Gating modifier toxins of voltage-gated calcium channels.

Some of the most potent and specific inhibitors of voltage-gated calcium channels are peptide toxins that inhibit channel function not by occlusion of the channel pore, but rather by interfering with the voltage dependence and kinetics of channel opening and closing. Many such gating modifier toxins conform to the inhibitor cystine knot structural family and have primary sequence or functional mechanism similar to toxins that target voltage-gated sodium or potassium channels. This review introduces known gating modifiers of calcium channels, discusses the selectivity, binding sites, and mechanism of the toxin-channel interaction, and reviews the usefulness of these toxins as research tools and as the basis for novel calcium channel pharmacology and therapeutics.     

Jingzhaotoxin-IX, a novel gating modifier of both sodium and potassium channels from Chinese tarantula Chilobrachys jingzhao

Tarantula Chilobrachys jingzhao is one of the most venomous species distributed in China. In this study, we have isolated and characterized a novel neurotoxin named Jingzhaotoxin-IX (JZTX-IX) from the venom of the tarantula. JZTX-IX is a C-terminally amidated peptide composed of 35 amino acid residues. The toxin shows 74% sequence identity with CcoTx3 from southeastern Africa tarantula Ceratogyrus cornuatus. JZTX-IX was found to interact with multiple types of ion channels including voltage-gated sodium channels (both tetrodotoxin-resistant and tetrodotoxin-sensitive isoforms) and Kv2.1 channel. The toxin had no effect on delayed rectifier potassium channel Kv1.1, 1.2 and 1.3. JZTX-IX shifted the voltage dependence of channel activation to more positive voltages, but binding of toxin to ion channels was not reversible by extreme depolarization. In addition, JZTX-IX could bias the activities of ion channels towards closed state because the time constant for decay (channel deactivation) of tail currents became faster in the presence of toxin. Taken together with the finding that 10 μM JZTX-IX completely blocked ion channels at resting potential without pulsing, we propose that JZTX-IX is a gating modifier showing low selectivity for ion channel types and trapping voltage sensor at closed state.     

The interaction of spider gating modifier peptides with voltage-gated potassium channels.

Gating modifier peptides bind to ion channels and alter the gating process of these molecules. One of the most extensively studied peptides, Hanatoxin (HaTx), isolated from a Chilean tarantula, has been used to characterize the blocking properties of the voltage-gated potassium channel Kv2.1. These studies have provided some insight into the gating mechanism in Kv channels. In this review we will discuss the interaction of HaTx and related spider peptides with Kv channels illustrating the properties of the binding surface of these peptides, their membrane partitioning characteristics, and will provide a working hypothesis for how the peptides inhibit gating of Kv channels. Advanced simulation results support the concept of mutual conformational changes upon peptide binding to the S3b region of the channel which will restrict movement of S4 and compromise coupling of the gating machinery to opening of the pore.     

Tarantulas: eight-legged pharmacists and combinatorial chemists.

Tarantula venoms represent a cornucopia of novel ligands for a variety of cell receptors and ion channels. The diversity of peptide toxin pharmacology has been barely explored as indicated by pharmacological, toxicological and mass spectrometry investigations on more than 55 tarantula venoms. MALDI-TOF MS analysis reveals that the pharmacological diversity is based on relatively small size peptides, which seem to fall into a limited number of structural patterns. Properties and biological activities of the 33 known peptide toxins from tarantula venoms are described. Most known toxins conform to the Inhibitory Cystine Knot (ICK) motif, with differences in the length of intercysteine loops. Recently described peptides show that tarantula toxins can fold according to an elaboration of the Disulfide-Directed beta-Hairpin (DDH) motif which is also the canonical motif for the ICK fold. The ICK fold itself offers many variations leading to differing toxin properties. Examination of pharmacological data gives insights on the possible conserved site of action of toxins acting on voltage-gated ion channels and other toxins acting by a pore-blocking mechanism. Structure-activity data shows the versatility of the toxin scaffolds and the importance of surface features in the selectivity and specificity of these toxins. Tarantulas appear to be a good model for the discovery of novel compounds with important therapeutic potential, and for the study of the molecular evolution of peptide toxins.     

