration of [14C]spermine and [3H]ifenprodil used for the binding assay was

20 µM and 0.2 µM, respectively. Where indicated, nonlabeled spermine (300 µM),

spermidine (1 mM), tribenzylspermidine (100 µM), ifenprodil (10 µM), glycine (100

µM) or glutamate (100 µM) was added to the reaction mixture. Data are shown as mean

± S. E. from three experiments. n.d., not determined

37

Table 2 Spermine and ifenprodil binding to purified NR2B-R

[14C]Spermine bound [3H]Ifenprodil bound Addition

nmol/mg protein % nmol/mg protein %

None

Spermine (300 µM)

Spermidine (1 mM)

Ifenprodil (10 µM)

Glycine (200 μM)

Glutamate (200 μM)

3.85

0.28

0.25

4.14

3.89

3.93

±

±

±

±

±

±

0.38

0.05

0.04

0.36

0.41

0.40

100

7

6

108

101

102

3.21

3.17

3.15

0.48

3.27

3.31

±

±

±

±

±

±

0.22

0.40

0.31

0.09

0.32

0.34

100

99

98

15

102

103

The concentration of [14C]spermine and [3H]ifenprodil used for the binding assay was

40 µM and 0.4 µM, respectively. Where indicated, nonlabeled spermine (300 µM),

spermidine (1 mM), ifenprodil (10 µM) glycine (200 μM) or glutamate (200 μM) was

added to the reaction mixture. Data are shown as mean ± S. E. from three

experiments.

38

Effect of aminoglycosides on spermine binding to R domain proteins

We have previously shown that a number of aminoglycosides potentiate

macroscopic currents at heteromeric NR1/NR2B receptors, probably through binding to

a stimulatory polyamine site on the receptor [82]. As shown in Fig. 13, binding of

spermine to NR1-R, NR2A-R and NR2B-R was inhibited by neomycin B,

paromomycin, kanamycin A and streptomycin, consistent with the idea that

aminoglycosides act at the stimulatory polyamine binding sites on the R domains of

NR1, NR2A and NR2B. The magnitude of inhibition of spermine binding to R

domain proteins by aminoglycosides paralleled potentiation of intact NR1/NR2B

receptors by aminoglycosides measured in functional studies, and occurred at similar

concentrations of aminoglycosides [82].

39

[14 C

]Spe

rmin

e bo

und

(nm

ol/m

g pr

otei

n)

1

2

3

0

75 mM 150 mM 300 mM

NR1-R NR2A-R NR2B-R4

Fig. 13 Effect of aminoglycosides on spermine binding to R domain proteins.

Binding of [14C]spermine was measured in the presence and absence of

aminoglycosides (75 to 300 µM) as described in Materials and methods.

[14C]Spermine used was 20 µM. Values are means ± S. D. from three experiments.

40

Discussion Several studies have reported the purification and properties of soluble S1-S2

domains of AMPA and kainate receptor proteins [69, 71]. The same approach has been

applied to NMDA receptor subunits. The characteristics of the glycine binding site on

the NR1 subunit have been reported from studies using a purified soluble S1-S2 domain

[75] and a membrane-bound fusion protein containing the entire extracellular portion (R

domain plus S1-S2) of NR1 [86]. In more recent studies, some characteristics of an

ifenprodil binding site on NR2B have been described using a purified soluble NR2B R

domain [89, 95]. We have used a similar approach to study the binding sites for

spermine and ifenprodil on the R domains of NR1 and NR2 subunits. These studies

indicate that spermine and ifenprodil can differentially bind to these domains. The

experiments led to several interesting findings as discussed below.

We found that spermine bound to the R domains of all three NMDA receptor

subunits. The affinity for spermine was NR1-R > NR2B-R > NR2A-R; the Kd values

for spermine at NR1-R, NR2A-R and NR2B-R were 19, 140 and 33 μM, respectively.

The results suggest that spermine stimulation of NR1/NR2A and NR1/NR2B may be

caused through spermine binding to the R domain of NR2A and NR2B together with

binding to the R domain of NR1. It is notable that the potency of spermine as

determined in functional assays was 3- to 4-fold lower at NR1/NR2A receptors (EC50,

278 µM) than at NR1/NR2B receptors (EC50, 78 µM), which correlates with the 4-fold

lower affinity for spermine of the NR2A R domain (Kd, 140 µM) compared to the

NR2B R domain (Kd, 33 µM). In any event, the results indicate that there are discrete

41

spermine binding sites on the three different R domain proteins, and are consistent with

our previous mutagenesis studies that identified residues important for spermine

stimulation in the R domains of NR1 and NR2B [63,64].

