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Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web-accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native-state energy. As a stringent test, we apply mPROSS to the Kv1.2-Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 9-26 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no significant change in the channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one-shot design calculations., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The serotonin receptor subtype 5-HTR 1B is widely distributed in the brain with an important role in various behavioral implications including neurological conditions and psychiatric disorders. The neuromodulatory action of 5-HTR 1B largely depends upon its arrestin mediated signaling pathway. In this study, we tried to investigate the role of unusually long intracellular loop 3 (ICL3) region of the serotonin receptor 5-HTR 1B in interaction with β-arrestin1 (Arr2) to compensate for the absence of the long cytoplasmic tail. Molecular modeling and docking tools were employed to obtain a suitable molecular conformation of the ICL3 region in complex with Arr2 which dictates the specific complex formation of 5-HTR 1B with Arr2. This reveals the novel molecular mechanism of phosphorylated ICL3 mediated GPCR-arrestin interaction in the absence of the long cytoplasmic tail. The in-cell disulfide cross-linking experiments and molecular dynamics simulations of the complex further validate the model of 5-HTR 1B -ICL3-Arr2 complex. Two serine residues (Ser281 and Ser295) within the 5-HTR 1B -ICL3 region were found to be occupying the electropositive pocket of Arr2 in our model and might be crucial for phosphorylation and specific Arr2 binding. The alignment studies of these residues showed them to be conserved only across 5-HTR 1B mammalian species. Thus, our studies were able to predict a molecular conformation of 5-HTR 1B -Arr2 and identify the role of long ICL3 in the signaling process which might be crucial in designing targeted drugs (biased agonists) that promote GPCR-Arr2 signaling to deter the effects of stress and anxiety-like disorders., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Store-operated calcium entry (SOCE) is a vital process aimed at refilling cellular internal Ca 2+ stores and a primary cellular signaling driver for transcription factors' entry to the nucleus. SOCE-associated regulatory factor (SARAF)/TMEM66 is an endoplasmic reticulum (ER)-resident transmembrane protein that promotes SOCE inactivation and prevents Ca 2+ overfilling of the cell. Here, we demonstrate that mice deficient in SARAF develop age-dependent sarcopenic obesity with decreased energy expenditure, lean mass, and locomotion without affecting food consumption. Moreover, SARAF ablation reduces hippocampal proliferation, modulates the activity of the hypothalamus-pituitary-adrenal (HPA) axis, and mediates changes in anxiety-related behaviors. Interestingly, selective SARAF ablation in the hypothalamus's paraventricular nucleus (PVN) neurons reduces old age-induced obesity and preserves locomotor activity, lean mass, and energy expenditure, suggesting a possible central control with a site-specific role for SARAF. At the cellular level, SARAF ablation in hepatocytes leads to elevated SOCE, elevated vasopressin-induced Ca 2+ oscillations, and an increased mitochondrial spare respiratory capacity (SPC), thus providing insights into the cellular mechanisms that may affect the global phenotypes. These effects may be mediated via the liver X receptor (LXR) and IL-1 signaling metabolic regulators explicitly altered in SARAF ablated cells. In short, our work supports both central and peripheral roles of SARAF in regulating metabolic, behavioral, and cellular responses., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Many venomous organisms carry in their arsenal short polypeptides that block K + channels in a highly selective manner. These toxins may compete with the permeating ions directly via a "plug" mechanism or indirectly via a "pore-collapse" mechanism. An alternative "lid" mechanism was proposed but remained poorly defined. Here we study the Drosophila Shaker channel block by Conkunitzin-S1 and Conkunitzin-C3, two highly similar toxins derived from cone venom. Despite their similarity, the two peptides exhibited differences in their binding poses and biophysical assays, implying discrete action modes. We show that while Conkunitzin-S1 binds tightly to the channel turret and acts via a "pore-collapse" mechanism, Conkunitzin-C3 does not contact this region. Instead, Conk-C3 uses a non-conserved Arg to divert the permeant ions and trap them in off-axis cryptic sites above the SF, a mechanism we term a "molecular-lid". Our study provides an atomic description of the "lid" K + blocking mode and offers valuable insights for the design of therapeutics based on venom peptides., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

