Isolation, synthesis, and pharmacological characterization of a short-structured peptide from the venom of Phidippus audax that affects potassium currents

Isolation, synthesis, and pharmacological characterization of a short-structured peptide from the venom of Phidippus audax that affects potassium currents | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation, synthesis, and pharmacological characterization of a short-structured peptide from the venom of Phidippus audax that affects potassium currents Emilio Salceda, Iván Arenas, Timoteo Olamendi-Portugal, Alma Reyes, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7076766/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Little is known about the venom of Salticidae spiders, so here, we look for venom peptides of the most cosmopolitan spider, Phidippus audax . The isolation, chemical synthesis, and pharmacological characterization of a short peptide from the venom of the spider Phidippus audax (Araneae: Salticidae) was unveiled. The peptide (Paudax1) consists of 22 residues and contains a single disulfide bridge. Paudax1 has paralytic activity against Acheta domesticus . It was synthesized in two N-terminal forms, Phi-Ala and Phi-Trp, which were also paralytic to house crickets. The pharmacology of both homologous peptides was evaluated in primary cultures of rat dorsal root ganglia neurons. Microperfusion of Phi-Ala [10 µM] (n = 6) resulted in a 17 ± 5% inhibition of the maximum amplitude of the outward current ( p 0.05) in the current amplitude at the end of the voltage pulse ( IK end ). Additionally, Phi-Ala did not affect the current inactivation time course (τ inact ). Meanwhile, the peptide Phi-Trp [10 µM] (n = 6) induced a 37 ± 3.6% inhibition (p ≤ 0.01) of the maximum amplitude of the outward current and a 44 ± 5% inhibition ( p ≤ 0.01) in IK end , with no change in τ inact . Although both peptides, Phi-Ala and Phi-Trp, displayed insect paralytic activity, they exhibited relatively low efficiency as blockers of the outward current at the concentrations used, and they did not affect the inward currents. Phi-Trp and Phi-Ala are worth investigating to explore their therapeutic potential. jumping spider potassium channel spider toxin sodium channel Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION It has been estimated that spider venoms may contain millions of unique peptides, derived from approximately 52,347 venomous spider species, with each species producing roughly 100 peptides (World Spider Catalog, 2025 ). However, only a small number of these peptides have been characterized. Most biochemically characterized peptides obtained from spider venoms block voltage-gated ion channels; for example, Hanatoxin (HaTx) inhibits the potassium channel Kv2.1, while Huwentoxin-IV blocks the TTX-sensitive sodium channel. One of the world's most diverse, cosmopolitan, and abundant spider species is Phidippus audax , also known as the “daring jumping spider". It belongs to the Salticidae family and is commonly associated with humans in warmer regions, often found in houses searching for small insects. They are solitary, like most spiders, and rely on their specialized eyesight to hunt and capture live prey. Jumping spiders prey on a variety of insects and other spiders (Wiggins and Wilder, 2022 ). Likewise, they are the most common spiders found in agricultural areas. They consume many crop pests, including bollworms, boll weevils, spotted cucumber beetles, sorghum midges, fall webworms, cotton leafworms, cotton flea-hoppers, tarnished plant bugs, stink bugs, lotus bugs, three-cornered alfalfa hoppers, and leafhoppers (Bailey, 1969 ). While searching the venom of Phidippus audax for TREK (TWIK Related K + channels), which play a role as mechano-transducers in different components of the cardiovascular system at central (heart) and peripheral (vascular) levels (Herrera-Perez and Lamas, 2023 ), we identified an interesting insecticidal peptide. Although this peptide with no action on TREK channels, it was synthesized in two homologous forms named Phi-Ala and Phi-Trp. In this work, we report the isolation, chemical synthesis, and pharmacological properties of voltage-dependent sodium and potassium currents of two synthetic peptides derived from P. audax venom. Materials and Methods Venom Crude spider venom from Phidippus audax was obtained by electrical stimulation from a laboratory colony of field-collected spiders from Veracruz, Mexico (Octolab Ltd.). Venom extraction was done, avoiding contamination by digestive fluids. Once collected, crude venom was centrifuged (14,000 rpm, 30 min) at 4°C, the small debris was discarded, and the venom was aliquoted, lyophilized, and stored at -70°C until further use. Peptide purification and N-terminal sequence A milligram of crude venom was diluted with 100 µL of 0.1% aqueous trifluoroacetic acid (TFA), centrifuged at 14,000 rpm for 5 min, and the supernatant was fractionated by reversed-phase HPLC on an analytical C 18 column (Vydac 214 TP 4.6 x 250 mm, USA). The column was equilibrated in 0.1% TFA and eluted with a 60 min linear gradient from 0 to 60% acetonitrile containing 0.1% TFA (1 mL/min). Fractions were collected manually by monitoring the absorbance at 230 nm. The fractions were vacuum-dried and stored at -20°C. Before sequencing, the peptide was reduced and alkylated simultaneously with DTT and iodoacetamide (Wako, Japan), respectively, in 0.5 M NaHCO 3 buffer (pH 8.3) for 2 h at 37°C in the dark. The reduced-alkylated peptide was then desalted by reversed-phase HPLC using a linear gradient of acetonitrile/water/0.1% TFA. Peptide sequencing was performed on a Shimadzu PPSQ-10 automated gas-phase sequencer. The peptide was dissolved in 20 µL HPLC-grade water and applied to TFA-treated glass fiber membranes precycled with Polybrene (Aldrich, USA). Peptide synthesis The peptide was chemically synthesized by the Fmoc solid-phase method using a Fmoc-Gln(tBu)-Wang resin to provide a free carboxyl at the C-terminal of the synthetic peptides (Calbiochem-Novabiochem Corp., CA, USA). (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, ChemPep Inc., FL, USA) and 1-hydroxybenzotriazole hydrate (HOBt, LC Sciences, TX, USA) were used as coupling reagents, and a two-fold excess of Fmoc amino acids was added during each coupling cycle. The Fmoc group was removed with 20% piperidine in N,N-dimethylformamide (DMF). Unreacted or deblocked free amines were monitored via ninhydrin after each cycle of peptide synthesis. Cleavage and deprotection of peptide resins were performed as described previously (Luna-Ramirez et al., 2020 ). The crude synthetic peptide was dissolved in 20% aqueous acetonitrile, and the reduced toxin was separated by RP-HPLC on an analytical C 18 column (Vydac 214 TP 4.6 x 250mm, USA). The free cysteine residues were allowed to oxidize by air exposure for 24 h, at 5°C in a 0.5 M aqueous ammonium acetate solution containing 1 mM reduced glutathione/0.1 mM oxidized glutathione. The biologically active synthetic peptides were purified on an analytical C 18 column (Agilent Zorbax SB-C18, USA) using a 35-min linear gradient from 0 to 35% aqueous acetonitrile/0.1% trifluoroacetic acid (1 mL/min). Mass spectrometry and N-terminal sequencing The protein fractions were reconstituted to a final concentration of 500 pmol/5 µL of 50% acetonitrile with 1% acetic acid and directly injected into a Thermo Scientific LCQ Fleet ion trap mass spectrometer (San Jose, CA) with a Surveyor MS syringe pump delivery system. The eluate at 10 µL/min was split out to introduce only 5% of the sample into the nanospray source (0.5 µL/min). The spray voltage was set at 1.5 kV, and the capillary temperature was set at 150°C. The fragmentation source was operated at 25–35 V of collision energy, 35–45% (arbitrary units) of normalized collision energy, and the scan with a wide band was activated. All spectra were obtained in positive-ion mode. The data acquisition and the deconvolution of data were performed using the Xcalibur Windows NT PC data system. Biological activity The insecticidal activity was assessed in vivo using house crickets, Acheta domesticus . House crickets were injected intrathoracically between the second and third pair of legs with 5 µL of five doses of each peptide dissolved in distilled water. Mouse toxicity was tested by intracranial injection in 20 g male mice (CD-1). Peptides were diluted to 5 µL with BSA solution (20 mg/mL in 0.9% NaCl) and injected using a 10 µL microsyringe fitted with a glass capillary. Controls were performed with saline solution only, and mice were placed in glass jars for observation of toxicity signs. Electrophysiological experiments Long Evans CII rats were provided by the laboratory animal facility ‘Claude Bernard’ of the Autonomous University of Puebla. The study was conducted in accordance with the recommendations outlined in the Guiding Principles for the Care and Use of Vertebrate Animals in Research and Training of the American Physiological Society, as well as the