C. elegans touch receptor neurons direct mechanosensory complex organization via repurposing conserved basal lamina proteins.

The sense of touch is conferred by the conjoint function of somatosensory neurons and skin cells. These cells meet across a gap filled by a basal lamina, an ancient structure found in metazoans. Using Caenorhabditis elegans, we investigate the composition and ultrastructure of the extracellular matrix at the epidermis and touch receptor neuron (TRN) interface. We show that membrane-matrix complexes containing laminin, nidogen, and the MEC-4 mechano-electrical transduction channel reside at this interface and are central to proper touch sensation. Interestingly, the dimensions and spacing of these complexes correspond with the discontinuous beam-like extracellular matrix structures observed in serial-section transmission electron micrographs. These complexes fail to coalesce in touch-insensitive extracellular matrix mutants and in dissociated neurons. Loss of nidogen reduces the density of mechanoreceptor complexes and the amplitude of the touch-evoked currents they carry. Thus, neuron-epithelium cell interfaces are instrumental in mechanosensory complex assembly and function. Unlike the basal lamina ensheathing the pharynx and body wall muscle, nidogen recruitment to the puncta along TRNs is not dependent upon laminin binding. MEC-4, but not laminin or nidogen, is destabilized by point mutations in the C-terminal Kunitz domain of the extracellular matrix component, MEC-1. These findings imply that somatosensory neurons secrete proteins that actively repurpose the basal lamina to generate special-purpose mechanosensory complexes responsible for vibrotactile sensing.

Controlled release of vancomycin from PEGylated fibrinogen polyethylene glycol diacrylate hydrogel.

Surgical site infection (SSI) is a common issue post-surgery which often prolongs hospitalization and can lead to serious complications such as sternal wound infection following cardiac surgery via median sternotomy. Controlled release of suitable antibiotics could allow maximizing drug efficacy and safety, and therefore achieving a desired therapeutic response. In this study, we have developed a vancomycin laden PEGylated fibrinogen-polyethylene glycol diacrylate (PF-PEGDA) hydrogel system that can release vancomycin at a controlled and predictable rate to be applied in SSI prevention. Two configurations were developed to study effect of the hydrogel on drug release, namely, vancomycin laden hydrogel and vancomycin solution on top of blank hydrogel. The relationship between the rigidity of the hydrogel and drug diffusion was found to comply with a universal power law, i.e., softer hydrogels result in a greater diffusion coefficient hence faster release rate. Besides, vancomycin laden hydrogels exhibited burst release, whereas the vancomycin solution on top of blank hydrogels exhibited lag release. A mathematical model was developed to simulate vancomycin permeation through the hydrogels. The permeation of vancomycin can be predicted accurately by using the mathematical model, which provided a useful tool to customize drug loading, hydrogel thickness and stiffness for personalized medication to manage SSI. To evaluate the potential of hydrogels for bone healing applications in cardiovascular medicine, we performed a proof-of-concept median sternotomy in rabbits and applied the hydrogels. The hydrogel formulations accelerated the onset of osteo-genetic processes in rabbits, demonstrating its potential to be used in human.

The Enhanced Performance of NiCuOOH/NiCu(OH)2 Electrode Using Pre-Conversion Treatment for the Electrochemical Oxidation of Ammonia.

Electrochemical oxidation of ammonia is an attractive process for wastewater treatment, hydrogen production, and ammonia fuel cells. However, the sluggish kinetics of the anode reaction has limited its applications, leading to a high demand for novel electrocatalysts. Herein, the electrode with the in situ growth of NiCu(OH)2 was partially transformed into the NiCuOOH phase by a pre-treatment using highly oxidative solutions. As revealed by SEM, XPS, and electrochemical analysis, such a strategy maintained the 3D structure, while inducing more active sites before the in situ generation of oxyhydroxide sites during the electrochemical reaction. The optimized NiCuOOH-1 sample exhibited the current density of 6.06 mA cm-2 at 0.5 V, which is 1.67 times higher than that of NiCu(OH)2 (3.63 mA cm-2). Moreover, the sample with a higher crystalline degree of the NiCuOOH phase exhibited lower performance, demonstrating the importance of a moderate treatment condition. In addition, the NiCuOOH-1 sample presented low selectivity (<20%) towards NO2- and stable activity during the long-term operation. The findings of this study would provide valuable insights into the development of transition metal electrocatalysts for ammonia oxidation.

