Painful toxins acting at TRPV1

Many plant and animal toxins cause aversive behaviors in animals due to their pungent or unpleasant taste or because they cause other unpleasant senstations like pain. This article reviews the current state of knowledge of toxins that act at the TRPV1 ion channel, which is expressed in primary sensory neurons, is activated by multiple painful stimuli and is thought to be a key pain sensor and integrator. The recent finding that painful peptide 'vanillotoxin' components of tarantula toxin activate the TRPV1 ion channel to cause pain led us to survey what is known about toxins that act at this receptor. Toxins from plants, spiders and jellyfish are considered. Where possible, structural information about sites of interaction is considered in relation to toxin-binding sites on the Kv ion channel, for which more structural information exists. We discuss a developing model where toxin agonists such as resiniferatoxin and vanillotoxins are proposed to interact with a region of TRPV1 that is homologous to the 'voltage sensor' in the Kv1.2 ion channel, to open the channel and activate primary sensory nerves, causing pain

Toxic level hypergolic vapor detection sensor development

Development of an electrochemical sensor technology capable of PPB level hypergolic vapor sensing is reported. A portable instrument capable of meeting the design goals is described

Inhibition of skeletal muscle CLC-1 chloride channels by low intracellular pH and ATP

Skeletal muscle acidosis during exercise has long been thought to be a cause of fatigue, but recent studies have shown that acidosis maintains muscle excitability and opposes fatigue by decreasing the sarcolemmal chloride conductance. ClC-1 is the primary sarcolemmal chloride channel and has a clear role in controlling muscle excitability, but recombinant ClC-1 has been reported to be activated by acidosis. Following our recent finding that intracellular ATP inhibits ClC-1, we investigated here the interaction between pH and ATP regulation of ClC-1. We found that, in the absence of ATP, intracellular acidosis frompH 7.2 to 6.2 inhibited ClC-1 slightly by shifting the voltage dependence of common gating to more positive potentials, similar to the effect of ATP. Importantly, the effects of ATP and acidosis were cooperative, such thatATPgreatly potentiated the effect of acidosis. Adenosine had a similar effect to ATP at pH 7.2, but acidosis did not potentiate this effect, indicating that the phosphates of ATP are important for this cooperativity, possibly due to electrostatic interactions with protonatable residues of ClC-1. A protonatable residue identified by molecular modeling, His-847, was found to be critical for both pH and ATP modulation and may be involved in such electrostatic interactions. These findings are now consistent with, and provide a molecular explanation for, acidosis opposing fatigue by decreasing the chloride conductance of skeletal muscle via inhibition of ClC-1. The modulation of ClC-1 by ATP is a key component of this molecular mechanism

Assembly, trafficking and function of a1ß2y2 GABA(A) receptors are regulated by N-terminal regions, in a subunit-specific manner

GABAA receptors are pentameric ligand-gated ion channels (pLGIC) that mediate inhibitory fast synaptic transmission in the central nervous system. Consistent with recent pLGIC structures, sequence analysis predicts an α-helix near the N-terminus of each GABAA receptor subunit. Preceding each α-helix are 8-36 additional residues, which we term the Nterminal extension. In homomeric GABAC receptors and nicotinic acetylcholine receptors (nAChR), the N-terminal α-helix is functionally essential. Here we determined the role of the N-terminal extension and putative α-helix in heteromeric α1β2γ2 GABAA receptors. This role was most prominent in the α1 subunit, with deletion of the N-terminal extension or further deletion of the putative α-helix both dramatically reduced the number of functional receptors at the cell surface. Conversely, deletion of the β2 or γ2 N-terminal extension had little effect on the number of functional cell-surface receptors. Additional deletion of the putative α-helix in the β2 or γ2 subunits did, however, decrease both functional cell surface receptors and incorporation of the γ2 subunit into mature receptors. In the β2 subunit only, α-helix deletions affected GABA sensitivity and desensitization. Our findings demonstrate that N-terminal extensions and α-helices make key subunit-specific contributions to assembly, consistent with both regions being involved in inter-subunit interactions