regulations established by NOM-062-ZOO-1999 of the Mexican Ministry of Agriculture, Livestock, Rural Development, Fisheries, and Food. The protocol was reviewed and approved by the Institutional Committee for Animal Care and Use (IACUC) of the Autonomous University of Puebla (SOEE-UALVIEP-17-4). All efforts were made to minimize animal suffering and to reduce the number of animals used. Cell preparation DRG neurons (DRGn) from Long Evans CII rats (P7–P10) of either sex were isolated and maintained in primary culture following the methodology previously described by Salceda et al. ( 2002 ). Electrophysiological Recording For patch clamping of isolated DRGn, a coverslip with attached neurons was transferred to a 500 µL perfusion chamber mounted on the stage of an inverted phase-contrast microscope (Nikon Diaphot, Tokyo, Japan). The solutions used in the experiments are detailed in Table 1 . Table 1 Solutions used in electrophysiological experiments (concentrations in mM). Solution KCl NaCl CsCl CsF MgCl 2 CaCl 2 Chol -Cl KF HEPES EGTA TEA-Cl 4-AP pH Extracellular I Na + - 20 - - 1 1.8 70 - 10 - 45 10 7.4 Intracellular I Na + - 10 30 100 - - - - 5 8 10 - 7.2 Extracellular I K + 10 - - - 1.2 1.8 130 - 10 - - - 7.4 Intracellular I K + 50 - - - - 0.1 50 40 5 5 - - 7.2 Extracellular solutions were supplemented with 10 mM glucose, while intracellular solutions were supplemented with 2 mM MgATP and 1 mM Na₂GTP. The pH of the extracellular solutions was adjusted to 7.4 with HCl, while that of the intracellular solutions was set to 7.2 with CsOH. Osmolarity was measured using a vapor pressure osmometer (Wescor, Logan, UT) and adjusted to 290 mOsm for extracellular solutions and 300 mOsm for intracellular solutions using dextrose. A gravity-driven perfusion system ensured a continuous flow of external solution into the chamber at approximately 100 µL/min. In addition, a double-barrel array made from borosilicate glass capillaries (TW120–3; WPI, Sarasota, FL) was positioned about 40 µm above the cell of interest. Each barrel was connected to a separate syringe operated by a Baby Bee pump (BAS, West Lafayette, IN). This setup allowed for continuous microperfusion of the neuron (20 µL/min) with either the external solution or the external solution containing the peptide. The tubing and syringe used were shielded from light due to the photosensitivity of the peptides. Patch pipettes were pulled from borosilicate glass capillaries (TW120 − 3; WPI), using a Flaming-Brown electrode puller (P80/PC; Sutter Instruments, San Rafael, CA). Electrodes had resistances of 0.9 to 1.8 MΩ when filled with internal solution. Whole-cell patch clamp recordings were performed with an Axopatch-1D amplifier (Molecular Devices, San Jose, CA, USA). Pulse generation and data sampling were controlled by Pclamp 9.2 software (Molecular Devices) via a 16-bit Digidata 1320A system (Molecular Devices). Experiments were conducted at room temperature (23–25°C). Leakage and capacitive currents were digitally subtracted using the P-P/n method, with capacitance and 80% of series resistance electronically compensated. Experiments were discarded if the voltage error exceeded 5 mV after series resistance compensation at peak current, but no corrections were applied for lower values. Data Analysis Recordings were analyzed offline with Clampfit 9.2 (Molecular Devices) and OriginPro 9.1 (OriginLab, Northampton, MA, USA). Numerical data are presented as means ± S.E.M. from at least four measurements. Statistical differences were assessed using Student’s t -test with p < 0.05. The parameters measured to characterize the ionic currents were: (a) the maximum peak amplitude ( I peak ), (b) the time-constant of the current inactivation ( τ inac ) as derived from an exponential fit, and (c) the current amplitude at the end of the voltage pulse ( I end ). Additionally, we calculated the ratio between the current amplitude at the end of the voltage pulse and the peak current amplitude ( I end / I max ) obtaining an estimate of the probability for the current inactivation process. Results Toxin purification and sequencing Following preliminary screening of some spider venoms (data not shown), the venom of Phidippus audax demonstrated paralytic activity against A. domesticus using intrathoracically injections; thence, it was selected for HPLC fractionation. Fractions were collected manually and assayed for paralytic activity toward A. domesticus (Fig. 1 ). Six out of twenty HPLC fractions (F1, F2, F3, F5, F7, and F9) were paralytic to crickets, but only fraction 9 was a peptide. The other tested fractions contained acyl-polyamines, non-peptide components in spider venoms that cause insect paralysis primarily by affecting ionotropic glutamate receptors (Estrada et al., 2007 ). Fraction 9 had an experimental molecular mass of 2,273.3 Da. The data obtained from automated Edman sequencing of the reduced-alkylated peptide allowed the complete determination of all twenty-two amino acids. The theoretical mass of the primary structure coincideed with the experimental mass spectrometry data for fraction 9, and the native peptide was named Paudax1 or Phi-Ala (Table 2 ). Table 2 Peptide sequences Peptide Amino acid sequence Molecular mass (Da) Phi-Ala AEGGKSRLPSKKNCPKADCGTQ 2,273.6 Phi-Trp WEGGKSRLPSKKNCPKADCGTQ 2,388.7 Peptide synthesis and paralytic activity Due to the small amount of Phi-Ala in the venom, it was synthesized in two isoforms using alanine (as in native Paudax1) and tryptophan as N-terminal amino acid for pharmacological characterization. Since Phi-Ala lacks an aromatic residue for its detection, the rationale for synthesizing a Phi-Ala variant with an N-terminal tryptophan was because Trp plays unique roles in "anchoring" membrane proteins within the cell membrane, and it is easily traceable by spectroscopy and fluorescence methods (Ghisaidoobe and Chung, 2014 ). Therefore, two peptides, Phi-Ala and Phi-Trp, were chemically synthesized (Fig. 2 ). The identities of synthetic Phi-Ala and Phi-Trp were compared to native Paudax1 by ESI mass spectrometry and HPLC retention times. The experimental molecular masses of Phi-Ala and Phi-Trp were 2,273.4 and 2,388.5 Da, respectively, which coincide with their theoretical molecular masses. The molecular mass of Phi-Ala also coincides with that of native Paudax1 (Table 2 ). Furthermore, the retention times of Phi-Ala and Phi-Trp were 24.9 and 27.9 min, respectively, under identical HPLC gradient conditions. The variant Phi-Trp was more hydrophobic than Phi-Ala, which had the same retention time as native Paudax1 (Fig. 2 ). Phi-Ala and Phi-Trp were paralytic to house crickets, Acheta domesticus (Table 3 ). The injected dose was 150 µg/g of crickets, and paralysis occurred within 2 min. Insect paralysis lasted for 5 min followed by recovery. When twice the concentration of Phi-Ala (300 µg/g) was injected, paralysis also occurred within 2 min, but lasted for 6 min before full recovery was observed. Similarly, an injection of 300 µg/g of Phi-Trp paralyzed crickets more rapidly (within 1.5 min), and for a longer duration (8 min). Table 3 Paralytical activities of Phi-Ala and Phi-Trp Peptide Dose (µg/g) Observations Phi-Ala 300 Paralysis was within 2 min and lasted for 6 min. Phi-Trp 300 Paralysis was within 1.5 min and lasted for 8 min. The control represents 5 µL of distilled water, n = 3. The paralytic activity of Phi-Ala and Phi-Trp was higher than that of some peptide toxins from arachnid venoms, which may belong to different pharmacological classes and have distinct modes of action. For example, excitatory and depressant scorpion toxins such as LqhαIT and LqhIT2 have insecticidal doses of 9.3 and 5.1 µg/g insect. Similarly, depressant spider toxins, like palutoxins, have insecticidal doses ranging from 9.5 to 24.7 µg/g (Corzo et al., 2000 ). Phi-Ala and Phi-Trp induced a temporary paralysis of the mice’s hind legs lasting no longer than 5 min, but were not lethal to mice via intracranial injection at 5 µg/20 g mouse. This prompted us to investigate their effects on DRG neurons. Effect of Phidippus audax peptides in DRGn currents A total of 67 DRGn were recorded using the whole-cell voltage-clamp technique. These cells had an average membrane capacitance (C m ) of 47 ± 3 pF, corresponding to an approximate cell diameter of 39 µm. Fifty neurons were recorded to study the effect of Phi-Ala and Phi-Trp on outward potassium currents, and 17 neurons were recorded to study isolated sodium currents. Outward current amplitude measured at the peak and the end of the responses elicited by depolarization to + 30 mV from a holding potential ( V hold ) of -60 mV, with an interpulse interval of 8 s. Recordings were performed under control conditions and in the presence of either Phi-Trp or Phi-Ala. Both peptides were tested at 1, 3, and 10 µM. To investigate the effects of Phi-Ala and Phi-Trp on inward sodium currents, a single-step voltage protocol was employed. A 40-ms test pulse to − 20 mV was applied from a holding potential of − 100 mV, with an