3D printing for personalised medicines: implications for policy and practice.

The current healthcare dynamic has shifted from one-size-fits-all to patient-centred care, with our increased understanding of pharmacokinetics and pharmacogenomics demanding a switch to more individualised therapies. As the pharmaceutical industry remains yet to succumb to the push of a technological paradigm shift, pharmacists lack the means to provide completely personalised medicine (PM) to their patients in a safe, affordable, and widely accessible manner. As additive manufacturing technology has already established its strength in producing pharmaceutical formulations, it is necessary to next consider methods by which this technology can create PM accessible from pharmacies. In this article, we reviewed the limitations of current pharmaceutical manufacturing methods for PMs, three-dimensional (3D) printing techniques that are most beneficial for PMs, implications of bringing this technology into pharmacy practice, and implications for policy surrounding 3D printing techniques in the manufacturing of PMs.

Recent progress in three-dimensionally-printed dosage forms from a pharmacist perspective.

OBJECTIVE: Additive manufacturing (AM), commonly known as 3D printing (3DP), has opened new frontiers in pharmaceutical applications. This review is aimed to summarise the recent development of 3D-printed dosage forms, from a pharmacists' perspective. METHODS: Keywords including additive manufacturing, 3D printing and drug delivery were used for literature search in PubMed, Excerpta Medica Database (EMBASE) and Web of Science, to identify articles published in the year 2020. RESULTS: For each 3DP study, the active pharmaceutical ingredients, 3D printers and materials used for the printing were tabulated and discussed. 3DP has found its applications in various dosage forms for oral delivery, transdermal delivery, rectal delivery, vaginal delivery, implant and bone scaffolding. Several topics were discussed in detail, namely patient-specific dosing, customisable drug administration, multidrug approach, varying drug release, compounding pharmacy, regulatory progress and future perspectives. AM is expected to become a common tool in compounding pharmacies to make polypills and personalised medications. CONCLUSION: 3DP is an enabling tool to fabricate dosage forms with intricate structure designs, tailored dosing, drug combinations and controlled release, all of which lend it to be highly conducive to personalisation, thereby revolutionising the future of pharmacy practice.

Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells.

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.

DEG/ENaC/ASIC channels vary in their sensitivity to anti-hypertensive and non-steroidal anti-inflammatory drugs.

The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) play important roles in sensing mechanical stimuli, regulating salt homeostasis, and responding to acidification in the nervous system. They have two transmembrane domains separated by a large extracellular domain and are believed to assemble as homomeric or heteromeric trimers. Based on studies of selected family members, these channels are assumed to form nonvoltage-gated and sodium-selective channels sensitive to the anti-hypertensive drug amiloride. They are also emerging as a target of nonsteroidal anti-inflammatory drugs (NSAIDs). Caenorhabditis elegans has more than two dozen genes encoding DEG/ENaC/ASIC subunits, providing an excellent opportunity to examine variations in drug sensitivity. Here, we analyze a subset of the C. elegans DEG/ENaC/ASIC proteins to test the hypothesis that individual family members vary not only in their ability to form homomeric channels but also in their drug sensitivity. We selected a panel of C. elegans DEG/ENaC/ASICs that are coexpressed in mechanosensory neurons and expressed gain-of-function or d mutants in Xenopus laevis oocytes. We found that only DEGT­1d, UNC­8d, and MEC­4d formed homomeric channels and that, unlike MEC­4d and UNC­8d, DEGT­1d channels were insensitive to amiloride and its analogues. As reported for rat ASIC1a, NSAIDs inhibit DEGT­1d and UNC­8d channels. Unexpectedly, MEC­4d was strongly potentiated by NSAIDs, an effect that was decreased by mutations in the putative NSAID-binding site in the extracellular domain. Collectively, these findings reveal that not all DEG/ENaC/ASIC channels are amiloride-sensitive and that NSAIDs can both inhibit and potentiate these channels.

PF-06526290 can both enhance and inhibit conduction through voltage-gated sodium channels.

