UNIVERISDAD DE COSTA RICA SISTEMA DE ESTUDIOS DE POSGRADO Caracterización del efecto del veneno del escorpión endémico de Costa Rica Tityus championi y sus neurotoxinas sobre la excitabilidad neuronal y las corrientes iónicas de sodio y potasio generadas por canales voltaje dependientes de moluscos, mamíferos e insectos Tesis sometida a la consideración de la Comisión del Programa de Estudios de Posgrado en Biología para optar al grado y título de Maestría Académica en Biología con énfasis en Genética y Biología Molecular CANDIDATA Johanna Galit Akerman Sánchez Ciudad Universitaria Rodrigo Facio, Costa Rica 2024 ii AGRADECIMIENTOS Hacer ciencia como la que se encuentra en esta tesis ha sido un verdadero privilegio y regalo. Puedo decir que, en su gran mayoría, me sentí plena y descubrí nuevas fases de mí. Esto jamás lo hubiera podido lograr sin la ayuda de muchas personas. Personas que me dieron algo que cambió una parte de mí. Primero, quisiera agradecer a mi papá y a mi mamá, los más importantes. A mi padre, Jorge, por llenarme de preguntas y curiosidad desde que estaba pequeña, viendo bichos en el bosque y células en el microscopio. No es casualidad que cada vez me convierta en la científica que inspiró mi padre. A mi madre, Lorena, por darme toda la fuerza y amor que una persona puede necesitar, con tan solo un abrazo. Por esas noches que nos acompañamos juntas estudiando, y me hiciste saber que era capaz de todo lo que quisiera lograr. A Daniel, mi querido novio, quien me ha apoyado incondicionalmente, desde trabajar en códigos, escucharme hablar por horas, hasta atravesar el océano Atlántico y disfrutar de la vida juntos. Gracias Dani, no hay palabras con las que pueda expresar mi gratitud hacia vos. Al Dr. Oscar Brenes, mi tutor y mentor, gracias por todo lo que ha compartido conmigo, gracias por darme la bienvenida al mundo de la electrofisiología y hacerla hermosa y cautivante sin importar cuan difícil sea. Comunicar la ciencia y lograr que las personas se enamoren así, es un don. Gracias por la confianza que ha depositado en mí, por las oportunidades que ha hecho florecer en mi camino. Por hacerme una mejor científica con cada conversación, mientras comemos pollo frito en el Mall San Pedro. Gracias a la Dra. Adarli Romero, por darme un ejemplo tan claro de la mujer fuerte científica que quiero ser. Por enseñarme a usar mis manos y construir lo que necesito. Gracias por esa primera clase de fisiología animal en donde con sus palabras yo dije, esto es lo que finalmente andaba buscando. Muchas personas pueden enseñar en la universidad, pero no todas van a cuidar y enseñar como usted lo hace, profe. También, quisiera agradecer a mi profesor en Bélgica, Dr. Jan Tytgat, quien abrió las puertas de su laboratorio para poder responder las preguntas con las cuales no dejaba dormir a Daniel. Esas respuestas que me apasionaba descubrir, las encontré a su lado, Dr. Jan. Gracias por su amabilidad, por las numerosas discusiones en el laboratorio al lado de registros, por todas las iii aventuras en bicicleta, donde me mostró realmente cómo es Leuven. Por abrir mi mente a una cantidad inmensurable de más preguntas, que no me paralizan, más bien me dan fuerza. Sin embargo, no me puedo ir sin mencionar a dos personas que siempre han celebrado mi camino en la ciencia. Ellas han sido mis abuelas, Diana y Zulma. Para el cielo, mando muchos besos hacia mi Dianita, quien siempre me escuchó con emoción cuando le decía que venía del laboratorio. A Zulma, quien siempre está ansiosa por escuchar lo que hago en la universidad. Siempre sentí que mis abuelas estaban orgullosas de mi con solo decirles que quería ser científica. De vez en cuando escuchaba decirse una a la otra “que dichosa, ¿verdad?”. Me decían sutilmente “aproveche mamita”, “me hubiera encantado…”. Esas frases siempre calaron en mí. Creo fielmente que, hoy tengo la posibilidad de hacer esto, gracias al camino que mujeres como mis abuelas han recorrido y pavimentado. Espero hacerla sentir orgullosa, Tita Diana. Espero hacerla sentir orgullosa, Zuli. Gracias por darme la vida, esta vida. Con gratitud y amor, Galit iv v RESUMEN El objetivo de esta investigación fue caracterizar electrofisiológicamente el veneno y las neurotoxinas del escorpión Tityus championi, una especie endémica de Costa Rica, enfocándose en los canales iónicos voltaje-dependientes de Na+ (NaV) y K+ (KV), que son los blancos selectivos de las neurotoxinas de escorpión. Se utilizaron dos modelos celulares: (i) una neurona del caracol Helix aspersa, que expresa el canal NaV1.7-like, relacionado con nociceptores, y (ii) ovocitos de rana Xenopus laevis, donde se expresaron de forma heteróloga isoformas de canales iónicos de humanos, ratas e insectos. Los resultados mostraron que el veneno disminuye la excitabilidad neuronal, y puede afectar tanto las corrientes de Na+ como de K+, en la neurona del caracol y en canales de mamíferos e insectos. Se identificaron toxinas que aumentan la sensibilidad de los NaVs y una toxina que los inhibe, siendo probablemente la toxina Tch3 una de las principales responsables de la inhibición de la corriente de Na+ y la disminución de la excitabilidad neuronal. Se identificó una nueva toxina selectiva para KVs, denominada TchKTx7, que mostró un bloqueo prácticamente completo en KV1.2 y parcial en ShakerIR. Además, se observaron efectos de α- toxina sobre los canales NaV1.6, NaV1.7 y BgNaV1. En el caso del NaV1.7, aunque presenta efectos de α -toxina, también se observó una inhibición de la corriente máxima, congruente con lo observado en los canales de caracol. Este estudio representa un avance significativo en el entendimiento de las neurotoxinas de T. championi, siendo el primero en caracterizar los efectos funcionales de estas toxinas en diferentes canales iónicos y especies, abriendo nuevas oportunidades para la prospección farmacológica de estas potentes moléculas. vi LISTA DE CUADROS CAPITULO I Table I. Neurotoxins purified from the venom of the scorpion T. championi and their primary structure………………………………………………………………………………………..….8 Table II. Open probability parameters of activation……………………………………………...24 Table III. Open probability parameters of steady-state inactivation……………………………...25 CAPITULO II Table I. Sequenced components and alignment of fractions 22 and 27…………………………...55 Table II. Sequenced components and alignment of fraction 25………………………………….56 Table III. Open probability biophysical parameters derived from the mean activation curve using Boltzmann equation for each channel tested……………………………………………………...62 Table IV. Steady-state inactivation biophysical parameters derived from the mean curve using Boltzmann equation for each channel tested……………………………………………………...62 ANEXOS CAPÍTULO I Table SI. Protein families, identified by mass spectrometry, present in the venom of the scorpion T. championi…………………………………………………………………………………...…76 Table SII. Comparative analysis of distinct properties of IT and IP Na+ currents in various organisms………………………………………………………………………………………...77 CAPÍTULO II Table SI. Isoforms of channels heterologously expressed in this study and their organism of origin……………………………………………………………………………………………..78 vii LISTA DE FIGURAS CAPITULO I Figure 1. Protein pattern of T. championi venom…………………………………………………11 Figure 2. Effect on the action potential firing upon exposure to T. championi venom (10 g/ml)..12 Figure 3. Effect of T. championi venom on the morphology of evoked action potentials……….14 Figure 4. Effect of the toxin Tch3 on the cellular firing frequency………………………………15 Figure 5. Effects of Tch3 on the evoked action potentials waveform……………………………..16 Figure 6. Macroscopic Na+ currents in the C1 neuron of the snail H. aspersa…………………..18 Figure 7. Tch2, Tch3, and Tch4 effects on transient and persistent currents……………………..19 Figure 8. Activation and inactivation kinetics under Tch2, Tch3, and Tch4 exposure……………22 Figure 9. Window current changes depending on the toxin exposition…………………………...23 Figure 10. Reduction in the recovery from inactivation by all toxins applied……………………27 Figure 11. Fast reduction of K+ current by toxin TchKTx5……………………………………….28 CAPITULO II Figure 1. Effect of Tityus championi venom on expressed isoforms of KV from human and insect……………………………………………………………………………………………..50 Figure 2. Representative currents of the KV1.2 and ShakerIR channels………………………...51 Figure 3. RP-HPLC separation of T. championi venom using a linear acetonitrile gradient……51 Figure 4. Effect of fractions 22 and 27 on KV1.2 and ShakerIR channel currents………………53 Figure 5. Combined effect of fractions 23, 24, and 25 on KV1.2 and ShakerIR channel currents…………………………………………………………………………………………..54 Figure 6. Effect of T. championi venom on expressed isoforms of NaV channels from mammals and insects……………………………………………………………………………………….59 Figure 7. The window current is altered by the venom in different ways depending on the target NaV expressed…………………………………………………………………………………...60 Figure 8. Inhibition of inactivation by venom in NaV1.6, NaV1.7, and BgNaV1 channels……...61 viii Figure 9. Differential effects of fractions 32, 33, and 35 on NaV1.6 and NaV1.7 channel currents………………………………………………………………………………………….64 ANEXOS CAPÍTULO I Figure S1. Voltage-clamp stimulation protocols on Helix aspersa C1 neurons in whole-cell patch- clamp configuration……………………………………………………………………………..74 Figure S2. Representation of some of the reported action potential parameters………………..74 ix LISTA DE ABREVIATURAS AIS: Axon Initial Segment AP: Action Potential BSA: Bovine Serum Albumin CNS: Central Nervous System G: Conductance GFL: Giant Fiber Lobe gNa: Na+ conductance I: Current IFF: Instantaneous Firing Frequency IP: Steady-state Persistent current IT: Transitory current KTx : K+ Toxin KV : Voltage-dependent K+ channel MFF: Mean Firing Frequency MWM: Molecular Weight Markers NaTx : Na+ Toxin NaV : Voltage-dependent Na+ channel PNS: Peripheral Nervous System Po: Channel opening probability PP: Propeptide s.e.m: Standard error of the mean TEVC: Two-Electrode Voltage Clamp V: Voltage 1 INTRODUCTION Scorpion envenomation is a global health concern, affecting over 1.2 million people annually and resulting in more than 3,000 deaths (Chippaux & Goyffon, 2008). Among the many scorpion species, the genus Tityus (Buthidae) is particularly notorious for causing severe envenomation in Central and South America, including the Amazon region (Borges et al., 2020). The venom of these scorpions contains peptide toxins that primarily target the nervous and muscular systems, altering excitability and leading to potentially life-threatening effects (Goudet et al., 2002). The bioactivity of scorpion neurotoxins is both potent and specific, enabling these toxins to effectively immobilize prey by targeting key cellular components such as ion channels (Gwee et al. 2002). These peptide toxins interact with excitable cells by modulating the activity of various ion channels, including those that regulate the flow of Na+, K+, Ca2+, and Cl- ions (Srinivasan et al., 2002). Scorpion toxins are known to modulate more than 40 types of ion channels, with more than 1.300 specific toxins identified to date (Housley et al., 2017; UniProtKB, 2024). This broad range of activity is what makes scorpion venom such a potent agent, capable of eliciting severe physiological responses. The primary molecular targets of scorpion toxins are voltage-dependent Na+ and K+ channels (NaV and KV, respectively), which play critical roles in cellular signaling and electrical activity, particularly in excitable cells, such as neurons and muscle fibers (García et al., 2001; de la Vega & Possani, 2007). NaV channels are transmembrane proteins essential for transmitting electrical signals in the central and peripheral nervous systems, with their rapid activation and inactivation regulating the flow of Na+ ions (Clairfeuille et al., 2019). Na+ channel toxins (NaTxs) from Tityus scorpions can disrupt this process by either delaying channel inactivation (α-toxins) or promoting channel opening at more negative voltages (β-toxins), mostly leading to increased neuronal excitability and altered muscle function (Chow et al., 2020). These disruptions can result in paralysis, cardiac arrhythmias, and even death (Hanck & Sheets, 2007). Similarly, K+ channel toxins (KTxs) from scorpions affect KV channels, which are essential for the cell's repolarization after action potentials (APs) (Kuang et al. 2015). These channels are highly selective for K+ ions due to their unique pore structure, which filters out other ions (Doyle et al., 1998). By binding to KV channels, KTxs block their function, prolonging APs and 2 contributing to abnormal cellular activity (Mouhat et al., 2008). Both NaTxs and KTxs are valuable tools in studying ion channel function and have potential therapeutic applications in treating conditions such as cancer, pain, and autoimmune diseases (Bergeron & Bingham, 2012). Given the medical relevance of scorpion toxins, particularly from the Tityus genus, which is responsible for the most severe and fatal envenomation in Latin America (Guerra-Duarte et al., 2023), it is crucial to expand our understanding of how these toxins affect ion channel function. In this study, we aimed to investigate the effects of the venom and toxins from Tityus championi, a species endemic to Costa Rica and Panama that is known to cause severe cases of envenomation in Panama (Salazar et al., 2018). The venom of T. championi likely targets Na+ and K+ channels, as seen in other Tityus species, and could provide insights into the mechanisms of toxin-channel interaction. This research could also contribute to therapeutic application development for conditions involving ion channel dysfunction. 