Aquatic Toxicology 69 (2004) 125–132
Characterization of a domoic acid binding site
from Pacific razor clam
Vera L. Trainer∗ , Brian D. Bill
NOAA Fisheries, Northwest Fisheries Science Center, Marine Biotoxin Program, 2725 Montlake Blvd. E., Seattle, WA 98112, USA
Received 5 November 2003; received in revised form 27 April 2004; accepted 27 April 2004
Abstract
The Pacific razor clam, Siliqua patula, is known to retain domoic acid, a water-soluble glutamate receptor agonist produced
by diatoms of the genus Pseudo-nitzschia. The mechanism by which razor clams tolerate high levels of the toxin, domoic acid,
in their tissues while still retaining normal nerve function is unknown. In our study, a domoic acid binding site was solubilized
from razor clam siphon using a combination of Triton X-100 and digitonin. In a Scatchard analysis using [3 H]kainic acid,
the partially-purified membrane showed two distinct receptor sites, a high affinity, low capacity site with a KD (mean ± S.E.)
of 28 ± 9.4 nM and a maximal binding capacity of 12 ± 3.8 pmol/mg protein and a low affinity, high capacity site with a
mM affinity for radiolabeled kainic acid, the latter site which was lost upon solubilization. Competition experiments showed
that the rank order potency for competitive ligands in displacing [3 H]kainate binding from the membrane-bound receptors was
quisqualate > ibotenate > iodowillardiine = AMPA = fluorowillardiine > domoate > kainate > l-glutamate. At high micromolar
concentrations, NBQX, NMDA and ATPA showed little or no ability to displace [3 H]kainate. In contrast, Scatchard analysis
using [3 H]glutamate showed linearity, indicating the presence of a single binding site with a KD and Bmax of 500 ± 50 nM and 14
± 0.8 pmol/mg protein, respectively. These results suggest that razor clam siphon contains both a high and low affinity receptor
site for kainic acid and may contain more than one subtype of glutamate receptor, thereby allowing the clam to function normally
in a marine environment that often contains high concentrations of domoic acid.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Domoic acid; Kainic acid; Razor clam; Glutamate receptor; Kainate binding protein; Receptor binding
1. Introduction
Abbreviations: fluorowillardiine, (S)-5-fluorowillardiine; iodowillardiine, (S)-5-iodowillardiine; NBQX, 1,2,3,4-tetrahydro-6nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium salt;
ATPA, (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid; AMPA, (±)-␣-amino-3-hydroxy-5-methylisoxazole-4propionic acid hydrate; NMDA, N-methyl-d-aspartic acid; CNQX,
6-cyano-7-nitroquinoxaline-2,3-dione disodium salt; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N ,N -tetraacetic acid.
∗ Corresponding author. Tel.: +1 206 8606788;
fax: +1 206 8603335.
E-mail address: [email protected] (V.L. Trainer).
Glutamate is an important excitatory amino acid
neurotransmitter that allows for normal function of
nerves. Our understanding of glutamate receptors
has increased over the past decades due to the synthesis and use of selective agonists and antagonists
that allow the pharmacological characterization of
glutamate-type receptors. The specific action of these
compounds has divulged the presence of three distinct
classes of excitatory ionotropic amino acid receptors,
0166-445X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2004.04.012
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V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132
namely kainate (KA), AMPA (previously known as
the quisqualate receptor), and N-methyl-d-aspartate
(NMDA) receptors (Watkins and Evans, 1981). Although KA and domoate (DA) display affinity for all
classes of ionotropic glutamate receptors, these toxins have high affinities for KA receptors, micromolar
affinities for AMPA receptors, and low affinity for
NMDA receptors.
