Distinct Receptor Sites Research Articles

In Silico Analysis of Limonoid-Based Antifeedants from Melia volkensii Targeting the Ryanodine Receptor in Spodoptera frugiperda

Spodoptera frugiperda is an invasive pest causing significant crop losses worldwide. Resistance development and health and environmental concerns associated with synthetic insecticides have prompted a search for eco-friendly biopesticides. Limonoids such as salannin, volkensin, and volkensinone, isolated from the East African plant Melia volkensii, show antifeedant activity against S. frugiperda larvae. Volkensin had an ED50 of 3.5 µg/cm², volkensinone (a lactone of volkensin) an ED50 of 6 µg/cm², and salannin an ED50 of 13 µg/cm². Additional limonoids from M. volkensii, including salanninolide and toosendanin, also displayed strong antifeedant effects. With toosendanin already used commercially, a re-evaluation of M. volkensii antifeedant compounds was conducted using in silico techniques. Docking simulations with 3D models of these limonoids and the S. frugiperda ryanodine receptor protein revealed binding affinities from -6.4 to -7.5 kcal/mol, comparable to those of chlorantraniliprole, a commercial insecticide targeting ryanodine receptors. These binding affinities at two distinct receptor sites align well with in-vitro antifeedant activity, underscoring M. volkensii’s potential for environmentally friendly, receptor-targeted biopesticide development against S. frugiperda.

Insecticidal activity of the spider neurotoxin PPTX-04 through modulating insect voltage-gated sodium channel

Ion channels on cell membrane are molecular targets of more than half peptide neurotoxins from spiders. From Pardosa pseudoannulata, a predatory spider on a range of insect pests, we characterized a peptide neurotoxin PPTX-04 with an insecticidal activity. PPTX-04 showed high toxicity to Nilaparvata lugens, a main prey of P. pseudoannulata, and the toxicity was not affected by the resistance to etofenprox (IUPAC chemical name:1-ethoxy-4-[2-methyl-1-[(3-phenoxyphenyl)methoxy]propan-2-yl]benzene, purity: 99%). On N. lugens voltage-gated sodium channel NlNav1 expressed in Xenopus oocytes, PPTX-04 prolonged the channel opening and induced tail currents, which is similar to pyrethroid insecticides. However, PPTX-04 potency on NlNav1 was not affected by mutations conferring pyrethroid resistance in insects, which revealed that PPTX-04 and pyrethroids should act on different receptors in NlNav1. In contrast, two mutations at the extracellular site 4 significantly reduced PPTX-04 potency, which indicated that PPTX-04 would act on a potential receptor containing the site 4 in NlNav1. The result from the molecular docking supported the conclusion that the binding pocket of PPTX-04 in NlNav1 should contain the site 4. In summary, PPTX-04 had high insecticidal activity through acting on a distinct receptor site in insect Nav, and was a potential resource to control insect pests and manage resistance to pyrethroids.

Efficient detoxification of Cr(VI)-containing effluents by sequential adsorption and reduction using a novel cysteine-doped PANi@faujasite composite: Experimental study supported by advanced statistical physics prediction

Nowadays, the global spreading of hazardous heavy metals becomes a top-priority environmental challenge, owing to its serious detrimental health outcomes. Herein, a novel cysteine-doped polyaniline@faujasite hybrid composite (Cys-PANi@FAU-50) was synthesized via a facile in-situ polymerization route for the effective detoxification of Cr(VI)-bearing wastewaters. The Cys-PANi@FAU-50 composite displayed an open mesoporous structure richly decorated with nitrogen/oxygen-containing functional groups, which consequently boosted the diffusion, adsorption and reduction of Cr(VI) oxyanions. The Cr(VI) adsorption behavior was satisfactorily tailored via pseudo-second-order law and Langmuir model with a maximum uptake capacity of 384.6 mg/g. Based on the advanced statistical physics theory, the monolayer model with two distinct receptor sites provided a reliable microscopic and macroscopic prediction of the Cr(VI) adsorption process. Stereographically, the Cr(VI) ions were adsorbed through horizontal multi-anchorage and vertical multi-molecular mechanisms on the amine and hydroxyl groups of Cys-PANi@FAU-50, respectively. The thermodynamic functions evidenced that the Cr(VI) adsorption was an endothermic spontaneous process. XPS analysis proved that Cr(VI) ions were electrostatically adsorbed, and subsequently reduced to Cr(III), which were in turn immobilized by chelation with imine/sulfonate groups and electrostatic interactions with carboxylate groups. The Cys-PANi@FAU-50 featured an effortless regenerability and good reusability. Overall, the Cys-PANi@FAU-50 composite owns outstanding potentiality for detoxifying Cr(VI)-laden effluents.

