picrotin and picrotoxinin

Among five hairy root lines of Picrorhiza kurrooa that were established through Agrobacterium rhizogenes, one (H7) was selected for encapsulation due to high accumulation of picrotin and picrotoxinin (8.3 and 47.6 μg/g DW, respectively). Re-grown encapsulated roots induced adventitious shoots with 73 % frequency on MS medium supplemented with 0.1 μM 6-benzylaminopurine, following 6 months of storage at 25 °C. Regenerated plantlets had 85 % survival after 2 months. Regenerants were of similar morphotype having increased leaf number and branched root system as compared to non-transformed plants. The transformed nature of the plants was confirmed through PCR and Southern blot analysis. Genetic fidelity analysis of transformed plants using RAPD and ISSR showed 5.2 and 3.6 % polymorphism, respectively. Phytochemical analysis also showed that picrotin and picrotoxinin content were similar in hairy root line and its regenerants.

Topics: Agrobacterium; Blotting, Southern; Cell Line; Culture Media; DNA Fingerprinting; Genotype; Picrorhiza; Picrotoxin; Plant Roots; Plant Shoots; Polymerase Chain Reaction; Random Amplified Polymorphic DNA Technique; Regeneration; Sesterterpenes; Temperature; Transformation, Genetic

Contrary to its effect on the gamma-aminobutyric acid type A and C receptors, picrotoxin antagonism of the alpha1 homomeric glycine receptors (GlyRs) has been shown to be non-use-dependent and nonselective between the picrotoxin components picrotoxinin and picrotin. Picrotoxin antagonism of the embryonic alpha2 homomeric GlyR is known to be use-dependent and reflects a channel-blocking mechanism, but the selectivity of picrotoxin antagonism of the embryonic alpha2 homomeric GlyRs between picrotoxinin and picrotin is unknown. Hence, we used the patch clamp recording technique in the outside-out configuration to investigate, at the single channel level, the mechanism of picrotin- and picrotoxinin-induced inhibition of currents, which were evoked by the activation of alpha2 homomeric GlyRs stably transfected into Chinese hamster ovary cells. Although both picrotoxinin and picrotin inhibited glycine-evoked outside-out currents, picrotin had a 30 times higher IC50 than picrotoxinin. Picrotin-evoked inhibition displayed voltage dependence, whereas picrotoxinin did not. Picrotoxinin and picrotin decreased the mean open time of the channel in a concentration-dependent manner, indicating that these picrotoxin components can bind to the receptor in its open state. When picrotin and glycine were co-applied, a large rebound current was observed at the end of the application. This rebound current was considerably smaller when picrotoxinin and glycine were co-applied. Both picrotin and picrotoxinin were unable to bind to the unbound conformation of the receptor, but both could be trapped at their binding site when the channel closed during glycine dissociation. Our data indicate that picrotoxinin and picrotin are not equivalent in blocking alpha2 homomeric GlyR.

Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Evoked Potentials; GABA Antagonists; Glycine; Humans; Membrane Potentials; Patch-Clamp Techniques; Picrotoxin; Protein Binding; Protein Conformation; Receptors, Glycine; Sesterterpenes; Transfection

Picrotoxin, an antagonist of structurally-rated GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs), is an equimolar mixture of picrotoxinin (PTXININ) and picrotin (PTN). These compounds share a common structure except that PTN contains a slightly larger dimethylmethanol in place of the PTXININ isopropenyl group. Although the homomeric alpha1 GlyR is equally sensitive to both compounds, we show here that homomeric alpha2 and alpha3 GlyRs, like most GABA(A)Rs, are selectively inhibited by PTXININ. As conservative mutations to pore-lining 6' threonines equally affect the sensitivity of the alpha1 GlyR to both compounds, we conclude that PTXININ and PTN bind to 6' threonines by hydrogen bonding with exocyclic oxygens common to both molecules. In contrast, substitution of the 2' pore-lining glycine by serine selectively reduces PTN sensitivity, whereas the introduction of 2' alanines selectively increases PTXININ sensitivity. These results define the orientation of PTXININ and PTN binding in the alpha1 GlyR pore and allow us to conclude that the relatively reduced sensitivity of PTN at GABA(A)Rs and alpha2 and alpha3 GlyRs is due predominantly to its larger size and reduced ability to form hydrophobic interactions with 2' alanines.

Topics: Data Interpretation, Statistical; DNA, Complementary; Electrophysiology; Glycine; Humans; Hydrogen Bonding; Models, Molecular; Mutagenesis; Neurons, Afferent; Pain; Patch-Clamp Techniques; Picrotoxin; Receptors, GABA-A; Receptors, Glycine; Sesterterpenes; Synapses

The anticonvulsant alpha-ethyl, alpha-methyl-gamma-thiobutyrolactone (alphaEMTBL) potentiates the response to a submaximal concentration of glycine produced by receptors composed of human glycine alpha1-subunits but reduces the response of receptors composed of rat glycine alpha3-subunits. Both the potentiating and blocking actions of alphaEMTBL are reduced by higher concentrations of glycine. The subunit specificity of alphaEMTBL block is conferred by a residue in the second membrane-spanning region (M2), which is alanine in the alpha3-subunit (A254) and glycine in the alpha1-subunit. The mutation A254G in the alpha3-subunit removes blocking by alphaEMTBL and reveals potentiation. Picrotin, a picrotoxinin analog, blocks responses of receptors composed of either alpha1 or alpha3-subunits. Blocking of alpha3 receptors by picrotin is reduced in the presence of alphaEMTBL, indicating that the mechanisms interact at some point, but the mutation alpha3 A254G does not remove block by picrotin. However, mutation of a nearby residue alpha3 T258F does remove block by picrotin, picrotoxinin and alphaEMTBL. These observations suggest that alphaEMTBL and picrotin act on glycine alpha3 receptors to produce block by an allosteric mechanism that involves overlapping sets of residues in the M2 region. Coexpression of the alpha3-subunit with the beta-subunit of the glycine receptor also removes block by alphaEMTBL and reveals potentiation, suggesting that receptors containing either alpha3 or alpha1 glycine receptor subunits are potentiated in the adult brain.

Topics: 4-Butyrolactone; Amino Acid Sequence; Amino Acids; Animals; Anticonvulsants; Humans; Membrane Proteins; Molecular Sequence Data; Mutation; Oocytes; Picrotoxin; Rats; Receptors, Glycine; Recombinant Proteins; Sequence Homology, Amino Acid; Sesterterpenes; Transfection; Xenopus

Topics: Picrotoxin; Sesterterpenes

Topics: Humans; Picrotoxin; Sesterterpenes