It has been reported earlier that the slow (C-type) inactivated conformation in Kv channels is stabilized by a multipoint hydrogen-bond network behind the selectivity filter. Furthermore, MD simulations revealed that structural water molecules are also involved in the formation of this network locking the selectivity filter in its inactive conformation. We found that the application of an extracellular, but not intracellular, solution based on heavy water (D2O) dramatically slowed entry into the slow inactivated state in Shaker-IR mutants (T449A, T449A/I470A, and T449K/I470C, displaying a wide range of inactivation kinetics), consistent with the proposed effect of the dynamics of structural water molecules on the conformational stability of the selectivity filter. Alternative hypotheses capable of explaining the observed effects of D2O were examined. Increased viscosity of the external solution mimicked by the addition of glycerol had a negligible effect on the rate of inactivation. In addition, the inactivation time constants of K+ currents in the outward and the inward directions in asymmetric solutions were not affected by a H2O/D2O exchange, negating an indirect effect of D2O on the rate of K+ rehydration. The elimination of the nonspecific effects of D2O on our macroscopic current measurements supports the hypothesis that the rate of structural water exchange at the region behind the selectivity filter determines the rate of slow inactivation, as proposed by molecular modeling., (© 2021 Szanto et al.)

Ion channels can tailor their activity to the particular cellular context by incorporating auxiliary subunits that are channel-type specific. In this issue of Cell, Ávalos Prado et al. now find that a well-characterized voltage-gated K + channel auxiliary subunit can also modulate the gating of Ca 2+ -activated Cl - channels., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Animal venom is a rich source for peptide toxins that bind and modulate the function of ion channels. Owing to their ability to bind receptor sites on the channel protein with high affinity and specificity, peptide neurotoxins have become an indispensable tool for ion channel research. Recent breakthroughs in structural biology and advances in computer simulations of biomolecules have sparked a new interest in animal toxins as probes of channel protein structure and function. Here, we focus on methods used to produce animal toxins for research purposes using recombinant expression. The specific challenges associated with heterologous production of venom peptides are discussed, and several methods targeting these issues are presented with an emphasis on E. coli based systems. An efficient protocol for the bacterial expression, folding, and purification of recombinant venom peptides is described., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Key Points: G-protein inwardly rectifying K + (GIRK) channels consist of four homologous subunits (GIRK1-4) and are essential regulators of electrical excitability in the nervous system. GIRK2-null mice have been widely investigated for their distinct behaviour and altered depotentiation following long-term potentiation (LTP), whereas GIRK1 mice are less well characterized. Here we utilize a novel knockin mouse strain in which the GIRK1 subunit is fluorescently tagged with yellow fluorescent protein (YFP-GIRK1) and the GIRK1-null mouse line to investigate the role of GIRK1 in neuronal processes such as spatial learning and memory, locomotion and depotentiation following LTP. Neurons dissected from YFP-GIRK1 mice had significantly reduced potassium currents and this mouse line phenotypically resembled GIRK1-null mice, making it a 'functional knockdown' model of GIRK1-containing channels. YFP-GIRK1 and GIRK1-null mice had increased locomotion, reduced spatial learning and memory and blunted depotentiation following LTP., Abstract: GIRK channels are essential for the slow inhibition of electrical activity in the nervous system and heart rate regulation via the parasympathetic system. The implications of individual GIRK isoforms in specific physiological activities are based primarily on studies conducted with GIRK-null mouse lines. Here we utilize a novel knockin mouse line in which YFP was fused in-frame to the N-terminus of GIRK1 (YFP-GIRK1) to correlate GIRK1 spatial distribution with physiological activities. These mice, however, displayed spontaneous seizure-like activity and thus were investigated for the origin of such activity. We show that GIRK tetramers containing YFP-GIRK1 are correctly assembled and trafficked to the plasma membrane, but are functionally impaired. A battery of behavioural assays conducted on YFP-GIRK1 and GIRK1-null (GIRK1 -/- ) mice revealed similar phenotypes, including impaired nociception, reduced anxiety and hyperactivity in an unfamiliar environment. However, YFP-GIRK1 mice exhibited increased home-cage locomotion while GIRK1 -/- mice did not. In addition, we show that the GIRK1 subunit is essential for intact spatial learning and memory and synaptic plasticity in hippocampal brain slices. This study expands our knowledge regarding the role of GIRK1 in neuronal processes and underlines the importance of GIRK1-containing heterotetramers., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Voltage-dependent potassium channels (K v s) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K + -selective pore. Animal toxins targeting K v s are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K + conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of K v 1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K + channels as a novel pharmacological target and provides a rational framework for drug design., Competing Interests: The authors declare no conflict of interest.

