The dispensability of 14-3-3 proteins for the regulation of human cardiac sodium channel Nav1.5.

BACKGROUND: 14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial. AIMS: In this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5. METHODS AND RESULTS: Amongst the seven 14-3-3 isoforms, only 14-3-3η (encoded by YWHAH gene) weakly co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Nav1.5 α-α subunits. The current-voltage relationship and the amplitude of Nav1.5-mediated sodium peak current density were also not changed. CONCLUSIONS: Our findings illustrate that the direct implication of 14-3-3 proteins in regulating Nav1.5 is not evident in a transformed human kidney cell line tsA201.

Dual receptor-sites reveal the structural basis for hyperactivation of sodium channels by poison-dart toxin batrachotoxin.

The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin's potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.

Experimental and computational evidence that Calpain-10 binds to the carboxy terminus of NaV1.2 and NaV1.6.

Voltage-gated sodium channels (NaV) are pivotal proteins responsible for initiating and transmitting action potentials. Emerging evidence suggests that proteolytic cleavage of sodium channels by calpains is pivotal in diverse physiological scenarios, including ischemia, brain injury, and neuropathic pain associated with diabetes. Despite this significance, the precise mechanism by which calpains recognize sodium channels, especially given the multiple calpain isoforms expressed in neurons, remains elusive. In this work, we show the interaction of Calpain-10 with NaV's C-terminus through a yeast 2-hybrid assay screening of a mouse brain cDNA library and in vitro by GST-pulldown. Later, we also obtained a structural and dynamic hypothesis of this interaction by modeling, docking, and molecular dynamics simulation. These results indicate that Calpain-10 interacts differentially with the C-terminus of NaV1.2 and NaV1.6. Calpain-10 interacts with NaV1.2 through domains III and T in a stable manner. In contrast, its interaction with NaV1.6 involves domains II and III, which could promote proteolysis through the Cys-catalytic site and C2 motifs.

Molecular Pharmacology of Selective NaV1.6 and Dual NaV1.6/NaV1.2 Channel Inhibitors that Suppress Excitatory Neuronal Activity Ex Vivo.

Voltage-gated sodium channel (NaV) inhibitors are used to treat neurological disorders of hyperexcitability such as epilepsy. These drugs act by attenuating neuronal action potential firing to reduce excitability in the brain. However, all currently available NaV-targeting antiseizure medications nonselectively inhibit the brain channels NaV1.1, NaV1.2, and NaV1.6, which potentially limits the efficacy and therapeutic safety margins of these drugs. Here, we report on XPC-7724 and XPC-5462, which represent a new class of small molecule NaV-targeting compounds. These compounds specifically target inhibition of the NaV1.6 and NaV1.2 channels, which are abundantly expressed in excitatory pyramidal neurons. They have a > 100-fold molecular selectivity against NaV1.1 channels, which are predominantly expressed in inhibitory neurons. Sparing NaV1.1 preserves the inhibitory activity in the brain. These compounds bind to and stabilize the inactivated state of the channels thereby reducing the activity of excitatory neurons. They have higher potency, with longer residency times and slower off-rates, than the clinically used antiseizure medications carbamazepine and phenytoin. The neuronal selectivity of these compounds is demonstrated in brain slices by inhibition of firing in cortical excitatory pyramidal neurons, without impacting fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model, whereas XPC-7224 does not, suggesting a possible requirement of Nav1.2 inhibition in 0-Mg2+- or 4-AP-induced brain slice seizure models. The profiles of these compounds will facilitate pharmacological dissection of the physiological roles of NaV1.2 and NaV1.6 in neurons and help define the role of specific channels in disease states. This unique selectivity profile provides a new approach to potentially treat disorders of neuronal hyperexcitability by selectively downregulating excitatory circuits.

Analgesic potential of voltage gated sodium channel modulators for the management of pain.

Neuronal electrochemical signals involve the flux of sodium ions through voltage-gated sodium channels (NaV) located in the neurolemma. Of the nine sodium channel subtypes, NaV-1.7, 1.8, and 1.9 are predominantly located on nociceptors, making them prime targets to control pain. This review highlights some of the latest discoveries targeting NaV channel activity, including: (1) charged local anaesthetic derivatives; (2) NaV channel toxins and associated small peptide blockers; (3) regulation of NaV channel accessory proteins; and (4) genetic manipulation of NaV channel function. While the translation of preclinical findings to a viable treatment in humans has remained a challenge, a greater understanding of NaV channel physiology could lead to the development of a new stream of therapies aimed at alleviating chronic pain.

