Nodulisporic acid opens insect glutamate-gated chloride channels: Identification of a new high affinity modulator

Nodulisporic acid (NA) is an indole diterpene fungal product with insectici dal activity. NA activates a glutamate-gated chloride channel (GluCl) in gr asshopper neurons and potentiates channel opening by glutamate. The endecto cide 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-H-3)nodulisporamide ([H-3]NAmide), binds to a single p opulation 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 KD 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 chlori de current in grasshopper neurons. Second, glutamate inhibits the binding o f [3H]NAmide by increasing the rate of dissociation 3-fold. Third, IVM pote ntly inhibits the binding of [3H]NAmide and IVM binds to GluCls. Finally, t he binding of [H-3]IVM is inhibited by NA. The B-max of [H-3]IVM is twice t hat of [3H]NAmide, and about half of the [3H]IVM binding sites are inhibite d by NA with high affinity (K-I = 25 pM). In contrast, [H-3]IVM binding to Caenorhabditis elegans membranes is not inhibited by NA at 100 nM, and ther e are no high affinity binding sites for NA on these membranes. Thus, half of the Drosophiln IVM receptors and all of the NA receptors are associated with GluCl. NA distinguishes between nematode and insect GluCls and identif ies subpopulations of IVM binding sites.