Tarantula Toxin Research Articles

Tarantula Toxin Transcripts and Toxin Structure Prediction by AlphaFold

Tarantula Toxin Transcripts and Toxin Structure Prediction by AlphaFold

Piezo activity levels need to be tightly regulated to maintain normal morphology and function in pericardial nephrocytes.

Due to their position on glomerular capillaries, podocytes are continuously counteracting biomechanical filtration forces. Most therapeutic interventions known to generally slow or prevent the progression of chronic kidney disease appear to lower these biomechanical forces on podocytes, highlighting the critical need to better understand podocyte mechano-signalling pathways. Here we investigated whether the mechanotransducer Piezo is involved in a mechanosensation pathway in Drosophila nephrocytes, the podocyte homologue in the fly. Loss of function analysis in Piezo depleted nephrocytes reveal a severe morphological and functional phenotype. Further, pharmacological activation of endogenous Piezo with Yoda1 causes a significant increase of intracellular Ca++ upon exposure to a mechanical stimulus in nephrocytes, as well as filtration disturbances. Elevated Piezo expression levels also result in a severe nephrocyte phenotype. Interestingly, expression of Piezo which lacks mechanosensitive channel activity, does not result in a severe nephrocyte phenotype, suggesting the observed changes in Piezo wildtype overexpressing cells are caused by the mechanosensitive channel activity. Moreover, blocking Piezo activity using the tarantula toxin GsMTx4 reverses the phenotypes observed in nephrocytes overexpressing Piezo. Taken together, here we provide evidence that Piezo activity levels need to be tightly regulated to maintain normal pericardial nephrocyte morphology and function.

Tarantula toxin inhibits potassium channels by the pore occlusion

Tarantula toxin inhibits potassium channels by the pore occlusion

Interaction of gating-modifier tarantula toxins with voltage-gated sodium channels.

Voltage-gated ion channels are membrane protein complexes. They allow the selective flow of their respective ions down an electrochemical gradient through a central ion conduction pore surrounded by four voltage sensing domains (VSDs). Gating-modifier toxins (GMTs) are peptide toxins found in different animal venoms that bind to these VSDs and change their gating properties. Our laboratory has determined that the GMT GsAF2, from the Chilean rose tarantula Grammostola rosea, binds to the VSD of the bacterial sodium channel NaChBac (called BHVSD). GsAF2, VSTx2 (another gating modifier toxin - the W residue at the C-terminus of GsAF2 is replaced by EG in VSTx2), and VSTx2-T8S have been synthesized recombinantly and their functionality has been tested through Automated Patch Clamp electrophysiology. Docking studies have been performed with a homology model of GsAF2 and the Cryo-EM structure of NaChBac to identify residues of the toxin and channel that could be involved in the interaction and guiding the design of suitable mutants to validate these interactions. The docked models were evaluated based on the presence of realistic and chemically feasible interactions. The best docked model based on these parameters will be energy-minimized in a membrane bilayer by molecular dynamics simulation. 15N-labeled GsAF2 has also been generated to perform Nuclear Magnetic Resonance (NMR) experiments for structure determination and to identify toxin residues involved in channel binding.

Neonatal NaV 1.5 channels: pharmacological distinctiveness of a cancer-related voltage-gated sodium channel splice variant.

Voltage-gated sodium (NaV ) channels are expressed de novo in carcinomas where their activity promotes invasiveness. Breast and colon cancer cells express the neonatal splice variant of NaV 1.5 (nNaV 1.5), which has several amino acid substitutions in the domain I voltage-sensor compared with its adult counterpart (aNaV 1.5). This study aimed to determine whether nNaV 1.5 channels could be distinguished pharmacologically from aNaV 1.5 channels. Cells expressing either nNaV 1.5 or aNaV 1.5 channels were exposed to low MW inhibitors, an antibody or natural toxins, and changes in electrophysiological parameters were measured. Stable expression in EBNA cells and transient expression in Xenopus laevis oocytes were used. Currents were recorded by whole-cell patch clamp and two-electrode voltage-clamp, respectively. Several clinically used blockers of NaV channels (lidocaine, procaine, phenytoin, mexiletine, ranolazine, and riluzole) could not distinguish between nNaV 1.5 or aNaV 1.5 channels. However, two tarantula toxins (HaTx and ProTx-II) and a polyclonal antibody (NESOpAb) preferentially inhibited currents elicited by either nNaV 1.5 or aNaV 1.5 channels by binding to the spliced region of the channel. Furthermore, the amino acid residue at position 211 (aspartate in aNaV 1.5/lysine in nNaV 1.5), that is, the charge reversal in the spliced region of the channel, played a key role in the selectivity, especially in antibody binding. We conclude that the cancer-related nNaV 1.5 channel can be distinguished pharmacologically from its nearest neighbour, aNaV 1.5 channels. Thus, it may be possible to design low MW compounds as antimetastatic drugs for non-toxic therapy of nNaV 1.5-expressing carcinomas.

Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties.

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.

EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues.

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane-endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

Structure and analysis of nanobody binding to the human ASIC1a ion channel.

ASIC1a is a proton-gated sodium channel involved in modulation of pain, fear, addiction, and ischemia-induced neuronal injury. We report isolation and characterization of alpaca-derived nanobodies (Nbs) that specifically target human ASIC1a. Cryo-electron microscopy of the human ASIC1a channel at pH 7.4 in complex with one of these, Nb.C1, yielded a structure at 2.9 Å resolution. It is revealed that Nb.C1 binds to a site overlapping with that of the Texas coral snake toxin (MitTx1) and the black mamba venom Mambalgin-1; however, the Nb.C1-binding site does not overlap with that of the inhibitory tarantula toxin psalmotoxin-1 (PcTx1). Fusion of Nb.C1 with PcTx1 in a single polypeptide markedly enhances the potency of PcTx1, whereas competition of Nb.C1 and MitTx1 for binding reduces channel activation by the toxin. Thus, Nb.C1 is a molecular tool for biochemical and structural studies of hASIC1a; a potential antidote to the pain-inducing component of coral snake bite; and a candidate to potentiate PcTx1-mediated inhibition of hASIC1a in vivo for therapeutic applications.

Chemical Synthesis and NMR Solution Structure of Conotoxin GXIA from Conus geographus.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded β-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

Structural Basis for High-Affinity Trapping of the NaV1.7 Channel in Its Resting State by Tarantula Toxin

Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel NaVAb with voltage-shifting mutations and the toxin-binding site of human NaV1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.

Transgenic Tarantula Toxin: A novel tool to study mechanosensitive ion channels in Drosophila

The tarantula venom toxin GsMTx4 is the only known specific inhibitor of cation-selective mechanosensitive ion channels (MSCs). Its specificity, potency, and ease of use on isolated tissues and cells have made it a powerful pharmacological tool to identify and probe the physiological function of MSCs. In some contexts, however, it would be desirable to deliver the toxin in a controlled way in vivo. Here we describe a novel tool to allow spatial and temporal control of GsMTx4 delivery in vivo in Drosophila. To test the tool, we targeted MSCs required for mechanical nociception in a specific subset of sensory neurons in intact larvae. Expression of GsMTx4 in these neurons results in robust inhibition of mechanical nociception, demonstrating the toxin is active when expressed in vivo. The tool will be particularly useful to manipulate MSC activity in a spatially and temporally-controlled manner to study their role in development, physiology and behaviour in intact, free moving animals.

Distinguishing Potassium Channel Resting State Conformations in Live Cells with Environment-Sensitive Fluorescence.

Ion channels are polymorphic membrane proteins whose high-resolution structures offer images of individual conformations, giving us starting points for identifying the complex and transient allosteric changes that give rise to channel physiology. Here, we report live-cell imaging of voltage-dependent structural changes of voltage-gated Kv2.1 channels using peptidyl tarantula toxins labeled with an environment-sensitive fluorophore, whose spectral shifts enable identification of voltage-dependent conformation changes in the resting voltage sensing domain (VSD) of the channel. We synthesize a new environment-sensitive, far-red fluorophore, julolidine phenoxazone (JP) azide, and conjugate it to tarantula toxin GxTX to characterize Kv2.1 VSD allostery during membrane depolarization. JP has an inherent response to the polarity of its immediate surroundings, offering site-specific structural insight into each channel conformation. Using voltage-clamp spectroscopy to collect emission spectra as a function of membrane potential, we find that they vary with toxin labeling site, the presence of Kv2 channels, and changes in membrane potential. With a high-affinity conjugate in which the fluorophore itself interacts closely with the channel, the emission shift midpoint is 50 mV more negative than the Kv2.1 gating current midpoint. This suggests that substantial conformational changes at the toxin-channel interface are associated with early gating charge transitions and these are not concerted with VSD motions at more depolarized potentials. These fluorescent probes enable study of conformational changes that can be correlated with electrophysiology, putting channel structures and models into a context of live-cell membranes and physiological states.