Background and Purpose: A second-generation antihistamine, terfenadine, is known to induce arrhythmia by blocking hERG channels. In this study, we have shown that terfenadine also inhibits the activity of G-protein-gated inwardly rectifying K + (GIRK) channels, which regulate the excitability of neurons and cardiomyocytes. To clarify the underlying mechanism(s), we examined the effects of several antihistamines on GIRK channels and identified the structural determinant for the inhibition., Experimental Approach: Electrophysiological recordings were made in Xenopus oocytes and rat atrial myocytes to analyse the effects of antihistamines on various GIRK subunits (K ir 3.x). Mutagenesis analyses identified the residues critical for inhibition by terfenadine and the regulation of ion selectivity. The potential docking site of terfenadine was analysed by molecular docking., Key Results: GIRK channels containing K ir 3.1 subunits heterologously expressed in oocytes and native GIRK channels in atrial myocytes were inhibited by terfenadine and other non-sedating antihistamines. In K ir 3.1 subunits, mutation of Phe137, located in the centre of the pore helix, to the corresponding Ser in K ir 3.2 subunits reduced the inhibition by terfenadine. Introduction of an amino acid with a large side chain in K ir 3.2 subunits at Ser148 increased the inhibition. When this residue was mutated to a non-polar amino acid, the channel became permeable to Na + . Phosphoinositide-mediated activity was also decreased by terfenadine., Conclusion and Implications: The Phe137 residue in K ir 3.1 subunits is critical for inhibition by terfenadine. This study provides novel insights into the regulation of GIRK channels by the pore helix and information for drug design., (© 2019 The British Pharmacological Society.)

Voltage sensing by ion channels is the key event enabling the generation and propagation of electrical activity in excitable cells. In this issue of Cell, Wisedchaisri et al. provide a structural view of a voltage-gated sodium channel in its resting closed conformation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca 2+ stores and generate intracellular Ca 2+ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the X-ray crystal structure of the SARAF luminal domain, SARAF L . This domain forms a novel 10-stranded β-sandwich fold that includes a set of three conserved disulfide bonds, denoted the "SARAF-fold." The structure reveals a domain-swapped dimer in which the last two β-strands (β9 and β10) are exchanged forming a region denoted the "SARAF luminal switch" that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Activation of voltage-gated sodium (Na(v)) channels initiates and propagates action potentials in electrically excitable cells. β-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of Na(V) channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIV(E15A). Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for β-scorpion toxins on Na(V) channels. Binding of α- and β-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on Na(V) channels acts synergistically to modify channel gating and paralyze prey.

Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.

Voltage-gated sodium (Na(v)) channels are the molecular targets of β-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of β-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins.

Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.

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.

