Picrotoxin

An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation.[Pubmed:25460510]

Insect Biochem Mol Biol. 2014 Dec;55:19-25.

GABA-activated RDL receptors are the insect equivalent of mammalian GABAA receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6'M) ablates Picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC50 of WT receptors, enhancing responses, but in T6'M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC50, the Picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6' residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.

The Effects of Repetitive Transcranial Magnetic Stimulation on Picrotoxin-Induced Convulsions in Mice.[Pubmed:27627566]

Adv Clin Exp Med. 2016 Mar-Apr;25(2):317-25.

BACKGROUND: Clinical and experimental results show that repetitive transcranial magnetic stimulation (rTMS) is effective and safe in the treatment of epilepsy. The characteristics of the impulse magnetic field (IMF) are empirical in these studies, making impossible to compare their effectiveness. OBJECTIVES: The article presents the results of a study of the anticonvulsive effects of different modes of IMF on the Picrotoxin seizure model, which is important for studying GABAergic brain mechanisms activated by rTMS. MATERIAL AND METHODS: The experiments were performed on outbred male mice (n = 597; 450 in the experimental group and 147 in the control group). The Picrotoxin was injected in a dose of 2.5 mg/kg subcutaneously after either rTMS or a placebo. The rTMS regimes varied in frequency (0.1, 0.3, 0.5, 1.0 and 10 Hz), intensity (10, 20 and 40% of maximal magnetic induction [MMI] of the big ring coil) and the number of procedures (1, 3, 10). The dependence of the criterial characteristics on the TMS parameters was shown in this convulsive model. RESULTS: The analysis of the data obtained showed that 10 rTMS sessions at an intensity of 20% of MMI in the range of frequencies from 0.5 to 1.0 Hz had the most stable and significant effect in terms of reducing the convulsive readiness of the brain, evaluated by the latent period of the myoclonuses in the Picrotoxin test. In all the regimes of exposure, rTMS decreased the number of seizures in the Picrotoxin model by 1.5-2 points; the most significant effect was obtained with 10 rTMS sessions in the 0.5-1.0 Hz frequency range and an intensity of 20% of the MMI (small er, Cyrillic < 0.01). CONCLUSIONS: The study results support the use of IMF as therapy for blocking convulsive attacks.

Anti-anhedonic effect of selective serotonin reuptake inhibitors with affinity for sigma-1 receptors in picrotoxin-treated mice.[Pubmed:27987210]

Br J Pharmacol. 2017 Feb;174(4):314-327.

BACKGROUND AND PURPOSE: Prefrontal dopamine release by the combined activation of 5-HT1A and sigma-1 (sigma1 ) receptors is enhanced by the GABAA receptor antagonist Picrotoxin in mice. Here, we examined whether this neurochemical event was accompanied by behavioural changes. EXPERIMENTAL APPROACH: Male mice were treated with Picrotoxin to decrease GABAA receptor function. Their anhedonic behaviour was measured using the female encounter test. The expression of c-Fos was determined immunohistochemically. KEY RESULTS: Picrotoxin caused an anxiogenic effect on three behavioural tests, but it did not affect the immobility time in the forced swim test. Picrotoxin decreased female preference in the female encounter test and attenuated the female encounter-induced increase in c-Fos expression in the nucleus accumbens. Picrotoxin-induced anhedonia was ameliorated by fluvoxamine and S-(+)-fluoxetine, selective serotonin reuptake inhibitors with high affinity for the sigma1 receptor. The effect of fluvoxamine was blocked by a 5-HT1A or a sigma1 receptor antagonist, and co-administration of the sigma1 receptor agonist (+)-SKF-10047 and the 5-HT1A receptor agonist osemozotan mimicked the effect of fluvoxamine. By contrast, desipramine, duloxetine and paroxetine, which have little affinity for the sigma1 receptor, did not affect Picrotoxin-induced anhedonia. The effect of fluvoxamine was blocked by a dopamine D2/3 receptor antagonist. Methylphenidate, an activator of the prefrontal dopamine system, ameliorated Picrotoxin-induced anhedonia. CONCLUSION AND IMPLICATIONS: Picrotoxin-treated mice show anhedonic behaviour that is ameliorated by simultaneous activation of 5-HT1A and sigma1 receptors. These findings suggest that the increased prefrontal dopamine release is associated with the anti-anhedonic effect observed in Picrotoxin-treated mice.

Picrotoxin blockade of invertebrate glutamate-gated chloride channels: subunit dependence and evidence for binding within the pore.[Pubmed:9886084]

J Neurochem. 1999 Jan;72(1):318-26.

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.

Enhancement by GABA of the association rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype.[Pubmed:7582470]

Br J Pharmacol. 1995 Jun;115(3):539-45.

1. We examined how gamma-aminobutyric acid (GABA) influences interaction of Picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, Picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for Picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance Picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of Picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the Picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors. 4. The time-dependent decay and preincubation time-dependent reduction of initial amplitude of GABA-induced Cl- currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 microM) enhanced the association rate for Picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from Picrotoxin or TBPS block yielded nearly identical values to the above analysis.5. We conclude that Picrotoxin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U-93631 improves our understanding of functional coupling between GABA and Picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various Picrotoxin site ligands.

On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat.[Pubmed:1317428]

J Physiol. 1992 Feb;447:191-213.

1. The mechanism of action of Picrotoxin on GABA receptor channels in rat sympathetic neurones has been investigated with whole-cell clamp. In addition, the action of Picrotoxin on single GABA channels has been examined in outside-out membrane patches from these cells. 2. Picrotoxin, at concentrations which dramatically reduced the amplitude of whole-cell GABA currents, did not alter the spectral time constants or single-channel conductance estimated by analysis of GABA-activated current noise. This was observed at potentials both negative and positive to the GABA reversal potential (i.e. for both inward and outward GABA currents). In control conditions, the slow and fast time constants from GABA noise were 40 +/- 14 ms and 2 +/- 0.4 ms, while the estimated single-channel conductance was 14 +/- 2 pS. In the presence of Picrotoxin, the time constants and estimated single-channel conductance were 41 +/- 5 ms, 2.7 +/- 0.6 ms and 15 +/- 2.3 pS. 3. Picrotoxin did not alter the shape of the whole-cell GABA current-voltage relationship, indicating that the steady-state block was not voltage dependent. The lack of effect of Picrotoxin on the GABA noise spectra and the lack of outward rectification makes it unlikely that Picrotoxin acts by a simple voltage-dependent (or voltage-independent) channel blocking mechanism. In the presence of Picrotoxin the reversal potential for GABA remained at approximately 0 mV in symmetrical chloride. 4. Distributions of total burst durations, obtained from single-channel records with low concentrations of GABA, were fitted with three or four exponential components. Picrotoxin had no consistent effect on the time constants of the total burst length distributions. It also did not alter the amplitude of the main conductance state. However, Picrotoxin did reduce the frequency of channel openings. 5. The application of brief ionophoretic pulses of GABA, to cells under whole-cell voltage clamp, revealed that the rate of onset of block by Picrotoxin was accelerated in the presence of GABA. In the absence of agonist, Picrotoxin produced a more slowly equilibrating block. 6. Our data are consistent with a mechanism whereby Picrotoxin binds preferentially to an agonist bound form of the receptor and stabilizes an agonist-bound shut state. This could, for example, mean that Picrotoxin enhances the occurrence of a desensitized state or an allosterically blocked state.

Picrotoxin (Cocculin) is a natural noncompetitive antagonist of GABAA receptor, binds to the interface pocket on the GABAR. Picrotoxin (Cocculin) causes overstimulation and induces convulsions.

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