Long-Range Coupled Motions Underlie Ligand Recognition by a Chemokine Receptor.

SummaryChemokines are unusual class-A G protein-coupled receptor agonists because of their large size (∼10 kDa) and binding at two distinct receptor sites: N-terminal domain (Site-I, unique to chemokines) and a groove defined by extracellular loop/transmembrane helices (Site-II, shared with all small molecule class-A ligands). Structures and sequence analysis reveal that the receptor N-terminal domains (N-domains) are flexible and contain intrinsic disorder. Using a hybrid NMR-MD approach, we characterized the role of Site-I interactions for the CXCL8-CXCR1 pair. NMR data indicate that the CXCR1 N-domain becomes structured on binding and that the binding interface is extensive with 30% CXCL8 residues participating in this initial interaction. MD simulations indicate that CXCL8 bound at Site-I undergoes extensive reorganization on engaging Site-II with several residues initially engaged at Site-I also engaging at Site-II. We conclude that structural plasticity of Site-I interactions plays an active role in driving ligand recognition by a chemokine receptor.

Molecular Mechanism of Action and Selectivity of Sodium Ch annel Blocker Insecticides.

Sodium channel blocker insecticides (SCBIs) are a relatively new class of insecticides that are represented by two commercially registered compounds, indoxacarb and metaflumizone. SCBIs, like pyrethroids and DDT, target voltage-gated sodium channels (VGSCs) to intoxicate insects. In contrast to pyrethroids, however, SCBIs inhibit VGSCs at a distinct receptor site that overlaps those of therapeutic inhibitors of sodium channels, such as local anesthetics, anticonvulsants and antiarrhythmics. This review will recount the development of the SCBI insecticide class from its roots as chitin synthesis inhibitors, discuss the symptoms of poisoning and evidence supporting inhibition of VGSCs as their mechanism of action, describe the current model for SCBI-induced inhibition of VGSCs, present a model for the receptor for SCBIs on VGSCs, and highlight differences between data collected from mammalian and insect experimental models.

Structural basis for inhibition of a voltage-gated Ca2+ channel by Ca2+ antagonist drugs.

Ca2+ antagonist drugs are widely used in therapy of cardiovascular disorders. Three chemical classes of drugs bind to three separate, but allosterically interacting, receptor sites on CaV1.2 channels, the most prominent voltage-gated Ca2+ (CaV) channel type in myocytes in cardiac and vascular smooth muscle. The 1,4-dihydropyridines are used primarily for treatment of hypertension and angina pectoris and are thought to act as allosteric modulators of voltage-dependent Ca2+ channel activation, whereas phenylalkylamines and benzothiazepines are used primarily for treatment of cardiac arrhythmias and are thought to physically block the pore. The structural basis for the different binding, action, and therapeutic uses of these drugs remains unknown. Here we present crystallographic and functional analyses of drug binding to the bacterial homotetrameric model CaV channel CaVAb, which is inhibited by dihydropyridines and phenylalkylamines with nanomolar affinity in a state-dependent manner. The binding site for amlodipine and other dihydropyridines is located on the external, lipid-facing surface of the pore module, positioned at the interface of two subunits. Dihydropyridine binding allosterically induces an asymmetric conformation of the selectivity filter, in which partially dehydrated Ca2+ interacts directly with one subunit and blocks the pore. In contrast, the phenylalkylamine Br-verapamil binds in the central cavity of the pore on the intracellular side of the selectivity filter, physically blocking the ion-conducting pathway. Structure-based mutations of key amino-acid residues confirm drug binding at both sites. Our results define the structural basis for binding of dihydropyridines and phenylalkylamines at their distinct receptor sites on CaV channels and offer key insights into their fundamental mechanisms of action and differential therapeutic uses in cardiovascular diseases.

