Picrotoxin blockade of invertebrate glutamate-gated chloride channels: Subunit dependence and evidence for binding within the pore

Glutamate-gated chloride channels have been described in nematodes, insects , crustaceans, and mollusks. Subunits from the nematode and insect channels have been cloned and are phylogenetically related to the GABA and glycine ligand-gated chloride channels. Ligand-gated chloride channels are blocked with variable potency by the nonselective blocker picrotoxin. The first two subunits of the glutamate-gated chloride channel family, GluCl alpha and G luCl beta, were cloned from the free living nematode Caenorhabditis elegans . In this study, we analyze the blockade of these novel channels by picroto xin. In vitro synthesized GluCl alpha and GluCl beta RNAs were injected ind ividually or coinjected into Xenopus oocytes. The EC50 values for picrotoxi n block of homomeric GluCl alpha and GluCl beta were 59 mu M and 77 nM, res pectively. Picrotoxin block of homomeric GluCl beta channels was promoted d uring activation of membrane current with glutamate, In addition, recovery from picrotoxin block was faster during current activation by glutamate. A chimeric channel between the N-terminal extracellular domain of GluCl alpha and the C-terminal membrane-spanning domain of GluCl beta localized the hi gher affinity picrotoxin binding site to the membrane-spanning domains of G luCl beta. A point mutation within the M2 membrane-spanning domain of GluCl beta reduced picrotoxin sensitivity >10,000-fold. We conclude that picroto xin blocks GluCl channels by binding to a site accessible when the channel is open.