Characterization of a Domoic Acid Binding Site from Pacific Razor Clam

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 [3H]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 [3H]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 [3H]kainate. In contrast, Scatchard analysis 3 using [ 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; io- Glutamate is an important excitatory amino acid dowillardiine, (S)-5-iodowillardiine; NBQX, 1,2,3,4-tetrahydro-6- nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium salt; neurotransmitter that allows for normal function of ATPA, (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)pro- nerves. Our understanding of glutamate receptors panoic acid; AMPA, (±)-␣-amino-3-hydroxy-5-methylisoxazole-4- has increased over the past decades due to the syn- propionic acid hydrate; NMDA, N-methyl-d-aspartic acid; CNQX, thesis and use of selective agonists and antagonists 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt; EGTA, ethy- that allow the pharmacological characterization of lene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid. ∗ Corresponding author. Tel.: +1 206 8606788; glutamate-type receptors. The specific action of these fax: +1 206 8603335. compounds has divulged the presence of three distinct E-mail address: [email protected] (V.L. Trainer). classes of excitatory ionotropic amino acid receptors,

0166-445X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2004.04.012 126 V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132 namely kainate (KA), AMPA (previously known as tors for feeding and other important physiological the quisqualate receptor), and N-methyl-d-aspartate processes. (NMDA) receptors (Watkins and Evans, 1981). Al- though KA and domoate (DA) display affinity for all classes of ionotropic glutamate receptors, these tox- 2. Materials and methods ins have high affinities for KA receptors, micromolar affinities for AMPA receptors, and low affinity for 2.1. Materials NMDA receptors. [3H]kainate ([3H]KA; 45 Ci/mmol), [3H]glutamate Phylogenetically, KA binding sites have been stud- (51 Ci/mmol), and kainic acid were purchased from ied in organisms ranging from bacteria to humans Perkin-Elmer Inc. (Shelton, CT). Ecolume was pur- (London et al., 1980). In radioligand binding assays, chased from ICN Biomedicals (Irvine, CA). Tris, KA and DA possess nanomolar affinity for KA recep- phenylmethylsulfonyl fluoride (PMSF), digitonin, Tri- tors in the mammalian brain (Hampson et al., 1987). In ton X-100, glutamate, quisqualate, ibotenate, EGTA, the mammalian nervous system, certain KA-sensitive, NMDA, ATPA, iodowillardiine, NBQX, AMPA and AMPA-type glutamate receptors (abbreviated GluR5 ␥-globulin were purchased from Sigma (St. Louis, through GluR7) and glutamate binding proteins (ab- MO). DA (CRM-DA-d) was obtained from National breviated KA1 and KA2) are found in high densities Research Council (Halifax, Nova Scotia, Canada). in hippocampal regions of the brain, an area associ- Fluorowillardiine was purchased from Tocris Cook- ated with memory and learning (Henley, 1994). How- son Inc. (Ellisville, MO). Razor clams were collected ever, it is known that GluR5-7 form functional recep- from central Washington beaches and transported on tors but no function has yet been assigned to KA1 ice to the lab where tissues were dissected and stored and KA2. Although it is clear that AMPA receptors at −80 ◦C until used in experiments. Clams were mediate fast excitatory transmission in the nervous collected during low tides in 2002 when DA was system, an unsolved puzzle in the glutamate recep- measurable in their tissues. tor field is the role of high affinity KA receptors both in synaptic transmission and in glutamate, KA, and 2.2. Membrane preparation DA-mediated neurotoxicity (Hampson and Manalo, 1998). Tissue from fresh or flash frozen razor clams (43 g A functional role for glutamate receptors in mol- siphon) was homogenized in a buffer containing lusks has been established in previous studies. A 50 mM Tris, pH 7.4, 0.5 mM PMSF, 1 mM EGTA glutamate receptor from the freshwater mollusk, Lym- (5 ml/g tissue) with an OMNI tissue homogenizer. The naea stagnalis, has been isolated and cloned (Stühmer homogenate was centrifuged at 800 × g for 20 min at et al., 1996). This polypeptide shows sequence iden- 4 ◦C. The pellet (P1) was rehomogenized with buffer tity to the