Functional and Molecular Interaction of Phenylalkylamines and Dihydropyridines with the Model Calcium Channel CavAb

Mammalian voltage-gated calcium channels (mCavs) are molecular targets of phenylalkylamines (PAAs) and dihydropyridines (DHPs), which are widely used antiarrhythmic and antihypertension drugs that block mCavs in a frequency- and voltage-dependent manner. The two distinct receptor sites for these drugs in mCavs have been elucidated through extensive photoaffinity labeling and mutagenesis studies, revealing key residues along the S5 and S6 transmembrane segments of domains III and IV of mCavs (Hockerman et al., 1997). We have recently reported the crystal structure of CavAb, which is a highly calcium-selective mutant of the prokaryotic sodium channel NavAb (Tang et al., 2014). Even though CavAb is separated evolutionarily from mCavs by more than one billion years, we found that PAAs and DHPs interact with CavAb in a similar state-dependent manner. The PAAs tested have Kd's in the range of 250 nM to 800 nM, and verapamil shows low-affinity block of the resting state and high-affinity use-dependent block of activated and/or inactivated states. Mutations of T206 in the center of segment S6 reduced PAA affinity by 20-fold. In the case of DHPs, nimodipine showed voltage-dependent block of activated CavAb with a Kd of 194 nM, but no resting-state block was observed in the tested concentration range. DHPs shift the voltage dependence of inactivation up to 100 mV negatively. This indicates that DHP interaction with CavAb is state-dependent and greatly favors the inactivated state, similar to mCavs. Mutation of I199 in the outer region of segment S6, which is critical for binding DHPs in mCavs, reduced binding affinity 40-fold. Our results reveal surprising similarity of drug action on CavAb and mCavs and thereby support use of CavAb as a molecular model to determine the three-dimensional structure of these important drug receptor sites.

Coevolution of the Ile1,016 and Cys1,534 Mutations in the Voltage Gated Sodium Channel Gene of Aedes aegypti in Mexico

BackgroundWorldwide the mosquito Aedes aegypti (L.) is the principal urban vector of dengue viruses. Currently 2.5 billion people are at risk for infection and reduction of Ae. aegypti populations is the most effective means to reduce the risk of transmission. Pyrethroids are used extensively for adult mosquito control, especially during dengue outbreaks. Pyrethroids promote activation and prolong the activation of the voltage gated sodium channel protein (VGSC) by interacting with two distinct pyrethroid receptor sites [1], formed by the interfaces of the transmembrane helix subunit 6 (S6) of domains II and III. Mutations of S6 in domains II and III synergize so that double mutants have higher pyrethroid resistance than mutants in either domain alone. Computer models predict an allosteric interaction between mutations in the two domains. In Ae. aegypti, a Ile1,016 mutation in the S6 of domain II was discovered in 2006 and found to be associated with pyrethroid resistance in field populations in Mexico. In 2010 a second mutation, Cys1,534 in the S6 of domain III was discovered and also found to be associated with pyrethroid resistance and correlated with the frequency of Ile1,016.Methodology/Principal FindingsA linkage disequilibrium analysis was performed on Ile1,016 and Cys1,534 in Ae. aegypti collected in Mexico from 2000–2012 to test for statistical associations between S6 in domains II and III in natural populations. We estimated the frequency of the four dilocus haplotypes in 1,016 and 1,534: Val1,016/Phe1,534 (susceptible), Val1,016/Cys1,534, Ile1,016/Phe1,534, and Ile1,016/Cys1,534 (resistant). The susceptible Val1,016/Phe1,534 haplotype went from near fixation to extinction and the resistant Ile1,016/Cys1,534 haplotype increased in all collections from a frequency close to zero to frequencies ranging from 0.5–0.9. The Val1,016/Cys1,534 haplotype increased in all collections until 2008 after which it began to decline as Ile1,016/Cys1,534 increased. However, the Ile1,016/Phe1,534 haplotype was rarely detected; it reached a frequency of only 0.09 in one collection and subsequently declined.Conclusion/SignificancePyrethroid resistance in the vgsc gene requires the sequential evolution of two mutations. The Ile1,016/Phe1,534 haplotype appears to have low fitness suggesting that Ile1,016 was unlikely to have evolved independently. Instead the Cys1,534 mutation evolved first but conferred only a low level of resistance. Ile1,016 in S6 of domain II then arose from the Val1,016/Cys1,534 haplotype and was rapidly selected because double mutants confer higher pyrethroid resistance. This pattern suggests that knowledge of the frequencies of mutations in both S6 in domains II and III are important to predict the potential of a population to evolve kdr. Susceptible populations with high Val1,016/Cys1,534 frequencies are at high risk for kdr evolution, whereas susceptible populations without either mutation are less likely to evolve high levels of kdr, at least over a 10 year period.

