Β-scorpion Toxin Research Articles

Exploring the Pivotal Components Influencing the Side Effects Induced by an Analgesic-Antitumor Peptide from Scorpion Venom on Human Voltage-Gated Sodium Channels 1.4 and 1.5 through Computational Simulation.

Voltage-gated sodium channels (VGSCs, or Nav) are important determinants of action potential generation and propagation. Efforts are underway to develop medicines targeting different channel subtypes for the treatment of related channelopathies. However, a high degree of conservation across its nine subtypes could lead to the off-target adverse effects on skeletal and cardiac muscles due to acting on primary skeletal muscle sodium channel Nav1.4 and cardiac muscle sodium channel Nav1.5, respectively. For a long evolutionary process, some peptide toxins from venoms have been found to be highly potent yet selective on ion channel subtypes and, therefore, hold the promising potential to be developed into therapeutic agents. In this research, all-atom molecular dynamic methods were used to elucidate the selective mechanisms of an analgesic-antitumor β-scorpion toxin (AGAP) with human Nav1.4 and Nav1.5 in order to unravel the primary reason for the production of its adverse reactions on the skeletal and cardiac muscles. Our results suggest that the rational distribution of residues with ring structures near position 38 and positive residues in the C-terminal on AGAP are critical factors to ensure its analgesic efficacy. Moreover, the substitution for residues with benzene is beneficial to reduce its side effects.

Pharmacological Dissection of the Crosstalk between NaV and CaV Channels in GH3b6 Cells.

Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.

Characterization of Synthetic Tf2 as a NaV1.3 Selective Pharmacological Probe.

NaV1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of Tityus fasciolatus, has been reported to selectively activate NaV1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for NaV1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of NaV1.3 activation in the hyperpolarizing direction by −11.3 mV, with no activity at NaV1.1, NaV1.2, and NaV1.4-NaV1.8. Additional activity was found at NaV1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by −6.3 mV. In an attempt to convert Tf2 into an NaV1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at NaV1.3, although it still caused a small −5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the NaV1.1/1.3 inhibitor ICA-121431 nor in NaV1.9-/- mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at NaV1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.

Tb1, a Neurotoxin from Tityus bahiensis Scorpion Venom, Induces Epileptic Seizures by Increasing Glutamate Release.

Here, we report the neurotoxic effects aroused by the intracerebral injection (in rats) of Tb1, which is a neurotoxin isolated from Tityus bahiensis scorpion venom. Biochemical analyses have demonstrated that this toxin is similar to the gamma toxin from T. serrulatus, which is a β-scorpion toxin that acts on sodium channels, causing the activation process to occur at more hyperpolarized membrane voltages. Male Wistar rats were stereotaxically implanted with intrahippocampal electrodes and cannulas for electroencephalographic recording and the evaluation of amino acid neurotransmitters levels. Treated animals displayed behavioral and electroencephalographic alterations similar to epileptiform activities, such as myoclonus, wet dog shakes, convulsion, strong discharges, neuronal loss, and increased intracerebral levels of glutamate. Scorpion toxins are important pharmacological tools that are widely employed in ion channel dysregulation studies. The current work contributes to the understanding of channelopathies, particularly epilepsy, which may originate, among other events, from dysfunctional sodium channels, which are the main target of the Tb1 toxin.

NaV 1.6 regulates excitability of mechanosensitive sensory neurons.

Voltage-gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders. The β-scorpion toxin, Cn2, is selective for NaV 1.6 in dorsal root ganglion neurons. NaV 1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon. NaV 1.6 activation also leads to enhanced response to mechanical stimulus in vivo. This works highlights the use of toxins in elucidating pain pathways moreover the importance of non-peripherally restricted NaV isoforms in pain generation. Peripheral sensory neurons express multiple voltage-gated sodium channels (NaV ) critical for the initiation and propagation of action potentials and transmission of sensory input. Three pore-forming sodium channel isoforms are primarily expressed in the peripheral nervous system (PNS): NaV 1.7, NaV 1.8 and NaV 1.9. These sodium channels have been implicated in painful and painless channelopathies and there has been intense interest in them as potential therapeutic targets in human pain. Emerging evidence suggests NaV 1.6 channels are an important isoform in pain sensing. This study aimed to assess, using pharmacological approaches, the function of NaV 1.6 channels in peripheral sensory neurons. The potent and NaV 1.6 selective β-scorpion toxin Cn2 was used to assess the effect of NaV 1.6 channel activation in the PNS. The multidisciplinary approach included Ca2+ imaging, whole-cell patch-clamp recordings, skin-nerve and gut-nerve preparations and in vivo behavioural assessment of pain. Cn2 facilitates NaV 1.6 early channel opening, and increased persistent and resurgent currents in large-diameter dorsal root ganglion (DRG) neurons. This promotes enhanced excitatory drive and tonic action potential firing in these neurons. In addition, NaV 1.6 channel activation in the skin and gut leads to increased response to mechanical stimuli. Finally, intra-plantar injection of Cn2 causes mechanical but not thermal allodynia. This study confirms selectivity of Cn2 on NaV 1.6 channels in sensory neurons. Activation of NaV 1.6 channels, in terminals of the skin and viscera, leads to profound changes in neuronal responses to mechanical stimuli. In conclusion, sensory neurons expressing NaV 1.6 are important for the transduction of mechanical information in sensory afferents innervating the skin and viscera.