Phylogenetically, KA binding sites have been studied in organisms ranging from bacteria to humans
(London et al., 1980). In radioligand binding assays,
KA and DA possess nanomolar affinity for KA receptors in the mammalian brain (Hampson et al., 1987). In
the mammalian nervous system, certain KA-sensitive,
AMPA-type glutamate receptors (abbreviated GluR5
through GluR7) and glutamate binding proteins (abbreviated KA1 and KA2) are found in high densities
in hippocampal regions of the brain, an area associated with memory and learning (Henley, 1994). However, it is known that GluR5-7 form functional receptors but no function has yet been assigned to KA1
and KA2. Although it is clear that AMPA receptors
mediate fast excitatory transmission in the nervous
system, an unsolved puzzle in the glutamate receptor field is the role of high affinity KA receptors both
in synaptic transmission and in glutamate, KA, and
DA-mediated neurotoxicity (Hampson and Manalo,
1998).
A functional role for glutamate receptors in mollusks has been established in previous studies. A
glutamate receptor from the freshwater mollusk, Lymnaea stagnalis, has been isolated and cloned (Stühmer
et al., 1996). This polypeptide shows sequence identity to the mammalian KA-sensitive glutamate receptor and has been shown to be important in feeding
responses (Hutton et al., 1991). However, a large gap
in our understanding exists regarding the characteristics of glutamate receptors in mollusks that are regularly exposed to the toxin, DA, which is produced by
diatoms of the genus, Pseudo-nitzschia. These marine
algae are a natural part of the assemblage on which the
Pacific razor clam, Siliqua patula, feeds. This clam is
not only routinely exposed to DA, but is also known
to retain this toxin in its tissues (up to 100 ␮M DA/g
tissue) for periods of over 1 year (Wekell et al., 1994;
Adams et al., 2000). Here we suggest a mechanism by
which these organisms survive in a toxic environment
and still retain active function of glutamate recep-
tors for feeding and other important physiological
processes.
2. Materials and methods
2.1. Materials
[3 H]kainate ([3 H]KA; 45 Ci/mmol), [3 H]glutamate
(51 Ci/mmol), and kainic acid were purchased from
Perkin-Elmer Inc. (Shelton, CT). Ecolume was purchased from ICN Biomedicals (Irvine, CA). Tris,
phenylmethylsulfonyl fluoride (PMSF), digitonin, Triton X-100, glutamate, quisqualate, ibotenate, EGTA,
NMDA, ATPA, iodowillardiine, NBQX, AMPA and
␥-globulin were purchased from Sigma (St. Louis,
MO). DA (CRM-DA-d) was obtained from National
Research Council (Halifax, Nova Scotia, Canada).
Fluorowillardiine was purchased from Tocris Cookson Inc. (Ellisville, MO). Razor clams were collected
from central Washington beaches and transported on
ice to the lab where tissues were dissected and stored
at −80 ◦ C until used in experiments. Clams were
collected during low tides in 2002 when DA was
measurable in their tissues.
2.2. Membrane preparation
Tissue from fresh or flash frozen razor clams (43 g
siphon) was homogenized in a buffer containing
50 mM Tris, pH 7.4, 0.5 mM PMSF, 1 mM EGTA
(5 ml/g tissue) with an OMNI tissue homogenizer. The
homogenate was centrifuged at 800 × g for 20 min at
4 ◦ C. The pellet (P1) was rehomogenized with buffer
(2 ml/g pellet), centrifuged again for 20 min, and the
combined supernatant fractions were centrifuged at
54,000 × g for 15 min at 4 ◦ C. The membrane pellet
was resuspended in 20 ml buffer using a hand-held
glass homogenizer and the membranes were centrifuged at 54,000 × g for 15 min. The supernatant
fraction was decanted and the partially-purified membranes (P2) were resuspended in 20 ml buffer using
a glass homogenizer. The washed pellet was stored at
–80 ◦ C.
