Nitza Ilan

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.

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.

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.

Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture

Gating modifiers of voltage-gated sodium channels (Navs) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion β-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNav1.2a) and skeletal muscle (rNav1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4F14A/E15A/E28R, of rNav1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4F14A/E15A/E28R was tested using an rNav1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4F14A/E15A/E28R and residues whose equivalents in the rat brain channel rNav1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1–2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Navs, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNav1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Navs resembles that of voltage-gated potassium channels.

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.

Mammalian skeletal muscle voltage-gated sodium channels are affected by scorpion depressant "insect-selective" toxins when preconditioned.

Among scorpion β- and α-toxins that modify the activation and inactivation of voltage-gated sodium channels (Navs), depressant β-toxins have traditionally been classified as anti-insect selective on the basis of toxicity assays and lack of binding and effect on mammalian Navs. Here we show that the depressant β-toxins LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus (Lqh) bind with nanomolar affinity to receptor site 4 on rat skeletal muscle Navs, but their effect on the gating properties can be viewed only after channel preconditioning, such as that rendered by a long depolarizing prepulse. This observation explains the lack of toxicity when depressant toxins are injected in mice. However, when the muscle channel rNav1.4, expressed in Xenopus laevis oocytes, was modulated by the site 3 α-toxin LqhαIT, LqhIT2 was capable of inducing a negative shift in the voltage-dependence of activation after a short prepulse, as was shown for other β-toxins. These unprecedented results suggest that depressant toxins may have a toxic impact on mammals in the context of the complete scorpion venom. To assess whether LqhIT2 and Lqh-dprIT3 interact with the insect and rat muscle channels in a similar manner, we examined the role of Glu24, a conserved “hot spot” at the bioactive surface of β-toxins. Whereas substitutions E24A/N abolished the activity of both LqhIT2 and Lqh-dprIT3 at insect Navs, they increased the affinity of the toxins for rat skeletal muscle channels. This result implies that depressant toxins interact differently with the two channel types and that substitution of Glu24 is essential for converting toxin selectivity.