The bioactive surface of scorpion beta-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved betaalphabetabeta core and the C-tail. In an attempt to evaluate the extent by which residues of the toxin core contribute to bioactivity, the anti-insect and anti-mammalian beta-toxins Bj-xtrIT and Css4 were truncated at their N and C termini, resulting in miniature peptides composed essentially of the core secondary structure motives. The truncated beta-toxins (DeltaDeltaBj-xtrIT and DeltaDeltaCss4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, DeltaDeltaBj-xtrIT and DeltaDeltaCss4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion alpha-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion alpha-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of LqhalphaIT at insect sodium channels. Co-application of DeltaDeltaBj-xtrIT or DeltaDeltaCss4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at DeltaDeltaBj-xtrIT and DeltaDeltaCss4 capable of interaction with sodium channels at a site other than sites 3, 4, or 5, which prior to the truncation was masked by the bioactive surface that interacts with receptor site-4. The disclosure of this hidden surface at both beta-toxins may be viewed as an exercise in "reverse evolution," providing a clue as to their evolution from a smaller ancestor of similar scaffold.

Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Na(v)s (voltage-gated Na+ channels) like the structurally dissimilar scorpion alpha-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Na(v)s, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65+/-0.46 pmol/100 mg), to compete well with the site-3 toxin LqhalphaIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (K(i)=21.4+/-7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNa(v)1, but not that of mammalian Na(v)s expressed in Xenopus oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion beta-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNa(v)1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.

The affinity of scorpion alpha-toxins for various voltage-gated sodium channels (Na(v)s) differs considerably despite similar structures and activities. It has been proposed that key bioactive residues of the five-residue-turn (residues 8-12) and the C-tail form the NC domain, whose topology is dictated by a cis or trans peptide-bond conformation between residues 9 and 10, which correlates with the potency on insect or mammalian Na(v)s. We examined this hypothesis using Lqh3, an alpha-like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Na(v)s compared with other alpha-toxins and its binding to insect Na(v)s is pH-dependent. Mutagenesis of Lqh3 revealed a bi-partite bioactive surface, composed of the Core and NC domains, as found in other alpha-toxins. Yet, substitutions at the five-residue turn and stabilization of the 9-10 bond in the cis conformation did not affect the activity. However, substitution of hydrogen-bond donors/acceptors at the NC domain reduced the pH-dependency of toxin binding, while retaining its high potency at Drosophila Na(v)s expressed in Xenopus oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident from the known solution structure, we suggest that acidic pH or specific mutations at the NC domain favor toxin conformations with high affinity for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and suggests a novel mechanism of dynamic conformer selection during toxin binding, enabling alpha-like toxins to affect a broad range of Na(v)s.

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.

Voltage-gated sodium channels are a major target for toxins and insecticides due to their central role in excitability, but due to the conservation of these channels in Animalia most insecticides do not distinguish between those of insects and mammals, thereby imposing risks to humans and livestock. Evidently, as long as modern agriculture depends heavily on the use of insecticides there is a great need for new substances capable of differentiating between sodium channel subtypes. Such substances exist in venomous animals, but ways for their exploitation have not yet been developed due to problems associated with manufacturing, degradation, and delivery to the target channels. Engineering of plants for expression of anti-insect toxins or use of natural vectors that express toxins near their target site (e.g. baculoviruses) are still problematic and raise public concern. In this problematic reality a rational approach might be to learn from nature how to design highly selective anti-insect compounds preferably in the form of peptidomimetics. This is a complex task that requires the elucidation of the face of interaction between insect-selective toxins and their sodium channel receptor sites. This review delineates current progress in: (i) elucidation of the bioactive surfaces of scorpion beta-toxins, especially the excitatory and depressant groups, which show high preference for insects and bind insect sodium channels with high affinity; (ii) studies of the mode of interaction of scorpion beta-toxins with receptor site-4 on voltage-gated sodium channels; and (iii) clarification of channel elements that constitute receptor site-4. This information may be useful in future attempts to mimic the bioactive surface of the toxins for the design of anti-insect selective peptidomimetics.

