Delta-theraphotoxin-Hm1a Antibody

What is Delta-theraphotoxin-Hm1a and why is it important in neuroscience research?

Delta-theraphotoxin-Hm1a (also known as Hm1a, κ-theraphotoxin-Hm1a, or Heteroscodratoxin-1) is a 35-amino acid peptide neurotoxin isolated from the Togo starburst tarantula. Its importance stems from its highly selective interaction with voltage-gated sodium channel Nav1.1, where it inhibits fast and slow inactivation with an EC50 of 38 ± 6 nM . Originally described as a Kv channel blocker, its specific action on Nav1.1 makes it a valuable tool for studying neuronal excitability, pain mechanisms, and neurological disorders such as epilepsy, Alzheimer's disease, and autism .

How does Delta-theraphotoxin-Hm1a differ from related spider toxins?

While Delta-theraphotoxin-Hm1a shares structural similarities with other tarantula toxins like Hm1b, scodratoxin, hanatoxin, and SGTx1, it has a unique pharmacological profile. Both Hm1a and Hm1b potently inhibit Nav1.1 inactivation, but Hm1b appears more stable in biological fluids . Unlike less selective peptide toxins, Hm1a exhibits remarkable selectivity for Nav1.1 over other sodium channel subtypes. It demonstrates substantially weaker effects on hNav1.2 and hNav1.3, and no effect on hNav1.4–1.8 . This selectivity profile differs from related toxins like SGTx1, which shows little selectivity among Nav subtypes .

What is the molecular structure of Delta-theraphotoxin-Hm1a?

Delta-theraphotoxin-Hm1a is a 35-amino acid peptide with the sequence ECRYLFGGCSSTSDCCKHLSCRSDWKYCAWDGTFS and a molecular weight of 3,995.61 Da . Structurally, it belongs to the inhibitory cysteine knot (ICK) family with three crossing disulfide bridges (Cys1-Cys4/Cys2-Cys5/Cys3-Cys6) . A homology model based on HwTx-IV shows dense packing of five aromatic residues with W6 and F7 at the tip of Loop 1 falling between W29 and Y32/Y21 on one face of the peptide . This overcrowding of bulky aromatic side chains likely contributes to the structural flexibility observed in both aqueous (NMR) and hydrophobic (RP-HPLC) conditions .

What are the key specifications of commercially available Delta-theraphotoxin-Hm1a antibodies?

Commercial Delta-theraphotoxin-Hm1a antibodies are typically polyclonal antibodies raised in rabbits against recombinant Heteroscodra maculata Delta-theraphotoxin-Hm1a (1-35AA) . A representative product (CSB-PA351577ZA01HGU) has the following specifications:

What experimental techniques are optimized for Delta-theraphotoxin-Hm1a antibody usage?

The Delta-theraphotoxin-Hm1a antibody has been validated for ELISA and Western blot applications for the identification of the antigen . While the search results don't explicitly mention other techniques, based on similar neurotoxin antibodies, potential applications could include:

• Immunohistochemistry (IHC) to visualize the distribution of toxin binding in tissue sections

• Immunoprecipitation to isolate Nav1.1 channel complexes

• Flow cytometry to quantify toxin binding in cell populations

• Immunofluorescence to visualize subcellular localization of toxin-channel interactions

For each application, optimization of antibody concentration is essential, with typical starting dilutions ranging from 1:500 to 1:2000 depending on the application and detection method.

How should researchers design experiments to study Nav1.1 modulation using Delta-theraphotoxin-Hm1a and its antibody?

When designing experiments to study Nav1.1 modulation, researchers should consider a multi-faceted approach:

• Electrophysiological characterization: Use patch-clamp recordings to measure changes in Nav1.1 channel properties upon Hm1a application. Typically, Hm1a is applied at 100-500 nM to observe effects on:

  • Channel inactivation kinetics

  • Current amplitude and persistence

  • Action potential threshold and firing frequency

• Binding studies: Employ the antibody in competition assays to:

  • Confirm specificity of toxin-channel interactions

  • Identify binding domains through mutagenesis studies

  • Quantify binding affinity through displacement curves

• Functional verification: Combine electrophysiology with antibody pre-incubation to:

  • Validate that antibody binding neutralizes toxin effects

  • Confirm specificity through lack of effect on other channel subtypes

  • Establish dose-response relationships for both toxin and antibody

Researchers should include appropriate controls, including vehicle (PBS) treatments, irrelevant antibodies of the same isotype, and comparative studies with other Nav subtypes to validate selectivity .

