title: Characterization of a domoic acid binding site from Pacific razor clam.
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 using [3H]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.
authors.affiliation: NOAA Fisheries, Northwest Fisheries Science Center, Marine Biotoxin Program, 2725 Montlake Blvd. E., Seattle, WA 98112, USA. vera.l.trainer@noaa.gov
authors.forename: Vera L
authors.initials: VL
authors.lastname: Trainer
authors.forename: Brian D
authors.initials: BD
authors.lastname: Bill
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D049872:Bivalvia / Q000378:metabolism* / Q000502:physiology; D004072:Digitonin; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D017830:Octoxynol; D017470:Receptors, Glutamate / Q000378:metabolism*; D012588:Scintillation Counting; D014316:Tritium; D014861:Washington
chemical_list: D017470:Receptors, Glutamate; D014316:Tritium; D017830:Octoxynol; D004072:Digitonin; C012301:domoic acid; D007608:Kainic Acid