Receptor site-3 on voltage-gated sodium channels is targeted by a variety of structurally distinct toxins from scorpions, sea anemones, and spiders whose typical action is the inhibition of sodium current inactivation. This site interacts allosterically with other topologically distinct receptors that bind alkaloids, lipophilic polyether toxins, pyrethroids, and site-4 scorpion toxins. These features suggest that design of insecticides with specificity for site-3 might be rewarding due to the positive cooperativity with other toxins or insecticidal agents. Yet, despite the central role of scorpion alpha-toxins in envenomation and their vast use in the study of channel functions, molecular details on site-3 are scarce. Scorpion alpha-toxins vary greatly in preference for sodium channels of insects and mammals, and some of them are highly active on insects. This implies that despite its commonality, receptor site-3 varies on insect vs. mammalian channels, and that elucidation of these differences could potentially be exploited for manipulation of toxin preference. This review provides current perspectives on (i) the classification of scorpion alpha-toxins, (ii) their mode of interaction with sodium channels and pharmacological divergence, (iii) molecular details on their bioactive surfaces and differences associated with preference for channel subtypes, as well as (iv) a summary of the present knowledge about elements involved in constituting receptor site-3. These details, combined with the variations in allosteric interactions between site-3 and the other receptor sites on insect and mammalian sodium channels, may be useful in new strategies of insect control and future design of anti-insect selective ligands.

Scorpion depressant beta-toxins show high preference for insect voltage-gated sodium channels (Na(v)s) and modulate their activation. Although their pharmacological and physiological effects were described, their three-dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatushebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 A resolution. The toxin molecule is composed of a conserved cysteine-stabilized alpha/beta-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na(v)s. The N-groove is flanked by Glu24 and Tyr28, which belong to the "pharmacophore" of scorpion beta-toxins, and by the side-chains of Trp53 and Asn58 that are important for receptor site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in "voltage-sensor trapping", the mode of action that typifies scorpion beta-toxins. The involvement of the N-groove in recognition of the receptor site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the receptor site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position.

In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon (2004) Nature 430, 232-235). This suggestion was adopted by Smith et al. (Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127-11133), who analyzed the partitioning of sodium channel modifiers in liposomes. They found that certain modifiers were able to partition in these artificial membranes, and on this basis, they have extrapolated that scorpion beta-toxins interact with their channel receptor in a similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually raised a new conception, we examined it in binding assays using a number of pharmacologically distinct scorpion beta-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNa(v)1 and rat brain rNa(v)1.2a develops. We show that in general, scorpion beta-toxins do not partition in neuronal membranes and that in the case in which a depressant beta-toxin partitions in insect neuronal membranes, this partitioning is unrelated to its interaction with the receptor site and the effect on the gating properties of the sodium channel. These results negate the hypothesis that the high affinity of beta-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion beta-toxins is accessible to the extracellular solvent.

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Na(v)s) and compete with the structurally dissimilar scorpion alpha-toxins on binding to receptor site-3. Although these features provide two structurally different probes for studying receptor site-3 and channel fast inactivation, the bioactive surface of sea anemone toxins has not been fully resolved. We established an efficient expression system for Av2 (known as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (previously named A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in toxicity and binding assays as well as by circular dichroism spectroscopy to discern the effects derived from structural perturbation from those related to bioactivity. Six residues were found to constitute the anti-insect bioactive surface of Av2 (Val-2, Leu-5, Asn-16, Leu-18, and Ile-41). Further analysis of nine Av2 mutants on the human heart channel Na(v)1.5 expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and mammals practically coincide but differ from the bioactive surface of a structurally similar sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica. Hence, our results not only demonstrate clear differences in the bioactive surfaces of Av2 and scorpion alpha-toxins but also indicate that despite the general conservation in structure and importance of the Arg-14 loop and its flanking residues Gly-10 and Gly-20 for function, the surface of interaction between different sea anemone toxins and Na(v)s varies.