2.3. Solubilization
Binding experiments using a variety of detergents
indicated that TX-100 solubilized the highest number
V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132
of binding sites, therefore this detergent was used
for further study. Solubilization buffer (0.5 M potassium phosphate, pH 7.0, 20% glycerol, 0.5 mM PMSF,
1 mM EGTA) containing Triton X-100 and digitonin
was added slowly on ice with constant stirring to the
thawed P2 membrane preparation (75 mg). The solubilized preparation had a final protein concentration of
5 mg/ml and final concentrations of Triton X-100 and
digitonin at 1.0 and 0.2% (w/v), respectively. The mixture was centrifuged at 54,000 × g for 15 min at 4 ◦ C.
The supernatant was removed and dialyzed against
three changes of 50 mM Tris citrate, pH 7.4, containing 10% glycerol and 0.5 mM PMSF.
2.4. Receptor binding assays
2.4.1. Membrane binding assay
Partially-purified membranes (P2 fractions) were
resuspended in 50 mM Tris citrate, pH 7.0 to give a
final protein concentration of 0.5 mg/ml. Assays were
carried out by incubating 5 nM [3 H]KA (50 ␮l) with
100 ␮l of the membrane suspension in 13 × 100 mM
glass tubes for 1 h on ice. [3 H]KA was used at a final
concentration of 5 nM at all times except in saturation analysis experiments. Nonspecific binding was
defined as the binding determined in the presence of
250 ␮M unlabeled KA (final concentration). In competitive binding assays, glutamatergic drugs (nM to
␮M concentrations) were used. Saturation binding
experiments were also performed using 0–950 nM
[3 H]glutamate in the presence and absence of 250 ␮M
l-glutamate (final concentration). The final assay volume in all tubes was 200 ␮l. The assay contents were
poured over Whatman GF/C filters under vacuum
and rinsed five times with 4 ml of ice-cold 50 mM
Tris citrate, pH 7.0. The radioactivity in each filter
was measured in Ecolume (10 ml) by counting in a
Packard 1900 TR scintillation counter.
2.4.2. Soluble binding assay
One hundred microliters of the solubilized membrane preparation (10 ␮g total) was added to 1.5 ml
polypropylene microfuge tubes with 5 nM [3 H]KA
(final concentration), except for saturation analyses
where 0.5–160 nM concentrations were used. Nonspecific binding was determined in the presence of
250 ␮M unlabeled KA. After incubation on ice for
1 h, samples were filtered on Whatman GF/B filters
127
that were pretreated with 0.3% polyethyleneimine
overnight at 4 ◦ C. Filters were rinsed five times with
4 ml ice-cold 50 mM Tris citrate, pH 7.0. The radioactivity in each filter was measured in Ecolume
(10 ml) by counting in a Packard 1900 TR scintillation
counter.
2.5. Protein determinations
Protein was determined by the BCA method (Pierce
Chemical Co., Rockford, IL) using bovine serum albumin as a standard.
2.6. Pharmacological analyses of membrane-bound
and solubilized receptors
Saturation analyses were performed using GraphPad Prism (San Diego, CA), a computerized nonlinear
curve-fitting program.
3. Results
3.1. Characterization of kainate binding sites
Binding experiments, using a final concentration of
5 nM [3 H]KA and with (nonspecific binding) or without (total binding) 250 ␮M cold KA, were performed
on crude membrane preparations of razor clam siphon,
adductor muscle, mantle, gill, viscera, and foot. The
highest specific binding, determined as the difference
between total and nonspecific binding, was seen in
siphon (2.4 pmol/mg protein), with lower levels of
binding observed in foot (0.5 pmol/mg) and adductor muscle (0.2 pmol/mg). All other tissues showed no
binding. Therefore subsequent experiments were performed using siphon tissue. Membranes from razor
clam siphon were solubilized using a modification of
the protocols developed for the solubilization of KA
receptors from rat brain (Hampson et al., 1987) and
frog brain (Hampson and Wenthold, 1988). This solubilization method resulted in an 85-fold purification
of the crude extract (Table 1).