Subtype-selective targeting of voltage-gated sodium channels

Voltage-gated sodium channels are key to the initiation and propagation of action potentials in electrically excitable cells. Molecular characterization has shown there to be nine functional members of the family, with a high degree of sequence homology between the channels. This homology translates into similar biophysical and pharmacological properties. Confidence in some of the channels as drug targets has been boosted by the discovery of human mutations in the genes encoding a number of them, which give rise to clinical conditions commensurate with the changes predicted from the altered channel biophysics. As a result, they have received much attention for their therapeutic potential. Sodium channels represent well-precedented drug targets as antidysrhythmics, anticonvulsants and local anaesthetics provide good clinical efficacy, driven through pharmacology at these channels. However, electrophysiological characterization of clinically useful compounds in recombinant expression systems shows them to be weak, with poor selectivity between channel types. This has led to the search for subtype-selective modulators, which offer the promise of treatments with improved clinical efficacy and better toleration. Despite developments in high-throughput electrophysiology platforms, this has proven very challenging. Structural biology is beginning to offer us a greater understanding of the three-dimensional structure of voltage-gated ion channels, bringing with it the opportunity to do real structure-based drug design in the future. This discipline is still in its infancy, but developments with the expression and purification of prokaryotic sodium channels offer the promise of structure-based drug design in the not too distant future.     

Spider-venom peptides that target voltage-gated sodium channels: Pharmacological tools and potential therapeutic leads

Voltage-gated sodium (Na_V) channels play a central role in the propagation of action potentials in excitable cells in both humans and insects. Many venomous animals have therefore evolved toxins that modulate the activity of Na_V channels in order to subdue their prey and deter predators. Spider venoms in particular are rich in Na_V channel modulators, with one-third of all known ion channel toxins from spider venoms acting on Na_V channels. Here we review the landscape of spider-venom peptides that have so far been described to target vertebrate or invertebrate Na_V channels. These peptides fall into 12 distinct families based on their primary structure and cysteine scaffold. Some of these peptides have become useful pharmacological tools, while others have potential as therapeutic leads because they target specific Na_V channel subtypes that are considered to be important analgesic targets. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides and so far only 0.01% of this diversity been characterised. Thus, it is likely that future research will reveal additional structural classes of spider-venom peptides that target Na_V channels.     

Heteropoda toxin 2 is a gating modifier toxin specific for voltage-gated K+ channels of the Kv4 family.

Kv4 voltage-gated K(+) channels are responsible for transient K(+) currents in the central nervous system and in the heart. HpTx2 is a peptide toxin that selectively inhibits these currents; making it a useful probe for understanding Kv4 channel structure and drug binding. Therefore, we developed a method to produce large amounts of recombinant HpTx2. Recombinant toxin inhibits all three Kv4 isoforms to the same degree; however, the voltage-dependence of inhibition is less apparent for Kv4.1 than for Kv4.3. Similarly, recombinant HpTx2(GS) effects gating characteristics of both channels, but Kv4.1 to a much lesser degree. The toxin lacks affinity for Kv1.4, Kv2.1, and Kv3.4. To locate the binding site, the amino acids linking the third and forth membrane spanning segments of Kv4.3 were replaced with analogous amino acids of Kv1.4. The chimeric K(+) channel was completely insensitive to block by rHpTx2, suggesting that its binding site is near the channel's voltage sensor. These data show that rHpTx2(GS) is a gating modifier toxin that binds to a site remote from the pore.     

Site-3 sea anemone toxins: molecular probes of gating mechanisms in voltage-dependent sodium channels.

Sea anemone toxins, whose biological function is the capture of marine prey, are invaluable tools for studying the structure and function of mammalian voltage-gated sodium channels. Their high degree of specificity and selectivity have allowed for detailed analysis of inactivation gating and assignment of molecular entities responsible for this process. Because of their ability to discriminate among channel isoforms, and their high degree of structural conservation, these toxins could serve as important lead compounds for future pharmaceutical design.     