A mutation in the intracellular loop III/IV of mosquito sodium channel synergizes the effect of mutations in helix IIS6 on pyrethroid resistance.

Activation and inactivation of voltage-gated sodium channels are critical for proper electrical signaling in excitable cells. Pyrethroid insecticides promote activation and inhibit inactivation of sodium channels, resulting in prolonged opening of sodium channels. They preferably bind to the open state of the sodium channel by interacting with two distinct receptor sites, pyrethroid receptor sites PyR1 and PyR2, formed by the interfaces of domains II/III and I/II, respectively. Specific mutations in PyR1 or PyR2 confer pyrethroid resistance in various arthropod pests and disease vectors. Recently, a unique mutation, N(1575)Y, in the cytoplasmic loop linking domains III and IV (LIII/IV) was found to coexist with a PyR2 mutation, L(1014)F in IIS6, in pyrethroid-resistant populations of Anopheles gambiae. To examine the role of this mutation in pyrethroid resistance, N(1575)Y alone or N(1575)Y + L(1014)F were introduced into an Aedes aegypti sodium channel, AaNav1-1, and the mutants were functionally examined in Xenopus oocytes. N(1575)Y did not alter AaNav1-1 sensitivity to pyrethroids. However, the N(1575)Y + L(1014)F double mutant was more resistant to pyrethroids than the L(1014)F mutant channel. Further mutational analysis showed that N(1575)Y could also synergize the effect of L(1014)S/W, but not L(1014)G or other pyrethroid-resistant mutations in IS6 or IIS6. Computer modeling predicts that N(1575)Y allosterically alters PyR2 via a small shift of IIS6. Our findings provide the molecular basis for the coexistence of N(1575)Y with L(1014)F in pyrethroid resistance, and suggest an allosteric interaction between IIS6 and LIII/IV in the sodium channel.

A negative charge in transmembrane segment 1 of domain II of the cockroach sodium channel is critical for channel gating and action of pyrethroid insecticides

Voltage-gated sodium channels are the primary target of pyrethroids, an important class of synthetic insecticides. Pyrethroids bind to a distinct receptor site on sodium channels and prolong the open state by inhibiting channel deactivation and inactivation. Recent studies have begun to reveal sodium channel residues important for pyrethroid binding. However, how pyrethroid binding leads to inhibition of sodium channel deactivation and inactivation remains elusive. In this study, we show that a negatively charged aspartic acid residue at position 802 (D802) located in the extracellular end of transmembrane segment 1 of domain II (IIS1) is critical for both the action of pyrethroids and the voltage dependence of channel activation. Charge-reversing or -neutralizing substitutions (K, G, or A) of D802 shifted the voltage dependence of activation in the depolarizing direction and reduced channel sensitivity to deltamethrin, a pyrethroid insecticide. The charge-reversing mutation D802K also accelerated open-state deactivation, which may have counteracted the inhibition of sodium channel deactivation by deltamethrin. In contrast, the D802G substitution slowed open-state deactivation, suggesting an additional mechanism for neutralizing the action of deltamethrin. Importantly, Schild analysis showed that D802 is not involved in pyrethroid binding. Thus, we have identified a sodium channel residue that is critical for regulating the action of pyrethroids on the sodium channel without affecting the receptor site of pyrethroids.

