Discrepancy between antennal and behavioural responses for enantiomers of -pinene: electrophysiology and behavior of Helicoverpa armigera (Lepidoptera)

The ability of adult cotton bollworm, Helicoverpa armigera (Hubner), to distinguish and respond to enantiomers of alpha-pinene was investigated with electrophysiological and behavioral methods. Electroantennogram recordings using mixtures of the enantiomers at saturating dose levels, and single unit electrophysiology, indicated that the two forms were detected by the same receptor neurons. The relative size of the electroantennogram response was higher for the (-) compared to the (+) form, indicating greater affinity for the (-) form at the level of the dendrites. Behavioral assays investigated the ability of moths to discriminate between, and respond to the (+) and (-) forms of alpha-pinene. Moths with no odor conditioning showed an innate preference for (+)-alpha-pinene. This preference displayed by naive moths was not significantly different from the preferences of moths conditioned on (+)-alpha-pinene. However, we found a significant difference in preference between moths conditioned on the (-) enantiomer compared to naive moths and moths conditioned on (+)-alpha-pinene, showing that learning plays an important role in the behavioral response. Moths are less able to distinguish between enantiomers of alpha-pinene than different odors (e.g., phenylacetaldehyde versus (-)-alpha-pinene) in learning experiments. The relevance of receptor discrimination of enantiomers and learning ability of the moths in host plant choice is discussed.

Molecular determinants of ginkgolide binding in the glycine receptor pore

Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha 1, alpha 2, alpha 1 beta and alpha 2 beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha 2 beta GlyR relative to the alpha 2 GlyR but not in the alpha 1 beta GlyR relative to the alpha 1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha 2 beta GlyR was transferred to the alpha 1 beta GlyR by the G2'A (alpha 1 to alpha 2 subunit) substitution. In addition, the alpha 1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha 1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.

mu O-conotoxin MrVIB selectively blocks Na(v)1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits

The tetroclotoxin-resistant voltage-gated sodium channel (VGSC) Na(v)1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that mu O-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Na(v)1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetroclotoxin-resistant current characteristic of Na(v)1.8 but not Na(v)1.9 or tetroclotoxin-sensitive VGSC currents. MrVIB blocked human Nav1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03-3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Na(v)1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.