The results demonstrate that ifenprodil binds to NR1-R and NR2B-R, but not

to NR2A-R. It has been reported that mutations at several residues in NR2B-R greatly

reduced sensitivity to ifenprodil [89]. These residues were in positions analogous to

residues in the NR2A subunit that affect sensitivity to Zn2+ and may form a Zn2+ binding

site on NR2A[94]. In the same report, ifenprodil was shown to retard protease

digestion of a soluble NR2B-R domain (residues 28 to 375) but not of an NR2A R

domain [89]. It was concluded that ifenprodil binds within the cleft of the bi-lobed R

domain of NR2B, analogous to binding of Zn2+ in the cleft of the NR2A R domain [89].

In this context, it should be noted that NR2A-R moves more rapidly on an SDS

polyacrylamide gel compared with NR1-R and NR2B-R (see Fig. 4), even though the

molecular masses of three regulatory domains were almost identical. This suggests

that the overall structure of NR2A-R may be different from that of NR1-R and NR2B-R,

accounting for differences in the binding sites for Zn2+ and ifenprodil even though these

two apparent binding sites share many homologous residues in NR2A and NR2B [88,

89].

In the present study, we observed binding of [3H]ifenprodil to NR1-R and

NR2B-R but not to NR2A-R. The Kd values for binding of ifenprodil to NR1-R and

NR2B-R (0.18 and 0.21 μM, respectively) were similar to the IC50 for ifenprodil

inhibition at intact, functional NR1/NR2B receptors (0.34 μM at pH 7.6) [55]. It has

42

also been reported that the binding constant for ifenprodil at NR2B-R was

0.13–0.17 μM when measured by circular dichroism [95] , a finding that we also

replicated in the current study. The finding that [3H]ifenprodil binds to NR1-R is

consistent with our previous finding that mutations in this region of the NR1 subunit

affect sensitivity to ifenprodil [64]. Although other reports have localized an ifenprodil

binding site to NR2B-R [89], our results suggest that the R domain of NR1 is also

capable of forming an ifenprodil binding site with a high affinity, similar to NR2B-R.

The finding that ifenprodil can bind to the R domain of NR1 is consistent with the high

affinity inhibition by ifenprodil of apparent "homomeric" NR1 receptors expressed in

oocytes [55]. It has been proposed that these receptors are not truly NR1 homomers and

that an endogenous 'NR2-like'Xenopus subunit can combine with NR1 to form these

receptors [100]. However, another study [101] has convincingly shown that this

endogenous subunit does not contribute to the formation of 'homomeric' NR1 receptors

in Xenopus oocytes, and the true nature of such receptors (NR1 homomers or NR1/NRX

heteromers) remains unresolved. There is no evidence for expression of functional

homomeric NR1 receptors in vivo, and ifenprodil appears to produce high affinity

inhibition (Kd less than 1 μM) only at heteromeric NR1/NR2 receptors containing

NR2B. Thus, even though the NR1 R domain is capable of forming a high affinity

ifenprodil binding site, the affinity for ifenprodil or the coupling of binding to channel

activity may be reduced in heteromeric receptors containing NR2A, NR2C, and NR2D,

but retained in receptors containing NR2B.

The results from this study are consistent with the idea that spermine and

ifenprodil act at distinct binding sites on the NMDA receptor [77, 64]. Amino acid

residues involved in ifenprodil inhibition in NR1-R and NR2B-R were mainly located

43

between residues 80 and 200[64, 89]. On the other hand, amino acid residues that

influence spermine stimulation in NR1 and NR2B were mainly located between

residues 170 and 350 [63, 64]. It may be that spermine binds within the cleft of the R

domain similar to polyamine binding to the bacterial protein PotD [90, 64]. In that case,

ifenprodil may bind to another site within the cleft [89] or elsewhere on the R domain.

Even though spermine and ifenprodil have separate binding sites, there are interactions

(presumably allosteric) between these two ligands at intact receptors [77].