Selective Closed-State Nav1.7 Blocker JZTX-34 Exhibits Analgesic Effects against Pain.

Jingzhaotoxin-34 (JZTX-34) is a selective inhibitor of tetrodotoxin-sensitive (TTX-S) sodium channels. In this study, we found that JZTX-34 selectively acted on Nav1.7 with little effect on other sodium channel subtypes including Nav1.5. If the DIIS3-S4 linker of Nav1.5 is substituted by the correspond linker of Nav1.7, the sensitivity of Nav1.5 to JZTX-34 extremely increases to 1.05 µM. Meanwhile, a mutant D816R in the DIIS3-S4 linker of Nav1.7 decreases binding affinity of Nav1.7 to JZTX-34 about 32-fold. The reverse mutant R800D at the corresponding position in Nav1.5 greatly increased its binding affinity to JZTX-34. This implies that JZTX-34 binds to DIIS3-S4 linker of Nav1.7 and the critical residue of Nav1.7 is D816. Unlike β-scorpion toxin trapping sodium channel in an open state, activity of JZTX-34 requires the sodium channel to be in a resting state. JZTX-34 exhibits an obvious analgesic effect in a rodent pain model. Especially, it shows a longer duration and is more effective than morphine in hot pain models. In a formalin-induced pain model, JZTX-34 at dose of 2 mg/kg is equipotent with morphine (5 mg/kg) in the first phase and several-fold more effective than morphine in second phase. Taken together, our data indicate that JZTX-34 releases pain by selectively binding to the domain II voltage sensor of Nav1.7 in a closed configuration.

Venom characterization of the Amazonian scorpion Tityus metuendus

The soluble venom from the scorpion Tityus metuendus was characterized by various methods. In vivo experiments with mice showed that it is lethal. Extended electrophysiological recordings using seven sub-types of human voltage gated sodium channels (hNav1.1 to 1.7) showed that it contains both α- and β-scorpion toxin types. Fingerprint analysis by mass spectrometry identified over 200 distinct molecular mass components. At least 60 sub-fractions were recovered from HPLC separation. Five purified peptides were sequenced by Edman degradation, and their complete primary structures were determined. Additionally, three other peptides have had their N-terminal amino acid sequences determined by Edman degradation and reported. Mass spectrometry analysis of tryptic digestion of the soluble venom permitted the identification of the amino acid sequence of 111 different peptides. Search for similarities of the sequences found indicated that they probably are: sodium and potassium channel toxins, metalloproteinases, hyaluronidases, endothelin and angiotensin-converting enzymes, bradykinin-potentiating peptide, hypothetical proteins, allergens, other enzymes, other proteins and peptides.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

Functional and immuno-reactive characterization of a previously undescribed peptide from the venom of the scorpion Centruroides limpidus

A previously undescribed toxic peptide named Cl13 was purified from the venom of the Mexican scorpion Centruroides limpidus. It contains 66 amino acid residues, including four disulfide bonds. The physiological effects assayed in 7 different subtypes of voltage gated Na+-channels, showed that it belongs to the β-scorpion toxin type. The most notorious effects were observed in subtypes Nav1.4, Nav1.5 and Nav1.6. Although having important sequence similarities with two other lethal toxins from this scorpion species (Cll1m and Cll2), the recently developed single chain antibody fragments (scFv) of human origin were not capable of protecting against Cl13. At the amino acid sequence level, in 3 stretches of peptide Cl13 (positions 7–9, 30–38 and 62–66) some differences with respect to other similar toxins are observed. Some of these differences coincide with contact points with the human antibody fragments.

The Scorpion Toxin Tf2 from Tityus fasciolatus Promotes Nav1.3 Opening.