Two novel sodium channel inhibitors from Heriaeus melloteei spider venom differentially interacting with mammalian channel's isoforms

Two new polypeptide toxins named Hm-1 and Hm-2 were isolated from the venom of the crab spider Heriaeus melloteei. These toxins consist of 37 and 40 amino acid residues, respectively, contain three intramolecular disulfide bonds, and presumably adopt the inhibitor cystine knot motif. Hm-1 is C-terminally amidated and shows a low degree of homology to spider toxins agelenin and mu-agatoxin-II, whereas Hm-2 has no relevantly related peptide sequences. Hm-1 and Hm-2 were found to act on mammalian voltage-gated Na(+) channels. Both toxins caused a strong decrease of Na(+) current peak amplitude, with IC(50) values of 336.4 and 154.8nM, respectively, on Na(V)1.4. Hm-1 and Hm-2 did not shift the voltage-dependence of activation, nor did they change the kinetics of fast inactivation of the Na(+) currents. Interestingly, both toxins negatively shifted the steady-state inactivation process, which might have important functional consequences in vivo. However, this hyperpolarizing shift cannot by itself explain the observed inhibition of the Na(+) current, indicating that the two presented toxins could provide important structural information about the interaction of polypeptide inhibitors with voltage-gated Na(+) channels.     

Site-3 toxins and cardiac sodium channels.

Site-3 toxins are small polypeptide venoms from scorpions, sea anemones, and spiders that bind with a high specificity to the extracellular surface of voltage-gated Na channels. After binding to a site near the S4 segment in domain IV the toxin causes disruption of the normal fast inactivation transition resulting in a marked prolongation of the action potentials of excitable tissues including those of cardiac and skeletal muscle and nerve. In this review we discuss the specific binding interactions between residues of the toxin and those of the Na channel, and the specific modification of Na channel kinetic behavior leading to a change in fast inactivation focusing on interactions deduced primarily from the study of sea anemone toxins and the cardiac Na channel (Na(V)1.5). We also illustrate the usefulness of site-3 toxins in the study of altered Na channel behavior by drug-modification.     

Differential interactions of lamotrigine and related drugs with transmembrane segment IVS6 of voltage-gated sodium channels

Voltage-gated sodium channels are blocked by local anesthetic and anticonvulsant drugs. A receptor site for local anesthetics has been defined in transmembrane segment S6 in domain IV (IVS6) of the alpha subunit, but the anticonvulsant lamotrigine and related compounds have more complex structures than local anesthetics and may interact with additional amino acid residues. Apparent K(D) values for inactivated-state block of rat brain type IIA sodium channels expressed in Xenopus oocytes were 31.9 micro M, 17.3 micro M, 3.7 micro M and 10.3 micro M for lamotrigine and compounds 227c89, 4030w92 and 619c89, respectively. Compound 619c89 was the strongest frequency-dependent blocker, which correlated with higher affinity and a five-fold slower recovery from drug block compared to lamotrigine. Examination of lamotrigine block of mutant sodium channel alpha subunits, in which alanine had been substituted for each individual amino acid in IVS6, identified mutations I1760A, F1764A and Y1771A as causing the largest reductions in affinity (six-, seven- and 12-fold, respectively). The ratios of effects of these three mutations differed for compounds 227c89, 4030w92, and 619c89. The amino acid residues interacting with these pore-blocking drugs define a surface of IVS6 that is exposed to the pore and may rotate during gating.     

JZTX-XIII, a Kv channel gating modifier toxin from Chinese tarantula Chilobrachys jingzhao

Jingzhaotoxin-XIII (JZTX-XIII), a 35 residue polypeptide, with the ability to inhibit voltage-dependent potassium channels in the shab (Kv2) and shal (Kv4) subfamilies, was purified from the venom of the Chinese tarantula Chilobrachys jingzhao. Electrophysiological recordings carried out in Xenopus laevis oocytes showed that JZTX-XIII acted as gating modifier of voltage-dependent K⁺ channels which inhibited the Kv2.1 channel and Kv4.1 channel, with the IC₅₀ value of 0.47 μM and 1.17 μM, respectively. JZTX-XIII shares high sequence similarity with gating modifier toxins inhibiting a wide variety of ion channels including Nav1.5 subtype, but it showed no Nav1.5 channel activity. Structure-function analysis indicates that the acidic residues of Glu10 and Glu17 in JZTX-XIII might be responsible for the loss of the Nav1.5 channel inhibitory potency for JZTX-XIII.     

