Recombinant Chilobrachys jingzhao Delta-theraphotoxin-Cj1a (JZTX-I)

What is JZTX-I and what are its structural characteristics?

JZTX-I is a 36-residue polypeptide neurotoxin secreted by the Chinese tarantula Chilobrachys jingzhao. It belongs to the jingzhaotoxin family of neurotoxins, which function as gating modifiers of ion channels. The toxin contains multiple stabilizing disulfide bridges that form an inhibitory cysteine knot (ICK) motif, characteristic of many spider toxins. This structural feature contributes to its high stability and specific binding properties to target channels. Like other spider venom peptides, JZTX-I has evolved to target neuronal and cardiac ion channels with high specificity .

What ion channels does JZTX-I target and with what affinity?

JZTX-I exhibits dual channel selectivity, targeting both voltage-gated sodium channels (VGSCs) and potassium channels. Specifically:

JZTX-I preferentially acts on cardiac sodium channels but also affects tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channels in dorsal root ganglion neurons. For potassium channels, it demonstrates affinity for Kv2.1 and Kv4.1 subtypes with IC50 values around 8 μM .

How does JZTX-I modify sodium channel function?

JZTX-I modifies sodium channel function through several mechanisms:

• It inhibits channel inactivation, prolonging the open state

• It speeds up recovery after inactivation

• It does not affect the activation threshold of sodium channels

These effects result in increased sodium currents and prolonged depolarization in affected neurons or cardiac cells. Unlike some other sodium channel toxins, JZTX-I does not block the channel pore but rather alters the gating kinetics by binding to specific voltage-sensing domains .

What is the mechanism of JZTX-I interaction with potassium channels?

JZTX-I interacts with potassium channels through the following mechanisms:

This gating modifier effect on potassium channels is consistent with its binding to voltage-sensing domains rather than physically occluding the channel pore. By slowing activation and accelerating deactivation, JZTX-I effectively reduces potassium currents, which contributes to increased cellular excitability .

How can researchers differentiate JZTX-I effects from other jingzhaotoxins?

To differentiate JZTX-I effects from other jingzhaotoxins, researchers should:

• Conduct comparative electrophysiological studies with selective concentration ranges (JZTX-I: IC50 of 8 μM for potassium channels)

• Perform channel subtype-specific assays (JZTX-I primarily affects Kv2.1, Kv4.1, and cardiac sodium channels)

• Analyze the kinetic signature of the toxin effect (JZTX-I distinctively inhibits inactivation while speeding recovery)

• Use control experiments with other characterized jingzhaotoxins like JZTX-X, which selectively blocks Kv4.2 and Kv4.3 but not other channels

• Implement competitive binding assays with known channel modulators

JZTX-I has a distinctive pharmacological profile compared to other family members; for instance, JZTX-X specifically targets Kv4.2 and Kv4.3 without affecting several other ion channels including Kv1.1-1.3, Kv2.1, and Nav1.5/1.7 .

What electrophysiological protocols are most effective for studying JZTX-I activity?

For optimal electrophysiological characterization of JZTX-I activity:

• Whole-cell voltage-clamp recordings are most appropriate, as they allow full assessment of activation, inactivation, and recovery parameters

• Use voltage step protocols from holding potentials of -80 to -100 mV to activate channels

• Implement double-pulse protocols to assess recovery from inactivation

• For sodium channels, include both depolarizing test pulses and strong conditioning pulses to evaluate state-dependent binding

• For potassium channels, use prolonged depolarizations to measure effects on activation kinetics and tail currents

• Employ voltage ramp protocols to generate I-V relationships and assess voltage-dependency of block

Similar protocols have been effective in characterizing related toxins like JZTX-X, which showed voltage-dependent inhibition of Kv4.2 and Kv4.3 channels .

How do recombinant versus native JZTX-I compare in experimental settings?

Recombinant JZTX-I (such as product CSB-EP543483DRU, UniProt B1P1B7) offers several advantages and considerations compared to native toxin:

• Consistent purity and composition: Recombinant production ensures uniform batches without contamination from other venom components

• Post-translational modifications: Native toxins may contain specific modifications absent in recombinant versions depending on expression systems

• Structural integrity: Proper disulfide bond formation is crucial and may vary between native and recombinant forms

• Activity profile: Systematic comparison studies have shown that properly folded recombinant toxins generally preserve the pharmacological profile of native toxins

• Storage stability: Recombinant JZTX-I typically maintains stability for 6 months in liquid form at -20°C/-80°C and 12 months in lyophilized form

For critical experiments, researchers should validate recombinant toxin activity against reference standards and potentially include native toxin controls.

What is known about structure-activity relationships of JZTX-I?

Structure-activity studies of JZTX-I reveal:

• The inhibitory cysteine knot (ICK) motif is essential for proper folding and stability

• Positively charged residues likely mediate initial electrostatic interactions with negatively charged channel regions

• Hydrophobic residues participate in binding to the voltage-sensing domains

• The specific arrangement of disulfide bridges contributes to the three-dimensional structure required for channel subtype selectivity

• The toxin likely binds to the S3-S4 linker regions of voltage sensors, similar to other gating modifier toxins

This binding mode is supported by studies of related toxins like JZTx-14, which has been shown to trap voltage sensors in deactivated states .

What are the in vivo toxicity parameters of JZTX-I?