U‐shaped dose‐dependent effects of BmK AS, a unique scorpion polypeptide toxin, on voltage‐gated sodium channels

Buthus martensi Karsch (BmK) AS is a scorpion polypeptide toxin, said to target the voltage-gated sodium channels (VGSCs). However, the mechanism of action of BmK AS on the VGSCs has yet to be defined. We examined the electrophysiological effects of BmK AS in a wide dose range on the rat brain-type VGSC alpha-subunit, rNav1.2a, heterologously expressed in Xenopus oocytes and on the VGSCs endogenously expressed in the dorsal root ganglion neuroblastoma ND7-23 cell line. In the oocytes, BmK AS depolarized the voltage dependence of activation and inactivation of rNav1.2a at 0.1 and 500 nM whereas these parameters were hyperpolarized at 1 nM. In ND7-23 cells, BmK AS hyperpolarized the voltage dependence of activation and inactivation at 0.1, 1 and 100 nM but not 10 nM. BmK AS also hyperpolarized the voltage dependence of recovery from inactivation at 0.1 and 100 nM and slowed the recovery kinetics at all concentrations, but the effects of 1 and 10 nM were relatively smaller than those at 0.1 and 100 nM. Moreover, the inactivation of VGSCs was potentiated by 10 nM BmK AS in both systems, whereas it was inhibited by 0.1 or 100 nM BmK AS in the oocytes or ND7-23 cells respectively. BmK AS modulated the VGSCs in a unique U-shaped dose-dependent manner, which could be due to the opposing effects of binding to two distinct receptor sites on the VGSCs.

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion α-Toxins

The scorpion alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na(v)s) and is inactive at insect Na(v)s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa(v)1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion alpha-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhalphaIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe(15) of the core domain in toxin potency at rNa(v)1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion alpha-toxins we were able to eliminate the activity of Lqh2 at rNa(v)1.4 (skeletal muscle), hNa(v)1.5 (cardiac), and rNa(v)1.6 channels, with no hindrance of its activity at Na(v)1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.

Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms

As voltage-gated Na + channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na + channels when exerting their different pharmacological effects, or at distinct receptor sites in different Na v channels subtypes whose particular properties lead to these pharmacological differences. The α-subunits of voltage-gated Na + channels (Na v1. x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Na v1. x channels and all are 46–49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Na v1.2-toxin interaction, developed for the α-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Na + channel protein. This region is indeed responsible for the normal Na + currents fast inactivation that is potently slowed by these toxins. This fundamental “gain-of-function” mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As most of the structural and electrophysiological studies were performed on type 1 sea anemone sodium channel toxins, we will present a comprehensive and updated review on the current understanding of the physiological actions of these Na channel modifiers.

Voltage-gated ion channels and gating modifier toxins

Voltage-gated sodium, calcium, and potassium channels generate electrical signals required for action potential generation and conduction and are the molecular targets for a broad range of potent neurotoxins. These channels are built on a common structural motif containing six transmembrane segments and a pore loop. Their pores are formed by the S5/S6 segments and the pore loop between them, and they are gated by bending of the S6 segments at a hinge glycine or proline residue. The voltage sensor domain consists of the S1–S4 segments, with positively charged residues in the S4 segment serving as gating charges. The diversity of toxin action on these channels is illustrated by sodium channels, which are the molecular targets for toxins that act at six or more distinct receptor sites on the channel protein. Both hydrophilic low molecular weight toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Hydrophobic alkaloid toxins and related lipid-soluble toxins act at intramembrane sites and alter voltage-dependent gating of sodium channels via an allosteric mechanism. In contrast, polypeptide toxins alter channel gating by voltage-sensor trapping through binding to extracellular receptor sites, and this toxin interaction has now been modeled at the atomic level for a β-scorpion toxin. The voltage-sensor trapping mechanism may be a common mode of action for polypeptide gating modifier toxins acting on all of the voltage-gated ion channels.