What are the optimal conditions for detecting Delta-theraphotoxin-Hm1a binding to Nav1.1 channels?

Optimal conditions for detecting Hm1a binding to Nav1.1 channels depend on the experimental system:

• Heterologous expression systems:

  • HEK293 cells expressing hNav1.1 show robust responses to Hm1a at 15-221 nM

  • Xenopus oocytes expressing hNav1.1 demonstrate toxin sensitivity at 38 nM (EC50)

  • Automated patch-clamp platforms (e.g., Qpatch16) provide high-throughput screening capabilities

• Neuronal preparations:

  • iPSC-derived neurons from Dravet syndrome patients show increased firing rates at 100-500 nM Hm1a

  • Trigeminal ganglion or dorsal root ganglia neurons from rodents respond to Hm1a (typically 0.5-1 μM)

  • Ex vivo skin-nerve preparations demonstrate enhanced AM fiber responses at 1 μM

• Detection methods:

  • Whole-cell patch clamp provides direct measurement of channel kinetics

  • Calcium imaging can reveal toxin-induced excitability changes

  • Multi-electrode array (MEA) recordings capture network-level effects of Nav1.1 modulation

Researchers should maintain consistent recording conditions (temperature, ionic composition, pH) across experiments to ensure reproducibility.

How can researchers distinguish between direct effects of Delta-theraphotoxin-Hm1a and secondary responses in neuronal networks?

Distinguishing direct from secondary effects requires careful experimental design:

• Isolate direct channel effects:

  • Use tetrodotoxin (TTX) to block Nav channels and determine if Hm1a effects persist

  • Apply channel subtype-selective blockers like ICA-121431 (Nav1.1/1.3 inhibitor) to confirm target specificity

  • Create chimeric channels containing specific Nav1.1 domains to validate binding sites

• Control for network effects:

  • Compare responses in isolated neurons versus intact networks

  • Apply synaptic blockers to eliminate network contributions

  • Perform time-resolved analyses to separate immediate (direct) from delayed (network) effects

• Cell-type specificity:

  • Use genetic markers to identify neuronal subtypes expressing Nav1.1

  • Compare Hm1a responses in excitatory versus inhibitory neurons

  • Correlate Hm1a sensitivity with Nav1.1 expression levels using the antibody

For example, in Dravet syndrome models, Hm1a selectively rescues function in GABAergic interneurons without affecting excitatory neurons, highlighting the importance of cell-type-specific analyses .

How can Delta-theraphotoxin-Hm1a antibodies be used to investigate the structural determinants of Nav1.1 selectivity?

The remarkable selectivity of Hm1a for Nav1.1 provides an opportunity to investigate structural determinants using antibody-based approaches:

• Epitope mapping: Use the antibody to:

  • Identify key binding residues through competitive binding assays

  • Perform alanine scanning mutagenesis of the toxin to identify critical interaction sites

  • Create toxin fragment libraries to define minimal binding domains

• Domain swapping experiments: The search results indicate that both the S1-S2 loops and the DIV S3-S4 loops are important for Hm1a selectivity . Researchers can:

  • Create chimeric channels with varying regions from Nav1.1 and insensitive channels

  • Use antibodies to confirm binding to chimeric constructs

  • Correlate structural elements with functional responses

• Structural analysis: Combined with computational approaches, researchers can:

  • Use antibody competition assays to validate docking simulations

  • Perform cross-linking studies to identify proximity relationships

  • Employ antibody fragments to stabilize toxin-channel complexes for structural studies

These approaches have revealed that the "variability of the S1-S2 loops between Nav channels contributes substantially to the selectivity profile observed for Pre1a [and related toxins], particularly with regards to fast inactivation" .

