Differential effects of modified batrachotoxins on voltage-gated sodium channel fast and slow inactivation.

Voltage-gated sodium channels (NaVs) are targets for a number of acute poisons. Many of these agents act as allosteric modulators of channel activity and serve as powerful chemical tools for understanding channel function. Herein, we detail studies with batrachotoxin (BTX), a potent steroidal amine, and three ester derivatives prepared through de novo synthesis against recombinant NaV subtypes (rNaV1.4 and hNaV1.5). Two of these compounds, BTX-B and BTX-cHx, are functionally equivalent to BTX, hyperpolarizing channel activation and blocking both fast and slow inactivation. BTX-yne-a C20-n-heptynoate ester-is a conspicuous outlier, eliminating fast but not slow inactivation. This property differentiates BTX-yne among other NaV modulators as a unique reagent that separates inactivation processes. These findings are supported by functional studies with bacterial NaVs (BacNaVs) that lack a fast inactivation gate. The availability of BTX-yne should advance future efforts aimed at understanding NaV gating mechanisms and designing allosteric regulators of NaV activity.

Evidence that toxin resistance in poison birds and frogs is not rooted in sodium channel mutations and may rely on "toxin sponge" proteins.

Many poisonous organisms carry small-molecule toxins that alter voltage-gated sodium channel (NaV) function. Among these, batrachotoxin (BTX) from Pitohui poison birds and Phyllobates poison frogs stands out because of its lethality and unusual effects on NaV function. How these toxin-bearing organisms avoid autointoxication remains poorly understood. In poison frogs, a NaV DIVS6 pore-forming helix N-to-T mutation has been proposed as the BTX resistance mechanism. Here, we show that this variant is absent from Pitohui and poison frog NaVs, incurs a strong cost compromising channel function, and fails to produce BTX-resistant channels in poison frog NaVs. We also show that captivity-raised poison frogs are resistant to two NaV-directed toxins, BTX and saxitoxin (STX), even though they bear NaVs sensitive to both. Moreover, we demonstrate that the amphibian STX "toxin sponge" protein saxiphilin is able to protect and rescue NaVs from block by STX. Taken together, our data contradict the hypothesis that BTX autoresistance is rooted in the DIVS6 N→T mutation, challenge the idea that ion channel mutations are a primary driver of toxin resistance, and suggest the possibility that toxin sequestration mechanisms may be key for protecting poisonous species from the action of small-molecule toxins.

Structure-Activity Relationship Evaluation of Wasp Toxin ß-PMTX Leads to Analogs with Superior Activity for Human Neuronal Sodium Channels.

Beta-pompilidotoxin (ß-PMTX) is a 13-amino acid wasp venom peptide that activates human neuronal sodium channel NaV1.1 with weak activity (40% activation at 3.3 µM of ß-PMTX). Through rational design of ß-PMTX analogs, we have identified peptides with significantly improved activity on human NaV1.1 (1170% activation at 3.3 µM of peptide 18). The underlying structure-activity relationship suggests importance of charge interactions (from residue Lys-3) and lipophilic interactions (from residue Phe-7 and Ser-11). Three top-ranked analogs showed parallel activity improvement for other neuronal sodium channels (human NaV1.2/1.3/1.6/1.7) but not muscular subtypes (NaV1.4/1.5). Finally, we found that analog 16 could partially rescue the pharmacological block imposed by NaV1.1/1.3 selective inhibitor ICA-121431 in cultured mouse cortical GABAergic neurons, demonstrating an activating effect of this peptide on native neuronal sodium channels and its potential utility as a neuropharmacological tool.

Veratridine: A Janus-Faced Modulator of Voltage-Gated Sodium Ion Channels.