Scorpion beta-toxins that affect the activation of mammalian voltage-gated sodium channels (Navs) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion beta-toxin, Css4, whose effects on rat brain Navs have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 A2 composed of the following: 1) a cluster of residues associated with the alpha-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion beta-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the beta2 and beta3 strands, which is likely to confer the specificity for mammalian Navs; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion beta-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus 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.

Scorpion alpha-toxins are similar in their mode of action and three-dimensional structure but differ considerably in affinity for various voltage-gated sodium channels (NaChs). To clarify the molecular basis of the high potency of the alpha-toxin LqhalphaIT (from Leiurus quinquestriatus hebraeus) for insect NaChs, we identified by mutagenesis the key residues important for activity. We have found that the functional surface is composed of two distinct domains: a conserved "Core-domain" formed by residues of the loops connecting the secondary structure elements of the molecule core and a variable "NC-domain" formed by a five-residue turn (residues 8-12) and a C-terminal segment (residues 56-64). We further analyzed the role of these domains in toxin activity on insects by their stepwise construction onto the scaffold of the anti-mammalian alpha-toxin, Aah2 (from Androctonus australis hector). The chimera harboring both domains, Aah2(LqhalphaIT(face)), was as active to insects as LqhalphaIT. Structure determination of Aah2(LqhalphaIT(face)) by x-ray crystallography revealed that the NC-domain deviates from that of Aah2 and forms an extended protrusion off the molecule core as appears in LqhalphaIT. Notably, such a protrusion is observed in all alpha-toxins active on insects. Altogether, the division of the functional surface into two domains and the unique configuration of the NC-domain illuminate the molecular basis of alpha-toxin specificity for insects and suggest a putative binding mechanism to insect NaChs.

Gating modifiers constitute a large group of polypeptide toxins that interact with the voltage-sensing module of ion channels. Among them, scorpion beta-toxins induce a negative shift in the voltage dependence of sodium channel activation. To explain their effect, a "voltage sensor trapping" model has been proposed in which the voltage sensor of domain-II (DIIS4) is trapped in an outward, activated position by a prebound beta-toxin upon membrane depolarization. Whereas toxin effect on channel activation was enhanced upon neutralization of the two outermost arginines in DIIS4, toxin residues involved in sensor trapping have not been identified. Using the scorpion excitatory beta-toxin, Bj-xtrIT, we found two conserved acidic residues, Glu15 and Glu30, mandatory for toxin action. Whereas mutagenesis of Glu30 affected both toxicity and binding affinity, substitutions E15A/F abolished activity but had minor effects on binding. Complete uncoupling of activity from binding was obtained with mutant E15R, acting as an efficient antagonist of Bj-xtrIT. On the basis of the voltage sensor trapping model and our results, we propose that Glu15 interacts with the emerging gating charges of DIIS4 upon membrane depolarization. Conserved acidic residues found in a variety of gating modifiers from scorpions and spiders may interact similarly with the voltage sensor.

Scorpion beta-toxins affect the activation of voltage-sensitive sodium channels (NaChs). Although these toxins have been instrumental in the study of channel gating and architecture, little is known about their active sites. By using an efficient system for the production of recombinant toxins, we analyzed by point mutagenesis the entire surface of the beta-toxin, Bj-xtrIT, an anti-insect selective excitatory toxin from the scorpion Buthotus judaicus. Each toxin mutant was purified and analyzed using toxicity and binding assays, as well as by circular dichroism spectroscopy to discern the differences among mutations that caused structural changes and those that specifically affected bioactivity. This analysis highlighted a functional discontinuous surface of 1405 A(2), which was composed of a number of non-polar and three charged amino acids clustered around the main alpha-helical motif and the C-tail. Among the charged residues, Glu(30) is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr(26) and Val(34)). Comparison of the Bj-xtrIT structure with that of other beta-toxins that are active on mammals suggests that the hot spot and an adjacent non-polar region are spatially conserved. These results highlight for the first time structural elements that constitute a putative "pharmacophore" involved in the interaction of beta-toxins with receptor site-4 on NaChs. Furthermore, the unique structure of the C-terminal region most likely determines the specificity of excitatory toxins for insect NaChs.

