Picrotoxin Blockade of Invertebrate Glutamate‐Gated Chloride Channels: Subunit Dependence and Evidence for Binding Within the Pore

Abstract

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, GluClalpha and GluClbeta, were cloned from the free living nematode Caenorhabditis elegans. In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluClalpha and GluClbeta RNAs were injected individually or coinjected into Xenopus oocytes. The EC50 values for picrotoxin block of homomeric GluClalpha and GluClbeta were 59 microM and 77 nM, respectively. Picrotoxin block of homomeric GluClbeta 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 GluClalpha and the C-terminal membrane-spanning domain of GluClbeta localized the higher affinity picrotoxin binding site to the membrane-spanning domains of GluClbeta. A point mutation within the M2 membrane-spanning domain of GluClbeta reduced picrotoxin sensitivity >10,000-fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.

Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors

“…Picrotoxin Can Access Its Site in the S15ЈQ Mutant in the Absence of Channel Opening-The ability of picrotoxin to access its site is poor in the absence of the agonist in GABA A and glutamate-gated Cl Ϫ channels (3,5). However, picrotoxin can efficiently access its site without channel opening in glycine ␣1 receptors (13); we confirmed this finding in the present investigation (data not shown).…”

“…However, single channel analysis has shown that picrotoxin lacks the flickery effect that is typical of a classical channel blocker (12). In addition, picrotoxin inhibition is voltage-independent in these channels (3,12,13). Furthermore, other reports have demonstrated little or no usefacilitated block by picrotoxin in glycine and GABA C receptors, respectively (13,21).…”

“…Based on the fact that the onset of picrotoxin block is facilitated in the presence of the agonist in GABA A and glutamate-gated Cl Ϫ channels, it has been suggested that picrotoxin acts as an open channel blocker (3,5). However, single channel analysis has shown that picrotoxin lacks the flickery effect that is typical of a classical channel blocker (12).…”

“…This trait is generally referred to as a use-dependent or use-facilitated block and suggests that the site of action of the antagonist may be in the channel lumen. Whereas picrotoxin block of GABA C and glycine ␣1 receptors displays weak or no use-facilitation (13, 21), picrotoxin inhibition of GABA A receptors and glutamate-gated Cl Ϫ channels is strongly use-facilitated (3,5,11,12). The molecular basis underlying this trait is unknown.…”

“…Picrotoxin inhibits all known anionic ligand-gated Cl Ϫ channels (3)(4)(5). The mechanism of action and the exact location of picrotoxin binding are still unknown (6 -12).…”