Izhar Karbat

Structure-Function Map of the Receptor Site for β-Scorpion Toxins in Domain II of Voltage-gated Sodium Channels

Voltage-gated sodium (Nav) 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 (Glu779 and Pro782) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly845, 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.

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

The scorpion α-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Navs) and is inactive at insect Navs. 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 rNav1.2a 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 α-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhαIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe15 of the core domain in toxin potency at rNav1.2a, 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 α-toxins we were able to eliminate the activity of Lqh2 at rNav1.4 (skeletal muscle), hNav1.5 (cardiac), and rNav1.6 channels, with no hindrance of its activity at Nav1.1–1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.

Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels

Background: β-Scorpion toxins enhance activation of voltage-gated sodium (NaV) channels. Results: Four amino acid residues in the IIISS2-S6 extracellular loop contribute to toxin binding and efficacy. Conclusion: The pore module of domain III and the voltage-sensing module of domain II form the receptor site. Significance: Scorpion toxins make a three-point interaction with NaV channels and alter voltage sensor function. Activation of voltage-gated sodium (Nav) channels initiates and propagates action potentials in electrically excitable cells. β-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of NaV 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 CssIVE15A. 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 NaV channels. Binding of α- and β-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on NaV channels acts synergistically to modify channel gating and paralyze prey.

The unique pharmacology of the scorpion α‐like toxin Lqh3 is associated with its flexible C‐tail

The affinity of scorpion α‐toxins for various voltage‐gated sodium channels (Navs) 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 Navs. We examined this hypothesis using Lqh3, an α‐like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Navs compared with other α‐toxins and its binding to insect Navs is pH‐dependent. Mutagenesis of Lqh3 revealed a bi‐partite bioactive surface, composed of the Core and NC domains, as found in other α‐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 Navs 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 α‐like toxins to affect a broad range of Navs.

Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels

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.

Elucidation of the Molecular Basis of Selective Recognition Uncovers the Interaction Site for the Core Domain of Scorpion α-Toxins on Sodium Channels

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 rNav1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNav1 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 rNav1.2a. 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 rNav1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNav1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNav1.2a 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.

Deletion of PsbM in Tobacco Alters the QB Site Properties and the Electron Flow within Photosystem II

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 ΔpsbM 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 ΔpsbM, 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 ΔpsbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in ΔpsbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher QB/Q –B ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the QA/QB sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the QB 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.

Direct evidence that receptor site-4 of sodium channel gating modifiers is not dipped in the phospholipid bilayer of neuronal membranes

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 β-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 β-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNav1 and rat brain rNav1.2a develops. We show that in general, scorpion β-toxins do not partition in neuronal membranes and that in the case in which a depressant β-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 β-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion β-toxins is accessible to the extracellular solvent.

Partial Agonist and Antagonist Activities of a Mutant Scorpion β-Toxin on Sodium Channels

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 Glu15 in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4E15R 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 Css4E15R 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 Css4E15R is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4E15R 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, Css4E15R 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.

Miniaturization of Scorpion β-Toxins Uncovers a Putative Ancestral Surface of Interaction with Voltage-gated Sodium Channels

The bioactive surface of scorpion β-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved βαββ 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 β-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 β-toxins (ΔΔBj-xtrIT and ΔΔCss4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, ΔΔBj-xtrIT and ΔΔCss4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion α-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion α-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of LqhαIT at insect sodium channels. Co-application of ΔΔBj-xtrIT or ΔΔCss4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at ΔΔBj-xtrIT and ΔΔCss4 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 β-toxins may be viewed as an exercise in “reverse evolution,” providing a clue as to their evolution from a smaller ancestor of similar scaffold.