Correlation between pyrethroid knockdown resistance and mutation frequency of voltage-gated sodium channel and its application in Aedes aegypti management.

Aedes aegypti, the primary vector responsible for transmitting dengue fever in southern Taiwan, has developed a relatively high resistance to synthetic pyrethroids. It has evolved four amino acid substitutions in the voltage-gated sodium channel (VGSC), namely S996P, V1023G, F1565C, and D1794Y. To unveil the distribution and correlation of VGSC mutations and pyrethroid resistance among different field populations, Ae. aegypti collected from various districts in Kaohsiung and Tainan Cities underwent tests for resistance development against different pyrethroids and frequency of S996P, V1023G, F1565C, and D1794Y substitutions. The adult knockdown assay revealed a relatively high knockdown resistance in the Ae. aegypti populations from Kaohsiung and Tainan against permethrin, cypermethrin, and fenvalerate (averaging >50-fold). Conversely, less resistance was observed against α-cypermethrin, deltamethrin, λ-cyhalothrin, cyfluthrin, and etofenprox (averaging <35-fold). Using Polymerase Chain Reaction/restriction fragment length polymorphism analysis, four mutant haplotypes were identified in these field populations. Notably, the SIAVFD and SIBVFD wild haplotypes were absent. Analysis utilizing IBM SPSS Statistics 20.0 and Spearman's rank correlation coefficient indicated that Haplotype C (PIAGFD), especially P allele, frequency displayed a significant positive correlation with five Type II pyrethroid resistance, while 1023G and 1023G/G exhibited a significant association with permethrin and fevalerate resistance. Conversely, Haplotype E (SIBVCD) negatively correlated with pyrethroid resistance, particularly fenvalerate resistance (-0.776). Haplotype C and E were the most prevalent and widely distributed among the investigated field populations. This prevalence of haplotype C is likely tied to the extensive and excessive use of Type II pyrethroids for dengue control over the past three decades. Given the significant positive correlation, the best-fit lines and R2 values were established to facilitate the swift prediction of knockdown resistance levels to various pyrethroids based on VGSC mutation frequency. This predictive approach aims to guide insecticide usage and the management of pyrethroid resistance in the field populations of Ae. aegypti in Taiwan.

The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction.

BACKGROUND: Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. METHODS: A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively. RESULTS: Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential. CONCLUSIONS: Genetic ablation of mapre2 led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.

Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

A structural atlas of druggable sites on Nav channels.

Voltage-gated sodium (Nav) channels govern membrane excitability by initiating and propagating action potentials. Consistent with their physiological significance, dysfunction, or mutations in these channels are associated with various channelopathies. Nav channels are thereby major targets for various clinical and investigational drugs. In addition, a large number of natural toxins, both small molecules and peptides, can bind to Nav channels and modulate their functions. Technological breakthrough in cryo-electron microscopy (cryo-EM) has enabled the determination of high-resolution structures of eukaryotic and eventually human Nav channels, alone or in complex with auxiliary subunits, toxins, and drugs. These studies have not only advanced our comprehension of channel architecture and working mechanisms but also afforded unprecedented clarity to the molecular basis for the binding and mechanism of action (MOA) of prototypical drugs and toxins. In this review, we will provide an overview of the recent advances in structural pharmacology of Nav channels, encompassing the structural map for ligand binding on Nav channels. These findings have established a vital groundwork for future drug development.

Expression and electrophysiological characteristics of VGSC during mouse myoblasts differentiation.

Voltage-gated sodium channels (VGSC) are essential for triggering and relaying action potentials (AP), which perform critical functions in a variety of physiological processes, such as controlling muscle contractions and facilitating the release of neurotransmitters. In this study, we used a mouse C2C12 cell differentiation model to study the molecular expression and channel dynamics of VGSC and to investigate the exact role of VGSC in the development of muscle regeneration. Immunofluorescence, Real-time quantitative polymerase chain reaction, Western blot, and whole-cell patch clamp were employed for this purpose in mouse myoblasts. The findings revealed an increase in intracellular sodium concentration, NaV1.4 gene expression, and protein expression with the progress of differentiation (days 0, 1, 3, 5 and 7). Furthermore, VGSC dynamics exhibit the following characteristics: ① The increase of sodium current (INa); ② The decrease in the activation threshold and the voltage trigger maximum of INa; ③ A positive shift in the steady-state inactivation curve; ④ The recovery of INa during repolarization is delayed, the activity-dependent decay rate of INa was accelerated, and the proportionate amount of the fraction of activated channels was reduced. Based on these results, it is postulated that the activation threshold of AP could be decreased, and the refractory period could be extended with the extension of differentiation duration, which may contribute to muscle contraction. Taken together, VGSC provides a theoretical and empirical basis for exploring potential targets for neuromuscular diseases and other therapeutic muscle regeneration dysfunctions.

LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons.

The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and selectively localized to the axon initial segment (AIS), the subcellular compartment triggering action potentials. Ectopic expression of LRRC37B in mouse CPNs in vivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. Molecularly, LRRC37B binds to the secreted ligand FGF13A and to the voltage-gated sodium channel (Nav) ß-subunit SCN1B. LRRC37B concentrates inhibitory effects of FGF13A on Nav channel function, thereby reducing excitability, specifically at the AIS level. Electrophysiological recordings in adult human cortical slices reveal lower neuronal excitability in human CPNs expressing LRRC37B. LRRC37B thus acts as a species-specific modifier of human neuron excitability, linking human genome and cell evolution, with important implications for human brain function and diseases.

Voltage gated sodium and calcium channels: Discovery, structure, function, and Pharmacology.

Voltage-gated sodium channels initiate action potentials in nerve and muscle, and voltage-gated calcium channels couple depolarization of the plasma membrane to intracellular events such as secretion, contraction, synaptic transmission, and gene expression. In this Review and Perspective article, I summarize early work that led to identification, purification, functional reconstitution, and determination of the amino acid sequence of the protein subunits of sodium and calcium channels and showed that their pore-forming subunits are closely related. Decades of study by antibody mapping, site-directed mutagenesis, and electrophysiological recording led to detailed two-dimensional structure-function maps of the amino acid residues involved in voltage-dependent activation and inactivation, ion permeation and selectivity, and pharmacological modulation. Most recently, high-resolution three-dimensional structure determination by X-ray crystallography and cryogenic electron microscopy has revealed the structural basis for sodium and calcium channel function and pharmacological modulation at the atomic level. These studies now define the chemical basis for electrical signaling and provide templates for future development of new therapeutic agents for a range of neurological and cardiovascular diseases.

The elusive Nav1.7: From pain to cancer.

Voltage-gated sodium channels (Nav) are protein complexes that play fundamental roles in the transmission of signals in the nervous system, at the neuromuscular junction and in the heart. They are mainly present in excitable cells where they are responsible for triggering action potentials. Dysfunctions in Nav ion conduction give rise to a wide range of conditions, including neurological disorders, hypertension, arrhythmia, pain and cancer. Nav family 1 is composed of nine members, named numerically from 1 to 9. A Nax family also exists and is involved in body-fluid homeostasis. Of particular interest is Nav1.7 which is highly expressed in the sensory neurons of the dorsal root ganglions, where it is involved in the propagation of pain sensation. Gain-of-function mutations in Nav1.7 cause pathologies associated with increased pain sensitivity, while loss-of-function mutations cause reduced sensitivity to pain. The last decade has seen considerable effort in developing highly specific Nav1.7 blockers as pain medications, nonetheless, sufficient efficacy has yet to be achieved. Evidence is now conclusively showing that Navs are also present in many types of cancer cells, where they are involved in cell migration and invasiveness. Nav1.7 is anomalously expressed in endometrial, ovarian and lung cancers. Nav1.7 is also involved in Chemotherapy Induced Peripheral Neuropathy (CIPN). We propose that the knowledge and tools developed to study the role of Nav1.7 in pain can be exploited to develop novel cancer therapies. In this chapter, we illustrate the various aspects of Nav1.7 function in pain, cancer and CIPN, and outline therapeutic approaches.

Voltage-gated sodium channels, sodium transport and progression of solid tumours.

Sodium (Na+) concentration in solid tumours of different origin is highly dysregulated, and this corresponds to the aberrant expression of Na+ transporters. In particular, the α subunits of voltage gated Na+ channels (VGSCs) raise intracellular Na+ concentration ([Na+]i) in malignant cells, which influences the progression of solid tumours, predominantly driving cancer cells towards a more aggressive and metastatic phenotype. Conversely, re-expression of VGSC ß subunits in cancer cells can either enhance tumour progression or promote anti-tumourigenic properties. Metastasis is the leading cause of cancer-related mortality, highlighting an important area of research which urgently requires improved therapeutic interventions. Here, we review the extent to which VGSC subunits are dysregulated in solid tumours, and consider the implications of such dysregulation on solid tumour progression. We discuss current understanding of VGSC-dependent mechanisms underlying increased invasive and metastatic potential of solid tumours, and how the complex relationship between the tumour microenvironment (TME) and VGSC expression may further drive tumour progression, in part due to the interplay of infiltrating immune cells, cancer-associated fibroblasts (CAFs) and insufficient supply of oxygen (hypoxia). Finally, we explore past and present clinical trials that investigate utilising existing VGSC modulators as potential pharmacological options to support adjuvant chemotherapies to prevent cancer recurrence. Such research demonstrates an exciting opportunity to repurpose therapeutics in order to improve the disease-free survival of patients with aggressive solid tumours.