interpulse interval of 8 s. Outward K + currents measured at peak ( IK max ) were reduced after Phi-Trp application, with maximum inhibition attained after approximately 1 min. This effect was statistically significant at concentrations of 3 µM (from 5.1 ± 0.8 nA to 4.7 ± 0.7 nA, corresponding to 6 ± 2% inhibition, n = 10, p = 0.04,) and 10 µM (from 8.4 ± 2.0 nA to 5.5 ± 1.6 nA, corresponding to 37 ± 4% inhibition of, n = 6, p = 0.004). Washout resulted in partial recovery of current amplitude, averaging 83 ± 7% across the three concentrations tested. The outward current measured at the end of the voltage pulse ( IK end ) was significantly reduced by 10.6 ± 3% (n = 10, p = 0.02) in the presence of Phi-Trp [3 µM]. Perfusion with higher Phi-Trp [10 µM] concentration caused a more pronounced decrease of 44 ± 5%, reducing the current from a control value of 5.4 ± 1.5 nA to 3.3 ± 1.0 nA (n = 6). The IK end / IK max ratio was also significantly affected by both 3 µM Phi-Trp (from 0.48 ± 0.03 to 0.46 ± 0.03, n = 10, p = 0.03) and 10 µM Phi-Trp (from 0.64 ± 0.07 to 0.58 ± 0.08, n = 6, p = 0.04). The time-constant of current inactivation τ inac was not significantly affected by Phi-Trp at any of the tested concentrations (Fig. 3 ). The Phi-Ala application also resulted in a significant reduction in IK max , with a temporal course similar to that exhibited by Phi-Trp (1 min after application). The inhibition was lower than that produced by Phi-Trp, and it was statistically significant only at 10 µM, reducing peak amplitude of the current from 6.7 ± 2.0 nA to 5.8 ± 2.0 nA, corresponding to a 17 ± 5% reduction (n = 4, p = 0.04). After washout, current amplitude partially recovered to 92 ± 6% of baseline. Phi-Ala [10 µM] caused a non-significant reduction in IK end with a 22 ± 7% decrease (from 4.83 ± 2.0 nA to 4.0 ± 1.9 nA; p = 0.05). Neither the IK end / IK max ratio nor τ inac were significantly affected by Phi-Ala [10 µM] (Fig. 3 ). To assess peptide selectivity, we also examined their effects on sodium currents. Mean control values for sodium current parameters were: INa max = 8.0 ± 1 nA; INa end = 0.43 ± 0.11 nA; τ inact = 0.99 ± 0.17 ms (n = 17). Neither Phi-Trp nor Phi-Ala [3 µM] significantly affected any of the current parameters studied (Fig. 4 ). Discussion One challenge in exploring biochemical diversity in arthropod venoms is the limited availability of biological material. However, the highly toxic activity of venom from small species often warrants investigation. Phi-Ala is a 22 aa acidic peptide, with one disulfide bridge, featuring a conserved motif (X)n-Cys-X 4 -Cys-(X)n where X designates variable positions. Phi-Ala does not belong to any known family of spider peptides. This core motif seems unique to this spider venom, and Phi-Ala is a basic (pH = 9.3) and hydrophilic (GRAVY = -1.291) peptide. Many spider toxins have been shown to produce paralysis in insects. Particularly in crickets, the mechanism of paralysis may be caused by different mechanisms, including excessive neuronal firing, synaptic blockade, or neuromuscular junction disruption preventing acetylcholine or glutamate release (Guo 2023). In the case of the Phidippus audax peptides analyzed in this work, the blockade of K + currents may cause a spastic paralysis due to sustained cell depolarization. The paralyzing effect of these peptides suggests they can be developed to use as bioinsecticides (Saez and -Hersing, 2019; Zhang et al., 2023 ). Electrophysiological findings in this study reveal that Phi-Trp and Phi-Ala, derived from Phidippus audax venom, modulate potassium currents in DRGn, without significantly affecting sodium currents. This suggests a degree of selectivity for potassium channels, highlighting their potential as targeted modulators of neuronal excitability, which could be relevant for future pharmacological applications. Phi-Trp exhibited concentration-dependent inhibition of potassium currents, with maximal reductions of 37% in IK max and 44% in IK end at 10 µM. This suggests a strong interaction with potassium channels involved in late repolarization. In contrast, Phi-Ala showed milder inhibition, reaching statistical significance at 10 µM (17% reduction in IK max and 22% in IK end ). Structural differences, particularly in side-chain residues, may contribute to their distinct efficacies and binding affinities. Similar observations have been reported for other venom-derived peptides, where minor structural variations significantly impact channel modulation (Li et al ., 2005). Partial recovery of potassium currents after washout suggests that peptide-channel interactions are not irreversible. This suggests a potential allosteric modulation or a voltage-dependent block rather than permanent modification (Vergara et al., 2020 ; Wulff & Zhorov, 2008 ). The lack of significant changes in the inactivation time constant (τ inac ) suggests that these peptides primarily affect channel activation or maximal conductance rather than inactivation kinetics, distinguishing them from classical pore-blocking toxins like dendrotoxins or kaliotoxins (Garcia et al., 1997 ; Yellen, 2002 ). In contrast, neither Phi-Trp nor Phi-Ala significantly altered sodium currents. Many spider-derived peptides target voltage-gated sodium channels, such as toxins from Phoneutria nigriventer , which shift activation or slow inactivation (Cestèle & Catterall, 2000 ; Bosmans & Tytgat, 2007 ; de Lima et al., 2015 ). The absence of sodium current effects suggests that Phidippus audax peptides exhibit functional selectivity, which could be advantageous for developing pharmacological tools that modulate neuronal excitability without disrupting action potential generation (Noreng et al., 2021 ; Bende et al., 2014 ). These findings contribute to the growing understanding of spider venom peptide diversity and their potential as selective ion channel modulators. Future studies should characterize the binding sites of Phi-Trp and Phi-Ala on potassium channels, investigate their effects in other cellular models, and explore their therapeutic potential in conditions involving altered neuronal excitability, such as chronic pain or epilepsy (Budde et al., 2015 ; Herrington & Gutman, 2011 ). Declarations Author Contributions Conception of the work: GC, ESo, and EV; collection of data: ESa, IA, TOP, AR, and GC; methodology and analysis: ESa, IA, TOP, AR, GC, ESo, and EV; writing end edition of manuscript: Esa, GC, ESo, and EV; funding acquisition: GC, ESo, and EV. Conflict of interest The authors declare no conflict of interest, financial or otherwise. Ethical statement No human experiments were performed. All applicable international, national, and institutional guidelines for the care and use of animals were followed. Procedures adhered to the bioethical standards of the “Instituto de Biotecnología - UNAM”. Consent to Participate All researchers listed in this work voluntarily agreed to participate. Consent for Publication All researchers agreed on publication. Availability of Data and Materials All data generated in this study are available upon request. Acknowledgments We thank Rosby Najera for preliminary peptide synthesis. This work was funded by the Dirección General de Asuntos del Personal Académico (DGAPA-UNAM) grant number IT200724 and the CONACyT/SECIHTI-PRONAII grant number 303045. References Bailey CL (1969) Life history of the spider, life history of Phidippus audax (Hentz), in relation to biological control of grain sorghum insects. Oklahoma State University. Bende NS, Dziemborowicz S, Mobli M, et al. (2014) A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a. Nature Communications. 5, 4350. doi:10.1038/ncomms5350 Bosmans F, Tytgat J (2007) Voltage-gated sodium channel modulation by scorpion α-toxins. Toxicon, 49(2), 142-158. 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Wiggins WD, Wilder SM (2022) Carbohydrates complement high-protein diets to maximize the growth of an actively hunting predator. Ecol Evol . 12, e9150. Wulff H, Zhorov BS (2008) K+ channel modulators and the therapeutic potential of small molecules targeting subtypes. Annu Rev Pharmacol Toxicol , 48, 431-458. Yellen G (2002) The voltage-gated potassium channels and their relatives. Nature , 419(6902), 35-42. Zhang YM, Ye DX, Liu Y, Zhang XY, Zhou YL, Zhang L, Yang XL (2023) Peptides, new tools for plant protection in eco-agriculture. Advanced Agrochem , 2(1), 2023, 58-78. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7076766","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":494599624,"identity":"c4e22b39-0bc0-4cad-bfdd-74cd014ab605","order_by":0,"name":"Emilio Salceda","email":"","orcid":"","institution":"Benemérita Universidad Autónoma de Puebla","correspondingAuthor":false,"prefix":"","firstName":"Emilio","middleName":"","lastName":"Salceda","suffix":""},{"id":494599625,"identity":"8a1959b2-f367-49a0-a5d3-b044965488b9","order_by":1,"name":"Iván Arenas","email":"","orcid":"","institution":"Universidad Nacional Autónoma de México","correspondingAuthor":false,"prefix":"","firstName":"Iván","middleName":"","lastName":"Arenas","suffix":""},{"id":494599626,"identity":"2c1f2a2a-6da4-4d99-9573-ed0e69213448","order_by":2,"name":"Timoteo Olamendi-Portugal","email":"","orcid":"","institution":"Universidad Nacional Autónoma de México","correspondingAuthor":false,"prefix":"","firstName":"Timoteo","middleName":"","lastName":"Olamendi-Portugal","suffix":""},{"id":494599629,"identity":"849ef293-6391-4bf8-8324-ea51c9397657","order_by":3,"name":"Alma Reyes","email":"","orcid":"","institution":"Benemérita Universidad Autónoma de Puebla","correspondingAuthor":false,"prefix":"","firstName":"Alma","middleName":"","lastName":"Reyes","suffix":""},{"id":494599631,"identity":"5c52d250-5a12-4060-a7a9-c067cfe2b665","order_by":4,"name":"Gerardo Corzo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYNACNhijQiKBVC1nJBJ4SNPC2MZAWIu8e+/DzxVlNgz87b2PP3ycZ5Fnz8Bj9uBnDkO0wQHsWgzPHDeWPHMujUHizHEDw5nbJIp5GHjMDXu3MeTObMChZUYag2Rj22EGA4k0hmTebRKJPUBbJHiBWvpxOMxw/jPmnzAth3nnQLRI/gVqacPlFwk2NpgtjM28DRAt0vhsMeBJY7NsOJfGI3HmGDPjjGNAvxxmK5OW3SaB0y/y7ceYbzaU2cjxt7cxf/hQU5fH3t68TfLtNpvcDThCDBaSSLHBDCYlcDgLaAsO20fBKBgFo2AUIAAAnzlOuE1SYf0AAAAASUVORK5CYII=","orcid":"","institution":"Universidad Nacional Autónoma de México","correspondingAuthor":true,"prefix":"","firstName":"Gerardo","middleName":"","lastName":"Corzo","suffix":""},{"id":494599633,"identity":"746e1bda-6f4d-4726-aa7a-7477a67b84c7","order_by":5,"name":"Enrique Soto","email":"","orcid":"","institution":"Benemérita Universidad Autónoma de Puebla","correspondingAuthor":false,"prefix":"","firstName":"Enrique","middleName":"","lastName":"Soto","suffix":""},{"id":494599637,"identity":"fa2b7cee-24ce-4aa4-be60-e8d89fb0b41c","order_by":6,"name":"Elba Villegas","email":"","orcid":"","institution":"Universidad Autónoma del Estado de Morelos","correspondingAuthor":false,"prefix":"","firstName":"Elba","middleName":"","lastName":"Villegas","suffix":""}],"badges":[],"createdAt":"2025-07-08 16:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7076766/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7076766/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88321731,"identity":"057cfd22-50f2-413f-ae26-6044cb767830","added_by":"auto","created_at":"2025-08-05 09:06:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131963,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC separation of \u003cem\u003ePhidippus audax \u003c/em\u003evenom. Twenty fractions were collected, dried, resuspended, and injected into house crickets.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7076766/v1/7ab51057daf589316ed505a0.png"},{"id":88321733,"identity":"4d993994-01a8-4e22-86b5-ac5ac3221cfa","added_by":"auto","created_at":"2025-08-05 09:06:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162451,"visible":true,"origin":"","legend":"\u003cp\u003eReverse-phase HPLC profiles of native Paudax 1 and synthetic Phi-Ala and Phi-Trp\u003cem\u003e.\u003c/em\u003e Retention times of native Paudax 1 (\u003cstrong\u003eA\u003c/strong\u003e) and synthetic Phi-Ala (\u003cstrong\u003eB\u003c/strong\u003e), and Phi-Trp (\u003cstrong\u003eC\u003c/strong\u003e). The peptides were separated using an analytical C\u003csub\u003e18\u003c/sub\u003e reverse-phase column (Agilent Zorbax SB-C18, USA) using 0.1% trifluoroacetic acid (TFA) in water as solvent A, and 0.1% TFA in acetonitrile as solvent B. The gradient was run from 0% to 35% solvent B for 35 min at 1 mL/min, and the peptides were detected at 230 nm. The dotted line shows the same retention time for Paudax and synthetic Phi-Ala.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7076766/v1/2e37da8aeae873c544039061.png"},{"id":88322937,"identity":"5e981562-0592-48cb-8e2f-00cf4887aef1","added_by":"auto","created_at":"2025-08-05 09:14:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":628214,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Phi-Ala and Phi-Trp on outward currents. Normalized traces showing the mean and standard deviation of the control recordings (blue traces) and after peptide application (red traces). In A, upper insert showing the pulse used to elicit the current traces for Phi-Trp (1, 3 and 10 µM). In B, the insert shows the pulse used to elicit the current traces for Phi-Ala (1, 3 and 10 µM). Both peptides produced significant inhibition of the outward K+ currents at 3 and 10 µM, without modifying the current time course.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7076766/v1/0d6b84038016e004877c5254.png"},{"id":88321737,"identity":"52185964-25d9-47b0-a430-2ca85d570476","added_by":"auto","created_at":"2025-08-05 09:06:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":399570,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Phi-Ala and Phi-Trp on the inward Na+ current. In A and B mean and standard deviation of the control recording (blue traces) and the peptide action (red traces). In A, the scheme of the pulse used to elicit the currents. In B, the use of 3 µM Phi-Ala (n = 4) produced no significant change on the current amplitude or kinetics. In C, the use of 3 µM Phi-Trp (N= 7) also produced non-significant actions in the current amplitude or kinetics.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7076766/v1/563770f51d005182734b6226.png"},{"id":89202639,"identity":"e46d1851-e5e9-4255-9594-a1e069a402d5","added_by":"auto","created_at":"2025-08-16 15:16:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2058105,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7076766/v1/53c3aeac-2091-4417-bcd4-2d03055ed1bc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation, synthesis, and pharmacological characterization of a short-structured peptide from the venom of Phidippus audax that affects potassium currents","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIt has been estimated that spider venoms may contain millions of unique peptides, derived from approximately 52,347 venomous spider species, with each species producing roughly 100 peptides (World Spider Catalog, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, only a small number of these peptides have been characterized. Most biochemically characterized peptides obtained from spider venoms block voltage-gated ion channels; for example, Hanatoxin (HaTx) inhibits the potassium channel Kv2.1, while Huwentoxin-IV blocks the TTX-sensitive sodium channel. One of the world's most diverse, cosmopolitan, and abundant spider species is \u003cem\u003ePhidippus audax\u003c/em\u003e, also known as the \u0026ldquo;daring jumping spider\". It belongs to the Salticidae family and is commonly associated with humans in warmer regions, often found in houses searching for small insects. They are solitary, like most spiders, and rely on their specialized eyesight to hunt and capture live prey. Jumping spiders prey on a variety of insects and other spiders (Wiggins and Wilder, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Likewise, they are the most common spiders found in agricultural areas. They consume many crop pests, including bollworms, boll weevils, spotted cucumber beetles, sorghum midges, fall webworms, cotton leafworms, cotton flea-hoppers, tarnished plant bugs, stink bugs, lotus bugs, three-cornered alfalfa hoppers, and leafhoppers (Bailey, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1969\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile searching the venom of \u003cem\u003ePhidippus audax\u003c/em\u003e for TREK (TWIK Related K\u003csup\u003e+\u003c/sup\u003e channels), which play a role as mechano-transducers in different components of the cardiovascular system at central (heart) and peripheral (vascular) levels (Herrera-Perez and Lamas, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we identified an interesting insecticidal peptide. Although this peptide with no action on TREK channels, it was synthesized in two homologous forms named Phi-Ala and Phi-Trp. In this work, we report the isolation, chemical synthesis, and pharmacological properties of voltage-dependent sodium and potassium currents of two synthetic peptides derived from \u003cem\u003eP. audax\u003c/em\u003e venom.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eVenom\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCrude spider venom from \u003cem\u003ePhidippus audax\u003c/em\u003e was obtained by electrical stimulation from a laboratory colony of field-collected spiders from Veracruz, Mexico (Octolab Ltd.). Venom extraction was done, avoiding contamination by digestive fluids. Once collected, crude venom was centrifuged (14,000 rpm, 30 min) at 4\u0026deg;C, the small debris was discarded, and the venom was aliquoted, lyophilized, and stored at -70\u0026deg;C until further use.