3 CAPÍTULO I 4 Effect of the venom and purified neurotoxins of Costa Rican scorpion Tityus championi on invertebrate cation channels Galit Akerman-Sánchez1,2, Cecilia Díaz3,4, Natalia Ortiz 3,4, Oscar Brenes1,5 1 Department of Physiology, School of Medicine, University of Costa Rica, San José, Costa Rica 2 SEP, School of Biology, University of Costa Rica, San José, Costa Rica 3Clodomiro Picado Institute, Faculty of Microbiology, University of Costa Rica, Coronado, Costa Rica 4 Department of Biochemistry, School of Medicine, University of Costa Rica, San José, Costa Rica 5 Neuroscience Research Center, University of Costa Rica, San José, Costa Rica ABSTRACT Scorpion neurotoxins are small peptides with significant bioactive potential, targeting ionic channels and offering opportunities for novel therapeutic discoveries. This study analyzed the functional electrophysiological effects of newly discovered toxins from the Costa Rican endemic scorpion Tityus championi. Using the C1 neuron from the land snail Helix aspersa as a cellular model, whole-cell patch-clamp techniques in both voltage- and current-clamp configurations were employed to analyze sodium and potassium currents, action potential generation and morphology in exposure to crude venom and purified toxins. Results revealed that scorpion crude venom was inhibitory on neuron activity, affecting both depolarization and repolarization. The venom toxins (2 M) exhibited different effects and mechanisms on sodium currents mediated by the NaV1.7- like channel. Specifically, Tch2 and Tch4 toxins increased both transient and persistent currents. In transient currents, both induced activation and inactivation changes in voltage sensitivity and dependency, ultimately widening the current window and increasing currents at more negative voltages but decreasing channel availability. Conversely, Tch3 behaved as an inhibitory toxin, affected only the transient current, closed the window current, and decreased cellular excitability. Since similar effects were also observed with the complete venom, results suggested that this inhibitory toxin is predominant within the venom’s effect. Additionally, partial blocking effects were noted for the K+ toxin TchKTx5. This study provides new insights into the toxins of Costa 5 Rican scorpions, elucidating their complex mechanisms of action and their effects, thus advancing our understanding of their bioactive potential. INTRODUCTION Scorpion neurotoxins are peptides that interfere with the normal function of the nervous system, exhibiting potent and specific activity to attack a possible offender or prey, by immobilizing or killing it (Gwee et al., 2002; Stevens et al., 2011). In scorpions, evolutionary pressure has selected these peptides to modulate different ion channels (Zhang et al., 2015), especially Na+ and K+ ion channels, which play crucial roles in numerous cellular processes, such as action potential (AP) generation and muscle contraction (García et al., 2001; de la Vega & Possani, 2007). Scorpion toxins selective for Na+ voltage-gated channels (NaV) (NaTxs) consist of 60-76 amino acids with disulfide bridges in their tertiary structure and can be distinguished into two types (Chow et al., 2020; Ghosh et al., 2019). The α-toxins (α-NaTxs) prevent rapid channel inactivation by binding to the extracellular loop called receptor site 3 (Cestéle et al., 1998). Due to their proximity to the S4 voltage sensor in the motive IV (IVS4), the toxin keeps the sensor in an internal, non-active position, delaying inactivation (Gwee et al., 2002). Functionally, this prolongs the duration of the AP and reduces cellular firing frequency (Clairfeuille et al., 2019; Zhu et al., 2020). On the other hand, the β-toxins (β-NaTxs), promote channel opening by binding to receptor site 4. During channel activation, the S4 voltage sensor of motive II is translocated extracellularly, where it interacts with the β-NaTxs, anchoring it in an external, active position (Gwee et al., 2002). This leads to increased channel activation and negative voltage dependence shift in future activations, opening at more negative voltages (Rogers et al., 1996). Functionally, a shift toward more negative membrane potentials results in increased AP firing frequency (Chow et al., 2020). These two types of NaTxs, with different action mechanisms, ultimately increase Na+ currents, probably causing massive neurotransmitter release (Gordon & Gurevitz, 2003; de la Vega & Possani, 2005). Consequently, α and β-NaTxs can cause paralysis, cardiac arrhythmias, and death (Hanck & Sheets, 2007). Scorpion toxins selective for K+ voltage-gated channels (KV) (KTxs) consist of 25-45 amino acid residues, with three or four disulfide bridges (Mouhat et al., 2008). KTxs reversibly 6 bind to the KV channel's negatively charged residues with their positively charged residues (Gwee et al., 2002). As a result, the channel is blocked, prolonging action potentials, and leading to abnormal nerve function (Mouhat et al., 2008). Together, NaTxs and KTxs modify the activity of their respective channels, collectively altering cell excitability, neurotransmitter release, and ultimately, neuronal function and animal behavior (Mouhat et al., 2008). In the present study, we aim to characterize the effect of the crude venom and isolated toxins from the scorpion Tityus championi (Pocock, 1898) on Na+ and K+ invertebrate ion channels and their impact on neuronal excitability. EXPERIMENTAL PROCEDURES Animals Juvenile specimens of terrestrial snails H. aspersa (NCBI taxonomic ID: 6535) were provided by local breeders. The snails were housed in plastic boxes in a room with a regulated temperature (20°C) and a 12:12 h light:dark cycle. Snails were fed once a week with a calcium- rich diet and lettuce or cucumber. Before procedures, a 0.1M MgCl2 solution was injected into the snail’s foot to anesthetize and induce muscle relaxation, subsequently, they were sacrificed by evisceration. All methodologies were previously validated by the Institutional Animal Care and Use Committee (CICUA-026) of the University of Costa Rica, and efforts were made to minimize the number and suffering of animals in accordance with guidelines established by the Ethical- Scientific Committee on the protection of animals for scientific purposes. C1 Neuron Isolation Cell isolation was performed as previously described (Brenes et al., 2015). Briefly, to isolate the cerebral ganglion, nerves, and surrounding tissue were carefully cut. Once isolated, the ganglia were incubated for enzymatic digestion using protease type XIV (Sigma-Aldrich) in L15 isotonic medium (0.4 U/mL) at 34°C for 4 h. Following digestion, the ganglia underwent two washes with L15 medium to proceed with the isolation of C1 neurons via microsurgery. This 7 neuron was identified by its position, size, and distinctive morphology in the cerebral ganglion. Neurons were gently isolated with segments of the axon still attached to the soma and subsequently placed in dishes pre-coated with 5% bovine serum albumin (BSA) until the axon was reabsorbed, resulting in a configuration known as the soma configuration as previously described (Fiumara et al. 2005). Venom The venom of T. championi was obtained from the Dangerous Animals Research Laboratory (LIAP) at the Clodomiro Picado Institute (ICP) through the sorting of specimens maintained in its animal facility. This part of the study was approved by the Biodiversity Commission of University of Costa Rica (No. 293-2021). The lyophilized venom (81 mg) was reconstituted in 1 mL of distilled water. Subsequently, protein quantification was performed in the Institute of Health Research (INISA), using the BCA method with the Thermo Scientific microBCA kit #23235 and the spectrophotometer (Nanodrop 2000, Thermo Scientific) in triplicate. Following this, venom samples were separated by SDS-PAGE (15% polyacrylamide) under reducing conditions (prior step of 10-min incubation at 98 degrees Celsius with 5% 2- mercaptoethanol) (Isotemp, Fischer Scientific). The low molecular weight ladder BLUeye Prestained Protein (94964, Sigma-Aldrich) was used to determine the mass of the main components. Subsequently, the gel was allowed to run for 1 h at 150 V using a Mini-Protean Tetra System coupled with PowerPac Basic power supply (BioRad), and proteins were stained with Coomassie Blue R-250. Toxins The toxins were isolated from the venom through reverse-phase HPLC and mass spectrometry was used to identify their sequences at the ICP. It was decided to proceed with the functional analysis of four of the most abundant toxins in the venom, Tch2, Tch3, Tch4, and TchKTx5. The amino acid sequence of these toxins is presented in Table I. 8 The putative toxins used in the electrophysiological studies were dissolved in distilled water and stored at -20°C until use. During experiments, each toxin was added directly to the culture dish where the cell was located in a 2 µM final concentration. Electrophysiology recordings Standard intracellular recording techniques, such as whole-cell patch-clamp, in current and voltage clamp configurations were used with isolated cells under a Nikon inverted microscope (Eclipse TS100). A borosilicate electrode manufactured at the time of use (with a puller Flaming- Brown P-1000), with a resistance between 1.5 and 2.8 MΩ, was brought close to the cell until a connection with the cytosol was established. Signals were amplified using a Multiclamp 700B amplifier (Axon Instruments) and converted to digital signals through the analog/digital interface Digidata 1322A (Axon Instruments). Data were collected using pClamp 11 software (Axon Instruments) on a personal computer. Series resistance compensation between 60 and 80% was performed before data collection, depending on each cell. Table I. Neurotoxins purified from the venom of the scorpion T. championi and their primary structure (Díaz et al., 2023). Neurotoxin Sequence of amino acids Tch2 EAIDGYPLSKNNYCKIYCPDDAVCKDTCKNRAGATNGKGDCINKGCY CYDVAPSTKMYPGRLPCNPY EALDGYPLSKNNYCKIYCPDDAVCKDTCKNRAGATNGKGDCINKGC YCYDVAPGTKMYPGRLPCNPY Tch3 EALDGYPLSKNNYCKIYCPNDEVCKDTCKHRAGATNGKGDCIWQTC