Voltage-gated sodium channels in cancer and their specific inhibitors.

Voltage-gated sodium channels (VGSCs) participate in generating and spreading action potentials in electrically excited cells such as neurons and muscle fibers. Abnormal expression of VGSCs has been observed in various types of tumors, while they are either not expressed or expressed at a low level in the matching normal tissue. Hence, this abnormal expression suggests that VGSCs confer some advantage or viability on tumor cells, making them a valuable indicator for identifying tumor cells. In addition, overexpression of VGSCs increased the ability of cancer cells to metastasize and invade, as well as correlated with the metastatic behavior of different cancers. Therefore, blocking VGSCs presents a new strategy for the treatment of cancers. A portion of this review summarizes the structure and function of VGSCs and also describes the correlation between VGSCs and cancers. Most importantly, we provide an overview of current research on various subtype-selective VGSC inhibitors and updates on ongoing clinical studies.

NaX Channel Is a Physiological [Na+] Detector in Oxytocin- and Vasopressin-Releasing Magnocellular Neurosecretory Cells of the Rat Supraoptic Nucleus.

The Scn7A gene encodes NaX, an atypical noninactivating Na+ channel, whose expression in sensory circumventricular organs is essential to maintain homeostatic responses for body fluid balance. However, NaX has also been detected in homeostatic effector neurons, such as vasopressin (VP)-releasing magnocellular neurosecretory cells (MNCVP) that secrete VP (antidiuretic hormone) into the bloodstream in response to hypertonicity and hypernatremia. Yet, the physiological relevance of NaX expression in these effector cells remains unclear. Here, we show that rat MNCVP in males and females is depolarized and excited in proportion with isosmotic increases in [Na+]. These responses were caused by an inward current resulting from a cell-autonomous increase in Na+ conductance. The Na+-evoked current was unaffected by blockers of other Na+-permeable ion channels but was significantly reduced by shRNA-mediated knockdown of Scn7A expression. Furthermore, reducing the density of NaX channels selectively impaired the activation of MNCVP by systemic hypernatremia without affecting their responsiveness to hypertonicity in vivo These results identify NaX as a physiological Na+ sensor, whose expression in MNCVP contributes to the generation of homeostatic responses to hypernatremia.SIGNIFICANCE STATEMENT In this study, we provide the first direct evidence showing that the sodium-sensing channel encoded by the Scn7A gene (NaX) mediates cell-autonomous sodium detection by MNCs in the low millimolar range and that selectively reducing the expression of these channels in MNCs impairs their activation in response to a physiologically relevant sodium stimulus in vitro and in vivo These data reveal that NaX operates as a sodium sensor in these cells and that the endogenous sensory properties of osmoregulatory effector neurons contribute to their homeostatic activation in vivo.

Trifunctional Saxitoxin Conjugates for Covalent Labeling of Voltage-Gated Sodium Channels.

Voltage-gated sodium ion channels (NaV s) are integral membrane protein complexes responsible for electrical signal conduction in excitable cells. Methods that enable selective labeling of NaV s hold potential value for understanding how channel regulation and post-translational modification are influenced during development and in response to diseases and disorders of the nervous system. We have developed chemical reagents patterned after (+)-saxitoxin (STX) - a potent and reversible inhibitor of multiple NaV isoforms - and affixed with a reactive electrophile and either a biotin cofactor, fluorophore, or 'click' functional group for labeling wild-type channels. Our studies reveal enigmatic structural effects of the probes on the potency and efficiency of covalent protein modification. Among the compounds analyzed, a STX-maleimide-coumarin derivative is most effective at irreversibly blocking Na+ conductance when applied to recombinant NaV s and endogenous channels expressed in hippocampal neurons. Mechanistic analysis supports the conclusion that high-affinity toxin binding is a prerequisite for covalent protein modification. Results from these studies are guiding the development of next-generation tool compounds for selective modification of NaV s expressed in the plasma membranes of cells.