3.2. Pharmacological analysis
Saturation analyses were performed on both the
partially-purified and soluble preparations (Fig. 1).
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Table 1
Semi-purification of a glutamate receptor from razor clam siphon
Fraction
Total protein (mg)
Binding (pmol)
Specific activity (pmol/mg)
Purification (fold)
Membrane-bound (P2)
soluble
120
42
4.8
142
0.04
3.4
1
85
Data are the average of three experiments in which ∼40 g of razor clam siphon was used for each. Using 5 nM [3 H]KA, specific binding
was determined as the difference between binding in the presence (nonspecific) and absence (total) of 250 ␮M cold KA.
Scatchard plots were curvilinear in the partiallypurified preparation, indicating the presence of two
binding sites. The KD and Bmax values (mean ± S.E.)
were 28 ± 9.4 nM and 12 ± 3.8 pmol/mg protein for
the high affinity/low capacity site and approximately
1 mM and 60 nmol/mg protein for the low affinity/high
capacity sites. The crude solubilized preparation
showed a similar affinity (KD = 35 ± 1.5 nM) and
number of binding sites (Bmax = 10 ± 0.1 pmol/mg
protein) as the low affinity site in the partially-purified
preparation. The pharmacological properties of the
partially-purified membrane preparation were further
analyzed in competition experiments (Fig. 2). The
IC50 values were in the nanomolar range for most
glutamatergic drugs with quisqualate being the most
potent inhibitor. Competition experiments showed that
the rank order potency for competitive ligands in displacing [3 H]KA binding from the membrane-bound
receptors was quisqualate > ibotenate > iodowillardine = AMPA = fluorowillardiine > domoate > KA
> l-glutamate. At high micromolar concentrations,
NBQX, NMDA, and ATPA and showed little or no
ability to displace [3 H]KA (Fig. 2). [3 H]glutamate
binding to partially-purified razor clam siphon tissue was up to 86% specific. This Scatchard analysis
showed linearity, indicating the presence of a single
binding site (Fig. 3). The KD and Bmax (mean ± S.E.)
were 500 ± 50 nM and 14 ± 0.8 pmol/mg protein,
respectively.
4. Discussion
4.1. Glutamate receptor subtypes in razor clam
Fig. 1. Saturation analysis of [3 H]KA binding to membrane-bound
and solubilized binding sites. Total binding (䊉) and nonspecific
binding (䊊) of 5 nM [3 H]KA to razor clam siphon tissue is shown.
Each point represents the average ± standard deviation of three
determinations. Each experiment was repeated three times with
similar results. Inset: Scatchard plots of the saturation data. The
binding maximum values (B) are expressed in pmol/mg protein.
We have characterized an AMPA/KA type receptor
in razor clam siphon that has specificity for the glutamate receptor ligands quisqualate, AMPA and KA, but
not NMDA. In addition, we have shown that both high
and low affinity receptors are present in razor clam
siphon tissue: one with low nM affinity for KA and
another with mM affinity. The pharmacological data
are consistent with the preparation being a part of the
physiologically active non-NMDA receptor complex.
However, this partially-purified receptor in razor clam
V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132
129
Fig. 2. Competition profiles for membrane-bound [3 H]KA binding sites. Membranes were incubated with 9–11 different concentrations
for each of the inhibitors. Binding assays were conducted using 5 nM [3 H]KA. Abbreviations are as follows: fluorowillardiine (Fwill),
iodowillardiine (Iwill), l-glutamate (l-glu), quisqualate (QUIS), ibotenate (IBO). Other abbreviations can be found in the footnote (above).
Each point represents the average ± standard deviation of three determinations. Experiments were performed three times with similar
results.
Fig. 3. Saturation analysis of [3 H]glutamate binding to membrane-bound razor clam siphon tissue. Total binding (䊉) and nonspecific
binding (䊊) of 50 nM [3 H]glutamate to razor clam siphon tissue is shown. Each point represents the average ± standard deviation of three
determinations. Each experiment was repeated three times with similar results. Inset: Scatchard plots of the saturation data. The binding
maximum values (B) are expressed in pmol/mg protein.