BACKGROUND AND PURPOSE: Pharmacological agents that either inhibit or enhance flux of ions through voltage-gated sodium (Nav ) channels may provide opportunities for treatment of human health disorders. During studies to characterize agents that modulate Nav 1.3 function, we identified a compound that appears to exhibit both enhancement and inhibition of sodium ion conduction that appeared to be dependent on the gating state that the channel was in. The objective of the current study was to determine if these different modulatory effects are mediated by the same or distinct interactions with the channel. EXPERIMENTAL APPROACH: Electrophysiology and site-directed mutation were used to investigate the effects of PF-06526290 on Nav channel function. KEY RESULTS: PF-06526290 greatly slows inactivation of Nav channels in a subtype-independent manner. However, upon prolonged depolarization to induce inactivation, PF-06526290 becomes a Nav subtype-selective inhibitor. Mutation of the domain 4 voltage sensor modulates inhibition of Nav 1.3 or Nav 1.7 channels by PF-06526290 but has no effect on PF-06526290 mediated slowing of inactivation. CONCLUSIONS AND IMPLICATIONS: These findings suggest that distinct interactions may underlie the two modes of Nav channel modulation by PF-06526290 and that a single compound can affect sodium channel function in several ways.

Addition of a single methyl group to a small molecule sodium channel inhibitor introduces a new mode of gating modulation.

BACKGROUND AND PURPOSE: Aryl sulfonamide Nav 1.3 or Nav 1.7 voltage-gated sodium (Nav ) channel inhibitors interact with the Domain 4 voltage sensor domain (D4 VSD). During studies to better understand the structure-activity relationship of this interaction, an additional mode of channel modulation, specifically slowing of inactivation, was revealed by addition of a single methyl moiety. The objective of the current study was to determine if these different modulatory effects are mediated by the same or distinct interactions with the channel. EXPERIMENTAL APPROACH: Electrophysiology and site-directed mutation were used to compare the effects of PF-06526290 and its desmethyl analogue PF-05661014 on Nav channel function. KEY RESULTS: PF-05661014 selectively inhibits Nav 1.3 versus Nav 1.7 currents by stabilizing inactivated channels via interaction with D4 VSD. In contrast, PF-06526290, which differs from PF-05661014 by a single methyl group, exhibits a dual effect. It greatly slows inactivation of Nav channels in a subtype-independent manner. However, upon prolonged depolarization to induce inactivation, PF-06526290 becomes a Nav subtype selective inhibitor similar to PF-05661014. Mutation of the D4 VSD modulates inhibition of Nav 1.3 or Nav 1.7 by both PF-05661014 and PF-06526290, but has no effect on the inactivation slowing produced by PF-06526290. This finding, along with the absence of functional inhibition of PF-06526290-induced inactivation slowing by PF-05661014, suggests that distinct interactions underlie the two modes of Nav channel modulation. CONCLUSIONS AND IMPLICATIONS: Addition of a methyl group to a Nav channel inhibitor introduces an additional mode of gating modulation, implying that a single compound can affect sodium channel function in multiple ways.

The Drosophila Sodium Channel 1 (DSC1): The founding member of a new family of voltage-gated cation channels.

It has been nearly three decades since the identification of the Drosophila Sodium Channel 1 (DSC1) gene from Drosophila melanogaster. The orthologs of the DSC1 gene have now been identified in other insect species including BSC1 from Blattella germanica. Functional analyses of DSC1/BSC1 channels in Xenopus oocytes reveal that DSC1 and BSC1 encode voltage-gated cation channels that are more permeable to Ca(2+) than to Na(+). Genetic and electrophysiological analyses show that knockout of the DSC1 gene in D. melanogaster causes behavioral and neurological modifications. In this review, we summarize major findings from recent studies and highlight a unique role of the DSC1 channel, distinct from that of the sodium channel, in regulating membrane excitability and modulating toxicity of pyrethroid insecticides.

Distinct modulating effects of TipE-homologs 2-4 on Drosophila sodium channel splice variants.

The Drosophila melanogaster TipE protein is thought to be an insect sodium channel auxiliary subunit functionally analogous to the ß subunits of mammalian sodium channels. Besides TipE, four TipE-homologous proteins (TEH1-4) have been identified. It has been reported that TipE and TEH1 have both common and distinct effects on the gating properties of splice variants of the Drosophila sodium channel, DmNav. However, limited information is available on the effects of TEH2, TEH3 and TEH4 on the function of DmNav channel variants. In this study, we found that TEH2 increased the amplitude of peak current, but did not alter the gating properties of three examined DmNav splice variants expressed in Xenopus oocytes. In contrast, TEH4 had no effect on peak current, yet altered the gating properties of all three channel variants. Furthermore, TEH4 enhanced persistent current and slowed sodium current decay. The effects of TEH3 on DmNav variants are similar to those of TEH4, but the data were collected from a small portion of oocytes because co-expression of TEH3 with DmNav variants generated a large leak current in the majority of oocytes examined. In addition, TEH3 and TEH4 enhanced the expression of endogenous currents in oocytes. Taken together, our results reveal distinct roles of TEH proteins in modulating the function of sodium channels and suggest that TEH proteins might provide an important layer of regulation of membrane excitability in vivo. Our results also raise an intriguing possibility of TEH3/TEH4 as auxiliary subunits of other voltage-gated ion channels besides sodium channels.