YCYDVAPGTKMYPGSSPCYA Tch4 GIKNGYPRDSKGCTFKCGQDAKHGDDYCDKMCKTTLKGEGGDCDFE YAECWCDNIPDTVVTWKNKEPK TchKTx5 MHFSGVAFILISMVLINSIFETTAEAGDGPKSDCKPDLCEKACKEEKGK PMDFCKGDICKCKD 9 Electrodes were filled with different solutions depending on the current of interest, Na+ or K+. The composition of the intracellular solution used for K+ current recording was as follows (mM): 100 KCl, 10 HEPES, 1 MgCl2 – 6H2O, 5 EGTA; while the extracellular solution in the dish was composed of (mM): 5 KCl, 90 cholineCl, 1 CaCl2-2H2O, 5 MgCl2-6H2O, 10 Tris-Cl. The composition of the intracellular solution used for Na+ current recording was (mM): 110 CsCl, 10 HEPES, 2 MgCl2, 1 NaCl, 2 EGTA; while the extracellular solution was (mM): 4 KCl, 90 NaCl, 1 CaCl2, 5 MgCl2, 10 Tris, 30 TEA-HCl, 4 4-AP. All solutions were adjusted to a pH of 7.4 before use. Ion Current Evaluation We evaluated the effects of the toxins on the three NaV gating processes (activation, inactivation, and recovery), and on KV activation. To evaluate the activation of NaV and KV channels, currents were measured at different voltages from -60 mV to +60 mV (ΔV =10 mV) for 200 ms, from a resting potential of -50 mV (Supplementary material Fig. S1A). For Na+ currents, data of the maximal transitory current (IT) and steady-state persistent current (IP) were extracted. Additionally, from these recordings, the inactivation time constants (τ of inactivation) were obtained by fitting the inactivation phase of the current curve to a standard exponential equation (Supplementary material SE.1) using Clampfit software (Axon Instruments). For K+ currents, data were extracted solely from IP, corresponding to the K+ current through KV channels. IT and IP were plotted against their respective voltage to generate standard IV curves. For the voltage-dependent activation, the conductance (gNa) and the channel opening probability (Po) were obtained using the standard approaches (Supplementary materials SE.2 and SE.3). Steady- state inactivation was evaluated by a two-pulse voltage protocol, quantifying the maximal Na+ current generated during a test pulse at -20 mV after channel inactivation caused by conditioning step pulses ranging from -60 mV to +60 mV (ΔV = 10 mV), each pulse lasting 200 ms (Supplementary material Fig. S1B) (Abd El-Aziz et al. 2021). Maximal test pulse IT was normalized to the maximal current (similar to SE.3). The voltage-dependent activation and steady-state inactivation were plotted as a function of voltage and the data were fitted using the Boltzmann equation (Supplementary materials SE.4), to obtain V1/2 and k values, where V1/2 represents the voltage of half-maximal 10 activation/inactivation and k is the slope factor of the voltage dependence of the open probability. The area subtended by the intersection of the activation and inactivation curves was calculated (window current). RStudio (Version 2023.06.0+421) was used for window current calculation (Calculation Code). The time required for the channel to recover from the inactive state (recovery from inactivation) was measured using two voltage steps at -20 mV, separated by an increasing time interval starting from 5 ms to 100 ms with a Δt = 5 ms (Supplementary material Fig. S1C) (Kiss 2003), assessing in this way the impact of toxins on the NaV channels availability. Action Potential Assessment The current-clamp technique was employed to identify possible changes in APs due to the effect of toxins. The stimulation protocol consisted of three stimuli of increasing intensity (0.5, 1.0, and 1.5 nA), each lasting 500 ms (Brenes et al., 2015). Mean Firing Frequency (MFF) was calculated as the average number of APs fired during the 500 ms stimulus, multiplied by two to report in Hz. Instantaneous Firing Frequency (IFF), where the IFF is the frequency between each AP. Also, several parameters of AP waveform morphology were reported (Supplementary material Fig. S2), the morphological variables of the AP analyzed include: (i) AP amplitude (mV), determined as the difference between the maximal depolarizing potential and the resting membrane potential before stimulation; (ii) half-width, measured as the duration (ms) at half of the maximal amplitude; (iii) maximal depolarization rate (mV/ms); (iv) depolarization rate (mV/ms) between 10% and 90%; (v) depolarization time constant (rise τ, ms), corresponding to the time required to reach 63% of the maximal magnitude of the AP. Data Data was reported as mean ± standard error of the mean (s.e.m.). Figures were made with GraphPad Prism version 10 (GraphPad Software). 11 RESULTS A clear inhibitory effect on neuronal excitability due to the cocktail of a complex mix of components and neurotoxins present in crude venom Starting with the crude venom, we observed a distinct pattern of proteins with varying molecular weights. The dominant components are low molecular weight peptides, primarily below 8 kDa, which are typically associated with small peptides present in the venom, such as neurotoxins (Fig. 1). We identified in this sense three prominent bands: two thicker bands representing peptides smaller than 8 kDa and a thinner band between 8 and 15 kDa. Discrete bands were also observed between 24 and 72 kDa, while bands representing higher molecular weights (>72 kDa) appeared faint, indicating lower concentrations of high molecular weight proteins in the venom. Given the dominant presence of low-weight peptides, including NaTxs and KTxs, we evaluated the Mean Firing Frequency (MFF) and the morphology of APs. Interestingly, exposing the cells to the complete venom (10 µg/ml) completely abolished firing induced by the smallest stimulus (0.5 nA), and decreased the firing frequency in a time-dependent manner with the highest stimulus (1.0 and 1.5 nA) (Fig. 2A). Figure 1. Protein pattern of T. championi venom. Complete venom was analyzed by SDS- PAGE (15% acrylamide) under reducing conditions. MWM = Molecular Weight Markers. 12 Figure 2. Effect on the action potential firing upon exposure to T. championi venom (10 g/ml). A. Mean Firing Frequency (MFF) (Hz) under stimuli of 0.5, 1.0, and 1.5 nA before (Control) and after 2 (grey), 5 (orange), 10 (red), and 20 (blue) min of venom exposure (n=4). B. Representative recording of the first action potential induced by 1.5 nA stimulus before (black) and after 20 min (purple) of venom exposure. Each value indicates mean ± s.e.m. Together with a decrease in the number of AP fired by the cells, we observed changes in AP waveform (Fig. 2B). Hence, we proceeded to examine in detail the effect on different parameters describing the morphology of individual AP induced by 1.5 nA current pulse (Fig. 3). The AP amplitude was not affected (Fig. 3A), and there was a slight reduction in half-width after 20 min of venom exposure (Fig. 3B). During depolarization, there was a small decrease in the rise slope from the second minute of exposure, consistent with an increase in rise time (Fig. 3C and D, respectively). On the other hand, during repolarization, no clear patterns were observed. In the first APs, there was a small increase followed by a small decrease in the decay slope during the first 2, 5, and 10 min of exposure, however with no strong changes in the decay time (Fig. 3E and F, respectively). Nevertheless, after 20 min of exposure, a clear increase in decay slope with a decreased decay time was evident. This suggests a reduction of AP duration, possibly explaining the smaller half-width after 20 min of venom exposure (Fig. 3B). The inhibitory effect of the crude venom is consistent with the effect of the predominant toxin Tch3 within it 13 To achieve a deeper understanding of the venom components effects on cells, we studied the effects of one of the most abundant toxins present in this venom, Tch3 (Diaz et al., 2023). Given that very little quantity of toxins can be isolated in a pure form from the venom, one single cell was used to test toxin effects on cell firing. In Figure 4A, it can be observed that, before toxin exposure, the cell fired APs in response to 1.0 and 1.5 nA. However, under the influence of the toxin, the cell only fired when the higher-intensity stimulus (1.5 nA) was applied. Similarly, it is noticeable that the IFF, decreased under toxin exposure, especially in the first APs, also decreasing AP adaptation (Fig. 4B). Regarding the action potential waveform, we observe a slight increase in the amplitude of APs, reaching a difference of approximately 10 mV between the control and the 30-min toxin exposure for the last AP generated (Fig. 5A). In the half-width, an increase in width was noted in the first APs fired (Fig. 5B). Since the sequence of Tch3 indicates that it is a putative NaTx, we analyzed the depolarization phase, which is governed by Na+ conductance. In the control cell, we observed a small decrease in the rise tau of each AP evoked. Regarding the maximal rise slope, the first AP was notably fast, but this slope decreased in the following four APs. A similar trend was observed in the rise slope 10-90%. In the presence of the toxin, the rise tau showed a progressive increase with the exposure time (Fig. 5C). The maximal rise slope of the first AP was lower in the presence of the toxin, and the pattern of slope reduction in the following four APs was not as pronounced. Finally, we observe that the rise slope 10-90% mostly decreased with exposure to the toxin (Fig. 5E). 14 Figure 3. Effect of T. championi venom on the morphology of evoked action potentials. All action potentials were evoked by a 1.5 nA stimulus for 500 ms, in control (black), and after 2 (grey), 5 (orange), 10 (red), and 20 (blue) min of venom exposure. The variables observed were Amplitude (A), Half-width (B), Rise Slope 10-90% (C), Rise Time 10-90% (D), Decay Slope 90-10% (E), and Decay Time 90-10% (F). Each value indicates mean ± s.e.m. 15 Figure 4. Effect of the toxin Tch3 on the cellular firing frequency. A. Mean Firing Frequency (MFF) (Hz) under increasing current stimuli, 0.5, 1.0, and 1.5 nA. In control (black), and after 10 (red), 20 (blue), and 30 min (dotted green) of toxin exposure. B. Instantaneous Firing Frequency (IFF) (Hz) in the intervals between action potentials. Firing frequency was measured before (control, black), and 10 (red), 20 (blue), and 30 min (green) after toxin exposure. 16 Figure 5. Effects of Tch3 on the evoked action potentials waveform. Action potentials were evoked by a stimulus of 1.5 nA for 500 ms, in control (black), and after 10 (red), 20 (blue), and 30 min (green) of toxin exposure. The variables measured were Amplitude (A), Half-width (B), rise tau (C), maximal rise slope (D), and rise slope from 10 to 90% of the action potential (E). 17 Distinction of two types of Na+ inward currents with distinct behavior in C1 neuron Being that we observe a noticeable effect on the depolarization, suggesting an alteration in Na+ conductance, and several neurotoxins in T. championi venom are putative NaTxs (Díaz et al., 2024), as a starting point, we characterized C1 Na+ currents. In this matter, different components were observed in the C1 macroscopic current (Figure 6A) probably composed of two distinct currents, a transient current called IT, and a steady-state persistent current called IP. The IV relationship of the IT current showed that activated from -30 mV, reached its maximal peak at -20 mV, with a maximal average amplitude of up to 10 nA, and its average reversal potential was around +40 mV (Figure 6B). On the other hand, the IP current showed smaller amplitudes (less than 2 nA), also started at -30 mV, but reached its peak at -10 mV and had a reversal potential close to 0 mV (Figure 6C). Using the IT current values, the relative conductance was plotted as a function of membrane potential (Figure 6D), and the mean activation of these channels was characterized by a V1/2 of - 29.7 mV and a k of 2.29. Regarding the inactivation of the IT, the relative current was plotted as a function of membrane potential (Figure 6E), reaching a complete inactivation at 0 mV, and with a V1/2 of -22.1 mV and a k of 6.63. By merging the activation and inactivation open probability curves, the window current reached an area value of 12.6. Comparative analysis of purified toxins on transient and persistent Na+ currents Subsequently, understanding the behavior of the Na+ currents in Helix snails, we proceed to analyze the effects of three purified toxins, which share primary structural homology with NaTxs, called Tch2, Tch3, and Tch4 (Díaz et al., 2023). As shown in Figure 7, the three NaTxs targeted the same channels but exhibited different effects. Tch2 and Tch4 increased transient current at -30 mV (Figure 7A and 7E, respectively). This effect becomes noticeable from five min of exposure. A slight increase in the Na+ IP was also observed at most of the voltages tested during Tch2 and Tch4 exposure (Figure 7B and 7F, respectively). 