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V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132
differs from other AMPA or KA specific subtypes due
to its inability to be blocked by the competitive antagonist NBQX. NBQX is the most potent and selective
AMPA receptor antagonist (Hawkins et al., 1995) that
has been shown to have no activity at NMDA receptors (Sheardown et al., 1990).
The razor clam receptor shows some similarities to
previously characterized glutamate receptors of both
the AMPA and KA subtypes. The rank order potency
of willardiines at KA receptors is the reverse of that
seen at AMPA receptors (Hawkins et al., 1995). AMPA
receptors have the highest affinity for fluorowillardiine, similar to what is seen in razor clams (Fig. 2).
Iodowillardiine has been shown to be selective for
native KA receptors compared with AMPA receptors
(Swanson et al., 1997, Patneau et al., 1992). On the
other hand, ATPA has low affinity for AMPA receptors due to its selectivity for GluR5 receptors of the
KA receptor subgroup (Clarke et al., 1997). As seen
in Fig. 2, ATPA shows little affinity for [3 H]KA binding sites. Although the high affinity of quisqualate
leads us to classify the razor clam siphon tissue as a
non-NMDA receptor, this tissue may contain a combination of functional and nonfunctional receptors of
the AMPA/KA and possibly NMDA subtype. In summary, razor clam siphon tissue shows characteristics
of KA receptors (due to its high affinity for iodowillardiine, DA and KA), AMPA receptors (due to its
high affinity for AMPA and 5-fluorowillardiine, and
low affinity for ATPA) and NMDA type receptors (due
to the inaction of NBQX), therefore it is likely that
more than one type of glutamate receptor is expressed
in this tissue.
4.1.1. Similarity of other glutamate receptors
The specific binding of [3 H]KA has been observed
to have a wide distribution in nervous tissue from vertebrates and invertebrates, indicating a broad phylogenetic conservation of these sites that have high density
in the brains of birds, fish and amphibians (London
et al., 1980). Ligand binding studies with [3 H]KA
have demonstrated specific, saturable and high affinity binding to brain membranes (Simon et al., 1976;
London and Coyle, 1979). Two specific sites in rat
brain have been described, a high affinity (KD = 5 nM)
and lower affinity (KD = 50 nM) site. The low affinity
site is detected in all major brain regions in the rat,
however the high affinity site appears to be focused
in the forebrain (London and Coyle, 1979). A high
affinity KA receptor in frog brain has also been characterized (KD = 5.5 nM and Bmax = 1700 pmol/mg,
Hampson and Wenthold, 1988). The razor clam glutamate receptor is also similar to insect and crustacean
receptors in that quisqualate is much more potent
than KA at insect and crustacean neuromuscular junctions (Gray et al., 1991; Schaeffer et al., 1989). A
quisqualate receptor in the nematode Caenorhabditis
elegans also bears similarity to the insect and crustacean glutamate receptor due to its selective diplacement of glutamate from the receptor by quisqualate
but not NMDA or KA (Schaeffer et al., 1989). The
razor clam siphon contains both a high affinity, low
capacity receptor and a low affinity, high capacity receptor, the former which is most similar to the rat and
frog brain in KA affinity and most similar in competitor selectivity to other invertebrate quisqualate-type
receptors.