A mutation in the intracellular loop III/IV of mosquito sodium channel synergizes the effect of mutations in helix IIS6 on pyrethroid resistance.

Activation and inactivation of voltage-gated sodium channels are critical for proper electrical signaling in excitable cells. Pyrethroid insecticides promote activation and inhibit inactivation of sodium channels, resulting in prolonged opening of sodium channels. They preferably bind to the open state of the sodium channel by interacting with two distinct receptor sites, pyrethroid receptor sites PyR1 and PyR2, formed by the interfaces of domains II/III and I/II, respectively. Specific mutations in PyR1 or PyR2 confer pyrethroid resistance in various arthropod pests and disease vectors. Recently, a unique mutation, N(1575)Y, in the cytoplasmic loop linking domains III and IV (LIII/IV) was found to coexist with a PyR2 mutation, L(1014)F in IIS6, in pyrethroid-resistant populations of Anopheles gambiae. To examine the role of this mutation in pyrethroid resistance, N(1575)Y alone or N(1575)Y + L(1014)F were introduced into an Aedes aegypti sodium channel, AaNav1-1, and the mutants were functionally examined in Xenopus oocytes. N(1575)Y did not alter AaNav1-1 sensitivity to pyrethroids. However, the N(1575)Y + L(1014)F double mutant was more resistant to pyrethroids than the L(1014)F mutant channel. Further mutational analysis showed that N(1575)Y could also synergize the effect of L(1014)S/W, but not L(1014)G or other pyrethroid-resistant mutations in IS6 or IIS6. Computer modeling predicts that N(1575)Y allosterically alters PyR2 via a small shift of IIS6. Our findings provide the molecular basis for the coexistence of N(1575)Y with L(1014)F in pyrethroid resistance, and suggest an allosteric interaction between IIS6 and LIII/IV in the sodium channel.

Molecular biology of insect sodium channels and pyrethroid resistance.

Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Many of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors.

[Simultaneous determination of seven constituents in Lamiophlomis rotata by HPLC].

OBJECTIVE: To establish a new method for simultaneous determination of shanzhiside methyl ester, chlorogenic acid, 8-O-acetyl shanzhiside methylester, forsythiaside B, rutin, acteoside and galuteolin in Lamiophlomis rotata. METHOD: Separation was performed on a Welchrom-C18 chromatographic column with acetonitrile-0.1% orthophosphoric acid as mobile phasewith gradient elution. The flow rate was 1.0 mL x min(-1). The column temperature was 30 degrees C, and the detection wavelength was set at 238 nm, 330 nm and 350 nm. RESULT: The seven compounds were well separated with good linear correlations. The mean recoveries of seven compounds were 96.47%-102.2% (RSD 0.70%-2.2%). CONCLUSION: There were good correlations among the seven compounds in the samples of aerial parts. The mean sum of shanzhiside methyl ester and 8-O-acetyl shanzhiside methylester in samples of aerial parts is 1.44%. The aerial parts have more kinds of composition and with higher content than that of underground parts in L. rotata, which was reasonable for the resonable use of the aerial part as medicinal part. The method was simple, repeatable and stable, which could be used for identification and quality evaluation of L. rotata.

Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+) currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an intron retention process in the transcription of the neuronal TEH1-like ancillary subunits of P. americana.

Differential effects of TipE and a TipE-homologous protein on modulation of gating properties of sodium channels from Drosophila melanogaster.