We identified Tf2, the first β-scorpion toxin from the venom of the Brazilian scorpion Tityus fasciolatus. Tf2 is identical to Tb2-II found in Tityus bahiensis. We found that Tf2 selectively activates human (h)Nav1.3, a neuronal voltage-gated sodium (Nav) subtype implicated in epilepsy and nociception. Tf2 shifts hNav1.3 activation voltage to more negative values, thereby opening the channel at resting membrane potentials. Seven other tested mammalian Nav channels (Nav1.1-1.2; Nav1.4-1.8) expressed in Xenopus oocytes are insensitive upon application of 1 μM Tf2. Therefore, the identification of Tf2 represents a unique addition to the repertoire of animal toxins that can be used to investigate Nav channel function.

Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of Escherichia coli

BackgroundAnimal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria. MethodsThe β-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined. ResultsWestern blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold. ConclusionsThe chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method. General significanceWe report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.

Outward stabilization of the voltage sensor in domain II but not domain I speeds inactivation of voltage-gated sodium channels

To determine the roles of the individual S4 segments in domains I and II to activation and inactivation kinetics of sodium current (INa) in NaV1.5, we used a tethered biotin and avidin approach after a site-directed cysteine substitution was made in the second outermost Arg in each S4 (DI-R2C and DII-R2C). We first determined the fraction of gating charge contributed by the individual S4's to maximal gating current (Qmax), and found that the outermost Arg residue in each S4 contributed ∼19% to Qmax with minimal contributions by other arginines. Stabilization of the S4's in DI-R2C and DII-R2C was confirmed by measuring the expected reduction in Qmax. In DI-R2C, stabilization resulted in a decrease in peak INa of ∼45%, while its peak current-voltage (I-V) and voltage-dependent Na channel availability (SSI) curves were nearly unchanged from wild type (WT). In contrast, stabilization of the DII-R2C enhanced activation with a negative shift in the peak I-V relationship by -7 mV and a larger -17 mV shift in the voltage-dependent SSI curve. Furthermore, its INa decay time constants and time-to-peak INa became more rapid than WT. An explanation for these results is that the depolarized conformation of DII-S4, but not DI-S4, affects the receptor for the inactivation particle formed by the interdomain linker between DIII and IV. In addition, the leftward shifts of both activation and inactivation and the decrease in Gmax after stabilization of the DII-S4 support previous studies that showed β-scorpion toxins trap the voltage sensor of DII in an activated conformation.

Structure and Function of Hainantoxin-III, a Selective Antagonist of Neuronal Tetrodotoxin-sensitive Voltage-gated Sodium Channels Isolated from the Chinese Bird Spider Ornithoctonus hainana

In the present study, we investigated the structure and function of hainantoxin-III (HNTX-III), a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. HNTX-III suppressed Nav1.7 current amplitude without significantly altering the activation, inactivation, and repriming kinetics. Short extreme depolarizations partially activated the toxin-bound channel, indicating voltage-dependent inhibition of HNTX-III. HNTX-III increased the deactivation of the Nav1.7 current after extreme depolarizations. The HNTX-III·Nav1.7 complex was gradually dissociated upon prolonged strong depolarizations in a voltage-dependent manner, and the unbound toxin rebound to Nav1.7 after a long repolarization. Moreover, analysis of chimeric channels showed that the DIIS3-S4 linker was critical for HNTX-III binding to Nav1.7. These data are consistent with HNTX-III interacting with Nav1.7 site 4 and trapping the domain II voltage sensor in the closed state. The solution structure of HNTX-III was determined by two-dimensional NMR and shown to possess an inhibitor cystine knot motif. Structural analysis indicated that certain basic, hydrophobic, and aromatic residues mainly localized in the C terminus may constitute an amphiphilic surface potentially involved in HNTX-III binding to Nav1.7. Taken together, our results show that HNTX-III is distinct from β-scorpion toxins and other β-spider toxins in its mechanism of action and binding specificity and affinity. The present findings contribute to our understanding of the mechanism of toxin-sodium channel interaction and provide a useful tool for the investigation of the structure and function of sodium channel isoforms and for the development of analgesics.