Allosteric interactions between scorpion toxin receptor sites on voltage‐gated Na channels imply a novel role for weakly active components in arthropod venom

Scorpion beta and alpha-toxins modify the activation and inactivation of voltage-gated sodium channels. Although the two types of toxin bind at two distinct receptor sites on the same sodium channel, they exhibit synergic effects when coinjected into insects. To clarify the basis of this synergism we examined the mutual effects of alpha and beta toxin representatives in radio-ligand binding assays. We found positive allosteric interactions between receptor site-4 of the excitatory Bj-xtrIT and the depressant LqhIT2 beta toxins and receptor site-3 of the alpha toxin LqhalphaIT, on locust neuronal membranes. Unexpectedly, a nontoxic mutant Bj-xtrIT-E15R, which binds with high affinity to receptor site-4, was able to enhance LqhalphaIT binding and toxicity similarly to the unmodified Bj-xtrIT. This result indicates that mere binding of a nontoxic ligand to receptor site-4 ("silent binding") induces a conformational change that does not alter channel gating, but influences toxin binding at receptor site-3 leading to enhanced toxicity. This finding suggests a new functional role for weakly toxic polypeptides in that they enhance the effect of other active neurotoxins in the arthropod venom. Such silent binding may have also valuable implications in attempts to improve drug efficacy by combining potent drugs with nonactive allosteric enhancers.

The endocannabinoid signalling system: Implications for anaesthesia and the pain clinic

Δ 9 -Tetrahydrocannabinol ( Δ 9 THC) is the major psychoactive ingredient of the plant Cannabis sativa that has been extensively used by humans for over 4000 years for both therapeutics and economic growth. Until a decade ago, the effects of Δ 9 THC were frequently attributed to non-specific alterations in membrane structure, based on its high lipophilicity, a theory similar to that proposed a century ago (by Meyer and Overton) for anaesthetics. Recent evidence identified a distinct protein receptor site (cannabinoid receptor, CB1) in the human brain activated not only by Δ 9 THC, but also by two endogenously produced lipid compounds, anandamide and 2-arachidonylglycerol. The cannabinoid has been subsequently tested in a plethora of in vitro, in vivo and clinical (to lesser extent) assays that revealed interesting features of their pharmacology and physiological roles. Cannabinoids have the potential to interact with a number of drugs that are used in clinical anaesthetic practice, while in turn anaesthetic agents may produce their effects through modulation of the endocannabinoid signalling system. Herein, we briefly review the discovery, pharmacological actions and putative physiological functions of endocannabinoids. We then discuss some of these finding in relation to anaesthesia in order to identify therapeutic potentials, highlight specific problems and stimulate further debate.

A Spider Toxin That Induces a Typical Effect of Scorpion α-Toxins but Competes with β-Toxins on Binding to Insect Sodium Channels

Delta-palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue long peptides that show preference for insect sodium channels (NaChs) and modulate their function. Although they slow NaCh inactivation in a fashion similar to that of receptor site 3 modifiers, such as scorpion alpha-toxins, they actually bind with high affinity to the topologically distinct receptor site 4 of scorpion beta-toxins. To resolve this riddle, we scanned by Ala mutagenesis the surface of delta-PaluIT2, a delta-palutoxin variant with the highest affinity for insect NaChs, and compared it to the bioactive surface of a scorpion beta-toxin. We found three regions on the surface of delta-PaluIT2 important for activity: the first consists of Tyr-22 and Tyr-30 (aromatic), Ser-24 and Met-28 (polar), and Arg-8, Arg-26, Arg-32, and Arg-34 (basic) residues; the second is made of Trp-12; and the third is made of Asp-19, whose substitution by Ala uncoupled the binding from toxicity to lepidopteran larvae. Although spider delta-palutoxins and scorpion beta-toxins have developed from different ancestors, they show some commonality in their bioactive surfaces, which may explain their ability to compete for an identical receptor (site 4) on voltage-gated NaChs. Yet, their different mode of channel modulation provides a novel perspective about the structural relatedness of receptor sites 3 and 4, which until now have been considered topologically distinct.