What insights can Delta-theraphotoxin-Hm1a provide into the role of Nav1.1 in pain processing and neurological disorders?

Delta-theraphotoxin-Hm1a has been instrumental in uncovering Nav1.1's role in several pathophysiological conditions:

• Mechanical pain processing:

  • Hm1a application to mechanonociceptive Aδ fibers increases firing rates in response to mechanical stimuli

  • Intraplantar injection of Hm1a (500nM) produces robust mechanical hypersensitivity without affecting heat sensitivity

  • Conditional Nav1.1 deletion confirms the channel's role in mechanical pain pathways

• Epilepsy models (Dravet syndrome):

  • In patient iPSC-derived neurons, Hm1a (100-500 nM) increases firing rates and intraburst frequency of GABAergic neurons

  • Treatment decreases neuronal network synchrony, potentially explaining its anti-seizure effects

  • Selective rescue of inhibitory interneuron function without affecting excitatory neurons supports Nav1.1's role in maintaining excitation/inhibition balance

• Visceral hypersensitivity:

  • In colonic afferents from chronic visceral hypersensitivity (CVH) mice, Hm1a enhances mechanically-evoked spiking

  • Nav1.1 inhibition reduces hypersensitivity in CVH models to baseline levels

  • These findings suggest upregulation of Nav1.1 function in irritable bowel syndrome

Researchers can use the antibody to correlate channel expression with these functional effects, potentially uncovering therapeutic targets for pain and neurological disorders.

How can Delta-theraphotoxin-Hm1a be utilized as a tool for developing novel therapeutics for neurological disorders?

Delta-theraphotoxin-Hm1a provides a valuable template for therapeutic development:

• Pharmacophore identification:

  • Researchers can use structure-activity relationship studies with the antibody to:

    • Identify essential binding motifs for Nav1.1 selectivity

    • Develop smaller, more stable mimetics that retain selectivity

    • Create penetrable analogues for CNS applications

• Target validation in disease models:

  • In Dravet syndrome, Hm1a and related toxins demonstrate that:

    • Selective Nav1.1 activation can rescue inhibitory neuron function

    • This approach reduces seizures and premature death in animal models

    • Hm1b's greater stability in biological fluids suggests directions for optimization

• Therapeutic development approaches:

  • Peptide engineering based on Hm1a structure to:

    • Enhance stability through non-natural amino acid incorporation

    • Improve blood-brain barrier penetration

    • Develop targeted delivery systems for specific neuron populations

• Diagnostic applications:

  • The antibody could be developed for:

    • Quantifying Nav1.1 expression in patient samples

    • Monitoring disease progression

    • Stratifying patients for clinical trials based on Nav1.1 status

While further optimization is required for clinical applications, these toxins demonstrate the value of Nav1.1 activation for restoring inhibitory interneuron activity in hyperexcitability disorders .

What are common technical challenges when working with Delta-theraphotoxin-Hm1a antibodies and how can they be addressed?

Researchers working with Delta-theraphotoxin-Hm1a antibodies may encounter several technical challenges:

• Structural heterogeneity: Synthetic Hm1a exhibits an unusual non-symmetrical chromatographic profile, suggesting conformational heterogeneity . To address this:

  • Validate antibody recognition of different conformational states

  • Consider using native toxin preparations as positive controls

  • Test antibody binding under various buffer conditions to optimize recognition

• Cross-reactivity concerns: The high sequence similarity with related toxins may lead to cross-reactivity. Researchers should:

  • Perform specificity tests against related toxins (Hm1b, SGTx1)

  • Include appropriate negative controls

  • Consider using epitope-specific antibodies for highest specificity

• Quantification challenges: When using the antibody for quantitative applications:

  • Develop standard curves using purified recombinant toxin

  • Optimize blocking conditions to minimize background

  • Validate linearity of detection across relevant concentration ranges

• Storage and stability: To maintain antibody activity:

  • Store at recommended temperatures (-20°C or -80°C)

  • Avoid repeated freeze-thaw cycles

  • Consider preparing working aliquots to minimize degradation

How should researchers address contradictory findings when comparing Delta-theraphotoxin-Hm1a effects across different experimental systems?