18 In contrast, the previously mentioned Tch3 was the only toxin that induced a trend of decrease in IT with exposure time, especially at 20 min (Figure 7C). However, when tested on IP Tch3 did not affect the currents, maintaining similar values at each voltage during exposure time (Figure 7D). Figure 6. Macroscopic Na+ currents in the C1 neuron of the snail H. aspersa. A. Representative macroscopic Na+ current, indicating the transient (IT) and persistent (IP) components. B. IV relationship of transient Na+ current (n=13). C. IV relationship of persistent Na+ current (IP) (n=12). D. Relative conductance as a function of membrane potential. E. Relative current as a function of the membrane potential of the preceding voltage pulse. Each value indicates mean ± s.e.m. Curve fittings in D and E were obtained by the Boltzmann equation. 19 Figure 7. Tch2, Tch3, and Tch4 effects on transient and persistent currents. IV relationship of IT is shown in A, C, and E. IV relationship of IP is shown in B, D, and F. Tch2 effect (A and B) Tch3 effect (C and D), and Tch4 effect (E and F) ware shown before (Control, black) and after 5 min (orange), 10 min (red), 15 min (purple) and 20 min (blue) of toxin exposure (n=2). Each value indicates mean ± s.e.m. 20 Effect of three different toxins on the activation and inactivation kinetics After observing the different effects on the Na+ IT when cells were exposed to the toxins, we proceeded to analyze the voltage dependency and sensibility by plotting the open probability of activation and inactivation particles as a function of voltage. With the exposure to Tch2, a ~10 mV negative shift in the activation curve was present after 5 and 10 min, which was then reversed by 5 mV at 20 min, and 30 min, a smaller voltage-dependency was also evident (bigger slope factor), especially at 30 min (Fig. 8A and Table II). Upon analyzing the inactivation curve, no strong effects of the toxin Tch2 were observed in voltage sensitivity (Fig. 8B, Table II). Still, a strong decrease in voltage dependency for inactivation was observed, evidenced by an increase in slope factor from 4.38 mV to 7.89 mV (Table III). These parallels with an increase in the inactivation time constant (τ) over exposure time (Table III), even leading to an incomplete inactivation (Fig. 8B, blue and green dots). To simultaneously analyze the effects of the toxin on both activation and inactivation, window current was calculated (Fig. 9). An increase in the window current was observed from the first ten min of exposure to Tch2, nearly doubling its value (Fig. 9, upper panel, red plot). However, as mentioned before, after 20 and 30 min of toxin exposure, a slight reversal in the effect was observed, evidenced by a reduction in the area, even though with the incomplete inactivation the window current is still more than 60% bigger than before toxin exposure (Fig. 9, upper panel, blue and green plots). Altogether, the increased currents reported at -30 mV in Figure 7A correlate with the positive shift observed in activation, and this shift increased the window current. Regarding the Tch3, the current decrease reported in Figure 7C was accompanied by a positive shift in voltage after 20 min of toxin exposure (Fig. 8C), the V1/2 shift from -32.08 to - 28.14 implied a decrease in voltage sensitivity with toxin exposure (Table II). Additionally, a strong decrease in the voltage dependency of the activation was identified (Table II). In the other way, in the inactivation curve, it was shown a negative shift in the relative current with the exposure time to Tch3 (Fig. 8D), with a strong change in V1/2 from -27.54 mV to -46.90 mV, a value very close to the resting membrane potential (Table III). Furthermore, the voltage dependence of the channel decreased, where the slope factor increased even after 5 min of toxin exposure (Table III). Finally, a change in the inactivation τ was observed, with a decrease also from 5 min of exposure (Table III). 21 It became evident that the current window gradually decreased with increasing exposure time, and the area was reduced by more than half at 20 min exposure (Fig. 9, middle panel). This reduction is consistent with previous observations, indicating that the toxin exerts an effect on the channel by decreasing its open probability and accelerating its inactivation at more negative membrane potentials, ultimately leading to the smaller currents reported in Figure 7C. When Tch4 was analyzed, a notable difference between the control activation curve and the toxin exposure curve was observed, with a negative shift (Fig. 8E). These changes are elucidated in Table II, where we can notice a change in the V1/2 value of -9 mV at 10 min of toxin exposure, that decreased to -4 mV after 20 min of exposure. Likewise, the slope factor decreased from 1.50 mV to 1.29 mV in the first 10 min of exposure, but then reversed and increased to 2.40 mV (Table II). There were also modifications in the inactivation curves compared to the control, not very clear in the plot of Figure 8F, however when analyzed through Boltzmann fitting, we saw a -6 mV negative shift, as well as a decrease in voltage dependency (increase in the k) and an increased inactivation τ (Table III). Analyzing the window current in the lower panel of Figure 9, a notable change was observed in the first few minutes, in detail, before toxin exposure, the activation open probability at -30 mV was around 50%, and the inactivation open probability at -10 mV was around 20%. However, after just 5 min of exposure, activation probability at -30 mV increased to 100% and inactivation probability at -10 mV decreased to 0%. Inactivation correlated with the value of the area under the curve, which decreased at 5 min. The effect persisted after 10 min of exposure, but reversed 10 min later, where the inactivation curve shows an open probability at the membrane potential of -10 mV exceeding 10%, with a larger area under the curves. Altogether, similar to Tch2, the increased currents reported at -30 mV in Figure 7E correlated with the positive shift observed in activation, however, this shift does not correlate with an increase in the window current. 22 Figure 8. Activation and inactivation kinetics under Tch2, Tch3, and Tch4 exposure. Relative conductance was plotted as a function of membrane potential (A, C, and E.), the curve represents the data fitted to the Boltzmann equation. Relative current was plotted as a function of the membrane potential of the preceding voltage pulse (B, D, and F), curves fitted to the Boltzmann equation. A-B. Exposure of Tch2. C-D. Exposure to Tch3. E-F. Exposure to Tch4. Cells were analyzed before (Control, black) and after toxin exposure for 5 min (orange), 10 min (red), 15 min (purple), 20 min (blue), and 30 min (green) (n=2). Each value indicates mean ± s.e.m. 23 Figure 9. Window current changes depending on the toxin exposition. The window current is observed by combining the activation curves (closed circles) generated as relative conductance (G/Gmax), and inactivation curves (open circles) calculated as relative current (I/Imax) in the same graph. The value of the window current area is denoted in each graph. The upper panel shows Tch2, the middle panel 24 shows Tch3, and the lower panel shows Tch4. Control (black) and toxin exposure for 10 min (red), 15 min (purple), 20 min (blue) (n=2). Each value indicates mean ± s.e.m. Table II. Open probability parameters of activation. V1/2 is the voltage corresponding to half-maximal activation, and k is the slope factor. All data are shown from n=2, as mean ± s.e.m. V1/2 (mV) k (mV) Toxin Time (min) 0 5 10 20 0 5 10 20 Tch2 -25.55 ± 0.52 -30.08 ± 3.09 -34.75 ± 1.58 -29.69 ± 3.64 1.5 ± 0.35 1.40 ± 0.09 1.29 ± 0.16 1.85 ± 0.32 Tch3 -32.08 ± 0.77 -31.85 ± 0.71 -28.07 ± 0.46 -28.14 ± 1.61 1.72 ± 0.21 1.62 ± 0.005 2.66 ± 0.07 3.18 ± 0.41 Tch4 -29.57 ± 4.84 -34.72 ± 0.14 -34.02 ± 0.57 -34.30 ± 0.36 2.03 ±0.65 1.27 ±0.02 1.23 ± 0.03 1.63 ± 0.23 25 Table III. Open probability parameters of steady-state inactivation. V1/2 is the voltage corresponding to half-maximal inactivation, and k is the slope factor. All data are shown from n=2, as mean ± s.e.m. V1/2 (mV) k (mV) Normalized τ Toxin Time (min) 0 5 10 20 0 5 10 20 0 5 10 2 0 Tch2 -16.22 ± 2.01 -16.77 ± 2.88 -17.82 ± 3.14 -18.79 ± 2.87 4.38 ± 1.25 4.72 ± 1.81 7.22 ± 0.43 7.89 ± 0.94 0.59 ± 0.15 0.79 ± 0.15 0.87 ± 0.11 1 Tch3 -27.54 ± 1.08 -32.00 ± 0.53 -38.00 ± 3.77 -46.90 ± 2.23 4.61 ± 0.20 6.24 ± 0.30 6.65 ± 1.71 6.85 ± 1.72 0.87 ± 0.13 0.49 ± 0.23 0.65 ± 0.35 Tch4 -16.33 ± 6.63 -26.24 ± 5.46 -22.33 ± 2.90 -22.64 ± 4.16 3.06 ± 2.73 5.19 ± 0.08 9.31 ± 1.44 10.55 ± 1.74 0.34 ± 0.03 0.53 ± 0.13 0.60 ± 0.17 1 26 All NaTxs analyzed from the venom reduced the probability of finding closed channels. The next step of the analysis was to examine whether the toxins affected the transition to the closed state using the degree of recovery from inactivation over the time spent at resting membrane potential. With the exposure to Tch2, an increase in the recovery was observed after 5 ms repolarization, reaching 50% of recovery within the first 10 min (Fig. 10A). This implies that the probability of finding closed channels, rather than inactive channels, is higher during this first 5 ms. However, a subsequent change in this pattern was observed, as the recovery percentage decreases with longer exposure time to Tch2, and longer times at resting membrane potentials, implying a 20% reduced availability of NaV channels on the membrane even after 75 ms in the long term. In the case of Tch3, a clear decrease in the recovery from inactivation was observed, especially after 20 min of toxin exposure (Fig. 10B), reaching almost 30% reduced availability of NaV channels. Then Tch4 was analyzed, and we saw a decrease in the recovery from inactivation after just 5 min of exposure, decreasing availability in the first milliseconds, however reaching a similar recovery to the control condition after 75 milliseconds at resting membrane potential (Figure 10 C). TchKTx5 is a toxin that blocks the macroscopic outward K+ current. Lastly, in the venom of the scorpion T. championi, the presence of a dominant toxin named TchKTx5 has been identified, which shows homology with KTxs in other Tityus venoms (Díaz et al., 2024). We tested the possible effects of this toxin on macroscopic K+ currents. The IV relationship of these currents showed that when exposed to the toxin a decrease in the steady-state recorded currents was evident after only 2 min of exposure (Figure 11). 27 Figure 10. Reduction in the recovery from inactivation by all toxins applied. A. Exposure to Tch2. B. Exposure to Tch3. C. Exposure to Tch4. Control (black) and toxin exposure for 5 min (orange), 10 min (red), 20 min (blue) and 30 min (green) (n=2). Each value indicates mean ± s.e.m. 28 Figure 11. Fast reduction of K+ current by toxin TchKTx5. Voltage-dependent K+ current was measured at different voltage steps (IV relationship) before (control, black) and after 2 min of exposure (red) to TchKTx5 (n=1). 