44

CHAPTER 3

Modeling of NR1A and NR2B

regulatory domain of the NMDA receptor

45

Introduction NMDA receptor function is modulated by a wide variety of compounds such

as polyamine, proton and ifenprodil, and findings show that the biding sites for these

modulators are found in the amino teminal domain of such receptors. In our previously

studies suggest that the biding sites of spermine and ifenprodil are on the R domain of

NMDA receptor. The R domain of NMDA receptors have not been crystallized but it

has been noted that there is homology with other periplasmic binding proteins (LIVBP),

polyamine binding protein (Pot D) and metabotropic glutamate receptors (mGluRs). In

the present study, the construction of a three-dimensional model of the NR1A-R and

NR2B-R is described using the closed agonist-binding domain of mGluR1 and docking

the R domain with spermine and ifenprodil. Through the simulation we found that there

are two binding sites of spermine in NR1A-R and one is into the cleft of the 3D model

of NR1A-R the other on the surface. But in the 3D model of NR2B-R there is only one

spermine binding site. Ifenprodil bound into the cleft of the 3D model of NR2B-R and

bound to surface of the3D model of NR 1A-R. The results suggest that the ligand which

bound to the cleft of the NR-R maybe regulate the NMDA receptor more than to the

surface.

46

Results Determination of amino acid residues at the R domain of NR1A and NR2B which

are involved in spermine stimulation at -20mV

Spermine is polycationic and we hypothesized that the amino groups of

spermine may interact with acidic residues on NMDA receptor subunits, as has been

found for the interaction of polyamines with the bacterial polyamine binding protein

PotD[64]. We previously found that mutations at NR1 (E181) and NR1 (E342)

influence spermine stimulation. In this study we made a series of individual point

mutations in the R domain of NR1A and NR2B. We screen each mutation by measuring

spermine stimulation at -20mV with 100 mM spermine [63, 64]. If spermine stimulation

decreased³20% in the mutants compared with those of wild type NMDA receptor, it is

judged that mutant amino acid residues are involved in spermine stimulation,

respectively.

Results on NR1A and NR2B mutants are shown in Fig. 14 and 16. Mutations at

the acidic residues of NR1A near NR1 (E181) and NR1 (E342) have a strong effect on

spermine stimulation. It suggests that there are two spermine binding sites in NR1A-R.

Mutation at acidic residues from 190-210 of NR2B show a strong effct on spermine

stimulation and indicate that there is one spermine binding site in NR2B-R.

Determination of amino acid residues at the R domain of NR1A and NR2B which

are involved in ifenprodil inhibition at -20mV

Ifenprodil is an atypical noncompetitive antagonist at the NR1/NR2B NMDA

receptor. We previously found that mutations at NR1 (D130) influence ifenprodil

47

inhibition [64]. In this study we screen each mutation by measuring ifenprodil inhibiton

at -20mV with 1 mM ifenprodil. If ifenprodil inhibition decreased³150% in the mutants

compared with those of wild type NMDA receptor, it is judged that mutant amino acid

residues are involved in ifenprodil inhibition, respectively.

Results on NR1A and NR2B mutants are shown in Fig. 15 and 17. Mutations

at the amino acid residues from 100 to 134 of NR1A near NR1 (D130) have a strong

effect on ifenprodil inhibition. It suggests that there is a ifenprodil binding sites in

NR1A-R. Mutation from 100 to 114 amino acid residues of NR2B show a strong effct

on ifenprodil inhibition and that indicate there is one ifenprodil binding site in NR2B-R.

Molecular modeling of the NR1A-R and NR2B-R and docking of spermine and

infenprodil

We built a 3D model of the R domain of NR1A and NR2B using the

agonist-binding domain (ABD) of the metabotropic glutamate receptor mGluR1 as a

template(PDB code 1EWK:A) [96]. Three dimensional structures of NR1A-R and

NR2B-R were constructed by homology modeling with the Amber99 force field in

MOE (version 2007.09, CCG Inc., Montreal, Canada). 3D model were generated using

the alignment shown in Fig. 18. Because the sequence identity between NR1A or NR2B

and mGlur1 ABD is very low (~18%), we base our alignment according to known

(mGluR1 ABD) and predicted NR1A-R and NR2B-R secondary structure elements (Fig.

18) . However , because the sequence of the NR1A-R and NR2B share such a low

sequence with mGluR1 ABD, the orientations of residues side-chains are rather

imprecise. Thorough functional validation of these model are therefore mandatory to

48

comfirm its biological relevance.