Scorpion toxins that affect sodium channel (NaCh) gating in excitable cells are divided into alpha- and beta-classes. Whereas alpha-toxins have been found in scorpions throughout the world, anti-mammalian beta-toxins have been assigned, thus far, to 'New World' scorpions while anti-insect selective beta-toxins (depressant and excitatory) have been described only in the 'Old World'. This distribution suggested that diversification of beta-toxins into distinct pharmacological groups occurred after the separation of the continents, 150 million years ago. We have characterized a unique toxin, Lqhbeta1, from the 'Old World' scorpion, Leiurus quinquestriatus hebraeus, that resembles in sequence and activity both 'New World'beta-toxins as well as 'Old World' depressant toxins. Lqhbeta1 competes, with apparent high affinity, with anti-insect and anti-mammalian beta-toxins for binding to cockroach and rat brain synaptosomes, respectively. Surprisingly, Lqhbeta1 also competes with an anti-mammalian alpha-toxin on binding to rat brain NaChs. Analysis of Lqhbeta1 effects on rat brain and Drosophila Para NaChs expressed in Xenopus oocytes revealed a shift in the voltage-dependence of activation to more negative membrane potentials and a reduction in sodium peak currents in a manner typifying beta-toxin activity. Moreover, Lqhbeta1 resembles beta-toxins by having a weak effect on cardiac NaChs and a marked effect on rat brain and skeletal muscle NaChs. These multifaceted features suggest that Lqhbeta1 may represent an ancestral beta-toxin group in 'Old World' scorpions that gave rise, after the separation of the continents, to depressant toxins in 'Old World' scorpions and to various beta-toxin subgroups in 'New World' scorpions.

Delta-atracotoxins (delta-ACTXs) from Australian funnel-web spiders differ structurally from scorpion alpha-toxins (Sc(alpha)Tx) but similarly slow sodium current inactivation and compete for their binding to sodium channels at receptor site-3. Characterization of the binding of 125I-labelled delta-ACTX-Hv1a to various sodium channels reveals a decrease in affinity for depolarized (0 mV; Kd=6.5 +/- 1.4 nm) vs.polarized (-55 mV; Kd=0.6 +/- 0.2 nm) rat brain synaptosomes. The increased Kd under depolarized conditions correlates with a 4.3-fold reduction in the association rate and a 1.8-increase in the dissociation rate. In comparison, Sc(alpha)Tx binding affinity decreased 33-fold under depolarized conditions due to a 48-fold reduction in the association rate. The binding of 125I-labelled delta-ACTX-Hv1a to rat brain synaptosomes is inhibited competitively by classical Sc(alpha)Txs and allosterically by brevetoxin-1, similar to Sc(alpha)Tx binding. However, in contrast with classical Sc(alpha)Txs, 125I-labelled delta-ACTX-Hv1a binds with high affinity to cockroach Na+ channels (Kd=0.42 +/- 0.1 nm) and is displaced by the Sc(alpha)Tx, Lqh(alpha)IT, a well-defined ligand of insect sodium channel receptor site-3. However, delta-ACTX-Hv1a exhibits a surprisingly low binding affinity to locust sodium channels. Thus, unlike Sc(alpha)Txs, which are capable of differentiating between mammalian and insect sodium channels, delta-ACTXs differentiate between various insect sodium channels but bind with similar high affinity to rat brain and cockroach channels. Structural comparison of delta-ACTX-Hv1a to Sc(alpha)Txs suggests a similar putative bioactive surface but a 'slimmer' overall shape of the spider toxin. A slimmer shape may ease the interaction with the cockroach and mammalian receptor site-3 and facilitate its association with different conformations of the rat brain receptor, correlated with closed/open and slow-inactivated channel states.