Recombinant Conus radiatus Iota-conotoxin-like R11.18

What is Recombinant Conus radiatus Iota-conotoxin-like R11.18?

Recombinant Conus radiatus Iota-conotoxin-like R11.18 belongs to the I-superfamily of conotoxins derived from the fish-hunting cone snail Conus radiatus. This peptide is closely related to ι-RXIA (previously known as r11a), which contains eight cysteine residues arranged in a pattern characteristic of I-superfamily conotoxins. Like other peptides in this family, R11.18 likely contains post-translational modifications including gamma-carboxyglutamate residues and C-terminal amidation, which contribute to its structural stability and functional properties . The recombinant form refers to the peptide produced through expression systems rather than directly isolated from venom, allowing for controlled production and potential modifications to study structure-function relationships.

Iota-conotoxins from Conus radiatus are excitatory peptides that affect voltage-gated sodium channels, primarily by shifting the voltage-dependence of activation to more hyperpolarized potentials . This mechanism differs from other conotoxins that block sodium channels (μ-conotoxins) or inhibit inactivation (δ-conotoxins), representing a distinct pharmacological class with unique research applications . The solution structure of related ι-RXIA has been determined, revealing important structural features that likely apply to R11.18 as well .

What expression systems are suitable for producing Recombinant R11.18?

The production of recombinant conotoxins, including R11.18, can be achieved through both prokaryotic and eukaryotic expression systems, each with distinct advantages for different research applications. Prokaryotic systems using E. coli provide high yields and cost-effectiveness but may struggle with proper disulfide bond formation, which is critical for the functional activity of cysteine-rich conotoxins . When using E. coli, researchers should consider specialized strains with enhanced disulfide isomerase activity or employ in vitro refolding protocols to achieve proper folding.

Eukaryotic expression systems, including yeast (P. pastoris), insect cells (Sf9, Sf21), or mammalian cells (CHO, HEK293), offer superior post-translational modification capabilities that more closely resemble native conotoxin processing . These systems are particularly valuable when studying R11.18, which likely contains important post-translational modifications such as gamma-carboxyglutamate residues that significantly affect its pharmacological properties . For analytical studies requiring isotopic labeling, minimal media expression in bacterial systems with 15N or 13C sources can provide material suitable for NMR structural determination.

How should R11.18 activity be assessed in experimental systems?

The assessment of R11.18 activity should employ multiple complementary approaches focusing on its effects on voltage-gated sodium channels. Electrophysiological techniques provide the gold standard for functional characterization. Two-electrode voltage clamp (TEVC) in Xenopus oocytes expressing specific sodium channel subtypes (particularly Nav1.6, Nav1.2, and Nav1.7) allows for precise measurement of R11.18 effects on channel activation parameters . Whole-cell patch clamp recording in mammalian cells offers higher temporal resolution for kinetic studies.

Researchers should measure multiple parameters affected by R11.18, including:

• Voltage-dependence of activation (shifts in V1/2)

• Dose-response relationships to determine EC50 values

• Association and dissociation kinetics to establish on/off rates

• Effects on inactivation properties and recovery from inactivation

Ex vivo preparations using sciatic nerve recordings can demonstrate the effects on action potential generation and propagation in both myelinated (A-fibers) and unmyelinated (C-fibers) axons . For in vivo assessment, intracranial injection in mice with monitoring for seizure activity provides a physiologically relevant readout of excitatory effects . These multi-level approaches ensure comprehensive characterization of R11.18's pharmacological profile.

What methods are effective for structural characterization of R11.18?

Structural characterization of R11.18 requires a multi-technique approach due to its complex disulfide-rich framework. Initial primary structure determination should employ reduction and alkylation followed by automatic Edman degradation to confirm the amino acid sequence . Mass spectrometry, particularly MALDI-TOF, provides precise molecular mass determination and can verify post-translational modifications by comparing observed mass with calculated values based on the amino acid sequence .