Furthermore, the structure of the docking complex between NR1A-R and

spermine, NR1A-R and ifenprodil, NR2B-R and spermine, and NR2B-R and ifenprodil

were predicted with ASEDock of MOE. The resulting three-dimensional structure of

each complex was displayed using MolFeat (Ver. 3.6, FiatLux, Tokyo, Japan). We

found that there are two spermine binding sites in the 3D model of NR1A-R, one binds

into the cleft of the 3D model of NR1A shown in Fig. 19 and the other binds on the

surface. While there is only one ifenprodil binding site on the surface of 3D model of

NR1A-R. These results indicate that there are two spermine binding sites and one

ifenprodil biding site at NR1A-R, respectively. We also found that there is one

sepermine binding site in the surface and one ifenprosil binding site in the cleft of the

3D model of NR2B-R. This result incicates that there is one spermine and one

ifenprodil binding site at NR2B-R, respectively.

49

0 50 100 150 200 250

E188QR187AE186QE185QE181QE172QD170ND169NW160LW160FY158LE153QW151LH146SY144LF137L

H134SS132AK131LD130NS129AY128AS126AY114LF113LT110AY109LS108LF102L

H101SD100N

Y88LD81NE80QS77AK68AS64AV46AE44QF42AE39QT35AD23N

WT

% of control

0 50 100 150 200 250

E387QE385QD376NY367LY351LF348AD345ND343NE342QF340AE339QD332NY330LY330FF321LW315LW315FD303NE299QE297QE294QD283NE277QD264NY261LE253QE251QW247LD227ND226NE225QE215QE213QE210QD198NF197L

E192QWT

% of control

Fig. 14 Effects of spermine at mutate NMDA receptors in NR1A.

Effect of spermine (100 mM) were detemined in oocytes expressing NR1A/NR2B

receptors with wild-type or mutant NR1A subunits, voltage-clamped at -20mV and

activate by 10 mM glutamate and 10 mM glycine . Values are mean ±S.E.M. from 20

oocytes for wild type and four oocytes for each mutant. Wild type are shown by red

boxes. Mutations that reduced stimulation by ³20% compared with wild type are shown

in yellow boxes.

50

0 50 100 150

E188QR187AE186QE185QE181QE172QD170ND169NW160LW160FY158LE153QW151LH146SY144LF137L

H134SS132AK131LD130NS129AY128AS126AY114LF113LT110AY109LS108LF102L

H101SD100N

Y88LD81NE80QS77AK68AS64AV46AE44QF42AE39QT35AD23N

WT

% of control0 50 100 150

E387QE385QD376NY367LY351LF348AD345ND343NE342QF340AE339QD332NY330LY330FF321L

W315lW315FD303NE299QE297QE294QD283NE277QD264NY261LE253QE251QW247LD227ND226NE225QE215QE213QE210QD198NF197L

E192QWT

% of control

Fig. 15 Effects of ifenprodil at mutate NMDA receptors in NR1A.

Effect of ifenprodil (1 mM) were detemined in oocytes expressing NR1A/NR2B

receptors with wild-type or mutant NR1A subunits, voltage-clamped at -20mV and

activate by 10 mM glutamate and 10 mM glycine . Values are mean ±S.E.M. from 20

oocytes for wild type and four oocytes for each mutant. Wild type is shown by red box.

Mutations that reduced inhibition by ³150% compared with wild type are shown in

yellow boxes.

51

0 50 100 150 200 250

K234LP226SQ224HN218VI216T

S214LD213LD210NL209FM207TD206NL204TE201QE200QE198QW197LE191QF182LD181NF176LY167LD165NE162QE151QF146LE139QD138ND136NS131LH127SS116AF114LD113ND101AE106QL70AE69QS63AF59LD46NS34A

WT

% of control0 50 100 150 200 250

E449QE448QD447NE443QE374QE370QK361AY356AD354NE353QF351LN348DE345QF344AY338FR337KR337AR337QS332LY330TI329AE326QS319AE316QE310KD306SP290EG288SS271KT268LD265NT255FY254HG253DV247FN245RV243EE242LF241LY239LT238V

WT

% of control

Fig. 16 Effects of spermine at mutate NMDA receptors in NR2B.