Characterization of a domoic acid binding site from Pacific razor clam

The Pacific razor clam, Siliqua patula, is known to retain domoic acid, a water-soluble glutamate receptor agonist produced by diatoms of the genus Pseudo-nitzschia. The mechanism by which razor clams tolerate high levels of the toxin, domoic acid, in their tissues while still retaining normal nerve function is unknown. In our study, a domoic acid binding site was solubilized from razor clam siphon using a combination of Triton X-100 and digitonin. In a Scatchard analysis using [ 3 H ]kainic acid, the partially-purified membrane showed two distinct receptor sites, a high affinity, low capacity site with a K D (mean ± S.E.) of 28 ± 9.4 nM and a maximal binding capacity of 12 ± 3.8 pmol/mg protein and a low affinity, high capacity site with a mM affinity for radiolabeled kainic acid, the latter site which was lost upon solubilization. Competition experiments showed that the rank order potency for competitive ligands in displacing [ 3 H ]kainate binding from the membrane-bound receptors was quisqualate > ibotenate > iodowillardiine = AMPA = fluorowillardiine > domoate > kainate > l-glutamate. At high micromolar concentrations, NBQX, NMDA and ATPA showed little or no ability to displace [ 3 H ]kainate. In contrast, Scatchard analysis using [ 3 H ]glutamate showed linearity, indicating the presence of a single binding site with a K D and B max of 500 ± 50 nM and 14 ± 0.8 pmol/mg protein, respectively. These results suggest that razor clam siphon contains both a high and low affinity receptor site for kainic acid and may contain more than one subtype of glutamate receptor, thereby allowing the clam to function normally in a marine environment that often contains high concentrations of domoic acid.

Allosteric interactions among pyrethroid, brevetoxin, and scorpion toxin receptors on insect sodium channels raise an alternative approach for insect control

Intensive pyrethroid use in insect control has led to resistance buildup among various pests. One alternative to battle this problem envisions the combined use of synergistically acting insecticidal compounds. Pyrethroids, scorpion α- and β-toxins, and brevetoxins bind to distinct receptor sites on voltage-gated sodium channels (NaChs) and modify their function. The binding affinity of scorpion α-toxins to locust, but not rat-brain NaChs, is allosterically increased by pyrethroids and by brevetoxin-1. Brevetoxin-1 also increases the binding of an excitatory β-toxin to insect NaChs. These results reveal differences between insect and mammalian NaChs and may be exploited in new strategies of insect control.

The binding of BmK abT, a unique neurotoxin, to mammal brain and insect Na + channels using biosensor

The binding properties of BmK abT (a novel neurotoxic polypeptide abT from Chinese scorpion Buthus martensi Karsch), a unique neurotoxin from Chinese scorpion, on mammal brain and insect sodium channels were investigated using the BIAcore assay. Results showed that BmK abT could bind to rat brain synaptosomes with an association rate constant of about 2.49×10 6 M −1 s −1 and a dissociation rate constant of about 1.57×10 −4 s −1, and to Heliothis nerve cord synaptosomes with an association rate constant of about 1.21×10 7 M −1 s −1 and a dissociation rate constant of about 0.99×10 −3 s −1. The binding of BmK abT to rat brain synaptosomes could be partially inhibited by increasing the membrane potential, but not by BmK AS (a novel active polypeptide AS from B. martensi Karsch), BmK IT2 (a depressant insect-selective toxin IT2 from B. martensi Karsch), and BmK I (an α-like anti-mammal toxin I from B. martensi Karsch). Binding was not modulated by veratridine. In addition, the binding of BmK abT to Heliothis nerve cord synaptosomes was significantly enhanced by increasing the membrane potential and veratridine concentration and was inhibited by BmK IT2, but not by BmK AS or BmK I. The results suggest that BmK abT binds to a distinct receptor site on mammal brain Na + channels and associates with a related site for depressant insect-selective toxins on insect sodium channels.