Functional Expression of a ß-Scorpion Toxin in the E.Coli Periplasm: A Tool for Exploring Sodium Channel/Ligand Interactions

β-scorpion toxins are polypeptides of 60-76 residues that bind to site 4 of voltage-gated sodium channels and induce a hyper-polarising shift in the voltage-dependence of activation. The domain II voltage sensor is trapped in its outward, activated conformation through interactions with the toxin. β-scorpion toxins represent lead candidates for novel therapeutics and insecticides but their expression in bacterial systems is not straight-forward; the reducing cytoplasmic environment hinders formation of four disulphide bridges that stabilise the toxin structure. Therefore reports to-date of recombinant β-toxin production include an oxidative-refolding step which follows initial purification of the mis-folded peptide.We report an expression strategy that produces correctly-folded β-toxin in E.coli. FPLC and HPLC analysis of β-toxin purified to homogeneity indicted that the protein adopts a single conformation. Crystallization and structure determination to 2.5 A resolution confirmed that the β-toxin adopts the functionally-active form. Additionally, thermal denaturation studies using synchrotron-radiation circular dichroism (SRCD) – the first application of this sensitive CD technique to this toxin class – demonstrated the stability of this cross-linked toxin.The methodology applied to produce this toxin may facilitate industrial scale-up or lab-based production of wild-type and mutant toxins for ligand interaction studies.

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

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.

Mapping the Receptor Sites for a β-Scorpion Toxin on the Pore Module in Domain III of Voltage-Gated Sodium Channels

Activation of voltage-gated sodium (Nav) channels initiates and propagates action potentials in electrically excitable cells. The -scorpion toxin CssIV traps a voltage-sensor of Nav channels in its activated state via a voltage-sensor trapping mechanism and thus shifts their voltage dependence of activation to more negative membrane potentials. The SS2-S6 linker of the pore domain in domain III (IIISS2-S6) is crucial in determining the action of CssIV upon Nav channels. We found that five substitutions at four amino acid residues in IIISS2-S6 markedly alter voltage-sensor trapping current (IVST) by a recombinant toxin derivative, CssIVE15A. These residues are concentrated in the region between N1436 and D1445 and form a discontinuous interaction site. Three of them (E1438A, D1445A and D1445Y) markedly decrease IVST, whereas the other two (N1436G and L1439A) markedly increase IVST. N1436G increases binding affinity of CssIVE15A to Nav channels in the resting state, whereas L1439A increases the efficacy of trapping the activated voltage-sensor by the prebound toxin. Time courses of voltage sensor trapping for the WT and mutant channels fit an allosteric kinetic model that includes a lower affinity resting state and a higher affinity activated/trapped state. Structural modeling suggests that the IIISS2-S6 is in close proximity to the IIS1-S2 and IIS3-S4 linkers in 3D space and that the bound toxin sits in a cleft formed by these three extracellular loops. Our results define the molecular map of a third interacting component of the -scorpion toxin receptor site of mammalian Nav channels and provide new molecular details of the voltage-sensor trapping mechanism of toxin action.

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

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.

Mapping the β-Scorpion Toxin Receptor Site on Voltage-Gated Sodium Channels

Voltage-gated sodium channels are molecular targets of β-scorpion toxins, which enhance excitability by shifting the voltage dependence of activation to more negative potentials. These effects result from a voltage sensor trapping mechanism, in which toxins trap the voltage sensor in its activated conformation. Determinants of β-scorpion toxin (CssIV) binding and action on sodium channel (Nav1.2) are located in the S1-S2 and S3-S4 extracellular linkers in the voltage-sensing module in domain II. To completely map these regions, we made substitutions for previously unstudied amino acid residues and examined modulation by CssIVE15A, a highly active toxin derivative. Of 11 positions studied in IIS1-S2, only one significantly altered the toxin effect from wild-type by reducing binding to the resting state and almost abolishing trapping activity. In IIS3-S4, five positions surrounding a previously identified key binding determinant, G845, define a hotspot of high impact residues. Three of these substitutions reduced toxin binding and voltage-sensor trapping. The other two, V843A and E844N, increased voltage-sensor trapping approximately 4-fold and decreased apparent EC50. The rate of voltage sensor trapping upon depolarization was unchanged for V843A and increased approximately 2.5-fold for E844N. The rate at which the toxin releases the voltage sensor upon repolarization was increased 2.2-fold for the V843A but was unchanged for E844N. Thus CssIVE15A interacts with a short segment of IIS1-S2 and a broader region of DIIS3-S4. The bidirectional effects of mutations on toxin efficacy suggest that native residues make both positive and negative interactions with the toxin. Substitutions that increase toxin effects do so by increasing affinity of resting channels for the toxin and further increasing the relative affinity of the activated voltage-sensor for the toxin. These results provide further support for the voltage sensor-trapping model.

β-Scorpion Toxin Modifies Gating Transitions in All Four Voltage Sensors of the Sodium Channel

Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here we have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the β-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge–voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge–voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of β-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels.

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.