29 DISCUSSION To analyze the electrophysiological effect of T. championi toxins, we used land snail C1 neurons as a cell model. Invertebrate neurons exhibit neuronal functioning similar to mammalian neurons (Lukyanetz & Sotkis, 1996). Given the high level of conservation in these processes, it is possible to study mechanisms in simpler systems and extrapolate them to more complex neuronal ones (Ierusalimsky et al., 2020). In this context, the serotonergic neuron C1 from the cerebral ganglion of Helix snails is commonly used due to its easy identification and high resistance to manipulation in vitro, and the identification of NaV1.7-like channel present in its membrane (Kiss et al., 2012; Brenes, 2022). The inhibitory effect on neuronal excitability of crude venom The protein pattern of the scorpion venom agrees with the findings reported by Díaz and colleagues (2023), describing the characteristic electrophoretic pattern of T. championi among other species within the Buthidae family. The bands with lower molecular weight likely correspond to several neurotoxins for Na+ and K+ channels, with amino acid sequences ranging between 30 and 70 residues (Borges et al., 2021), such as the toxins studied in the present work (Table I). The intense staining observed in these bands could suggest a higher concentration of these proteins or peptides in the sample, consistent with their attributed role as the primary molecules responsible for clinical effects in Tityus scorpion envenomation (Abroug et al., 2020; de Oliveira et al., 2018). On the other hand, in the middle section of the gel, distinct bands can be identified as hyaluronidases (46 kDa) and various proteases (37 kDa). And the higher molecular weight bands are probably enzymes, such as metalloproteases, serine proteases, endothelin-converting enzymes, angiotensin-converting enzymes, endopeptidases, among others identified in this venom through mass spectrometry (Supplementary Table SI). These enzymes may be associated with improved toxin dispersion in tissues, facilitating the degradation of extracellular matrix and enhancing the efficiency of toxin delivery to their target channels (Guerra-Duarte et al., 2019; Díaz et al., 2023). Initially, what caught our attention, regarding the effects of complete venom, was the reduction in the MFF of the cells (Figure 2A). This effect has been documented in the literature, where venom from other scorpion species has been shown to decrease firing frequency (Narahashi et al., 1972). Analyzing the major effect on AP morphology, a clear impact on depolarization was 30 observed (Figure 3C and D). In addition, when we tested the most abundant toxin in the venom (the NaTx Tch3) its effect on excitability agreed with the venom effect, decreasing Mean Firing Frequency and exhibiting reduced rise slope and increased depolarization time (Fig. 4 and 5). Since Nav1.7 channels have been identified as threshold channels for AP initiation in cells as nociceptors (McDermott et al., 2019; Alexandrou et al., 2016), it is reasonable that the impact of Tch3 and venom on these channels holds the potential to influence cellular firing. This reduction in MFF can also be attributed to the slow recovery from inactivation, or slow repriming, characteristic of this isoform, reported in mouse spinal sensory neurons (Herzog et al., 2003), with the venom making it harder for the channel to reopen, thereby reducing the macroscopic Na+ current. Collaço et al. (2019) tested venom from a sister species, T. bahiensis, at low (1 µg/ml) and high (10 µg/ml) concentrations, where low concentrations led to prolonged depolarization, enhancing neurotransmitter release and facilitating nerve-evoked muscle contraction. Conversely, at high concentrations, there was a blockade in AP generation and muscle twitches (Collaço et al., 2019), consistent with our findings. Some of these findings also agree with observations from studies on synaptic transmission inhibitors derived from tarantula toxins, which evoked reductions in AP slopes (Schmalhofer et al., 2008). On the contrary, there has been reported the induction of repetitive firing in response to short currents in the presence of the venom of scorpions as Centruroides suffusus, which contains β-NaTx in its venom (Couraud et al., 1982). Upon scrutinizing AP morphology, we note an alteration in repolarization when the venom was tested, characterized by an increase in slope and a decrease in duration (Figure 3E and F). However, it could be expected that the repolarization slope should decrease rather than increase because of TchKTx5 presence. This outcome diverges from the hypothesized result, yet it is conceivable that the influence of other toxins may obscure the overall repolarization effect of the venom. In conclusion of this part, the decline in AP firing highlights the intricacies and potency of scorpion venom in disrupting neural excitability and, therefore, nervous system function. Results suggested that the most affected phase is depolarization, which agrees with the fact that most of the identified toxins so far in T. championi are putative NaTx (Díaz et al., 2023). 31 Two types of Na+ inward currents were identified in C1 neuron The macroscopic Na+ currents generated appeared to be composed by two different currents, each of them with different behavior, a faster transient current and a slower persistent one, the IT and IP, respectively. In our work, the IT began to activate from -30 mV and reached its maximal magnitude at -20 mV, with a current close to 10 nA, which then becomes an output current with the Erev of +40 mV (Fig. 6B). in the other hand, IP has ten times less current (1 nA), its peak is at -10 mV, and its Erev is close to 0 mV (Fig. 6C). This IP is also found through NaV1.8- and NaV1.9-like channels in snail neurons, where the IT is significantly larger, up to more than six times the IP. Additionally, there is a notable shift in the Erev, which is more positive in IT than IP. Emphasizing that IT exhibits greater selectivity to Na+ compared to IP (Kiss et al., 2012). In fact, IP has been observed in gastropod neurons (Kiss, 2003; Staras et al., 2002) and giant squid axons (Clay, 2003). Similarly, a slow inward current, referred to as IB in some literature, was identified in mollusk cells, characterized by its significantly smaller amplitude, up to 100 times smaller than IT, and a prolonged decay, lasting several seconds (Adams et al., 1980). This current activates at more depolarized voltages, and run in parallel with larger currents, making individual observation challenging (Adams et al., 1980). C1 in H. aspersa, these two inward currents overlapped. It is inferred that this current might be generated by a different channel with low or mixed specificity (Adams et al., 1980), explaining the Erev close to 0 mV. And they can contribute to a prolonged depolarization, leading to a faster frequency of spikes, and can increase excitatory stimulus through the summation of excitatory postsynaptic currents (Adams et al., 1980). In vertebrates, particularly in mammals, functional diversity arises from multiple genes. This situation contrasts with invertebrates, where fewer genes are present, and diversity is attributed mainly to alternative splicing and RNA editing (Goldin, 2001; Zakon, 2012). This could explain why there is just one reported isoform expressed, because of slight differences in sequences that can result in extensive changes in channel behavior. This observation correlates with findings from a previous study (Magistretti et al., 1999) in which different channels, demonstrated by biophysical differences, mediate the IP current, channels exhibiting fast kinetics possess a conductivity of approximately ~15 pS, whereas those with slower kinetics exhibit ~20 pS conductivity. It is well known that scorpion toxins have been invaluable tools for the purification, identification, and functional characterization of channels (García et al., 2001). In this research, 32 we can also highlight the usefulness of T. championi toxins in detecting two currents, based on their different effects. For example, for Tch2 and Tch4, the IT's voltage sensitivity was affected (Fig. 7A and E), which did not happen in the IP (Fig. 7B and F). Furthermore, this difference between IT and IP becomes more noticeable when we see how Tch3 clearly affected the IT but not the IP (Fig. 7C and D). In general, IP can significantly influence several physiological functions (Goldin et al., 2001), it is suggested to possess both a commanding and intrinsic modulatory role. The IP current can enhance excitatory synaptic inputs, primarily by increasing both the amplitude and, more notably, the duration of the inputs. The IP recorded in the C1 neuron, an interneuron within a neural network involved in foraging and salivation, may contribute significantly to signal integration and response modulation (Kiss, 2003). The results obtained for IT and IP of C1 neurons resemble those obtained in other species. The supplementary Table SII summarizes several properties of both currents in different organisms. For example, for IT, snails of the same genius, H. pomatia expressed NaV1.7-like channels that showed similar behavior to the present study, a fast current with a maximal amplitude of approximately -24 nA at -20 mV and an Erev of +20 mV (Kiss et al., 2012). Other mollusks, such as freshwater snail Lymnaea sp., also exhibited a transient macroscopic Na+ current of at least 40 nA at a membrane potential of -35 mV and an Erev of +25 mV (Staras et al., 2002). These Na+ currents have also been studied in Aplysia sp. neurons and in the giant fiber lobe (GFL) neuron of the cephalopod Loligo sp., where they were activated at -30 mV, as in the case of this study, and reversed between +30 and +20 mV, respectively (Gilly et al., 1997). Hence, it can be shown that the behavior of this IT seems conserved in several orders of the phylum Mollusca. Looking at the activation and inactivation kinetics reported in some studies in more detail, the LPa3 and RPa3 neurons of H. pomatia, have different activation voltage dependence and sensitivity, with a V1/2 of -43 mV, and a k of 3.5 (Kiss, 2003), in comparison with -29.7 mV and 2.3, respectively in the present study. This negative shift in voltage sensitivity is also present in the inactivation kinetics, having a V1/2 of -43.2 mV (Kiss, 2003) compared to a V1/2 of -22.1 mV from our data. These variabilities may be due to differences in the cells studied, as there are differences in the expression of channel isoforms by cells in the ganglia (Kiss et al., 2012). 