mammalian KA-sensitive glutamate recep- (2 ml/g pellet), centrifuged again for 20 min, and the tor and has been shown to be important in feeding combined supernatant fractions were centrifuged at responses (Hutton et al., 1991). However, a large gap 54,000 × g for 15 min at 4 ◦C. The membrane pellet in our understanding exists regarding the characteris- was resuspended in 20 ml buffer using a hand-held tics of glutamate receptors in mollusks that are regu- glass homogenizer and the membranes were cen- larly exposed to the toxin, DA, which is produced by trifuged at 54,000 × g for 15 min. The supernatant diatoms of the genus, Pseudo-nitzschia. These marine fraction was decanted and the partially-purified mem- algae are a natural part of the assemblage on which the branes (P2) were resuspended in 20 ml buffer using Pacific razor clam, Siliqua patula, feeds. This clam is a glass homogenizer. The washed pellet was stored at not only routinely exposed to DA, but is also known –80 ◦C. to retain this toxin in its tissues (up to 100 ␮M DA/g tissue) for periods of over 1 year (Wekell et al., 1994; 2.3. Solubilization Adams et al., 2000). Here we suggest a mechanism by which these organisms survive in a toxic environment Binding experiments using a variety of detergents and still retain active function of glutamate recep- indicated that TX-100 solubilized the highest number V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132 127 of binding sites, therefore this detergent was used that were pretreated with 0.3% polyethyleneimine for further study. Solubilization buffer (0.5 M potas- overnight at 4 ◦C. Filters were rinsed five times with sium phosphate, pH 7.0, 20% glycerol, 0.5 mM PMSF, 4 ml ice-cold 50 mM Tris citrate, pH 7.0. The ra- 1 mM EGTA) containing Triton X-100 and digitonin dioactivity in each filter was measured in Ecolume was added slowly on ice with constant stirring to the (10 ml) by counting in a Packard 1900 TR scintillation thawed P2 membrane preparation (75 mg). The solu- counter. bilized preparation had a final protein concentration of 5 mg/ml and final concentrations of Triton X-100 and 2.5. Protein determinations digitonin at 1.0 and 0.2% (w/v), respectively. The mix- ture was centrifuged at 54,000 × g for 15 min at 4 ◦C. Protein was determined by the BCA method (Pierce The supernatant was removed and dialyzed against Chemical Co., Rockford, IL) using bovine serum al- three changes of 50 mM Tris citrate, pH 7.4, contain- bumin as a standard. ing 10% glycerol and 0.5 mM PMSF. 2.6. Pharmacological analyses of membrane-bound 2.4. Receptor binding assays and solubilized receptors

2.4.1. Membrane binding assay Saturation analyses were performed using Graph- Partially-purified membranes (P2 fractions) were Pad Prism (San Diego, CA), a computerized nonlinear resuspended in 50 mM Tris citrate, pH 7.0 to give a curve-fitting program. final protein concentration of 0.5 mg/ml. Assays were carried out by incubating 5 nM [3H]KA (50 ␮l) with 100 ␮l of the membrane suspension in 13 × 100 mM 3. Results glass tubes for 1 h on ice. [3H]KA was used at a final concentration of 5 nM at all times except in satura- 3.1. Characterization of kainate binding sites tion analysis experiments. Nonspecific binding was defined as the binding determined in the presence of Binding experiments, using a final concentration of 250 ␮M unlabeled KA (final concentration). In com- 5nM[3H]KA and with (nonspecific binding) or with- petitive binding assays, glutamatergic drugs (nM to out (total binding) 250 ␮M cold KA, were performed ␮M concentrations) were used. Saturation binding on crude membrane preparations of razor clam siphon, experiments were also performed using 0–950 nM adductor muscle, mantle, gill, viscera, and foot. The [3H]glutamate in the presence and absence of 250 ␮M highest specific binding, determined as the difference l-glutamate (final concentration). The final assay vol- between total and nonspecific binding, was seen in ume in all tubes was 200 ␮l. The assay contents were siphon (2.4 pmol/mg protein), with lower levels of poured over Whatman GF/C filters under vacuum binding observed in foot (0.5 pmol/mg) and adduc- and rinsed five times with 4 ml of ice-cold 50 mM tor muscle (0.2 pmol/mg). All other tissues showed no Tris citrate, pH 7.0. The radioactivity in each filter binding. Therefore subsequent experiments were per- was