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeptide purification and N-terminal sequence\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA milligram of crude venom was diluted with 100 \u0026micro;L of 0.1% aqueous trifluoroacetic acid (TFA), centrifuged at 14,000 rpm for 5 min, and the supernatant was fractionated by reversed-phase HPLC on an analytical C\u003csub\u003e18\u003c/sub\u003e column (Vydac 214 TP 4.6 x 250 mm, USA). The column was equilibrated in 0.1% TFA and eluted with a 60 min linear gradient from 0 to 60% acetonitrile containing 0.1% TFA (1 mL/min). Fractions were collected manually by monitoring the absorbance at 230 nm. The fractions were vacuum-dried and stored at -20\u0026deg;C. Before sequencing, the peptide was reduced and alkylated simultaneously with DTT and iodoacetamide (Wako, Japan), respectively, in 0.5 M NaHCO\u003csub\u003e3\u003c/sub\u003e buffer (pH 8.3) for 2 h at 37\u0026deg;C in the dark. The reduced-alkylated peptide was then desalted by reversed-phase HPLC using a linear gradient of acetonitrile/water/0.1% TFA. Peptide sequencing was performed on a Shimadzu PPSQ-10 automated gas-phase sequencer. The peptide was dissolved in 20 \u0026micro;L HPLC-grade water and applied to TFA-treated glass fiber membranes precycled with Polybrene (Aldrich, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeptide synthesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe peptide was chemically synthesized by the Fmoc solid-phase method using a Fmoc-Gln(tBu)-Wang resin to provide a free carboxyl at the C-terminal of the synthetic peptides (Calbiochem-Novabiochem Corp., CA, USA). (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, ChemPep Inc., FL, USA) and 1-hydroxybenzotriazole hydrate (HOBt, LC Sciences, TX, USA) were used as coupling reagents, and a two-fold excess of Fmoc amino acids was added during each coupling cycle. The Fmoc group was removed with 20% piperidine in N,N-dimethylformamide (DMF). Unreacted or deblocked free amines were monitored via ninhydrin after each cycle of peptide synthesis. Cleavage and deprotection of peptide resins were performed as described previously (Luna-Ramirez et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The crude synthetic peptide was dissolved in 20% aqueous acetonitrile, and the reduced toxin was separated by RP-HPLC on an analytical C\u003csub\u003e18\u003c/sub\u003e column (Vydac 214 TP 4.6 x 250mm, USA). The free cysteine residues were allowed to oxidize by air exposure for 24 h, at 5\u0026deg;C in a 0.5 M aqueous ammonium acetate solution containing 1 mM reduced glutathione/0.1 mM oxidized glutathione. The biologically active synthetic peptides were purified on an analytical C\u003csub\u003e18\u003c/sub\u003e column (Agilent Zorbax SB-C18, USA) using a 35-min linear gradient from 0 to 35% aqueous acetonitrile/0.1% trifluoroacetic acid (1 mL/min).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMass spectrometry and N-terminal sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe protein fractions were reconstituted to a final concentration of 500 pmol/5 \u0026micro;L of 50% acetonitrile with 1% acetic acid and directly injected into a Thermo Scientific LCQ Fleet ion trap mass spectrometer (San Jose, CA) with a Surveyor MS syringe pump delivery system. The eluate at 10 \u0026micro;L/min was split out to introduce only 5% of the sample into the nanospray source (0.5 \u0026micro;L/min). The spray voltage was set at 1.5 kV, and the capillary temperature was set at 150\u0026deg;C. The fragmentation source was operated at 25\u0026ndash;35 V of collision energy, 35\u0026ndash;45% (arbitrary units) of normalized collision energy, and the scan with a wide band was activated. All spectra were obtained in positive-ion mode. The data acquisition and the deconvolution of data were performed using the Xcalibur Windows NT PC data system.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBiological activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe insecticidal activity was assessed \u003cem\u003ein vivo\u003c/em\u003e using house crickets, \u003cem\u003eAcheta domesticus\u003c/em\u003e. House crickets were injected intrathoracically between the second and third pair of legs with 5 \u0026micro;L of five doses of each peptide dissolved in distilled water. Mouse toxicity was tested by intracranial injection in 20 g male mice (CD-1). Peptides were diluted to 5 \u0026micro;L with BSA solution (20 mg/mL in 0.9% NaCl) and injected using a 10 \u0026micro;L microsyringe fitted with a glass capillary. Controls were performed with saline solution only, and mice were placed in glass jars for observation of toxicity signs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eElectrophysiological experiments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLong Evans CII rats were provided by the laboratory animal facility \u0026lsquo;Claude Bernard\u0026rsquo; of the Autonomous University of Puebla. The study was conducted in accordance with the recommendations outlined in the Guiding Principles for the Care and Use of Vertebrate Animals in Research and Training of the American Physiological Society, as well as the regulations established by NOM-062-ZOO-1999 of the Mexican Ministry of Agriculture, Livestock, Rural Development, Fisheries, and Food. The protocol was reviewed and approved by the Institutional Committee for Animal Care and Use (IACUC) of the Autonomous University of Puebla (SOEE-UALVIEP-17-4). All efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDRG neurons (DRGn) from Long Evans CII rats (P7\u0026ndash;P10) of either sex were isolated and maintained in primary culture following the methodology previously described by Salceda et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eElectrophysiological Recording\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor patch clamping of isolated DRGn, a coverslip with attached neurons was transferred to a 500 \u0026micro;L perfusion chamber mounted on the stage of an inverted phase-contrast microscope (Nikon Diaphot, Tokyo, Japan). The solutions used in the experiments are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSolutions used in electrophysiological experiments (concentrations in mM).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSolution\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKCl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNaCl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCsCl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCsF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMgCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eChol -Cl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eKF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eHEPES\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eEGTA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eTEA-Cl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003e4-AP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtracellular \u003cem\u003eI\u003c/em\u003e\u003csub\u003eNa\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e7.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntracellular \u003cem\u003eI\u003c/em\u003e\u003csub\u003eNa\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e7.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtracellular \u003cem\u003eI\u003c/em\u003e\u003csub\u003eK\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e7.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntracellular \u003cem\u003eI\u003c/em\u003e\u003csub\u003eK\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e7.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eExtracellular solutions were supplemented with 10 mM glucose, while intracellular solutions were supplemented with 2 mM MgATP and 1 mM Na₂GTP. The pH of the extracellular solutions was adjusted to 7.4 with HCl, while that of the intracellular solutions was set to 7.2 with CsOH. Osmolarity was measured using a vapor pressure osmometer (Wescor, Logan, UT) and adjusted to 290 mOsm for extracellular solutions and 300 mOsm for intracellular solutions using dextrose.\u003c/p\u003e\u003cp\u003eA gravity-driven perfusion system ensured a continuous flow of external solution into the chamber at approximately 100 \u0026micro;L/min. In addition, a double-barrel array made from borosilicate glass capillaries (TW120\u0026ndash;3; WPI, Sarasota, FL) was positioned about 40 \u0026micro;m above the cell of interest. Each barrel was connected to a separate syringe operated by a Baby Bee pump (BAS, West Lafayette, IN). This setup allowed for continuous microperfusion of the neuron (20 \u0026micro;L/min) with either the external solution or the external solution containing the peptide. The tubing and syringe used were shielded from light due to the photosensitivity of the peptides.\u003c/p\u003e\u003cp\u003ePatch pipettes were pulled from borosilicate glass capillaries (TW120 \u0026minus;\u0026thinsp;3; WPI), using a Flaming-Brown electrode puller (P80/PC; Sutter Instruments, San Rafael, CA). Electrodes had resistances of 0.9 to 1.8 MΩ when filled with internal solution. Whole-cell patch clamp recordings were performed with an Axopatch-1D amplifier (Molecular Devices, San Jose, CA, USA). Pulse generation and data sampling were controlled by Pclamp 9.2 software (Molecular Devices) via a 16-bit Digidata 1320A system (Molecular Devices). Experiments were conducted at room temperature (23\u0026ndash;25\u0026deg;C). Leakage and capacitive currents were digitally subtracted using the P-P/n method, with capacitance and 80% of series resistance electronically compensated. Experiments were discarded if the voltage error exceeded 5 mV after series resistance compensation at peak current, but no corrections were applied for lower values.