33 Looking at the inactivation characteristic within other gastropods several similarities can be found with our data. For example, inactivation V1/2 in Aplysia sp. had a value of -30 (Adams et al., 1980), in Pleurobranchea sp. a value of -23.7 and in Doripsilla sp. a value of -25.3 mV (Gilly et al., 1997), compared to the V1/2 of -22.1 mV from our study. Toxins effects on transient and persistent Na+ currents We will now discuss the activities of the toxins under investigation. Notably, Tch2 and Tch4 exhibited similar behaviors, both inducing a negative voltage shift. For instance, Tch2 was also found in the scorpions T. jaimei (called Tja2) and T. desdoslargos (called Tde2), and shares 91% homology with To6, a putative -NaTxs (Guerrero-Vargas et al., 2012; Diaz et al., 2023). However, despite the high homology with -NaTxs, our study suggests that it behaves more like a β-NaTxs. Both Tch2 and Tch4 evoked a shift in activation to more negative voltages after 5 and 10 min of treatment (Fig. 8A and C, Table II), a functional characteristic of β-NaTx (Catterall et al., 2007). In T. obscurus this kind of effect was described for To4, classified as a β-NaTx, where there is a shift in V1/2 to more hyperpolarized values in all human NaV isoforms (Duque et al., 2017). Also, in the β-NaTxs, there is an additional reported effect, a reduction in Na+ conductance (Duque et al., 2017). That is also evident for To1, which shows a negative shift and a decrease in macroscopic Na+ current (Tibery et al., 2019). In our study, when the cells were exposed to Tch2 and Tch4 instead of a decrease, we saw a small increase in IT (at -30 mV) and IP. Intriguingly, there was an increase in IP, in this matter, it has been reported that the toxin Tst1 causes incomplete inactivation of the NaV1.6 isoform, resulting in a different behavior by slowing down the inactivation process (da Mata et al., 2023). If this was our case, an incomplete inactivation in the NaV1.7-like channel could contaminate the current from the IP, which could explain why an increase in IP was observed. A similar case, a toxin that shared sequence homology with -NaTx but presented β-NaTx effect has been illustrated by the case of Ts17 toxin from T. serrulatus venom. Despite Ts17 sequence identity with Ts5 and Ts3 (both -NaTx) from the same species, detailed electrophysiological investigations unambiguously characterized Ts17 as a β-NaTx (Menezes et al., 2023). This divergence was attributed to the highly conserved three-dimensional structure of 34 both scorpion toxin groups, characterized by -helices and 3 to 4 antiparallel β-sheets, featuring cysteine residues critical for disulfide bridge formation (Quintero-Hernández et al., 2013). Noteworthy insights arise from molecular dynamics simulations conducted by Chen and Chung, indicating that both - and β-NaTx bind to receptor sites 3 and 4 respectively, in a consistent orientation within the binding pocket (Chen & Chung, 2012). This underscores that distinctions in functional outcomes may be more closely associated with specific amino acid residues pivotal for interactions in the specific receptor site. Upon comparison with toxins such as AaHIT from Androctonus australis, recognized as a β-NaTx selective for insects, CssII and CssIV from C. suffusus, β-NaTx selective for mammals, and TsVII from T. serrulatus, which is β-like (selective for mammals and insects), all of these demonstrate, an increase in the inward current by up to 5, 3, 9, and 6 times more than the control at -40 mV (Bosmans et al., 2007). Similarly, in our data Tch2 and Tch4 toxins induced Na+ current increase at a fixed voltage of −30 mV. The mechanism by which β-NaTx interacts with NaV channels is known as the voltage sensor-trapping model. In this model, the toxin anchors to the loops between segments S4 and S5 of motive II, keeping the channel in a pre-active state, and making it more sensitive to voltage depolarization (Zhang et al., 2012). Indeed, there is an increase in V1/2 by up to 10 and 5 mV more negative for Tch2 and Tch4, respectively, thereby making it more responsive to voltage changes and consequently inducing channel opening earlier than the control. This increase in the probability of channel opening could be associated with an excitatory effect of the toxin, similar to the effects observed with toxins like AaHIT from Androctonus australis and Bj-xtrlT from Buthotus judaicus (Bosmans et al., 2007). In these cases, an immediate and reversible effect of rapid contraction paralysis occurs, inducing spastic paralysis attributed to the general activation of skeletal musculature due to an excitatory presynaptic action on motor nerves that leads to repetitive firing (Oren et al., 1998). We observed that the window current increases but reverses its effect after 20 min of exposure to Tch2, exhibiting a fast and reversible effect, however, it does not return to a control state. This partial recovery behavior has also been reported for some -NaTxs, such as Tc49b and Ts4 (Batista et al., 2002b; Pucca et al., 2015). From an evolutionary point of view, it can be associated with the immediate time of the effect being sufficient for the scorpion to immobilize its 35 invertebrate prey with its pincers, and there might not be a strong selection for insect-selective toxins with excessively high binding affinity. However, it cannot be ruled out that Tch2 and Tch4 toxins can affect also mammals, as the effect of β-NaTx has been linked to defensive behavior, affecting channels expressed in humans such as NaV1.4 in skeletal muscle and NaV1.6 in the central nervous system, as seen with To1, Tma1, and Tpa2 (Rincón-Cortés et al., 2019). The reversibility of the toxins is consistent with observations that venom from T. obscurus shows a systemic effect hours after injection and reverses after 3 h in over 50% of animals (Santos da Silva et al., 2017). The inactivation of the current was influenced by the Tch2 and Tch4 toxins, with an increase in the k, indicating a reduced voltage dependence for inactivation. Simultaneously, a higher sensitivity was observed, with more negative V1/2 values and increased time constants (Table III). In a study of the β-NaTx Tst1 purified from T. stigmurus venom on different NaV isoforms, it was found that in three out of seven isoforms, V1/2 becomes more negative than in control (da Mata et al., 2023), resembling the case of Tch2 and Tch4. Additionally, for the Ts17 toxin, a more hyperpolarized V1/2 is observed for NaV1.7 channels (Menezes et al., 2023). An interesting observation is that in various studies, the k of inactivation, associated with voltage dependence, increases (da Mata et al., 2023; Menezes et al., 2023). Consistently, in our two β-NaTx, Tch2 and Tch4, there is an increase in the k, thus a decrease in voltage dependence, as evidenced by the less steep slope of the inactivation curve. It is noteworthy that, in most NaV isoforms exposed to the Ts17 toxin (except for hNaV1.5), the inactivation curve shows a decrease in k (Menezes et al., 2023). However, in the case of the Tst1 toxin, most isoforms exhibit an increase in k (da Mata et al., 2023). This suggests that despite the conservation of the sequence or the proximity of species, there is a specificity of action and isoform tart of these toxins. Now, analyzing Tch3 toxin, we observed a remarkable one-third reduction in the IT after a 20-min exposure at -30 and -20 mV, while the inward IP remained mostly unaffected. This reduction can be elucidated by studying open probability graphs, revealing a diminished probability of channel opening, due to a positive shift, causing channels to open at more depolarized potentials. There was also a reduction in the voltage dependence, requiring a greater voltage change to transition between states. Furthermore, not only did it require a larger change to initiate channel opening, but inactivation occurred at more negative potentials, evident as a 36 negative shift and a less steep slope. Collectively, these alterations led to less than half of the window current, culminating in a reduction in macroscopic IT. Discovered by Díaz and colleagues, Tch3 was proposed to function as a modulator of NaVs, as confirmed in this study. In their investigation, the toxin was also found in T. jaimei (called Tja3) and T.dedoslargos (called Tde3), and an 88% homology was found with To7 toxin from T. obscurus (Díaz et al., 2023). To7 shares 69 and 63% identity with the neurotoxins TdNa10 and TdNa9 from T. discrepans (Guerrero-Vargas et al., 2012). All these sequences display a resemblance to -NaTx. These toxins have a core domain, similar to Old World scorpion toxins (Gordon et al., 2007). These Old World buthid species, that possess -NaTx and high specificity for receptor site 3, induce the most significant harmful effects in children (Amr et al., 2021). However, it is important to highlight that the -NaTx characterization was made mostly regarding effects on mammalian channels, not in mollusk channels, so, we cannot categorize the effect of our Tch3 toxin as an -NaTx, even though there are sequence similarities with various scorpions from the same genus. This underscores the significance of the development of functional electrophysiological research on different animal models, like our research, and the improvement of toxins classification. Investigations involving T. obscurus shed light on the behavior of the Tc49b toxin, which deviates from the conventional characteristics of a typical scorpion -NaTx. Notably, it exerts an almost complete blocking effect on the current at a concentration of 100 nM in rat cerebellar granule cells (Batista et al., 2002b). This agrees with our findings, where we observe a reduction in current. This suggests that the ambiguity in classification is not exclusive to Tch3. Drawing parallels with pore-blocking peptides, we could conceptualize Tch3 as a blocking peptide, akin to the m-conotoxin. This peptide impacts Na+ permeation through the membrane and exhibits an affinity with another receptor site in the channel, specifically receptor site number 1 situated in the pore-forming loop responsible for the ionic selectivity filter (Moczydlowski et al., 1986; Chahine et al., 1995; Dudley et al., 1995). The incompleteness of the effect can be explained by variations in toxin binding within specific receptor site residues (Dudley et al., 1995; Chahine et al., 1995; Moczydlowski et al., 1986; Yanagawa et al., 1986). However, we cannot conclude that blocking is the main mechanism since this toxin affected biophysical channel characteristics. A distinct toxin, derived from a tarantula, named ProTx-II, provides intriguing insights. Classified as a Na+ channel blocker, ProTx-II exhibits noteworthy specificity for human NaV1.7 37 channels in comparison to other isoforms. Notably, it induces a significant shift in activation towards more positive potentials, similar to the impact observed with our Tch3 toxin. Moreover, it substantially reduces channel conductance (Schmalhofer et al., 2008). In 2019, Xu and collaborators proposed that ProTx-II acts as an electrostatic gating modifier. This mechanism involves the introduction of positive charges into the S3-S4 loop of the voltage sensor domain 2 (VSD2), identified as receptor site 4. The toxin neutralizes acidic residues with basic side chains, impeding the movement of S4 (Xu et al., 2019). This revelation unveils the binding mechanisms between toxins and NaV1.7, elucidating how a toxin can influence kinetics while simultaneously exerting an overall blocking effect. A similar trend is observed with the local anesthetic QX-314, a derivative of lidocaine. QX-314 induces a shift in the activation V1/2 to more positive voltages while simultaneously capable of inhibiting peak current in NaV1.7 channels (Klasfauseweh et al., 2022). Through mutant analysis, Klasfauseweh and colleagues conclude that pore blocking and restriction of the voltage sensor S4 movement can occur through multiple interactions between the positive charges of QX- 314 and the negatively charged residues of the channel (Klasfauseweh et al., 2022). Such convergence in the mechanisms of action between analgesics and scorpion toxins opens possibilities, proposing these toxins as potential agents for pathologies associated with pain and nociception. Regarding the time required to obtain these effects, Tch3 induced a significant change in activation and inactivation after 20 min of exposure, indicating a non-immediate response (Krisch et al., 1989). This latency in the toxin's effect has also been identified in studies with prey insects, exposing crickets to Tma2 and Tma3 (classified as insect -NaTx), where initial symptoms of disorientation and paralysis are observed, and insect death is recorded up to 4 h later (Rincón- Cortés et al., 2019). This effect is non-lethal at the moment but sufficient to facilitate predation on the animal. Moreover, the recovery from inactivation was examined after exposure to the three toxins, and it was concluded that all of them affect the availability of closed channels (Fig. 10). Similar observations were made for the Ts17 toxin, where recovery from