Functional diversity of sodium channel variants in common eastern bumblebee, Bombus impatiens.

For the past decade, Colony Collapse Disorder has been reported worldwide. Insecticides containing pyrethroids may be responsible for a decline in bees, which are more sensitive to pyrethroids compared with other insects. Voltage-gated sodium channels (Nav ) are the major target sites of pyrethroids, and the sodium channel diversity is generated through extensive alternative splicing and RNA editing. In this study, we cloned and analyzed the function of variants of the Nav channel, BiNav , from Bombus impatiens. BiNav covers a 46 kb genome region including 30 exons. Sequence analysis of 56 clones showed that the clones can be grouped into 22 splice types with 11 optional exons (exons j, w, p, q, r, b, e, t, l/k, and z). Here, a special alternative exon w is identified, encoding a stretch of 31 amino acid resides in domain I between S3 and S4. RNA editing generates 18 amino acid changes in different positions in individual variants. Among 56 variants examined, only six variants generated sufficient sodium currents for functional characterization in Xenopus oocytes. In the presence of B. impatiens TipE and TEH1, the sodium current amplitude of BiNav 1-1 increased by fourfold, while TipE of other insect species had no effect on the expression. Abundant alternative splicing and RNA editing of BiNav suggests the molecular and functional pharmacology diversity of the Nav channel for bumblebees. This study provides a theoretical basis for the design of insecticides that specifically target pests without affecting beneficial insects.

Prokaryotic voltage-gated sodium channels are more effective than endogenous Nav1.5 channels in rescuing cardiac action potential conduction: an in silico study.

Methods to augment Na+ current in cardiomyocytes hold potential for the treatment of various cardiac arrhythmias involving conduction slowing. Because the gene coding cardiac Na+ channel (Nav1.5) is too large to fit in a single adeno-associated virus (AAV) vector, new gene therapies are being developed to enhance endogenous Nav1.5 current (by overexpression of chaperon molecules or use of multiple AAV vectors) or to exogenously introduce prokaryotic voltage-gated Na+ channels (BacNav) whose gene size is significantly smaller than that of the Nav1.5. In this study, based on experimental measurements in heterologous expression systems, we developed an improved computational model of the BacNav channel, NavSheP D60A. We then compared in silico how NavSheP D60A expression vs. Nav1.5 augmentation affects the electrophysiology of cardiac tissue. We found that the incorporation of BacNav channels in both adult guinea pig and human cardiomyocyte models increased their excitability and reduced action potential duration. When compared with equivalent augmentation of Nav1.5 current in simulated settings of reduced tissue excitability, the addition of the BacNav current was superior in improving the safety of conduction under conditions of current source-load mismatch, reducing the vulnerability to unidirectional conduction block during premature pacing, preventing the instability and breakup of spiral waves, and normalizing the conduction and ECG in Brugada syndrome tissues with mutated Nav1.5. Overall, our studies show that compared with a potential enhancement of the endogenous Nav1.5 current, expression of the BacNav channels with their slower inactivation kinetics can provide greater anti-arrhythmic benefits in hearts with compromised action potential conduction.NEW & NOTEWORTHY Slow action potential conduction is a common cause of various cardiac arrhythmias; yet, current pharmacotherapies cannot augment cardiac conduction. This in silico study compared the efficacy of recently proposed antiarrhythmic gene therapy approaches that increase peak sodium current in cardiomyocytes. When compared with the augmentation of endogenous sodium current, expression of slower-inactivating bacterial sodium channels was superior in preventing conduction block and arrhythmia induction. These results further the promise of antiarrhythmic gene therapies targeting sodium channels.

A scorpion toxin affecting sodium channels shows double cis-trans isomerism.

Scorpion α-toxins (α-NaTx) inhibiting the inactivation of voltage-gated sodium channels (Nav ) are a well-studied family of small proteins. We previously showed that the structure of α-NaTx specificity module responsible for selective Nav binding is governed by an interplay between the nest and niche protein motifs. Here, we report the solution structure of the toxin Lqq4 from the venom of the scorpion Leiurus quinquestriatus. Unexpectedly, we find that this toxin presents an ensemble of long-lived structurally distinct states. We unequivocally assign these states to the alternative configurations (cis-trans isomers) of two peptide bonds: V56-P57 and C17-G18; neither of the cis isomers has been described in α-NaTx so far. We argue that the native conformational space of α-NaTx is wider than assumed previously.