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 alpha1, alpha2, alpha1beta and alpha2beta 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 alpha2beta GlyR relative to the alpha2 GlyR but not in the alpha1beta GlyR relative to the alpha1 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 alpha2beta GlyR was transferred to the alpha1beta GlyR by the G2'A (alpha1 to alpha2 subunit) substitution. In addition, the alpha1 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 alpha1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position

Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn 168 in NS1 and Asn 459 in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg 368 and Arg 87 respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn 168 /Arg 368 and Asn 459 /Arg 87 residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn 168 /Arg 368 and Asn 459 /Arg 87 pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discusse

A loss-of-function polymorphism in the human P2X4 receptor is associated with increased pulse pressure

The P2X4 receptor is involved in endothelium-dependent changes in large arterial tone in response to shear stress and is, therefore, potentially relevant to arterial compliance and pulse pressure. Four identified nonsynonymous polymorphisms in P2RX4 were reproduced in recombinantly expressed human P2X4. Electrophysiological studies showed that one of these, the Tyr315>Cys mutation (rs28360472), significantly reduced the peak amplitude of the ATP-induced inward current to 10.9% of wild-type P2X4 receptors in transfected HEK-293 cells (10 µmol/L of ATP; n=4-8 cells; P<0.001). Concentration-response curves for ATP and the partial agonist BzATP demonstrate that the 315Cys-P2X4 mutant had an increased EC50 value for both ligands. Mutation of Tyr315>Cys likely disrupts the agonist binding site of P2X4 receptors, a finding supported by molecular modeling based on the zebrafish P2X4 receptor crystal structure. We tested inheritance of rs28360472 encoding the Tyr315>Cys mutation in P2RX4 against pulse pressure in 2874 subjects from the Victorian Family Heart Study. The minor allele frequency was 0.014 (1.4%). In a variance components analysis we found significant association with pulse pressure (P=0.023 for total association) where 1 minor allele increased pulse pressure by 2.84 mm Hg (95% CI: 0.41-5.27). This increase in pulse pressure associated with inheritance of 315Cys-P2X4 receptors might reflect reduced large arterial compliance as a result of impaired endothelium-dependent vasodilation in large arteries

Design, synthesis, and subtype selectivity of 3,6-disubstituted b-carbolines at Bz/GABA(A)ergic receptors. SAR and studies directed toward agents for treatment of alcohol abuse

A series of 3,6-disubstituted ß-carbolines was synthesized and evaluated for their in vitro affinities at axß3V2 GABAA/benzodiazepine receptor subtypes by radioligand binding assays in search of a1 subtype selective ligands to treat alcohol abuse. Analogues of ß-carboline-3-carboxylate-t-butyl ester (ßCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted ß-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo-ßCCt (5). The bivalent ligands of ßCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure-activity relationships (SAR) to larger ligands

Axon initial segment dysfunction in a mouse model of human genetic epilepsy with febrile seizures plus

Febrile seizures are a common childhood seizure disorder and a defining feature of genetic epilepsy with febrile seizures plus (GEFS+), a syndrome frequently associated with Na+ channel mutations. Here, we describe the creation of a knockin mouse heterozygous for the C121W mutation of the ß1 Na+ channel accessory subunit seen in patients with GEFS+. Heterozygous mice with increased core temperature displayed behavioral arrest and were more susceptible to thermal challenge than wild-type mice. Wild-type ß1 was most concentrated in the membrane of axon initial segments (AIS) of pyramidal neurons, while the ß1(C121W) mutant subunit was excluded from AIS membranes. In addition, AIS function, an indicator of neuronal excitability, was substantially enhanced in hippocampal pyramidal neurons of the heterozygous mouse specifically at higher temperatures. Computational modeling predicted that this enhanced excitability was caused by hyperpolarized voltage activation of AIS Na+ channels. This heat-sensitive increased neuronal excitability presumably contributed to the heightened thermal seizure susceptibility and epileptiform discharges seen in patients and mice with ß1(C121W) subunits. We therefore conclude that Na+ channel ß1 subunits modulate AIS excitability and that epilepsy can arise if this modulation is impaired