ß subunits of mammalian sodium channels play important roles in modulating the expression and gating of mammalian sodium channels. However, there are no orthologs of ß subunits in insects. Instead, an unrelated protein, TipE in Drosophila melanogaster and its orthologs in other insects, is thought to be a sodium channel auxiliary subunit. In addition, there are four TipE-homologous genes (TEH1-4) in D. melanogaster and three to four orthologs in other insect species. TipE and TEH1-3 have been shown to enhance the peak current of various insect sodium channels expressed in Xenopus oocytes. However, limited information is available on how these proteins modulate the gating of sodium channels, particularly sodium channel variants generated by alternative splicing and RNA editing. In this study, we compared the effects of TEH1 and TipE on the function of three Drosophila sodium channel splice variants, DmNav9-1, DmNav22, and DmNav26, in Xenopus oocytes. Both TipE and TEH1 enhanced the amplitude of sodium current and accelerated current decay of all three sodium channels tested. Strikingly, TEH1 caused hyperpolarizing shifts in the voltage-dependence of activation, fast inactivation and slow inactivation of all three variants. In contrast, TipE did not alter these gating properties except for a hyperpolarizing shift in the voltage-dependence of fast inactivation of DmNav26. Further analysis of the gating kinetics of DmNav9-1 revealed that TEH1 accelerated the entry of sodium channels into the fast inactivated state and slowed the recovery from both fast- and slow-inactivated states, thereby, enhancing both fast and slow inactivation. These results highlight the differential effects of TipE and TEH1 on the gating of insect sodium channels and suggest that TEH1 may play a broader role than TipE in regulating sodium channel function and neuronal excitability in vivo.

Role of the DSC1 channel in regulating neuronal excitability in Drosophila melanogaster: extending nervous system stability under stress.

Voltage-gated ion channels are essential for electrical signaling in neurons and other excitable cells. Among them, voltage-gated sodium and calcium channels are four-domain proteins, and ion selectivity is strongly influenced by a ring of amino acids in the pore regions of these channels. Sodium channels contain a DEKA motif (i.e., amino acids D, E, K, and A at the pore positions of domains I, II, III, and IV, respectively), whereas voltage-gated calcium channels contain an EEEE motif (i.e., acidic residues, E, at all four positions). Recently, a novel family of ion channel proteins that contain an intermediate DEEA motif has been found in a variety of invertebrate species. However, the physiological role of this new family of ion channels in animal biology remains elusive. DSC1 in Drosophila melanogaster is a prototype of this new family of ion channels. In this study, we generated two DSC1 knockout lines using ends-out gene targeting via homologous recombination. DSC1 mutant flies exhibited impaired olfaction and a distinct jumpy phenotype that is intensified by heat shock and starvation. Electrophysiological analysis of the giant fiber system (GFS), a well-defined central neural circuit, revealed that DSC1 mutants are altered in the activities of the GFS, including the ability of the GFS to follow repetitive stimulation (i.e., following ability) and response to heat shock, starvation, and pyrethroid insecticides. These results reveal an important role of the DSC1 channel in modulating the stability of neural circuits, particularly under environmental stresses, likely by maintaining the sustainability of synaptic transmission.

Identification of new batrachotoxin-sensing residues in segment IIIS6 of the sodium channel.

Ion permeation through voltage-gated sodium channels is modulated by various drugs and toxins. The atomistic mechanisms of action of many toxins are poorly understood. A steroidal alkaloid batrachotoxin (BTX) causes persistent channel activation by inhibiting inactivation and shifting the voltage dependence of activation to more negative potentials. Traditionally, BTX is considered to bind at the channel-lipid interface and allosterically modulate the ion permeation. However, amino acid residues critical for BTX action are found in the inner helices of all four repeats, suggesting that BTX binds in the pore. In the octapeptide segment IFGSFFTL in IIIS6 of a cockroach sodium channel BgNa(V), besides Ser_3i15 and Leu_3i19, which correspond to known BTX-sensing residues of mammalian sodium channels, we found that Gly_3i14 and Phe_3i16 are critical for BTX action. Using these data along with published data as distance constraints, we docked BTX in the Kv1.2-based homology model of the open BgNa(V) channel. We arrived at a model in which BTX adopts a horseshoe conformation with the horseshoe plane normal to the pore axis. The BTX ammonium group is engaged in cation-π interactions with Phe_3i16 and BTX moieties interact with known BTX-sensing residues in all four repeats. Oxygen atoms at the horseshoe inner surface constitute a transient binding site for permeating cations, whereas the bulky BTX molecule would resist the pore closure, thus causing persistent channel activation. Our study reinforces the concept that steroidal sodium channel agonists bind in the inner pore of sodium channels and elaborates the atomistic mechanism of BTX action.