inactivation was affected (Menezes et al., 2023). This indicates that akin to Ts17, these toxins stabilize a population of 38 channels in an inactive state, hindering their recovery to the closed state and subsequent opening upon further depolarizations. Interestingly, even if Tch2 and Tch4 increase NaVs voltage sensitivity, decreasing recovery from inactivation their final effect will be a reduction in cellular AP firing. Together with the inhibitory effects of the Tch3 toxin, all these T. championi NaTxs reduce cellular excitability. TchKTx5 effect on macroscopic K+ current. Lastly, it is known that the venom of T. championi does not exclusively contain NaTxs; rather, it is a complex mixture of more than 10 different molecules (Supplementary Table SI), including KTxs. In our case, we attested the dominant K+ toxin, called TchKTx5 (Díaz et al., 2023). The same sequence is found in TjaKTx5 of T. jaimei and TdeKTx5 of T. dedoslargos, with homology to peptides from T. obscurus (Díaz et al., 2023). A decrease in the K+ outward current was evident with just 2 min of exposure, a fast effect compared with the NaTxs presented here. The presence of KV channels in C1 metaneuron of Helix is known (Brenes et al., 2022), suggesting that the macroscopic K+ current in the steady-state is mainly generated from voltage-dependent channels, and we showed how the toxin affects these channels. This agrees with literature reports, where KTxs primarily act as blockers of the current from KVs. For instance, Ts6, known as butantoxin, has a blocking effect on more than five different KV isoforms (the most affected were Kv1.1, 1.2, and 1.3). In this Ts6 toxin, a shift in the activation curve to more depolarized potentials was observed, accompanied by a decrease in k (Cerni et al., 2014). It has been proposed that these toxins block conductance by occluding the extracellular region of the channel pore, involving a lysine and a hydrophobic residue that are fully exposed to interact with the channel (Srinivasan et al., 2002). This is a well-known molecular couple that disrupts K+ conduction (Banerjee et al., 2013), where electrostatic and hydrophobic interactions occlude the channel pore with their side chain (Lange et al., 2006). Over 120 K+ blockers from scorpion venom have been identified and classified into around 22 families with distinct primary and secondary structures and distinct target channels have been described (Cologna et al., 2011). We can theorize that the overall effect is similar, a blockage. 39 Some families exhibit higher affinity, resulting in complete blockage (-KTx) while others, like in our case, show lower affinity causing partial blockage (k-KTx) in specific regions where the target channels are expressed (Jiménez-Vargas et al., 2017). Martin-Euclaire and collaborators reviewed that depending on the toxin and its target (i.e., specific isoforms of K+ channels affected), the phenotypic effects can range from cardiovascular and muscular to immune system impacts (Martin-Eauclaire et al., 2016). Therefore, NaV and KV toxins in the venom do not compete; rather, they affect both Na+ and K+ currents in different ways, ultimately having different functional and pathological effects that serve the scorpion in its attack or defense mechanisms. Any disruption in the normal functioning of these channels can lead to neural activity disorders, muscle contraction issues, and nociceptor activity, etc. (Santos da Silva et al., 2017). The sequences of these toxins show homology with those from scorpions such as T. obscurus, known for causing a significant number of sting incidents in the Amazon (Pardal et al., 2003, 2014). While there are differences in epidemiology—mainly due to the lower probability of encounters between scorpions and humans in Costa Rica—the toxins and their effects are similar. This supports the hypothesis by Díaz and colleagues, who attribute the low incidence of severe scorpionism cases in Costa Rica to this reduced likelihood of interaction, as these species are allopatric in Costa Rican forests (Díaz et al., 2023). However, it cannot be ruled out that, interspecific variations caused by geographical isolation could influence the severity of stings, as these variations may be reflected in differences in specific amino acids (Nishikawa et al., 1994) or in the presence of distinct toxin profiles within the venom of Tityus species. CONCLUSIONS This study provides functional evidence of the effects of venom and specific toxins from the Costa Rican scorpion T. championi on mollusk neurons and their NaV and KV channels, which had not been previously reported in the literature. We showed how the T. championi venom exerted an inhibitory effect on cellular excitability, primarily through changes in the AP time course, at least partially induced by the Tch3 toxin, and during repetitive firing affected by Tch2 and Tch4 toxins. 40 We showed that Tch3 toxin exhibited an inhibitory effect in all the NaV characteristics analyzed. While Tch2 and Tch4 demonstrated excitatory effects in IV relationship, but an inhibitory effect of channel availability, with one of them (Tch2) showing reversible actions. Additionally, we confirmed the presence of a functional inhibitory KTx (TchKTx5) in T. championi venom, although this toxin seems to have no contributions to the venom effects on C1 AP repolarization. Furthermore, our findings suggest that the conventional classification of scorpion toxins targeting NaV may be inadequate to fully explain the toxin effects on different channels. Finally, this research described the macroscopic Na+ currents in the C1 neuron of H. aspersa, allowing a better understanding of the cellular functioning and thereby enhancing its value as a model for future studies, such as pharmacological screening. 41 CAPÍTULO II 42 Effect of the venom and selected fractions of Costa Rican scorpion Tityus championi on voltage-gated ion channels from mammals and insects Galit Akerman-Sánchez1,2, Steve Peigneur3, Kathleen Carter3, Cecilia Díaz4,5, Jan Tytgat3, Oscar Brenes1,6 1 Department of Physiology, School of Medicine, University of Costa Rica, San José, Costa Rica 2 Postgraduate Study System, School of Biology, University of Costa Rica, San José, Costa Rica 3 Toxicology and Pharmacology, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven (KU Leuven), Leuven, Belgium 4 Clodomiro Picado Institute, Faculty of Microbiology, Univesity of Costa Rica, Coronado, Costa Rica 5 Department of Biochemistry, School of Medicine, University of Costa Rica, San José, Costa Rica 6 Neuroscience Research Center, University of Costa Rica, San José, Costa Rica ABSTRACT Identifying the target channel isoforms of scorpion toxins is crucial for unraveling the mechanisms by which scorpions utilize their venom, as well as for channeling their potential pharmaceutical applications. In this study, we expressed various isoforms of voltage-gated sodium and potassium channels in Xenopus laevis oocytes to investigate the effects of Tityus championi´s whole venom and fractions separated by reverse-phase HPLC. The protein sequences of key components were identified through mass spectrometry analysis. Our findings revealed that the venom of Tityus championi exerts a potent blocking effect on the human Kv1.2 channel and a partial blockage of the Drosophila ShakerIR potassium channel. These effects were primarily attributed to the scorpion toxins TchKTx3, and a novel toxin identified in this study, named TchKTx7. Additionally, the venom contained classical scorpion alpha-toxins that affect NaV1.6, NaV1.7, and the cockroach BgNaV1 channels. It is worth noticing that the venom exhibited both excitatory and inhibitory effects, either enhancing and reducing the current depending on the channel and the venom fraction tested. These results provide better insights on the functional role of the venom in natural conditions for T. championi, and the complex interplay between venom components and ion 43 channels. Finally, we point to Tch3 as, at least, one of the responsible peptides for the inhibitory activity on nociceptive channel NaV1.7 and highlight its potential pharmacological application. INTRODUCTION Tityus championi, a scorpion endemic to Costa Rica and Panama, is a compelling subject of study due to its distinct ecological distribution and the varying severity of scorpionism cases in these two regions. In 2023, Díaz and colleagues, presented the first list of toxins found in T. championi venom from Costa Rica, identifying multiple putative sodium (NaTx) and potassium (KTx) channel toxins. That same year, Salazar et al., demonstrated that both the venom and the toxin-containing fractions from T. championi from Panama exhibit toxicity in mammals and insects (Salazar et al., 2023). While severe cases of scorpionism in Costa Rica are rare, likely due to the low frequency of human-scorpion encounters (Díaz et al., 2023), Panama has seen an increase in morbidity and fatalities, with T. championi recognized as a medically significant species (Ministry of Health, 2017). The venom’s LD50 of T. championi from Panama is lower (3 mg/kg) compared to Costa Rica (4 mg/kg) (Brenes & Gómez, 2016), and human-scorpion interactions are more frequent in Panama (Salazar et al., 2023). These findings, alongside the growing number of severe scorpionism cases in Panama, make investigating the venom of T. championi particularly relevant. Although it is known from our work (Akerman-Sánchez et al., unpublished), that the toxins in T. championi venom target sodium (Nav) and potassium (Kv) channels in mollusks, the isoforms these toxins act upon in mammals and insects remain unidentified. Determining these molecular targets is crucial for understanding the mechanisms behind envenomation's neuronal and systemic effects (Isbister & Bawaskar, 2014; Godoy et al., 2021) and the identification of these targets could open the way for exploring potential clinical applications. Several scorpion toxins, such as chlorotoxin, maurotoxin, and BmK AGAP, have followed this research path and now show therapeutic potential, offering promising insights into treatments for conditions such as cancer, autoimmune diseases, and neurological disorders (Molavinia et al., 2024; Shakeel et al., 2023; Todesca et al., 2024; Kampo et al., 2019). This study aims to characterize the electrophysiological 44 impact of T. championi venom and some of its fractions on mammal and insect NaV and KV channel isoforms expressed in a heterologous model. EXPERIMENTAL PROCEDURES Venom The venom of T. championi was obtained from the Dangerous Animals Research Laboratory (LIAP) at the Clodomiro Picado Institute (ICP). This part of the study was approved by the Biodiversity Commission of University of Costa Rica (No. 293-2021). Venom recollected from several specimens maintained in their animal facility was pulled, lyophilized, and diluted in autoclaved distilled water to achieve a concentration of 7.5 mg/ml. Separation of the major components of the venom through RP-HPLC A total amount of 7.5 µg crude venom of T. championi was further separated by reversed- phase HPLC using a Shim-pack Arata C18 column with dimensions of 4.6 × 250 mm and particle size of 5 µM with a linear gradient from 0% to 60% of solution B (0.1% TFA in ACN, vol/vol) in solution A (0.1% TFA in water, vol/vol) at a flow rate of 1 mL/min over 80 min. The absorbance was monitored at wavelengths of 214 and 280 nm. Eluting compounds were collected in fractions and dried with the Speed-Vac (Genevac™ model miVac DNA 23050-B0). Each dried fraction was then resuspended in 30 uL of ND96 buffer (96 mM NaCl, 2 mM MgCl2, 2 mM KCl, and 5 mM HEPES; pH 7.5), and kept at -20 °C until electrophysiological evaluations. Identification of the major components of the venom Following HPLC, venom fractions were separated by SDS-PAGE (15-20% gradient polyacrylamide commercial gel) under reducing conditions (prior step of 10 min at 98 °C with 5% 2-mercaptoethanol) (Isotemp, Fischer Scientific). The low molecular weight ladder BLUeye Prestained Protein (94964, Sigma-Aldrich) was used. Subsequently, the gel was allowed to run for one hour at 150 V using a Mini-Protean Tetra System coupled with PowerPac Basic power supply (BioRad), and proteins were stained with Coomassie Blue R-250. 