JZTX-I demonstrates significant but not extreme toxicity in animal models:

• The intraperitoneal LD50 in mice is 1.48 mg/kg, which is relatively high compared to other jingzhaotoxins

• For context, the crude venom of Chilobrachys jingzhao has an LD50 of 4.4 mg/kg

• Other jingzhaotoxins show varying toxicity levels; for example, JZTX-IX has an LD50 of 0.23 mg/kg

• JZTX-X has been shown to cause hyperalgesia (increased sensitivity to pain) without affecting motor function when administered intrathecally or via intraplantar injection

These toxicity parameters should guide safety protocols when handling the toxin in laboratory settings.

What biosafety precautions should be implemented when working with JZTX-I?

When working with JZTX-I, implement the following safety measures:

• Always handle in a certified biosafety cabinet (minimum BSL-2)

• Wear appropriate personal protective equipment including gloves, lab coat, and eye protection

• Avoid generating aerosols during preparation and handling

• Implement strict waste disposal protocols according to institutional guidelines for toxic compounds

• Store securely in properly labeled containers at recommended temperatures (-20°C/-80°C)

• Develop emergency protocols for accidental exposure including specific neutralization steps if available

• Provide staff training on toxin handling and emergency procedures

These measures reflect standard protocols for handling peptide neurotoxins with known toxicity profiles.

How can researchers address potential experimental artifacts when using JZTX-I?

To minimize artifacts in JZTX-I experiments:

• Include vehicle controls with all buffer components except the toxin

• Use concentration series rather than single-dose experiments to establish dose-response relationships

• Account for potential toxin adsorption to plasticware by using low-binding tubes and pipette tips

• Ensure proper oxygenation of solutions in electrophysiological experiments

• Monitor seal stability in patch-clamp experiments as changes may be misinterpreted as toxin effects

• Implement reversibility tests by washout to confirm specific binding

• Use positive control toxins with well-established effects (e.g., TTX for sodium channels)

These approaches help distinguish true pharmacological effects from technical artifacts.

What are the key considerations for storage and handling of recombinant JZTX-I?

For optimal stability and activity of recombinant JZTX-I:

• Store lyophilized toxin at -20°C/-80°C where it typically maintains stability for up to 12 months

• For liquid preparations, maintain at -20°C/-80°C with expected stability of approximately 6 months

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Reconstitute in appropriate buffers according to experimental requirements (typically phosphate-buffered solutions)

• Verify activity periodically using standardized assays

• Consider the addition of carrier proteins (e.g., BSA) for very dilute solutions to prevent adsorption losses

• Document lot numbers and preparation dates for experimental reproducibility

These handling practices maximize toxin stability and experimental consistency.

How does JZTX-I compare with other voltage-gated channel toxins from spider venoms?

Comparative analysis of JZTX-I with other spider toxins reveals:

• JZTX-I exhibits dual-target specificity (sodium and potassium channels) unlike many toxins that show higher selectivity for specific channel subtypes

• Compared to HNTx-III, JZTX-I shows more state-independent binding to sodium channels

• While many spider toxins like HWTx-IV show reversible binding upon strong depolarization, JZTx-14 (another jingzhaotoxin family member) demonstrates irreversible blocking of Nav1.2 currents

• JZTX-I has modest potency (IC50 in μM range) against potassium channels, whereas some spider toxins achieve nM potency

• JZTX-X, another member of the jingzhaotoxin family, shows higher selectivity, targeting only Kv4.2 and Kv4.3 channels

This comparative profile positions JZTX-I as a valuable tool for understanding channel gating mechanisms.

How might JZTX-I be used in pain research applications?

JZTX-I offers several applications in pain research:

• As a probe for studying cardiac and neuronal voltage-gated sodium channel contributions to pain signaling

• For investigating the role of Kv2.1 and Kv4.1 channels in modulating neuronal excitability in pain pathways

• As a comparative tool alongside JZTX-X, which causes hyperalgesia when administered intrathecally or via intraplantar injection

• For developing structure-based analogues with enhanced specificity for pain-relevant channel subtypes

• In mechanistic studies of sodium channel inactivation as a potential analgesic target

Evidence from related toxins like JZTX-X demonstrates that modulation of certain potassium channels can significantly alter nociceptive thresholds without affecting motor function, suggesting potential therapeutic applications .

What are promising avenues for structure-guided development of JZTX-I derivatives?

Future structure-guided research on JZTX-I could focus on:

• Rational design of JZTX-I variants with enhanced selectivity for specific channel subtypes by modifying surface residues

• Development of minimized peptides retaining key pharmacophore elements but with improved synthetic feasibility

• Conjugation strategies to improve blood-brain barrier penetration for potential CNS applications

• Identification of specific residues mediating interaction with the S3-S4 voltage sensor paddle

• Creation of fluorescently labeled derivatives for real-time binding studies while preserving pharmacological activity

• Computational modeling of toxin-channel interactions to predict modifications enhancing subtype selectivity

These approaches would build upon successful strategies applied to other ICK peptides from spider venoms.

How might JZTX-I contribute to understanding voltage-gated channels in cancer research?

Emerging research suggests potential applications of JZTX-I in cancer studies:

• As a tool for investigating the role of voltage-gated sodium channels, particularly TTX-sensitive subtypes, in cancer cell migration and invasion

• For studying Kv2.1 channel contributions to cancer cell proliferation, as this channel has been implicated in several cancer types

• As a comparative probe alongside other channel modulators to map channel expression profiles in different cancer models

• For investigating the mechanistic connections between ion channel activity and oncogenic signaling pathways like JAK-STAT

• In developing screening platforms for channel-targeted cancer therapeutics

Recent evidence has demonstrated that voltage-gated sodium channels play roles in various cancer types, including thyroid cancer where Nav1.6 mediates proliferation and invasion through the JAK-STAT pathway .