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eRecordings were analyzed offline with Clampfit 9.2 (Molecular Devices) and OriginPro 9.1 (OriginLab, Northampton, MA, USA). Numerical data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. from at least four measurements. Statistical differences were assessed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003eThe parameters measured to characterize the ionic currents were: (a) the maximum peak amplitude (\u003cem\u003eI\u003c/em\u003e\u003csub\u003epeak\u003c/sub\u003e), (b) the time-constant of the current inactivation (\u003cem\u003eτ\u003c/em\u003e\u003csub\u003einac\u003c/sub\u003e) as derived from an exponential fit, and (c) the current amplitude at the end of the voltage pulse (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e). Additionally, we calculated the ratio between the current amplitude at the end of the voltage pulse and the peak current amplitude (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) obtaining an estimate of the probability for the current inactivation process.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eToxin purification and sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing preliminary screening of some spider venoms (data not shown), the venom of \u003cem\u003ePhidippus audax\u003c/em\u003e demonstrated paralytic activity against \u003cem\u003eA. domesticus\u003c/em\u003e using intrathoracically injections; thence, it was selected for HPLC fractionation. Fractions were collected manually and assayed for paralytic activity toward \u003cem\u003eA. domesticus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSix out of twenty HPLC fractions (F1, F2, F3, F5, F7, and F9) were paralytic to crickets, but only fraction 9 was a peptide. The other tested fractions contained acyl-polyamines, non-peptide components in spider venoms that cause insect paralysis primarily by affecting ionotropic glutamate receptors (Estrada et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Fraction 9 had an experimental molecular mass of 2,273.3 Da. The data obtained from automated Edman sequencing of the reduced-alkylated peptide allowed the complete determination of all twenty-two amino acids. The theoretical mass of the primary structure coincideed with the experimental mass spectrometry data for fraction 9, and the native peptide was named Paudax1 or Phi-Ala (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePeptide sequences\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeptide\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmino acid sequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMolecular mass (Da)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhi-Ala\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAEGGKSRLPSKKNCPKADCGTQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2,273.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhi-Trp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWEGGKSRLPSKKNCPKADCGTQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2,388.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeptide synthesis and paralytic activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDue to the small amount of Phi-Ala in the venom, it was synthesized in two isoforms using alanine (as in native Paudax1) and tryptophan as N-terminal amino acid for pharmacological characterization. Since Phi-Ala lacks an aromatic residue for its detection, the rationale for synthesizing a Phi-Ala variant with an N-terminal tryptophan was because Trp plays unique roles in \"anchoring\" membrane proteins within the cell membrane, and it is easily traceable by spectroscopy and fluorescence methods (Ghisaidoobe and Chung, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, two peptides, Phi-Ala and Phi-Trp, were chemically synthesized (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The identities of synthetic Phi-Ala and Phi-Trp were compared to native Paudax1 by ESI mass spectrometry and HPLC retention times. The experimental molecular masses of Phi-Ala and Phi-Trp were 2,273.4 and 2,388.5 Da, respectively, which coincide with their theoretical molecular masses. The molecular mass of Phi-Ala also coincides with that of native Paudax1 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, the retention times of Phi-Ala and Phi-Trp were 24.9 and 27.9 min, respectively, under identical HPLC gradient conditions. The variant Phi-Trp was more hydrophobic than Phi-Ala, which had the same retention time as native Paudax1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePhi-Ala and Phi-Trp were paralytic to house crickets, \u003cem\u003eAcheta domesticus\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The injected dose was 150 \u0026micro;g/g of crickets, and paralysis occurred within 2 min. Insect paralysis lasted for 5 min followed by recovery. When twice the concentration of Phi-Ala (300 \u0026micro;g/g) was injected, paralysis also occurred within 2 min, but lasted for 6 min before full recovery was observed. Similarly, an injection of 300 \u0026micro;g/g of Phi-Trp paralyzed crickets more rapidly (within 1.5 min), and for a longer duration (8 min).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eParalytical activities of Phi-Ala and Phi-Trp\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeptide\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDose (\u0026micro;g/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eObservations\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhi-Ala\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eParalysis was within 2 min and lasted for 6 min.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhi-Trp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eParalysis was within 1.5 min and lasted for 8 min.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe control represents 5 \u0026micro;L of distilled water, n\u0026thinsp;=\u0026thinsp;3.\u003c/p\u003e\u003cp\u003eThe paralytic activity of Phi-Ala and Phi-Trp was higher than that of some peptide toxins from arachnid venoms, which may belong to different pharmacological classes and have distinct modes of action. For example, excitatory and depressant scorpion toxins such as LqhαIT and LqhIT2 have insecticidal doses of 9.3 and 5.1 \u0026micro;g/g insect. Similarly, depressant spider toxins, like palutoxins, have insecticidal doses ranging from 9.5 to 24.7 \u0026micro;g/g (Corzo et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Phi-Ala and Phi-Trp induced a temporary paralysis of the mice\u0026rsquo;s hind legs lasting no longer than 5 min, but were not lethal to mice via intracranial injection at 5 \u0026micro;g/20 g mouse. This prompted us to investigate their effects on DRG neurons.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003ePhidippus audax\u003c/b\u003e \u003cb\u003epeptides in DRGn currents\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 67 DRGn were recorded using the whole-cell voltage-clamp technique. These cells had an average membrane capacitance (C\u003csub\u003em\u003c/sub\u003e) of 47\u0026thinsp;\u0026plusmn;\u0026thinsp;3 pF, corresponding to an approximate cell diameter of 39 \u0026micro;m. Fifty neurons were recorded to study the effect of Phi-Ala and Phi-Trp on outward potassium currents, and 17 neurons were recorded to study isolated sodium currents.\u003c/p\u003e\u003cp\u003eOutward current amplitude measured at the peak and the end of the responses elicited by depolarization to +\u0026thinsp;30 mV from a holding potential (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003ehold\u003c/em\u003e\u003c/sub\u003e) of -60 mV, with an interpulse interval of 8 s. Recordings were performed under control conditions and in the presence of either Phi-Trp or Phi-Ala. Both peptides were tested at 1, 3, and 10 \u0026micro;M.\u003c/p\u003e\u003cp\u003eTo investigate the effects of Phi-Ala and Phi-Trp on inward sodium currents, a single-step voltage protocol was employed. A 40-ms test pulse to \u0026minus;\u0026thinsp;20 mV was applied from a holding potential of \u0026minus;\u0026thinsp;100 mV, with an interpulse interval of 8 s.