4.2. Molluscan glutamate receptors
Glutamate receptors have been shown previously to
be important in the molluscan feeding system (Katz
and Levitan, 1993; Quinlan and Murphy, 1991). In addition, the Lymnaea glutamate receptor may be related
to the vertebrate non-NMDA, AMPA/quisqualate type
of ionotropic receptor because a cDNA clone of Lymnaea shows strong sequence homology to mammalian
KA and DA sensitive glutamate receptors (Stühmer
et al., 1996, Brierley et al., 1997). The main synaptic responses in feeding neurons in Lymnaea are due
to non-NMDA receptors, because CNQX, a specific
non-NMDA glutamate receptor antagonist, effectively
blocks most of the excitatory response on the mollusk neuron (Brierley et al., 1997). In the snail, Helix aspersa, a single high affinity KA binding site
has been identified in ganglia preparations (Pin et al.,
1986). The pharmacological characteristics of the snail
KA binding site were similar to the rat CNS KA
sites (London and Coyle, 1979). The excitatory effect
of glutamate has also been demonstrated on buccal
(Taraskevich et al., 1977) and aorta (Sawada et al.,
1984) muscle fibers of Aplysia, further demonstrating
the presence of glutamate receptors on molluscan muscle fibers. Similarly, our study suggests the presence
of functional glutamate receptors that are essential for
proper muscle response in razor clams.
V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132
4.3. Razor clam tolerance of DA
A strategy by which razor clams prevent toxification may be through tissue-specific expression of high
and low affinity receptor sites. The razor clam may selectively express high affinity glutamate receptors in a
manner similar to that observed in rat brain (London
and Coyle, 1979) by expressing low affinity receptors
in all tissues, and selectively expressing high affinity
sites in tissues such as siphon. It may be via these
low affinity, high capacity sites that razor clams retain
DA for long periods of time. In addition, the razor
clam may protect itself from domoic acid in its environment through the differential expression of different types of glutamate receptors. Although binding
of [3 H]glutamate to razor clam siphon showed similar
affinity (Fig. 3) to that observed in [3 H]KA diplacement assays (Fig. 2), this binding may also indicate
the presence of another glutamate receptor subtype.
Because of the low affinity of [3 H]glutamate to razor clam siphon, filtration assays could not be used to
determine the precise affinities of other glutamatergic
ligands. However, assays using such ligands did indicate that the receptor characterized by [3 H]glutamate
binding showed different rank order potencies (not
shown) than that characterized by [3 H]KA displacement assays (Fig. 2) suggesting that more than one
glutamate receptor subtype is present in this tissue.
No function has yet been assigned to the KA binding
proteins, KA1 and KA2 (Henley, 1994). It is thought
that the mammalian brain KA receptor may be derived from a primitive receptor still present in mollusks
(Coyle et al., 1980). Because this protein appears to
have survived the evolutionary process, it may play an
important role not yet elucidated in mammalian brain.
Proteins that bind KA with high affinity but do not
form functional channels have also been isolated from
the brains of frogs (Hampson and Wenthold, 1988),
birds (Gregor et al., 1988) and fish (Wo and Oswald,
1994). A glutamate receptor in the freshwater mollusk, Lymnae stagalis is KA specific (Stühmer et al.,
1996), but when expressed in Xenopus oocytes, does
not form a functional ion channel (Hutton et al., 1991;
Schuster et al., 1991; Ultsch et al., 1993). In razor
clams, KA binding proteins may be present to sequester DA from sensitive nerve tissue.
Determining the relationship between the partiallypurified razor clam receptors and those in mammalian
131
brain may provide insight into the function of KA receptors. Could KA receptors in mammals be derived
from an invertebrate toxin sequestration tool? It has
been shown that there is a remarkable similarity between the neurotoxic effects of KA and the alterations
seen in the neurogenerative disorder, Huntington’s disease (Coyle and Schwarcz, 1976) and hereditary spinal
degenerative disorders (Herndon et al., 1980). Further
understanding of KA receptors may help in our understanding of these debilitating disorders and in providing appropriate therapies for these diseases.
Acknowledgements
We extend our thanks to Anthony Odell for collection of razor clams used in these studies. We acknowledge the Ecology and Oceanography of Harmful Algal
Blooms (ECOHAB) program for their financial assistance for the grant “Mechanisms and control of toxin
accumulation in shellfish”. This is ECOHAB publication 100.
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