Effect of spermine (100 mM) were detemined in oocytes expressing NR1A/NR2B

receptors with wild-type or mutant NR2B subunits, voltage-clamped at -20mV and

activate by 10 mM glutamate and 10 mM glycine . Values are mean ±S.E.M. from 20

oocytes for wild type and four oocytes for each mutant. Wild type are shown by red

boxes. Mutations that reduced stimulation by ³20% compared with wild type are shown

in yellow boxes.

52

0 50 100 150

E449QE448QD447NE443QE374QE370QK361AY356AD354NE353QF351LN348DE345QF344AY338FR337KR337AR337QS332LY330TI329A

E326QS319AE316QE310KD306SP290EG288SS271KT268LD265NT255FY254HG253DV247FN245RV243EE242LF241LY239LT238V

WT

% of control0 50 100 150

K234LP226SQ224HN218V

I216TS214LD213LD210NL209FM207TD206NL204TE201QE200QE198QW197

E191QF182LD181NF176L

Y167LD165NE162QE151QF146LE139QD138ND136NS131LH127SS116AF114LD113NT103AD101AE106Q

T76CL70AE69QS63AF59LD46N

V42AS34A

WT

% of control

Fig. 17 Effects of ifenprodil at mutate NMDA receptors in NR2B.

Effect of ifenprodil (1 mM) were detemined in oocytes expressing NR1A/NR2B

receptors with wild-type or mutant NR2B subunits, voltage-clamped at -20mV and

activate by 10 mM glutamate and 10 mM glycine . Values are mean ±S.E.M. from 20

oocytes for wild type and four oocytes for each mutant. Wild type is shown by red box.

Mutation that reduced inhibition by ³150% compared with wild type are shown in

yellow boxes.

53

RKCGEIREQYGIQRVEAMFHTLDKINADPVLLPNITLGSEIRDSCWHSSVALEQSIEFIR -------------KPIAGVIGPGKIVNIGAVLSTRKHEQMFREAVNQANKRHGSWKIQLNATSVTHKPNAIQMALSVCEDLI SSQVYAILVSHPPTPNSQKSPPSIGIAVILVGTSDEVAIKDAHEKDDFHHLSVVPRVELVAMNETDPKSIITRICDLMSDRKIQGVVFADDTD

SSSVAIQVQNLLQLFDIPQIAYSATSIDLSDKTLYKYFLRVVPSDTLQARAMLDIVKRYNWTYVSAVHTEGNYGESDHFTPTPVSYTAGFYRIPVLGLTTRMSIYSDKSIHLSFLRTVPPYSHQSSVWFEMMRVYNWNHIILLVSDDHEGRQEAIAQILDFISAQTLTPILGIHGGSSMIMADKDESSMFFQFGPSIEQQASVMLNIMEEYDWYIFSIVTTYFPGYQD

13199

104

mGluR1NR1ANR2B

239174181

mGluR1NR1ANR2B

GMDAFKELAAQEGLCIAHSDKIYSNAGEKSFDRLLRKLRERLPKARVVVCFCEGMTVRGLLSAMRRLGVVGEFSAAQKRLETLLEERE-------SKAEKVLQFDPGTKNVTALLMEAREL-EARVIILSASEDDAATVYRAAAMLNMTGSGYFVNKIRSTIENS----FVGWELEEVLLLDMSLDDGDSKIQNQLKKLQSPIILLYCTKEEATYIFEVANSVGLTGYGYTW

mGluR1NR1ANR2B

313245256

LIGSDGWADRDEVIEGYEVEANGGITIKLQSPEVRSFDDYFLKLRLDTNTRNPWFPEFWQHRFQCRLPGHLLENVWLVGEREISGNAL---------RYAPDGIIGLQLINGKNESAHISDAVGVVAQAVHELLEKENITDPPRGCVGNTNIWKIVPSLVAGTDTVPSEFPTGLISVSYDEWD-------YGLPARVRDGIAIITTAASDMLSEHSFIPEPKSSCYNTHEKRIY

mGluR1NR1ANR2B

387313330

PNFKKVCTGNESLEENYVQDSKMGFVINAIYAMAHGLQNMHHALCPGHVGLCDAMKPIDGRKLTGPLFKRVLMSSKYADGVTGRVEFNEDGDRKFANYSIMNLQNRKLVQVGIYNGTHVIPNDRKIIQSNMLNRYLINVTFEGRNLSFSEDGYQMHPKLVIILLNKERKWERVGKWKDKSLQMKYYV

mGluR1NR1ANR2B

450380390

Fig. 18 Multiple amino acid sequence alignment of NR1A-R and NR2B-R with the

agonist-binding domain of mGluR1.