Inhibition of Inositol Monophosphatase Enhances TRPV1 Function in vivo

Plasma membrane lipid changes play an essential role in regulating various cellular processes including ion channel function. Membrane remodeling by inflammatory mediators exerts an extra level of modulation in these proteins. The transient receptor potential vanilloid 1 (TRPV1) is an excitatory cation channel involved in neurogenic inflammation and pain hypersensitivity, and its function is regulated by phosphoinositides content. Here, we determined the effect of phosphoinositides on TRPV1 activity in Caenorhabditis elegans using genetic dissection, diet supplementation, and functional analyses. As in mammalian cells, transgenic TRPV1 worms’ aversive response can be positively or negatively modulated with arachidonic acid, a tarantula toxin, and capsazepine. Chemical inhibition of the inositol monophosphatase enzyme (TTX-7) with lithium chloride enhances TRPV1 function in vivo. Moreover, TRPV1 worms lacking the function of TTX-7 have low level of phosphoinositide lipids and display enhanced aversive behavior to capsaicin. On the contrary, TRPV1 worms supplemented with phosphoinositides displayed reduced aversive behavior. Calcium imaging in TRPV1-expressing neurons of live worms shows that capsaicin elicited robust Ca2+ transients in TRPV1 worms fed with control diet, but significantly less changes in worms grown in phosphoinositide supplemented media. Behavior analysis of a TRPV1 worm mutant lacking the C terminus phosphoinositide-interaction site demonstrates that this region modulates channel agonist sensitivity. Our results support that TRPV1 function is negatively regulated by phosphoinositide lipids in vivo.

Structural Basis of Nav1.7 Inhibition by the Tarantula Toxin Protoxin-II

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Neurotoxins have provided important insights into Nav channels, but the structural basis for toxin modulation of channel gating is not well-defined. Protoxin-II (ProTx2) is an inhibitor cystine-knot peptide and a selective antagonist of the human Nav1.7 channel. Here, using X-ray crystallography and cryo-electron microscopy, we have captured ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct structural features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule of VSD2 to antagonize S4 gating charge movement through an electrostatic mechanism. ProTx2 engages a small interaction surface and few direct contacts on VSD2, allowing the S4 helix to be visualized in both activated and deactivated states. Our results illuminate pharmacological concepts employed by membrane-partitioning peptide toxins, clarify the structural basis of electromechanical coupling in voltage-gated ion channels, and provide new templates to design selective Nav channel antagonists.

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel

The tarantula toxin GxTx detains K+ channel gating charges in their resting conformation.

Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K+ channel's voltage sensor allosterically modulates opening of its K+-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K+ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K+-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K+-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K+ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors.

Using Unnatural Amino Acids to Probe the Interaction between Tarantula Toxins and Voltage Sensing Domains in Kv Channels

Using Unnatural Amino Acids to Probe the Interaction between Tarantula Toxins and Voltage Sensing Domains in Kv Channels

Imaging Voltage Gating of Endogenous Neuronal Ion Channels with Fluorescent Tarantula Toxin

Imaging Voltage Gating of Endogenous Neuronal Ion Channels with Fluorescent Tarantula Toxin

Heat activation is intrinsic to the pore domain of TRPV1

The TRPV1 channel is a sensitive detector of pain-producing stimuli, including noxious heat, acid, inflammatory mediators, and vanilloid compounds. Although binding sites for some activators have been identified, the location of the temperature sensor remains elusive. Using available structures of TRPV1 and voltage-activated potassium channels, we engineered chimeras wherein transmembrane regions of TRPV1 were transplanted into the Shaker Kv channel. Here we show that transplanting the pore domain of TRPV1 into Shaker gives rise to functional channels that can be activated by a TRPV1-selective tarantula toxin that binds to the outer pore of the channel. This pore-domain chimera is permeable to Na+, K+, and Ca2+ ions, and remarkably, is also robustly activated by noxious heat. Our results demonstrate that the pore of TRPV1 is a transportable domain that contains the structural elements sufficient for activation by noxious heat.

The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel Nav1.7.

Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Nav1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.