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).