measured in Ecolume (10 ml) by counting in a formed using siphon tissue. Membranes from razor Packard 1900 TR scintillation counter. clam siphon were solubilized using a modification of the protocols developed for the solubilization of KA 2.4.2. Soluble binding assay receptors from rat brain (Hampson et al., 1987) and One hundred microliters of the solubilized mem- frog brain (Hampson and Wenthold, 1988). This sol- brane preparation (10 ␮g total) was added to 1.5 ml ubilization method resulted in an 85-fold purification polypropylene microfuge tubes with 5 nM [3H]KA of the crude extract (Table 1). (final concentration), except for saturation analyses where 0.5–160 nM concentrations were used. Non- 3.2. Pharmacological analysis specific binding was determined in the presence of 250 ␮M unlabeled KA. After incubation on ice for Saturation analyses were performed on both the 1 h, samples were filtered on Whatman GF/B filters partially-purified and soluble preparations (Fig. 1). 128 V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132

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) 120 4.8 0.04 1 soluble 42 142 3.4 85

Data are the average of three experiments in which ∼40 g of razor clam siphon was used for each. Using 5 nM [3H]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 partially- capacity sites. The crude solubilized preparation purified preparation, indicating the presence of two showed a similar affinity (KD = 35 ± 1.5 nM) and binding sites. The KD and Bmax values (mean ± S.E.) number of binding sites (Bmax = 10 ± 0.1 pmol/mg were 28 ± 9.4 nM and 12 ± 3.8 pmol/mg protein for protein) as the low affinity site in the partially-purified the high affinity/low capacity site and approximately preparation. The pharmacological properties of the 1 mM and 60 nmol/mg protein for the low affinity/high 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 [3H]KA binding from the membrane-bound receptors was quisqualate > ibotenate > iodowillar- dine = AMPA = fluorowillardiine > domoate > KA > l-glutamate. At high micromolar concentrations, NBQX, NMDA, and ATPA and showed little or no ability to displace [3H]KA (Fig. 2). [3H]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

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 Fig. 1. Saturation analysis of [3H]KA binding to membrane-bound and low affinity receptors are present in razor clam and solubilized binding sites. Total binding (᭹) and nonspecific siphon tissue: one with low nM affinity for KA and ᭺ 3 binding ( )of5nM[ H]KA to razor clam siphon tissue is shown. another with mM affinity. The pharmacological data Each point represents the average ± standard deviation of three determinations. Each experiment was repeated three times with are consistent with the preparation being a part of the similar results. Inset: Scatchard plots of the saturation data. The physiologically active non-NMDA receptor complex. binding maximum values (B) are expressed in pmol/mg protein. 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 [3H]KA binding sites. Membranes were incubated with 9–11 different concentrations for each of the inhibitors. Binding assays were conducted using 5 nM [3H]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 [3H]glutamate binding to membrane-bound razor clam siphon tissue. Total binding (᭹) and nonspecific binding (᭺)of50nM[3H]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. 130 V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132 differs from other AMPA or KA specific subtypes due in the forebrain (London and Coyle, 1979). A high to its inability to be blocked by the competitive antag- affinity KA receptor in frog brain has also been char- onist NBQX. NBQX is the most potent and selective acterized (KD = 5.5 nM and Bmax = 1700 pmol/mg, AMPA receptor antagonist (Hawkins et al., 1995) that Hampson and Wenthold, 1988). The razor clam gluta- has been shown to have no activity at NMDA recep- mate receptor is also similar to insect and crustacean tors (Sheardown et al., 1990). receptors in that quisqualate is much more potent The razor clam receptor shows some similarities to than KA at insect and crustacean neuromuscular junc- previously characterized glutamate receptors of both tions (Gray et al., 1991; Schaeffer et al., 1989). A the AMPA and KA subtypes. The rank order potency quisqualate receptor in the nematode Caenorhabditis of