For tertiary structure elucidation, solution NMR spectroscopy represents the method of choice for disulfide-rich peptides like R11.18. This approach has successfully determined the structural features of related iota-conotoxins, revealing their three-dimensional conformation and disulfide connectivity patterns . Circular dichroism (CD) spectroscopy offers complementary information about secondary structural elements and can monitor proper folding during production and purification processes.

Computational approaches, including homology modeling based on structurally characterized conotoxins, provide preliminary structural insights, especially when experimental data is limited . Graph-based approaches have been developed to identify optimal templates for modeling conotoxins with similar cysteine frameworks, improving the quality of predicted structures . The combination of experimental and computational methods yields the most comprehensive structural characterization of R11.18.

How does R11.18 selectivity compare to other iota-conotoxins?

The sodium channel subtype selectivity of R11.18 likely follows patterns observed with related iota-conotoxins from Conus radiatus, particularly ι-RXIA. Careful voltage-clamp studies have demonstrated that ι-RXIA exhibits a distinct selectivity profile among rodent sodium channel subtypes, with an order of sensitivity: Nav1.6 > Nav1.2 > Nav1.7, while Nav1.1, Nav1.3, Nav1.4, Nav1.5, and Nav1.8 show minimal sensitivity . This selectivity pattern provides important insights into structure-activity relationships that likely apply to R11.18 as well.

The molecular determinants of this selectivity involve specific interactions between the toxin and the voltage-sensing domains of sodium channels. Research approaches to analyze R11.18 selectivity should include:

• Systematic testing against all sodium channel subtypes expressed in standardized systems (e.g., Xenopus oocytes)

• Chimeric channel constructs to identify critical regions for toxin interaction

• Site-directed mutagenesis of both the toxin and channels to pinpoint key residues involved in binding

• Computational docking studies to model interaction surfaces

Comparison with other conotoxins that target sodium channels through different mechanisms (μ-conotoxins and δ-conotoxins) can further highlight the unique properties of iota-conotoxins like R11.18 . This cross-mechanistic analysis advances understanding of voltage-sensing domain pharmacology and facilitates rational design of subtype-selective channel modulators.

What is the significance of D-Phe in R11.18 and how can its role be investigated?

The presence of D-phenylalanine, an unconventional D-amino acid, near the C-terminus of iota-conotoxins like ι-RXIA represents a rare post-translational modification that significantly impacts pharmacological properties . Studies comparing ι-RXIA with its L-Phe analog demonstrated that the D-isomer confers both higher affinity (two-fold) and slower off-rate (two-fold) for Nav1.6 channels . Additionally, the D-Phe appears critical for activity against Nav1.2, as the L-Phe variant loses activity at this channel subtype .

To investigate the role of D-Phe in R11.18, researchers should consider several experimental approaches:

• Synthesis of R11.18 variants with substitution of D-Phe with L-Phe and electrophysiological comparison

• Alanine-scanning mutagenesis to assess the importance of aromatic properties versus stereochemistry

• NMR solution structure determination of both D-Phe and L-Phe variants to identify conformational differences

• Molecular dynamics simulations to examine how the D-amino acid influences peptide flexibility and binding orientation

This stereochemical feature likely represents an evolutionary adaptation that enhances target specificity and binding kinetics. Understanding its role advances both basic knowledge of peptide-channel interactions and informs design of improved research tools for sodium channel targeting.

What are the optimal methodologies for investigating structure-activity relationships of R11.18?