\u003c/p\u003e\u003cp\u003eOutward K\u003csup\u003e+\u003c/sup\u003e currents measured at peak (\u003cem\u003eIK\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) were reduced after Phi-Trp application, with maximum inhibition attained after approximately 1 min. This effect was statistically significant at concentrations of 3 \u0026micro;M (from 5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 nA to 4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 nA, corresponding to 6\u0026thinsp;\u0026plusmn;\u0026thinsp;2% inhibition, n\u0026thinsp;=\u0026thinsp;10, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04,) and 10 \u0026micro;M (from 8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 nA to 5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 nA, corresponding to 37\u0026thinsp;\u0026plusmn;\u0026thinsp;4% inhibition of, n\u0026thinsp;=\u0026thinsp;6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004). Washout resulted in partial recovery of current amplitude, averaging 83\u0026thinsp;\u0026plusmn;\u0026thinsp;7% across the three concentrations tested. The outward current measured at the end of the voltage pulse (\u003cem\u003eIK\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e) was significantly reduced by 10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3% (n\u0026thinsp;=\u0026thinsp;10, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) in the presence of Phi-Trp [3 \u0026micro;M]. Perfusion with higher Phi-Trp [10 \u0026micro;M] concentration caused a more pronounced decrease of 44\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, reducing the current from a control value of 5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 nA to 3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 nA (n\u0026thinsp;=\u0026thinsp;6). The \u003cem\u003eIK\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e/\u003cem\u003eIK\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e ratio was also significantly affected by both 3 \u0026micro;M Phi-Trp (from 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 to 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, n\u0026thinsp;=\u0026thinsp;10, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) and 10 \u0026micro;M Phi-Trp (from 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 to 0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, n\u0026thinsp;=\u0026thinsp;6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). The time-constant of current inactivation \u003cem\u003eτ\u003c/em\u003e\u003csub\u003einac\u003c/sub\u003e was not significantly affected by Phi-Trp at any of the tested concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe Phi-Ala application also resulted in a significant reduction in \u003cem\u003eIK\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e, with a temporal course similar to that exhibited by Phi-Trp (1 min after application). The inhibition was lower than that produced by Phi-Trp, and it was statistically significant only at 10 \u0026micro;M, reducing peak amplitude of the current from 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 nA to 5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 nA, corresponding to a 17\u0026thinsp;\u0026plusmn;\u0026thinsp;5% reduction (n\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). After washout, current amplitude partially recovered to 92\u0026thinsp;\u0026plusmn;\u0026thinsp;6% of baseline. Phi-Ala [10 \u0026micro;M] caused a non-significant reduction in \u003cem\u003eIK\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e with a 22\u0026thinsp;\u0026plusmn;\u0026thinsp;7% decrease (from 4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 nA to 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 nA; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05). Neither the \u003cem\u003eIK\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e/\u003cem\u003eIK\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e ratio nor \u003cem\u003eτ\u003c/em\u003e\u003csub\u003einac\u003c/sub\u003e were significantly affected by Phi-Ala [10 \u0026micro;M] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo assess peptide selectivity, we also examined their effects on sodium currents. Mean control values for sodium current parameters were: \u003cem\u003eINa\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e = 8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nA; \u003cem\u003eINa\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e = 0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 nA; \u003cem\u003eτ\u003c/em\u003e\u003csub\u003einact\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 ms (n\u0026thinsp;=\u0026thinsp;17). Neither Phi-Trp nor Phi-Ala [3 \u0026micro;M] significantly affected any of the current parameters studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOne challenge in exploring biochemical diversity in arthropod venoms is the limited availability of biological material. However, the highly toxic activity of venom from small species often warrants investigation. Phi-Ala is a 22 aa acidic peptide, with one disulfide bridge, featuring a conserved motif (X)n-Cys-X\u003csub\u003e4\u003c/sub\u003e-Cys-(X)n where X designates variable positions. Phi-Ala does not belong to any known family of spider peptides. This core motif seems unique to this spider venom, and Phi-Ala is a basic (pH\u0026thinsp;=\u0026thinsp;9.3) and hydrophilic (GRAVY = -1.291) peptide.\u003c/p\u003e\u003cp\u003eMany spider toxins have been shown to produce paralysis in insects. Particularly in crickets, the mechanism of paralysis may be caused by different mechanisms, including excessive neuronal firing, synaptic blockade, or neuromuscular junction disruption preventing acetylcholine or glutamate release (Guo 2023). In the case of the \u003cem\u003ePhidippus audax\u003c/em\u003e peptides analyzed in this work, the blockade of K\u003csup\u003e+\u003c/sup\u003e currents may cause a spastic paralysis due to sustained cell depolarization. The paralyzing effect of these peptides suggests they can be developed to use as bioinsecticides (Saez and -Hersing, 2019; Zhang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eElectrophysiological findings in this study reveal that Phi-Trp and Phi-Ala, derived from \u003cem\u003ePhidippus audax\u003c/em\u003e venom, modulate potassium currents in DRGn, without significantly affecting sodium currents. This suggests a degree of selectivity for potassium channels, highlighting their potential as targeted modulators of neuronal excitability, which could be relevant for future pharmacological applications.\u003c/p\u003e\u003cp\u003ePhi-Trp exhibited concentration-dependent inhibition of potassium currents, with maximal reductions of 37% in \u003cem\u003eIK\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e and 44% in \u003cem\u003eIK\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e at 10 \u0026micro;M. This suggests a strong interaction with potassium channels involved in late repolarization. In contrast, Phi-Ala showed milder inhibition, reaching statistical significance at 10 \u0026micro;M (17% reduction in \u003cem\u003eIK\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e and 22% in \u003cem\u003eIK\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e). Structural differences, particularly in side-chain residues, may contribute to their distinct efficacies and binding affinities. Similar observations have been reported for other venom-derived peptides, where minor structural variations significantly impact channel modulation (Li \u003cem\u003eet al\u003c/em\u003e., 2005).\u003c/p\u003e\u003cp\u003ePartial recovery of potassium currents after washout suggests that peptide-channel interactions are not irreversible. This suggests a potential allosteric modulation or a voltage-dependent block rather than permanent modification (Vergara et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wulff \u0026amp; Zhorov, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The lack of significant changes in the inactivation time constant (τ\u003csub\u003einac\u003c/sub\u003e) suggests that these peptides primarily affect channel activation or maximal conductance rather than inactivation kinetics, distinguishing them from classical pore-blocking toxins like dendrotoxins or kaliotoxins (Garcia et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Yellen, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, neither Phi-Trp nor Phi-Ala significantly altered sodium currents. Many spider-derived peptides target voltage-gated sodium channels, such as toxins from \u003cem\u003ePhoneutria nigriventer\u003c/em\u003e, which shift activation or slow inactivation (Cest\u0026egrave;le \u0026amp; Catterall, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Bosmans \u0026amp; Tytgat, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; de Lima et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The absence of sodium current effects suggests that \u003cem\u003ePhidippus audax\u003c/em\u003e peptides exhibit functional selectivity, which could be advantageous for developing pharmacological tools that modulate neuronal excitability without disrupting action potential generation (Noreng et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bende et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese findings contribute to the growing understanding of spider venom peptide diversity and their potential as selective ion channel modulators. Future studies should characterize the binding sites of Phi-Trp and Phi-Ala on potassium channels, investigate their effects in other cellular models, and explore their therapeutic potential in conditions involving altered neuronal excitability, such as chronic pain or epilepsy (Budde et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Herrington \u0026amp; Gutman, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception of the work: GC, ESo, and EV; collection of data: ESa, IA, TOP, AR, and GC; methodology and analysis: ESa, IA, TOP, AR, GC, ESo, and EV; writing end edition of manuscript: Esa, GC, ESo, and EV; funding acquisition: GC, ESo, and EV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest, financial or otherwise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human experiments were performed. All applicable international, national, and institutional guidelines for the care and use of animals were followed. Procedures adhered to the bioethical standards of the “Instituto de Biotecnología - UNAM”.