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Picrotoxin Blockade of Invertebrate Glutamate‐Gated Chloride Channels: Subunit Dependence and Evidence for Binding Within the Pore
Author(s) -
Etter A.,
Cully D. F.,
Liu K. K.,
Reiss B.,
Vassilatis D. K.,
Schaeffer J. M.,
Arena J. P.
Publication year - 1999
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.1999.72.1.318
Subject(s) - picrotoxin , homomeric , chloride channel , ligand gated ion channel , biophysics , glutamate receptor , chemistry , biology , biochemistry , protein subunit , ion channel , gabaa receptor , receptor , gene
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 α and GIuCl β , were cloned from the free living nematode Caenorhabditls elegans . In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluCl α and GluCl β RNAs were injected individually or coinjected into Xenopus oocytes. The EC 50 values for picrotoxin block of homomeric GluCl α and GluCl β were 59 μM and 77 n M , respectively. Picrotoxin block of homomeric GluCl β channels was promoted during 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 α and the C‐terminal membrane‐spanning domain of GIuCl β localized the higher affinity picrotoxin binding site to the membrane‐spanning domains of GluCl β . A point mutation within the M2 membrane‐spanning domain of GluCl β reduced picrotoxin sensitivity >10,000‐fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.