Animal toxins that modulate the activity of voltage-gated sodium (Na) channels are broadly divided into two categories-pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Na channel NaPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSD and the pore of NaPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Na channel drugs.

Temple-Baraitser syndrome (TBS) is a multisystem developmental disorder characterized by intellectual disability, epilepsy, and hypoplasia or aplasia of the nails of the thumb and great toe. Here we report damaging de novo mutations in KCNH1 (encoding a protein called ether à go-go, EAG1 or KV10.1), a voltage-gated potassium channel that is predominantly expressed in the central nervous system (CNS), in six individuals with TBS. Characterization of the mutant channels in both Xenopus laevis oocytes and human HEK293T cells showed a decreased threshold of activation and delayed deactivation, demonstrating that TBS-associated KCNH1 mutations lead to deleterious gain of function. Consistent with this result, we find that two mothers of children with TBS, who have epilepsy but are otherwise healthy, are low-level (10% and 27%) mosaic carriers of pathogenic KCNH1 mutations. Consistent with recent reports, this finding demonstrates that the etiology of many unresolved CNS disorders, including epilepsies, might be explained by pathogenic mosaic mutations.

Supplementary data are available at Bioinformatics online.

Venomous animals have evolved toxins that interfere with specific components of their victim's core physiological systems, thereby causing biological dysfunction that aids in prey capture, defense against predators, or other roles such as intraspecific competition. Many animal lineages evolved venom systems independently, highlighting the success of this strategy. Over the course of evolution, toxins with exceptional specificity and high potency for their intended molecular targets have prevailed, making venoms an invaluable and almost inexhaustible source of bioactive molecules, some of which have found use as pharmacological tools, human therapeutics, and bioinsecticides. Current biomedically-focused research on venoms is directed towards their use in delineating the physiological role of toxin molecular targets such as ion channels and receptors, studying or treating human diseases, targeting vectors of human diseases, and treating microbial and parasitic infections. We provide examples of each of these areas of venom research, highlighting the potential that venom molecules hold for basic research and drug development.

Please cite this article as: Cristofori-Armstrong, B., Rash, L.D., Acid-sensing ion channel (ASIC) structure and function: Insights from spider, snake and sea anemone venoms, Neuropharmacology (2017), doi: 10.1016/j.neuropharm.2017.04.042. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT2 ABSTRACT Acid-sensing ion channels (ASICs) are proton-activated cation channels that are expressed in a variety of neuronal and non-neuronal tissues. As proton-gated channels, they have been implicated in many pathophysiological conditions where pH is perturbed. Venom derived compounds represent the most potent and selective modulators of ASICs described to date, and thus have been invaluable as pharmacological tools to study ASIC structure, function, and biological roles. There are now ten ASIC modulators described from animal venoms, with those from snakes and spiders favouring ASIC1, while the sea anemones preferentially target ASIC3. Some modulators, such as the prototypical ASIC1 modulator PcTx1 have been studied in great detail, while some of the newer members of the club remain largely unstudied. Here we review the current state of knowledge on venom derived ASIC modulators, with a particular focus on their molecular interaction with ASICs, what they have taught us about channel structure, and what they might still reveal about ASIC function and pathophysiological roles.

BACKGROUND AND PURPOSEThe spider-venom peptide PcTx1 is the most potent and selective inhibitor of acid-sensing ion channel (ASIC) 1a. It has centrally acting analgesic activity and is neuroprotective in rodent models of ischaemic stroke. Understanding the molecular details of the PcTx1 : ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. Previously, we showed that several key pharmacophore residues of PcTx1 reside in a dynamic β-hairpin loop; conclusions confirmed by recent crystal structures of the complex formed between PcTx1 and chicken ASIC1 (cASIC1). Numerous peptide : channel contacts were observed in these crystal structures, but it remains unclear which of these are functionally important. EXPERIMENTAL APPROACHWe combined molecular dynamics (MD) simulations of the PcTx1 : cASIC1 complex with mutagenesis of PcTx1 and rat ASIC1a. KEY RESULTSCrystal structures of the PcTx1 : cASIC1 complex indicated that 15 PcTx1 residues form a total of 57 pairwise intermolecular contacts (<5 Å) with 32 channel residues. MD simulations, however, suggested that about half of these interactions do not persist in solution. Mutation to alanine of only eight of 15 PcTx1 contact residues substantially altered ASIC1a inhibition by PcTx1. Our data reveal that many of the peptide-channel interactions observed in the PcTx1 : cASIC1 crystal structures are not important for PcTx1 inhibition of rat ASIC1a. CONCLUSIONS AND IMPLICATIONSWe identified the atomic interactions that are critical for PcTx1 inhibition of ASIC1a. Our data highlight the value of combining structural information, MD and functional experiments to obtain detailed insight into the molecular basis of protein : protein interactions. Non-standard AbbreviationsASIC, acid-sensing ion channel; βHL, β-hairpin loop; cASIC1, chicken acid-sensing ion channel, isoform 1; ECD, extracellular domain; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; MALDI-TOF, matrix-assisted laser desorption/ionization/time-of-flight; MBP, maltose binding protein; MD, molecular dynamics; PcTx1, psalmotoxin 1 (also known as π-theraphotoxin-Pc1a); rASIC1a, rat acid-sensing ion channel, isoform 1a; RP-HPLC, reversed-phase HPLC; TEV, tobacco etch virus; TEVC, two-electrode voltage clamp BJP British Journal of Pharmacology

Snake venom α-neurotoxins potently inhibit rodent nicotinic acetylcholine receptors (nAChRs), but their activity on human receptors and their role in human paralysis from snakebite remain unclear. We demonstrate that two short-chain α-neurotoxins (SαNTx) functionally inhibit human muscle-type nAChR, but are markedly more reversible than against rat receptors. In contrast, two long-chain α-neurotoxins (LαNTx) show no species differences in potency or reversibility. Mutant studies identified two key residues accounting for this. Proteomic and clinical data suggest that paralysis in human snakebites is not associated with SαNTx, but with LαNTx, such as in cobras. Neuromuscular blockade produced by both subclasses of α-neurotoxins was reversed by antivenom in rat nerve-muscle preparations, supporting its effectiveness in human post-synaptic paralysis.

This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.