Using the deadly mu-conotoxins as probes of voltage-gated sodium channels.

Mu-Conotoxins (mu-CTX) are potent Na channel inhibitory peptides isolated from the venom of the predatory marine snail Conus geographus. Mu-CTXs exert their biological action by physically occluding the ion-conducting pore of voltage-gated Na (Na(v)) channels with a 1:1 stoichiometry in an all-or-none fashion. This article reviews our current knowledge of the mechanism of mu-CTX and the associated structural and functional insights into its molecular target--Na(v) channels.     

Insect-selective spider toxins targeting voltage-gated sodium channels.

The voltage-gated sodium (Na(v)) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate Na(v) channel gating. A number of peptide neurotoxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized and determined to interact with several of these sites. These all conform to an 'inhibitor cystine-knot' motif with structural, but not sequence homology, to a variety of other spider and marine snail toxins. Of these, spider toxins several show phyla-specificity and are being considered as lead compounds for the development of biopesticides. Hainantoxin-I appears to target site-1 to block Na(v) channel conductance. Magi 2 and Tx4(6-1) slow Na(v) channel inactivation via an interaction with site-3. The delta-palutoxins, and most likely mu-agatoxins and curtatoxins, target site-4. However, their action is complex with the mu-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion beta-toxins, but with both delta-palutoxins and mu-agatoxins slowing Na(v) channel inactivation, a site-3-like action. In addition, several other spider neurotoxins, such as delta-atracotoxins, are known to target both insect and vertebrate Na(v) channels most likely as a result of the conserved structures within domains of voltage-gated ion channels across phyla. These toxins may provide tools to establish the molecular determinants of invertebrate selectivity. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the above areas remains underdeveloped.     

Inhibition of the activation pathway of the T-type calcium channel CaV3.1 by ProTxII

Toxins have been used extensively to probe the gating mechanisms of voltage-gated ion channels. Relatively few such tools are available to study the low-voltage activated T-type Ca channels, which underlie thalamic neuron firing and affect sleep, resistance to seizures, and weight gain. Here we show that ProTxII, a peptide toxin recently isolated from the venom of the tarantula spider Thrixopelma pruriens, dose-dependently inhibited Ca_V3.1 causing a decrease in current (81.6% ± 3.1% at −30 mV in 5 μM toxin) and a positive shift in the voltage range of activation (+34.5 mV ± 4.4 mV). Toxin-modified currents were slower to activate and faster to deactivate and they displayed a longer lag in the onset of current, i.e. the Cole-Moore shift, consistent with the inhibition of gating transitions along the activation pathway, particularly the final opening transition. Single-channel current amplitude and total gating charge were unaffected by toxin, ruling out a change in ion flux or channel dropout as mechanisms for the decrease in macroscopic conductance. A positive shift in the voltage range of gating charge movement (+30.6 mV ± 2.6 mV shift in the voltage of half maximal charge movement in the presence of 5 μM toxin) confirmed that ProTxII-induced gating perturbations in this channel occur at the level of the voltage sensors, and kinetic modeling based on these findings suggested that reductions in current magnitude could be largely accounted for by kinetic perturbations of activation.     

Type B brevetoxins show tissue selectivity for voltage-gated sodium channels: comparison of brain, skeletal muscle and cardiac sodium channels.