The indicated a-helix (red) and b-strands (blue) of mGluR1 are from the X-ray structure

of the agonist-binding domain of mGluR1 (PDB: 1EWK: A). Secondary structure

elements of NR1A-R and NR2B-R and the sequence alignment s were predicated using

MOE.

54

SPM

Ifenprodil

SPM

Fig. 19 3D model of spermine and ifenprodil binding into the R-domain of NR-1A.

The 3D model of NR1A-R shows a projected structure based on the known crystal

structure of agonist-binding domain of mGluR1.

55

SPM

Ifenprodil

Fig. 20 3D model of spermine and ifenprodil binding into the R-domain of NR-2B.

The 3D model of NR2B-R shows a projected structure based on the known crystal

structure of agonist-binding domain of mGluR1.

56

Discussion Several studies have reported the homology modeling of R domain of the

NMDA receptors. [97, 98, 99] In more recent studies NR1A-R or NR2B 3D model was

described with periplasmic binding proteins (LIVBP) and metabotropic glutamate

receptors (mGluRs) as template. [97, 98, 99] Their studies on homology modeling show

that ifenprodil binds into the cleft of the NR2B-R. We have used a similar approach to

study the homology modeling of NR1A-R and NR2B-R, then spermine and ifenprodil

were docked into the homology model. In our study we found that there are two

spermine binding sites ( one is on surface and the other is into the cleft of NR1A-R 3D

model) and one ifenprodil binding site at NR1A-R, but one spermine binding site on the

surface and one ifenprodil biding site into the cleft of NR2B-R 3D model. It is reported

that ifenprodil is a novel NMDA antagonist that selectively inhibits NR1/NR2 receptors

containing the NR2B subunit and binding of ifenprodil into the cleft of NR2B-R

controls the high-affinity ifenprodil inhibition. [98, 99] It suggest that binding into the

cleft will get a more conformation change of the NMDA receptor than biding on surface.

Therefore our results suggest that it is important for sepermine binding to NR1A-R

because one spermine binding site is in the cleft. It also indicates that it is important for

ifenprodil biding to NR2B-R.

57

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LIST OF PUBLICATIONS

Han, X., Tomitori, H., Mizuno, S., Higashi, K., Full, C., Fukiwake, T., Terui, Y., Leewanich, P., Nishimura, K., Toida, T., WIlliams, K., Kashiwagi, K., and Igarashi, K.: Binding of spermine and ifenprodil to a purified, soluble regulatory domain of the N-methyl-D-aspartate receptor. J. Neurochem. (2008)107, 1566-1577

71

ACKNOWLEDGEMENTS

I wish to express my great thanks to Professor Kazuei Igarashi for his constant

guidance , kind encouragement and valuable discussions in the course of this study.

I would like to acknowledge Professor Keiko Kashiwagi and Professor

Toshihiko Toida, and Associate Professor Kazuhiro Nishimura for their encouragement,

support and helpful advice. I also thanks for Professor Yu Tamura for his for his help on

the homology modeling and docking of spermine and ifenprodil to the R domain of the

NMDA receptor. Thanks are also due to Drs Hideyuki Tomitori, Yusuke Terui, Takeshi

Uemura, Kyouhei Higashi for their precious help and support.

I would also like to greatly appreciate Ms Satomi Mizuno, Lihua Jin, Hiromi

Sugiyama, Miki Takigawa and Mr Ryotaro Saiki for their kind experimental assistance

and continuous encouragement. If with out their help, it was not possible to accomplish

this work.

Deep appreciation is also expressed to our laboratory members and everyone

whom I have had the opportunity to share time with for their friendly collaboration

kindness and help.

Finally, I am deeply grateful to my friends and family for their understanding

patience support and constant encouragement.

72

THESIS COMMITTEE

This thesis was evaluated by the following committee authorized by the

Graduate School of Pharmaceutical Science, Chiba University.

Professor Kouichi Ueno, Ph.D. Chairman

(Graduate School of Pharmaceutical Science, Chiba University)

Professor Hiroshi Kobayashi, Ph.D.

(Graduate School of Pharmaceutical Science, Chiba University)

Professor Toshihiko Murayama, Ph.D.

(Graduate School of Pharmaceutical Science, Chiba University)