willardiines at KA receptors is the reverse of that elegans also bears similarity to the insect and crus- seen at AMPA receptors (Hawkins et al., 1995). AMPA tacean glutamate receptor due to its selective diplace- receptors have the highest affinity for fluorowillardi- ment of glutamate from the receptor by quisqualate ine, similar to what is seen in razor clams (Fig. 2). but not NMDA or KA (Schaeffer et al., 1989). The Iodowillardiine has been shown to be selective for razor clam siphon contains both a high affinity, low native KA receptors compared with AMPA receptors capacity receptor and a low affinity, high capacity re- (Swanson et al., 1997, Patneau et al., 1992). On the ceptor, the former which is most similar to the rat and other hand, ATPA has low affinity for AMPA recep- frog brain in KA affinity and most similar in competi- tors due to its selectivity for GluR5 receptors of the tor selectivity to other invertebrate quisqualate-type KA receptor subgroup (Clarke et al., 1997). As seen receptors. in Fig. 2, ATPA shows little affinity for [3H]KA binding sites. Although the high affinity of quisqualate 4.2. Molluscan glutamate receptors leads us to classify the razor clam siphon tissue as a non-NMDA receptor, this tissue may contain a com- Glutamate receptors have been shown previously to bination of functional and nonfunctional receptors of be important in the molluscan feeding system (Katz the AMPA/KA and possibly NMDA subtype. In sum- and Levitan, 1993; Quinlan and Murphy, 1991). In ad- mary, razor clam siphon tissue shows characteristics dition, the Lymnaea glutamate receptor may be related of KA receptors (due to its high affinity for iodow- to the vertebrate non-NMDA, AMPA/quisqualate type illardiine, DA and KA), AMPA receptors (due to its of ionotropic receptor because a cDNA clone of Lym- high affinity for AMPA and 5-fluorowillardiine, and naea shows strong sequence homology to mammalian low affinity for ATPA) and NMDA type receptors (due KA and DA sensitive glutamate receptors (Stühmer to the inaction of NBQX), therefore it is likely that et al., 1996, Brierley et al., 1997). The main synap- more than one type of glutamate receptor is expressed tic responses in feeding neurons in Lymnaea are due in this tissue. to non-NMDA receptors, because CNQX, a specific non-NMDA glutamate receptor antagonist, effectively 4.1.1. Similarity of other glutamate receptors blocks most of the excitatory response on the mol- The specific binding of [3H]KA has been observed lusk neuron (Brierley et al., 1997). In the snail, He- to have a wide distribution in nervous tissue from ver- lix aspersa, a single high affinity KA binding site tebrates and invertebrates, indicating a broad phyloge- has been identified in ganglia preparations (Pin et al., netic conservation of these sites that have high density 1986). The pharmacological characteristics of the snail in the brains of birds, fish and amphibians (London KA binding site were similar to the rat CNS KA et al., 1980). Ligand binding studies with [3H]KA sites (London and Coyle, 1979). The excitatory effect have demonstrated specific, saturable and high affin- of glutamate has also been demonstrated on buccal ity binding to brain membranes (Simon et al., 1976; (Taraskevich et al., 1977) and aorta (Sawada et al., London and Coyle, 1979). Two specific sites in rat 1984) muscle fibers of Aplysia, further demonstrating brain have been described, a high affinity (KD = 5 nM) the presence of glutamate receptors on molluscan mus- and lower affinity (KD = 50 nM) site. The low affinity cle fibers. Similarly, our study suggests the presence site is detected in all major brain regions in the rat, of functional glutamate receptors that are essential for however the high affinity site appears to be focused proper muscle response in razor clams. V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132 131

