title: Nodulisporic acid opens insect glutamate-gated chloride channels: identification of a new high affinity modulator.
abstract: Nodulisporic acid (NA) is an indole diterpene fungal product with insecticidal activity. NA activates a glutamate-gated chloride channel (GluCl) in grasshopper neurons and potentiates channel opening by glutamate. The endectocide ivermectin (IVM) induces a similar, but larger current than NA. Using Drosophila melanogaster head membranes, a high affinity binding site for NA was identified. Equilibrium binding studies show that an amide analogue, N-(2-hydroxyethyl-2,2-(3)H)nodulisporamide ([(3)H]NAmide), binds to a single population of sites in head membranes with a K(D) of 12 pM and a B(max) of 1.4 pmol/mg of protein. A similar K(D) is determined from the kinetics of ligand binding and dissociation. Four lines of evidence indicate that the binding site is a GluCl. First, NA potentiates opening of a glutamate-gated chloride current in grasshopper neurons. Second, glutamate inhibits the binding of [(3)H]NAmide by increasing the rate of dissociation 3-fold. Third, IVM potently inhibits the binding of [(3)H]NAmide and IVM binds to GluCls. Finally, the binding of [(3)H]IVM is inhibited by NA. The B(max) of [(3)H]IVM is twice that of [(3)H]NAmide, and about half of the [(3)H]IVM binding sites are inhibited by NA with high affinity (K(I) = 25 pM). In contrast, [(3)H]IVM binding to Caenorhabditis elegans membranes is not inhibited by NA at 100 nM, and there are no high affinity binding sites for NA on these membranes. Thus, half of the Drosophila IVM receptors and all of the NA receptors are associated with GluCl. NA distinguishes between nematode and insect GluCls and identifies subpopulations of IVM binding sites.
authors.affiliation: Department of Membrane Biochemistry and Biophysics, Merck Research Laboratories, RY80C31N, Rahway, New Jersey 07065-0900, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Smith
authors.forename: V A
authors.initials: VA
authors.lastname: Warren
authors.forename: B S
authors.initials: BS
authors.lastname: Thomas
authors.forename: R M
authors.initials: RM
authors.lastname: Brochu
authors.forename: E A
authors.initials: EA
authors.lastname: Ertel
authors.forename: S
authors.initials: S
authors.lastname: Rohrer
authors.forename: J
authors.initials: J
authors.lastname: Schaeffer
authors.forename: D
authors.initials: D
authors.lastname: Schmatz
authors.forename: B R
authors.initials: BR
authors.lastname: Petuch
authors.forename: Y S
authors.initials: YS
authors.lastname: Tang
authors.forename: P T
authors.initials: PT
authors.lastname: Meinke
authors.forename: G J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: C J
authors.initials: CJ
authors.lastname: Cohen
mesh_terms: D000577:Amides / Q000138:chemical synthesis / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D017173:Caenorhabditis elegans; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism; D004594:Electrophysiology; D006110:Grasshoppers; D007211:Indoles / Q000494:pharmacology*; D007306:Insecticides / Q000494:pharmacology*; D015640:Ion Channel Gating / Q000187:drug effects*; D007559:Ivermectin / Q000494:pharmacology; D007700:Kinetics; D015394:Molecular Structure; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding
chemical_list: D000577:Amides; D018118:Chloride Channels; D007211:Indoles; D007306:Insecticides; C410124:N-2-hydroxyethyl-2,2-nodulisporamide N amide; C109777:glutamate-gated chloride channels; C110703:nodulisporic acid A; D007559:Ivermectin