Brevetoxins and ciguatoxins are two classes of phycotoxins which exert their toxic effect by binding to site-5 of voltage-gated sodium channels. Sodium channels, a family of at least 10 structurally different proteins, are responsible for the rising phase of the action potential in membranes of neuronal, cardiac and muscular excitable cells. This work is a comparative study of the binding properties and the cytotoxic effects of ciguatoxins and brevetoxins on human embryonic cells (HEK) stably expressing either the skeletal muscle (Na(v)1.4), or the cardiac (Na(v)1.5) sodium channel alpha-subunit isoforms. We report that type A (PbTx-1) and type B (PbTx-3 and PbTx-2) brevetoxins as well as ciguatoxins target both cardiac and muscle channels; type B brevetoxins show isoform selectivity, presenting a lower affinity for the heart than the skeletal muscle channel. The lower selectivity of type B brevetoxins for heart sodium channels may result from a more rigid backbone structure than is found in type A brevetoxins and ciguatoxins.     

omega-Lsp-IA, a novel modulator of P-type Ca2+ channels.

A novel polypeptide, designated omega-Lsp-IA, which modulates P-type Ca(2+) channels, was purified from the venom of the spider Geolycosa sp. omega-Lsp-IA contains 47 amino acid residues and 4 intramolecular disulfide bridges. It belongs to a group of spider toxins affecting Ca(2+) channels and presumably forms the inhibitor cystine knot (ICK) fold. Peculiar structural features (a cluster of positively charged residues in the C-terminal loop of the peptide and a regular distribution of hydrophobic residues) that may play a decisive role in the omega-Lsp-IA mechanism of action were located. Recombinant omega-Lsp-IA was produced in prokaryotic expression system and was shown to be structurally and functionally identical to the native toxin. At saturating concentration (10nM), the peptide clearly slows down the activation kinetics and partially inhibits the amplitude of P-current in rat cerebellar Purkinje neurons. Prominent deceleration of the activation kinetics is manifested as the appearance of a five-fold slower component of the current activation. The specificity of action of omega-Lsp-IA on different Ca(2+) channel types was studied in isolated hippocampal neurons of rat. omega-Agatoxin IVA completely removed the effect of omega-Lsp-IA on the whole-cell Ca(2+) current. Therefore, omega-Lsp-IA appears to act specifically on P-type Ca(2+) channels.     

电压门控钠通道在慢性疼痛药物发现中的研究进展

由于发病率高、药物效果有限或治疗药物受限等原因,慢性疼痛的治疗一直是世界范围内研究人员关注的难题。电压门控钠通道( VGSCs)阻滞剂有较为显著的镇痛作用,目前已知与慢性疼痛相关的钠通道亚型主要有 Nav1.3、Nav1.7、Nav1.8、 Nav1.9。2021年 8—9月进行了该研究,全面概括了上述钠通道亚型与慢性疼痛的关系,归纳出潜在候选药物临床前研究方法,以及已被证实安全有效的选择性钠通道阻滞剂品种,为选择性钠通道阻滞剂的开发提供参考。     

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 beta-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.     

Precious Natural Peptides from Spider Venoms: New Tools for Studying Potassium Channels

Among the large variety of animal toxins that target potassium channels, spider peptides constitute an unique class of voltage-dependent K⁺ (Kv) current inhibitors according to their structure and pharmacological properties. Spider toxins that block Kv currents are small basic peptides the include three disuflide bridges and belong to the family of inhibitor cystine knot (ICK) molecules. Unlike snake, bee, scorpion, or sea anemone toxins that block Kv1 or Kv3 channels, ICK spider toxins target Kv2 and Kv4 channels, which are expressed in the central nervous system (CNS) and in the cardiovascular system. Their selective affinities for Kv2 and/or Kv4 subfamilies are very useful for dissecting these currents in neuronal and cardiac cells and for the determination of their contribution in physiological processes. Their mode of action is also original, since they induce a shift of channel opening to more depolarized potentials that alter the voltage-dependent properties of K currents. Then they are called gating modifiers. Structure-function studies of these gating modifiers were recently facilitated by solving their tridimensional structure together with the crystallization of prokaryotic K⁺ channels. Spider toxins present an active molecular surface, including a hydrophobic patch surrounded by charged residues, which are important for their binding on Kv channels. Gating modifiers interact with important residues in the S3C-S4 external loop via both hydrophobic and electrostatic interactions. Several dynamic interaction models were proposed, but all of them remain putative.