45 SDS-PAGE bands were excised from gels and subjected to reduction with dithiothreitol (10 mM) and alkylation with iodoacetamide (50 mM), followed by overnight in-gel digestion with sequencing-grade bovine trypsin (Sigma Chemical Co.) in an automated workstation (Intavis). The resulting peptides were analyzed by nESI--MS/MS using a nano-Easy® 1200 chromatograph and a Q-Exactive Plus® mass spectrometer (Thermo). 5 µL of each tryptic digest were loaded on a C18 trap column (75 μm × 2 cm, 3 μm particle; PepMap, Thermo), washed with 0.1% formic acid (solution A), and separated at 200 nL/min with a 3 µm particle, 15 cm × 75 µm C18 Easy-spray® analytical column using the following gradient toward solution B (80% acetonitrile, 0.1% formic acid): 1-5% B in 1 min, 5-25% B in 30 min, 25-79% B in 6 min, 79-99% B in 2 min, and 99% B in 6 min, for a total time of 45 min (Lomonte and Fernández, 2022). MS spectra were acquired in positive mode at 1.9 kV, with a capillary temperature of 200°C, using 1 scan at 400-1600 m/z, maximum injection time of 100 msec, AGC target of 3×106, and orbitrap resolution of 70,000. The top 10 ions with 2-5 positive charges were fragmented with AGC target of 1×105, maximum injection time of 110 msec, resolution of 17,500, loop count of 10, isolation window of 1.4 m/z, and a dynamic exclusion time of 5 sec. MS/MS spectra were processed for the assignment of peptide matches to known protein families by similarity with sequences contained in the UniProt/SwissProt database (Scorpion, 2024) using Peaks X® (Bioinformatics Solutions). Cysteine carbamidomethylation was set as a fixed modification, while deamidation of asparagine or glutamine and methionine oxidation were set as variable modifications, allowing up to 3 missed cleavages by trypsin. Xenopus laevis frogs and the isolation of oocytes by partial ovariectomy All experiments conducted in this study received approval from the Ethical Committee for Animal Research at KU Leuven. Overall experiments and procedures involving Xenopus laevis frogs were performed as previously described (Peigneur et al., 2012b). Briefly, before harvesting stage V-VI oocytes from ovarian tissue, adult female X. laevis frogs underwent immersion in an aqueous solution containing 0.1% buffered tricaine (ethyl 3-aminobenzoate methanesulfonate, 1 g/L, Sigma-Aldrich, USA) and NaHCO3 (sodium bicarbonate, 1 g/L; Sigma-Aldrich, USA) in aquarium water (pH 7.5) for a duration of 20 min. Following the recovery period, the frogs were monitored daily and returned to their tanks at the Aquatic Facility of KU Leuven. The surgically 46 removed ovarian lobes underwent enzymatic defolliculation in a Ca2+-free ND96 solution (96 mM NaCl, 2 mM KCl, 2 mM MgCl2, and 5 mM HEPES), supplemented with collagenase from Clostridium histolyticum type IA, 1.5 mg/mL; Sigma-Aldrich, USA) on a rocker platform at 16 °C for 2.5 h. Following enzymatic defolliculation, the oocytes were transferred to a calcium containing ND96 buffer (96 mM NaCl, 2 mM MgCl2, 2 mM KCl, 5 mM HEPES, and 1.8 mM CaCl2; pH 7.5) supplemented with gentamicin (100 mg/L; Schering-Plough, Belgium) and theophylline (90 mg/L; ABC chemicals, Belgium) at 16 °C. In vitro transcription of cDNA clones Molecular biology techniques (subcloning, transformation, linearization and transcription) were used to make RNA encoding: (1) voltage-gated sodium channels (rNav1.2, rNav1.3, rNav1.4, hNav1.5, rNav1.6, rNav1.7, and BgNav) and their auxiliary subunits rβ1, hβ1, and TiPE β and, (2) potassium channels (hKv1.1, hKv1.2, hKv1.3, hKv1.4, hKv1.5, hKv1.6, hKv10.1, and ShakerIR) (Table SI). Plasmids corresponding to each channel underwent linearization using specific restriction enzymes and subsequent transcription with the T7 or SP6 mMESSAGE mMACHINE transcription kit (Ambion, USA). Depending on the channel type, defolliculated oocytes were injected with 5–20 nL of cRNA at a concentration of 1 ng/nL using a micro-injector (Micro2T SmarTouchTM, World Precision Instruments). Electrophysiological experiments were conducted following cRNA injection, with an incubation period of 1–5 days at 16 °C in ND96 buffer. Electrophysiological recordings with a two‑electrode voltage-clamp (TEVC) A two-electrode voltage-clamp (TEVC) system was performed with a GeneClamp 500 amplifier (Molecular Devices, Downingtown, PA, USA), under the control of pClamp data acquisition system (Axon Instruments, USA), and pClamp Clampex 10.4 software (Axon Instruments®, USA). This setup was employed to measure currents across the cell membrane. The whole-cell currents from the oocytes were recorded at room temperature (18-22 °C). Two microelectrodes, comprising voltage and current electrodes, were crafted from borosilicate glass capillaries (1.14 mm outside diameter, 0.7 mm inside diameter), pulled using a microelectrode puller, PUL-1 (World Precision Instruments, USA). These electrodes were filled with 3 M KCl 47 using a MicroFill needle, and their resistance was maintained between 0.5 and 1.5 MΩ. Throughout the measurements, oocytes were positioned in a 200 µL recording chamber. A membrane test was initially conducted to adjust measurement parameters based on the membrane quality. After the expression check of the voltage-gated ion channel, the venom of its fractions was applied until a stable effect on the channel was reached. In all experiments, 0.6 µg of the venom from T. championi was directly pipetted into the bath, resulting in a final concentration of 0.15 µg/µL tested and the fractions were tested at a final concentration of 2 µM. For voltage-gated sodium and potassium channel protocols, the recorded currents were sampled at 20 kHz for NaVs and BgNav1, and 10 kHz for KV and ShakerIR. Via a four-pole low- pass Bessel filter, the currents were filtered at 2 kHz for NaVs, and 500 MHz for KVs, and ShakerIR. Leak subtraction was performed using a −P/4 protocol. NaV traces used to monitor effects were generated through 100 ms depolarizations to 0 mV from a baseline of -90 mV. Standard current- voltage (IV) relationships for Na+ and K+ channels, were performed using 50 ms step depolarizations ranging from −90 to +65 mV with 5 and 10 mV increments, respectively from a baseline of -90 mV. A two-step protocol was used for steady-state inactivation analyzes, involving a 100 ms pulse from −90 to 0 mV with a 5 mV step, followed immediately by a test pulse to 0 mV. To obtain the channel opening probability, the conductance (gNa) was calculated as 𝑔 = ( ) , where Ipeak is the peak current recorded, Vm is the voltage step and Vrev is the reversal potential. The opening probability (Po) was calculated by the normalization of the conductance with the maximum conductance and was plotted as a function of voltage. The data were fitted using the Boltzmann equation 𝐼(𝑉) = / , where V1/2 represents the voltage where half of the channels are activated, and k is the slope factor. Data and statistical analysis of the electrophysiological experiments All data were presented as the means ± standard error of the mean (s.e.m.). All experiments were replicated at least three times (n≥3). GraphPad Prism version 10 (GraphPad Software, USA) was employed for parametric statistical analysis. The normal distribution was assessed by 48 Kolmogorov–Smirnov and Shapiro–Wilk normality tests and the two-way ANOVA followed by Bonferroni post-hoc test was performed to assess significance between groups. Non-parametric statistical analysis was performed in RStudio (Version 2023.06.0+421) using the paired Wilcoxon test. Significance level was set at p≤0.05, and denoted as follows: *p≤0.05, **p<0.01, ***p<0.001, and ****p<0.0001. The specific results of the statistical tests, including F values, degrees of freedom, and p-values, are reported in the text as “exp” for venom exposure and “int” for the interaction between voltage and venom exposure. RESULTS Using heterologous expression in frog oocytes and TEVC, the effect of venom at an approximate concentration of 2 µM was evaluated on different isoforms of ion channels. For KV channels, the venom had a significant impact on six of the eight tested channels (KV1.1-1.3, KV1.5- 1.6, and ShakerIR) (Fig. 1). In the case of the KV1.1 channel, exposure to the venom resulted in up to 7% reduction in current compared the control (F(1,112) exp = 40.8, p < 0.0001; F(15,112) int = 2.37, p = 0.0052). The KV1.2 channel exhibited the most substantial effect, with up to a 70% reduction in current compared to the control (p wilcoxon = 0.0006) (Fig. 1 and Fig. 2, left panel). For the KV1.3 channel, only half of the venom concentration was applied, leading up to a 12% decrease in current at the highest depolarized voltages (F(1,16) exp = 146, p < 0.0001; F(15,16) int = 4.95, p = 0.0014). The venom's effect on the KV1.5 channel was distinct from the others in two key ways: first, the current was only affected at positive voltages, and second, the venom increased the outward current, with an up to 9% change (F(1,88) exp = 19.5, p < 0.0001; F(15,88) int = 1.17, p = 0.3124). For the KV1.6 channel, differences in current amplitude were observed from -20 mV, with the inhibition becoming more pronounced as the cell depolarized (F(1,91) exp = 147, p < 0.0001; F(15,91) int = 12.1, p < 0.0001), reaching up to a 10% reduction. In the case of KV10.1, no effects were observed. The venom's effect was also tested on an invertebrate KV channel, the ShakerIR from D. melanogaster, where a pronounced inhibition was observed, with up to 31% reduction in current compared to control conditions (F(1,48) exp = 95.5, p < 0.0001; F(15,48) int = 5.53, p < 0.0001). In some cells, the current was inhibited up to 50% following venom exposure (Fig. 2, right panel). 49 After studying the effect of the complete venom, the next step was to separate some of its principal fractions to identify the toxins that could been generating the effect observed on KV1.2 and ShakerIR channels. Chromatographic runs were carried out, successfully separating the components into fractions. One representative chromatogram is shown in Figure 3, small individual peaks were observed with an elution time from minute 29 to minute 37. After this, additional peaks with higher absorbance are detected until minute 48. 50 Figure 1. Effect of Tityus championi venom on expressed isoforms of KV from human and insect. All channels were tested using a concentration of 0.15 µg/µL (approximately 2 µM), except for the KV1.3 channel, where half of the venom concentration, 0.075 µg/µL (approximately 1 µM), was used. Control conditions are shown in black and venom exposure in blue. KV1.1 (n=8), KV1.2 (n=3), KV1.3 (n=2), KV1.4 (n=4), KV1.5 (n=7), KV1.6 (n=7), KV10.1 (n=3), and ShakerIR (n=4). Each value indicates mean ± s.e.m. 51 Figure 2. Representative currents of the KV1.2 and ShakerIR channels. These two channels exhibited the most significant reduction in current following venom exposure (blue recording) regarding control conditions (black recording). Figure 3. RP-HPLC separation of T. championi venom using a linear acetonitrile gradient. Panels A and B present two independent chromatographic runs conducted under identical conditions. The labeled peaks correspond to the fractions that were selected for further analysis. KV1.2 ShakerIR 52 Seven selected fractions were analyzed using TEVC and SDS-PAGE polyacrylamide gel electrophoresis. This allowed the correlation of the functional effects with the qualitative comparison of the molecular weights of the components in these fractions and an initial assessment of their purity. The main protein components of