Voltage-gated sodium ion channels (NaVs) are integral to both neuronal and muscular signaling and are a primary target for a number of proteinaceous and small molecule toxins. Included among these neurotoxins is veratridine (VTD), a C-nor-D homosteroidal alkaloid from the seeds of members of the Veratrum genus. VTD binds to NaV within the pore region, causing a hyperpolarizing shift in the activation threshold in addition to reducing peak current. We have characterized the activity of VTD against heterologously expressed rat NaV1.4 and have demonstrated that VTD acts on the channel as either an agonist or antagonist depending on the nature of the electrophysiological stimulation protocol. Structure-activity studies with VTD and VTD derivatives against NaV mutants show that the functional duality of VTD can be decoupled. These findings suggest that the dichotomous activity of VTD may derive from two distinct, use-dependent binding orientations of the toxin.

Striatal Cholinergic Interneurons Modulate Spike-Timing in Striosomes and Matrix by an Amphetamine-Sensitive Mechanism.

The striatum is key for action-selection and the motivation to move. Dopamine and acetylcholine release sites are enriched in the striatum and are cross-regulated, possibly to achieve optimal behavior. Drugs of abuse, which promote abnormally high dopamine release, disrupt normal action-selection and drive restricted, repetitive behaviors (stereotypies). Stereotypies occur in a variety of disorders including obsessive-compulsive disorder, autism, schizophrenia and Huntington's disease, as well as in addictive states. The severity of drug-induced stereotypy is correlated with induction of c-Fos expression in striosomes, a striatal compartment that is related to the limbic system and that directly projects to dopamine-producing neurons of the substantia nigra. These characteristics of striosomes contrast with the properties of the extra-striosomal matrix, which has strong sensorimotor and associative circuit inputs and outputs. Disruption of acetylcholine signaling in the striatum blocks the striosome-predominant c-Fos expression pattern induced by drugs of abuse and alters drug-induced stereotypy. The activity of striatal cholinergic interneurons is associated with behaviors related to sensory cues, and cortical inputs to striosomes can bias action-selection in the face of conflicting cues. The neurons and neuropil of striosomes and matrix neurons have observably separate distributions, both at the input level in the striatum and at the output level in the substantia nigra. Notably, cholinergic axons readily cross compartment borders, providing a potential route for local cross-compartment communication to maintain a balance between striosomal and matrix activity. We show here, by slice electrophysiology in transgenic mice, that repetitive evoked firing patterns in striosomal and matrix striatal projection neurons (SPNs) are interrupted by optogenetic activation of cholinergic interneurons either by the addition or the deletion of spikes. We demonstrate that this cholinergic modulation of projection neurons is blocked in brain slices taken from mice exposed to amphetamine and engaged in amphetamine-induced stereotypy, and lacking responsiveness to salient cues. Our findings support a model whereby activity in striosomes is normally under strong regulation by cholinergic interneurons, favoring behavioral flexibility, but that in animals with drug-induced stereotypy, this cholinergic signaling breaks down, resulting in differential modulation of striosomal activity and an inability to bias action-selection according to relevant sensory cues.

Evaluation of Bio-VOC Sampler for Analysis of Volatile Organic Compounds in Exhaled Breath.

Monitoring volatile organic compounds (VOCs) from exhaled breath has been used to determine exposures of humans to chemicals. Prior to analysis of VOCs, breath samples are often collected with canisters or bags and concentrated. The Bio-VOC breath sampler, a commercial sampling device, has been recently introduced to the market with growing use. The main advantage for this sampler is to collect the last portion of exhaled breath, which is more likely to represent the air deep in the lungs. However, information about the Bio-VOC sampler is somewhat limited. Therefore, we have thoroughly evaluated the sampler here. We determined the volume of the breath air collected in the sampler was approximately 88 mL. When sampling was repeated multiple times, with the succeeding exhalations applied to a single sorbent tube, we observed linear relationships between the normalized peak intensity and the number of repeated collections with the sampler in many of the breath VOCs detected. No moisture effect was observed on the Tenax sorbent tubes used. However, due to the limitation in the collection volume, the use of the Bio-VOC sampler is recommended only for detection of VOCs present at high concentrations unless repeated collections of breath samples on the sampler are conducted.