Discrepancies in observed effects across experimental systems are common. To address these:

• System-specific differences:

  • HEK cells vs. oocytes: Studies show approximately 7-fold difference in potency between these systems . Researchers should:

    • Directly compare systems in parallel experiments

    • Report EC50/IC50 values specific to each system

    • Consider differences in membrane composition and auxiliary subunits

• Age-dependent effects:

  • Toxin responses are more robust in embryonic or newborn-derived neurons compared to adult preparations . Researchers should:

    • Carefully document and report developmental stage

    • Consider age-dependent expression of Nav1.1 and regulatory proteins

    • Use age-matched controls for comparative studies

• Experimental protocol variations:

  • Consider differences in:

    • Recording solutions (ionic composition, pH)

    • Temperature (room temperature vs. physiological)

    • Application methods (bath application vs. focal delivery)

    • Holding potentials and stimulation protocols

• Data integration approaches:

  • Use multiple complementary techniques (electrophysiology, calcium imaging, antibody labeling)

  • Employ consistent positive and negative controls across systems

  • Consider mathematical modeling to reconcile apparently contradictory findings

What emerging technologies could enhance the utility of Delta-theraphotoxin-Hm1a antibodies in neuroscience research?

Several emerging technologies could expand the applications of Delta-theraphotoxin-Hm1a antibodies:

• High-throughput screening platforms:

  • Microfluidic-based antibody arrays for rapid toxin variant testing

  • Automated patch-clamp systems for functional validation at scale

  • High-density microelectrode array (HD-MEA) platforms for network-level analyses

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize toxin-channel interactions at nanometer scale

  • Voltage-sensitive fluorescent proteins to correlate binding with functional effects

  • Multiplexed imaging to simultaneously track multiple channel subtypes

• Toxin engineering platforms:

  • Phage display libraries incorporating antibody-derived binding motifs

  • Computational design of toxin-antibody fusion proteins for targeted delivery

  • Click chemistry approaches for site-specific toxin modification

• Patient-derived models:

  • iPSC-derived neurons from patients with Nav1.1-related disorders

  • Organoid models to study toxin effects in tissue-like environments

  • In vivo genetic models with humanized Nav1.1 channels

These technologies could accelerate the development of Nav1.1-targeted therapeutics for epilepsy, pain, and neurodegenerative disorders .

How might understanding the binding mechanism of Delta-theraphotoxin-Hm1a inform the development of next-generation Nav channel modulators?

The unique binding mechanism of Hm1a provides valuable insights for developing novel Nav channel modulators:

• Structure-guided drug design:

  • The identification of both DIV S3-S4 loops and S1-S2 loops as critical for toxin binding suggests novel targeting strategies

  • Understanding how Hm1a selectively modulates inactivation without affecting activation could inform design of modulators with specific kinetic effects

  • The structural flexibility of Hm1a suggests that conformational dynamics may be important for function

• Allosteric modulation strategies:

  • Hm1a's mechanism of inhibiting fast and slow inactivation without blocking the pore represents an elegant approach to channel modulation

  • This suggests development of positive allosteric modulators rather than traditional blockers or openers

  • Targeting voltage sensor movements specifically could provide greater subtype selectivity than pore-directed approaches

• Modality-specific therapeutics:

  • Hm1a's selective effect on mechanical but not thermal pain suggests the possibility of developing pain modulators without affecting normal sensation

  • This could lead to analgesics with improved side effect profiles

  • Understanding how Nav1.1 contributes to specific pain modalities could inform targeted therapeutic approaches

• Combination approaches:

  • The distinct binding sites and mechanisms of Hm1a compared to small molecule Nav modulators suggest potential for combination therapies

  • Antibody-guided targeting could enhance delivery specificity of small molecule modulators

  • Bispecific antibodies incorporating Hm1a-binding domains could provide novel therapeutic strategies

Understanding these mechanisms will be crucial for developing next-generation modulators for epilepsy, pain, and neurodegenerative disorders .