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll researchers listed in this work voluntarily agreed to participate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll researchers agreed on publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated in this study are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Rosby Najera for preliminary peptide synthesis. This work was funded by the Dirección General de Asuntos del Personal Académico (DGAPA-UNAM) grant number IT200724 and the CONACyT/SECIHTI-PRONAII grant number 303045.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBailey CL (1969) Life history of the spider, life history of \u003cem\u003ePhidippus audax\u003c/em\u003e (Hentz), in relation to biological control of grain sorghum insects. Oklahoma State University.\u003c/li\u003e\n\u003cli\u003eBende NS, Dziemborowicz S, Mobli M, et al. (2014) A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a. Nature Communications. 5, 4350. doi:10.1038/ncomms5350\u003c/li\u003e\n\u003cli\u003eBosmans F, Tytgat J (2007) Voltage-gated sodium channel modulation by scorpion \u0026alpha;-toxins. Toxicon, 49(2), 142-158.\u003c/li\u003e\n\u003cli\u003eBudde T, Daut J, Kurtz A, Pape HC (2015) Two-pore-domain potassium channels: regulators of many cellular functions. Pfl\u0026uuml;gers Archiv\u003cem\u003e - \u003c/em\u003eEuropean Journal of Physiology, 467(5), 865-866.\u003c/li\u003e\n\u003cli\u003eHerrington J, Gutman GA (2011) BK channels and neurological disease. Frontiers in Physiology, 2, 1-10.\u003c/li\u003e\n\u003cli\u003eCest\u0026egrave;le S, Catterall WA (2000) Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie, 82(9-10), 883-892.\u003c/li\u003e\n\u003cli\u003eCorzo G, Escoubas P, Stankiewicz M, et al. (2000). Isolation, synthesis and pharmacological characterization of delta-palutoxins IT, novel insecticidal toxins from the spider \u003cem\u003eParacoelotes luctuosus \u003c/em\u003e(Amaurobiidae). Eur. J. Biochem. 267, 5783-5795.\u003c/li\u003e\n\u003cli\u003ede Lima ME et al. (2015). Phoneutria nigriventer Venom and Toxins: A Review. In: Gopalakrishnakone, P., Corzo, G., Diego-Garcia, E., de Lima, M. (eds) Spider Venoms. Toxinology. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-6646-4_6-1\u003c/li\u003e\n\u003cli\u003eEstrada G, Villegas E, Corzo G. (2007) Spider venoms: a rich source of acylpolyamines and peptides as new leads for CNS drugs. Nat Prod Rep 24, 145-161.\u003c/li\u003e\n\u003cli\u003eGarcia ML, Kaczorowski GJ, Glimour R, et al. (1997) Potassium channels: From scorpion venoms to high-resolution structure. Toxicon, 35(5), 745-752.\u003c/li\u003e\n\u003cli\u003eGhisaidoobe ABT, Chung SJ (2014) Intrinsic tryptophan fluorescence in the detection and analysis of proteins: a focus on F\u0026ouml;rster resonance energy transfer techniques. Int J Mol Sci, 15(12), 22518-22538.\u003c/li\u003e\n\u003cli\u003eGuo R, Guo G, Wang A, Xu G, et al. (2023) Spider-venom peptides: structure, bioactivity, strategy, and research applications. Molecules 29(1), 35.\u003c/li\u003e\n\u003cli\u003eHerrera-Perez S, Lamas JA (2023) TREK channels in mechanotransduction: a focus on the cardiovascular system. 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Biol. 433(17), 166967.\u003c/li\u003e\n\u003cli\u003eSaez NJ, Herzig V (2019) Versatile spider venom peptides and their medical and agricultural applications. \u003cem\u003eToxicon\u003c/em\u003e, 158 109-126.\u003c/li\u003e\n\u003cli\u003eSalceda E, Garateix A, Soto E (2002) The Sea Anemone Toxins BgII and BgIII Prolong the Inactivation Time Course of the Tetrodotoxin-Sensitive Sodium Current in Rat Dorsal Root Ganglion Neurons. \u003cem\u003eJ Pharmacol Exp Ther\u003c/em\u003e 303, 1067.\u003c/li\u003e\n\u003cli\u003eWorld Spider Catalog (2025) World Spider Catalog. Version 26. Natural History Museum Bern, online at http://wsc.nmbe.ch, accessed on {date of access}. doi: 10.24436/2\u003c/li\u003e\n\u003cli\u003eVergara C, Latorre R, Marrion NV, et al. (2020) Calcium-activated potassium channels. \u003cem\u003ePhysiol Revs\u003c/em\u003e, 101(1), 531-606.\u003c/li\u003e\n\u003cli\u003eWiggins WD, Wilder SM (2022) Carbohydrates complement high-protein diets to maximize the growth of an actively hunting predator. \u003cem\u003eEcol Evol\u003c/em\u003e. 12, e9150.\u003c/li\u003e\n\u003cli\u003eWulff H, Zhorov BS (2008) K+ channel modulators and the therapeutic potential of small molecules targeting subtypes. \u003cem\u003eAnnu Rev Pharmacol Toxicol\u003c/em\u003e, 48, 431-458.\u003c/li\u003e\n\u003cli\u003eYellen G (2002) The voltage-gated potassium channels and their relatives. \u003cem\u003eNature\u003c/em\u003e, 419(6902), 35-42.\u003c/li\u003e\n\u003cli\u003eZhang YM, Ye DX, Liu Y, Zhang XY, Zhou YL, Zhang L, Yang XL (2023) Peptides, new tools for plant protection in eco-agriculture. \u003cem\u003eAdvanced Agrochem\u003c/em\u003e, 2(1), 2023, 58-78.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"jumping spider, potassium channel, spider toxin, sodium channel","lastPublishedDoi":"10.21203/rs.3.rs-7076766/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7076766/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLittle is known about the venom of Salticidae spiders, so here, we look for venom peptides of the most cosmopolitan spider, \u003cem\u003ePhidippus audax\u003c/em\u003e. The isolation, chemical synthesis, and pharmacological characterization of a short peptide from the venom of the spider \u003cem\u003ePhidippus audax\u003c/em\u003e (Araneae: Salticidae) was unveiled. The peptide (Paudax1) consists of 22 residues and contains a single disulfide bridge. Paudax1 has paralytic activity against \u003cem\u003eAcheta domesticus\u003c/em\u003e. It was synthesized in two N-terminal forms, Phi-Ala and Phi-Trp, which were also paralytic to house crickets. The pharmacology of both homologous peptides was evaluated in primary cultures of rat dorsal root ganglia neurons. Microperfusion of Phi-Ala [10 \u0026micro;M] (n\u0026thinsp;=\u0026thinsp;6) resulted in a 17\u0026thinsp;\u0026plusmn;\u0026thinsp;5% inhibition of the maximum amplitude of the outward current (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a non-significant decrease of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;7% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the current amplitude at the end of the voltage pulse (\u003cem\u003eIK\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e). Additionally, Phi-Ala did not affect the current inactivation time course (τ\u003csub\u003einact\u003c/sub\u003e). Meanwhile, the peptide Phi-Trp [10 \u0026micro;M] (n\u0026thinsp;=\u0026thinsp;6) induced a 37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6% inhibition (p\u0026thinsp;\u0026le;\u0026thinsp;0.01) of the maximum amplitude of the outward current and a 44\u0026thinsp;\u0026plusmn;\u0026thinsp;5% inhibition (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01) in \u003cem\u003eIK\u003c/em\u003e\u003csub\u003eend\u003c/sub\u003e, with no change in τ\u003csub\u003einact\u003c/sub\u003e. Although both peptides, Phi-Ala and Phi-Trp, displayed insect paralytic activity, they exhibited relatively low efficiency as blockers of the outward current at the concentrations used, and they did not affect the inward currents. Phi-Trp and Phi-Ala are worth investigating to explore their therapeutic potential.\u003c/p\u003e","manuscriptTitle":"Isolation, synthesis, and pharmacological characterization of a short-structured peptide from the venom of Phidippus audax that affects potassium currents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 09:06:24","doi":"10.21203/rs.3.rs-7076766/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"84f9e230-dca5-4430-9205-9745efe93ce0","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-16T15:08:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-05 09:06:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7076766","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7076766","identity":"rs-7076766","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}