Investigation of structure-activity relationships (SAR) for R11.18 requires integrated approaches combining molecular, structural, and functional techniques. Alanine-scanning mutagenesis provides the foundation for SAR studies by systematically replacing each non-cysteine residue with alanine to identify positions critical for activity . For comprehensive SAR analysis, researchers should implement:

• Conservative and non-conservative substitutions at positions identified as important by alanine scanning

• Cysteine framework modifications to evaluate disulfide bond patterns and their contribution to structural stability

• C-terminal amidation variants to assess the importance of this post-translational modification

• Truncation analogs to identify the minimal active sequence

• Chimeric peptides combining segments from different conotoxins to transfer selectivity properties

Functional evaluation of each variant should employ consistent electrophysiological protocols measuring effects on sodium channel activation parameters, particularly the shift in voltage-dependence of activation (ΔV1/2) at Nav1.6 channels . Binding affinity and kinetics should be determined using the linear relationship between observed rate constants (kobs) and peptide concentration, which has been validated for related iota-conotoxins .

The following table summarizes key SAR elements to investigate for R11.18:

This systematic approach identifies the pharmacophore elements essential for R11.18 activity and guides rational design of optimized variants for research applications.

How should researchers approach the challenge of proper folding and disulfide bond formation in recombinant R11.18?

The correct formation of disulfide bonds represents a critical challenge in recombinant production of cysteine-rich peptides like R11.18. With eight cysteines in the I-superfamily framework, there are 105 theoretical disulfide bonding patterns possible, but only one native configuration confers proper biological activity . Researchers should implement a multi-faceted strategy to address this challenge:

For expression systems, consider:

• Specialized E. coli strains with oxidizing cytoplasmic environments (SHuffle, Origami)

• Co-expression with disulfide isomerases (DsbC, PDI)

• Periplasmic expression using appropriate signal sequences

• Eukaryotic systems with natural disulfide-forming machinery

• Fusion partners that enhance solubility and facilitate correct folding (thioredoxin, MBP)

For in vitro refolding approaches:

• Systematic optimization of redox buffer conditions (GSH/GSSG ratios)

• Presence of folding additives (arginine, detergents)

• Gradual removal of denaturants via dialysis or dilution

• Temperature and pH optimization

• Chaperone-assisted refolding with PDI

Validation of correct folding should employ orthogonal analytical techniques including:

• Reversed-phase HPLC comparing retention time with native peptide

• Circular dichroism spectroscopy for secondary structure analysis

• Limited proteolysis resistance compared to scrambled disulfide isomers

• Bioactivity assays correlating structure with function

• Direct disulfide mapping using targeted proteolysis and mass spectrometry

This comprehensive approach maximizes the likelihood of obtaining correctly folded R11.18 with native-like activity for research applications.

What are the most effective experimental designs for electrophysiological characterization of R11.18?

Electrophysiological characterization of R11.18 requires carefully designed protocols that capture its unique mechanism of action on voltage-gated sodium channels. Based on studies with related iota-conotoxins, researchers should implement the following experimental designs:

• Voltage-protocol optimization:

  • Wide range of test potentials (-80 to +60 mV) to fully capture the shift in activation

  • Standard holding potential of -100 mV to prevent inactivation

  • Multiple pulse protocols to assess use-dependence effects

  • Conductance-voltage (G-V) relationships before and after toxin application

• Concentration-response studies:

  • Sequential application of increasing concentrations (0.1-30 µM) to single cells/oocytes

  • Time course measurements at each concentration until steady-state

  • Calculation of observed rate constants (kobs) for different concentrations

  • Plot of kobs versus concentration to determine binding kinetics

• Mathematical analysis:

  • Boltzmann equation fitting for activation and inactivation curves:
    Y = 1/(1 + exp[(Vstep − V1/2)/k])

  • Quantification of toxin effect using:
    a) Shift in V1/2 of activation
    b) Changes in slope factor (k)
    c) Effects on peak current amplitude

• Comparative subtype studies:

  • Standard expression of Nav1.1-1.9 with β1 subunit

  • Identical recording conditions and voltage protocols

  • Matched expression levels where possible

  • Control applications of established subtype-selective toxins

These methodological approaches provide a comprehensive electrophysiological profile of R11.18 and allow direct comparison with other sodium channel-targeting toxins.