4.3. Razor clam tolerance of DA brain may provide insight into the function of KA receptors. Could KA receptors in mammals be derived A strategy by which razor clams prevent toxifica- from an invertebrate toxin sequestration tool? It has tion may be through tissue-specific expression of high been shown that there is a remarkable similarity be- and low affinity receptor sites. The razor clam may se- tween the neurotoxic effects of KA and the alterations lectively express high affinity glutamate receptors in a seen in the neurogenerative disorder, Huntington’s dis- manner similar to that observed in rat brain (London ease (Coyle and Schwarcz, 1976) and hereditary spinal and Coyle, 1979) by expressing low affinity receptors degenerative disorders (Herndon et al., 1980). Further in all tissues, and selectively expressing high affinity understanding of KA receptors may help in our un- sites in tissues such as siphon. It may be via these derstanding of these debilitating disorders and in pro- low affinity, high capacity sites that razor clams retain viding appropriate therapies for these diseases. 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 dif- Acknowledgements ferent types of glutamate receptors. Although binding of [3H]glutamate to razor clam siphon showed similar We extend our thanks to Anthony Odell for collec- affinity (Fig. 3) to that observed in [3H]KA diplace- tion of razor clams used in these studies. We acknowl- ment assays (Fig. 2), this binding may also indicate edge the Ecology and Oceanography of Harmful Algal the presence of another glutamate receptor subtype. Blooms (ECOHAB) program for their financial assis- Because of the low affinity of [3H]glutamate to ra- tance for the grant “Mechanisms and control of toxin zor clam siphon, filtration assays could not be used to accumulation in shellfish”. This is ECOHAB publica- determine the precise affinities of other glutamatergic tion 100. ligands. However, assays using such ligands did indicate that the receptor characterized by [3H]glutamate binding showed different rank order potencies (not References shown) than that characterized by [3H]KA displace- ment assays (Fig. 2) suggesting that more than one Adams, N.G., Lesoing, M.L., Trainer, V.L., 2000. Environmen- glutamate receptor subtype is present in this tissue. tal conditions associated with domoic acid in razor clams No function has yet been assigned to the KA binding on the Washington coast. J. Shellfish Res. 19 (2), 1007– proteins, KA1 and KA2 (Henley, 1994). It is thought 1015. Brierley, M.J., Yeoman, M.S., Benjamin, P.R., 1997. Glutamate that the mammalian brain KA receptor may be de- is the transmitter for N2v retraction phase interneurons of rived from a primitive receptor still present in mollusks the Lymnaea feeding system. J. Neurophysiol. 78 (6), 3408– (Coyle et al., 1980). Because this protein appears to 3414. have survived the evolutionary process, it may play an Clarke, V.R., Ballyk, B.A., Hoo, K.H., Mandelzys, A., Pellizzari, important role not yet elucidated in mammalian brain. A., Bath, C.P., Thomas, J., Sharpe, E.F., Davies, C.H., Ornstein, P.L., Schoepp, D.D., Kamboj, R.K., Collingridge, G.L., Lodge, Proteins that bind KA with high affinity but do not D., Bleakman, D., 1997. A hippocampal GluR5 kainate form functional channels have also been isolated from receptor regulating inhibitory synaptic transmission. Nature the brains of frogs (Hampson and Wenthold, 1988), (Lond.) 389, 599–603. birds (Gregor et al., 1988) and fish (Wo and Oswald, Coyle, J.T., Schwarcz, R., 1976. Lesion of striatal neurons with 1994). A glutamate receptor in the freshwater mol- kainic acid provides a model for Huntington’s chorea. Nature (Lond.) 263, 244–246. lusk, Lymnae stagalis is KA specific (Stühmer et al., Coyle, J.T., Slevin, J., London, E.D., Biziere, K., Collins, J., 1980. 1996), but when expressed in Xenopus oocytes, does Characterization of neuronal recognition sites for [3H]-kainic not form a functional ion channel (Hutton et al., 1991; acid. In: Yamamura, H.W., Olsen, R.W., Usdin, E. (Eds.), Schuster et al., 1991; Ultsch et al., 1993). In razor Psychopharmacology and Biochemistry of Neurotransmitter clams, KA binding proteins may be present to se- Recepetors. Elsevier, Amsterdam, pp. 501–513. Gray, S.R., Batstone, F.R., Satiapillae, N.F., Richardson, P.J., quester DA from sensitive nerve tissue. 1991. Solubilization and purification of a putative quisqualate- Determining the relationship between the partially- sensitive glutamate receptor from crustacean muscle. Biochem. purified razor clam receptors and those in mammalian J. 273, 165–171. 132 V.L. Trainer, B.D. Bill / Aquatic Toxicology 69 (2004) 125–132

