Assessing the Binding of Venoms from Aquatic Elapids to the Nicotinic Acetylcholine Receptor Orthosteric Site of Different Prey Models

Abstract

The evolution of an aquatic lifestyle from land dwelling venomous elapids is a radical ecological modification, bringing about many evolutionary changes from morphology to diet. Diet is an important ecological facet which can play a key role in regulating functional traits such as venom composition and prey-specific targeting of venom. In addition to predating upon novel prey (e.g., fish, fish eggs and invertebrates), the venoms of aquatic elapids also face the challenge of increased prey-escape potential in the aquatic environment. Thus, despite the independent radiation into an aquatic niche on four separate occasions, the venoms of aquatic elapids are evolving under convergent selection pressures. Utilising a biolayer interferometry binding assay, this study set out to elucidate whether crude venoms from representative aquatic elapids were target-specific to the orthosteric site of postsynaptic nicotinic acetylcholine receptor mimotopes of fish compared to other terrestrial prey types. Representatives of the four aquatic lineages were: aquatic coral snakes representative was Micrurus surinamensis;, sea kraits representative was Laticauda colubrina; sea snakes representatives were two Aipysurus spp. and eight Hydrophis spp; and water cobras representative was Naja annulata. No prey-specific differences in crude venom binding were observed from any species tested, except for Aipysurus laevis, which showed slight evidence of prey-potency differences. For Hydrophis caerulescens, H. peronii, H. schistosus and M. surinamensis, there was a lack of binding to the orthosteric site of any target lineage. Subsequent testing on the in vitro chick-biventer cervicis muscle preparation suggested that, while the venoms of these species bound postsynaptically, they bound to allosteric sites rather than orthosteric. Allosteric binding is potentially a weaker but faster-acting form of neurotoxicity and we hypothesise that the switch to allosteric binding is likely due to selection pressures related to prey-escape potential. This research has potentially opened up the possibility of a new functional class of toxins which have never been assessed previously while shedding light on the selection pressures shaping venom evolution.

Monkeying around with venom: an increased resistance to α-neurotoxins supports an evolutionary arms race between Afro-Asian primates and sympatric cobras

“…We tested the binding of these venoms upon mimotopes that represent the orthosteric site loop C region of α-1 nAChRs from different primate clades (Table S1). We utilized a biolayer interferometry (BLI) assay, which has been previously validated to assess the binding of α-neurotoxins to taxa-specific orthosteric mimotopes [ 37 , 46 – 48 ], to determine if primates might have evolved some resistance elements toward α-neurotoxins.…”

“…The α-1 nAChR orthosteric site of humans has been shown to be much less susceptible to binding of α-neurotoxins [ 37 – 40 ] in contrast to the same site binding of other taxa [ 41 – 45 ]. Thus, while neurotoxic effects of elapid venoms may be a lethal outcome in some human envenomations (but not always), it is possible that in these instances, binding allosterically (to a receptor region other than the orthosteric site) might be a more derived method [ 46 ], rather than binding to the orthosteric site.…”

The Search for Natural and Synthetic Inhibitors That Would Complement Antivenoms as Therapeutics for Snakebite Envenoming

“…A high throughput methodology has been applied to address this issue, i.e., label-free bio-layer interferometry, which enables the study of the binding of different venoms and neurotoxins to various molecular regions of the receptor [ 196 ]. This has allowed the recognition of orthosteric and allosteric sites in the receptor responsible for neurotoxin binding [ 197 ]. The identification of these sites may lead to the synthesis of a diverse set of mimotopes that could bind and block a wide variety of α-neurotoxins.…”

The Dragon’s Paralysing Spell: Evidence of Sodium and Calcium Ion Channel Binding Neurotoxins in Helodermatid and Varanid Lizard Venoms

“…To observe the effects on whole receptors, we used the Gallus gallus chick biventer nerve-muscle preparation assay, an avian skeletal muscle preparation well-validated through numerous studies on snake venoms [18,[53][54][55][56]. To determine the binding affinity of the venoms for various ion channels, we used biolayer interferometry to ascertain binding to the voltage-sensing domain IV S3-S4 extracellular loop mimotopes for human sodium and calcium channels, an approach validated using venoms as diverse as snakes and stonefish [57][58][59][60][61][62][63]. The present study represents the most rigorous investigation of the ion channel neurotoxicity of the Heloderma genus, and the first for varanid lizard venoms.…”