Gregor, P., Eshhar, N., Ortega, A., Teichberg, V.I., 1988. Isolation, Quinlan, E.M., Murphy, A.D., 1991. Glutamate as a putative immunochemical characterization and localization of the neurotransmitter in the buccal central pattern generator of kainate sub-class of glutamate receptor from chick cerebellum. Helisoma trivolvis. J. Neurophysiol. 66, 1264–1271. EMBO J. 7 (9), 2673–2679. Sawada, M., Gibson, D., McAdoo, D.J., 1984. l-Glutamic acid, Hampson, D.R., Huie, D., Wenthold, R.J., 1987. Solubilization of a possible neurotransmitter to anterior aorta of Aplysia. J. kainic acid binding sites from rat brain. J. Neurochem. 49, Neurophysiol. 51, 375–386. 1209–1215. Schaeffer, J.M., Ruiz-Sanchez, J., Shih, T.L., 1989. Argiopine Hampson, D.R., Wenthold, R.J., 1988. A kainic acid receptor from differentiates between vertebrate and invertebrate glutamate frog brain purified using domoic acid affinity chromatography. binding sites. Pesticide Biochem. Physiol. 34, 124–132. J. Biol. Chem. 263 (5), 2500–2505. Schuster, C.M., Ultsch, A., Schloss, P., Cox, J.A., Schmitt, B., Hampson, D.R., Manalo, J.L., 1998. The activation of glutamate Betz, H., 1991. Molecular cloning of an invertebrate glutamate receptors by kainic acid and domoic acid. Nat. Toxins 6, 153– receptor subunit expressed in Drosophila muscle. Science 254, 158. 112–114. Hawkins, L.M., Beaver, K.M., Jane, D.E., Taylor, P.M., Sunter, Sheardown, M.J., Nielsen, E.O., Jacobsen, P., Honore, T., 1990. D.C., Toberts, P.J., 1995. Binding of the new radioligand 2,3-Dihydroxy-6-nitro-benzo[f]-quinoxaline: a neuroprotectant (S)-[3H]AMPA to rat brain synaptic membranes: effects of for cerebral ischemia. Science 247, 571. a series of structural analogues of the non-NMDA receptor Simon, J.R., Contrera, J.F., Kuhar, J.J., 1976. Binding of agonist willardiine. Neuropharmacology 34 (4), 405–410. [3H]-kainic acid and the analogue of l-glutamate to brain Henley, J.M., 1994. Kainate-binding proteins: phylogeny, structures membranes. J. Neurochem. 26, 141–146. and possible functions. Trends Pharmacol. Sci. 15 (6), 182– Stühmer, T., Amar, M., Harvey, R.J., Bermudez, I., van Minner, J., 190. Darlison, M.G., 1996. Structure and pharmacological properties Herndon, R.M., Addicks, E., Coyle, J.T., 1980. Ultrastructural of a molluscan glutamate-gated cation channel and its likely analysis of kainic acid lesion to cerebellar cortex. Neuroscience role in feeding behavior. J. Neurosci. 16 (9), 2869–2880. 5, 1015–1026. Swanson, G.T., Gereau, R.W., Green IV, T., Henemann, S.F., 1997. Hutton, M.L., Harvey, R.J., Barnard, E.A., Darlison, M.G., 1991. Identification of amino acid residues that control functional Cloning of a cDNA that encodes an invertebrate glutamate behavior in GluR5 and GluR6 kainate receptors. Neuron 19, receptor subunit. FEBS Lett. 292, 111–114. 913–926. Katz, P.S., Levitan, I.B., 1993. Quisqualate and ACPD are agonists Taraskevich, P.S., Gibbs, D., Schmued, L., Orkand, R.K., 1977. for a glutamate-activated current in identified Aplysia neurons. Excitatory effects of cholinergic, adrenergic and glutaminergic J. Neurophysiol. 69, 143–150. agonists on a buccal muscle of Aplysia. J. Neurobiol. 8, 325– London, E.D., Klemm, N., Coyle, J.T., 1980. Phylogenetic 335. distribution of [3H] labeled kainic acid receptor-binding sites Ultsch, A., Schuster, C.M., Laube, B., Betz, H., Schmitt, B., 1993. in neuronal tissue. Brain Res. 192, 463–476. Glutamate receptors of Drosophila melanogaster: primary London, E.D., Coyle, J.T., 1979. Specific binding of [3H]-kainic structure of a putative NMDA receptor protein expressed in acid to receptor sites in rat brain. Mol. Pharmacol. 15, 492– the head of the adult fly. FEBS Lett. 324, 171–177. 505. Watkins, J.C., Evans, R.H., 1981. Excitatory amino acid Patneau, D.K., Mayer, M.L., Jane, D.E., Watkins, J.C., 1992. transmitters. Ann. Rev. Pharmacol. Toxicol. 21, 165–204. Activation and desensitization of AMPA/kainate receptors by Wekell, J.C., Gauglitz Jr., E.J., Barnett, H.J., Hatfield, C.L., novel derivatives of willardiine. J. Neurosci. 12 (2), 595– Simons, D., Ayres, D., 1994. Occurrence of domoic acid 606. in Washington State razor clams (Siliqua patula) during Pin, J.-P., Bockaert, J., Recasens, M., 1986. The binding of 1991–1993. Nat. Toxins 2, 197–205. acidic amino acids to snail, Helix aspersa, periesophagic ring Wo, Z.G., Oswald, R.E., 1994. Transmembrane topology of two membranes reveals a single high-affinity glutamate/kainate site. kainate receptor subunits revealed by N-glycosylation. Proc. Brain Res. 366, 290–299. Natl. Acad. Sci. U.S.A. 91, 7154–7158.