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Voltage-gated sodium (Na v ) channels have been implicated in cardiac and neurological disorders. There are many subtypes of these channels, making it challenging to develop specific therapeutics. A core α subunit is sufficient for voltage sensing and ion conductance, but function is modulated by β subunits and by natural toxins that can either act as pore blockers or gating modifiers (see the Perspective by Chowdhury and Chanda). Shen et al. present the structures of Na v 1.7 in complex with both β1 and β2 subunits and with animal toxins. Pan et al. present the structure of Na v 1.2 bound to β2 and a toxic peptide, the µ-conotoxin KIIIA. The structure shows why KIIIA is specific for Na v 1.2. These and other recently determined Na v structures provide a framework for targeted drug development. Science , this issue p. 1303 , p. 1309 ; see also p. 1278 Structural insight into subtype-specific modulation of voltage-gated sodium channels provides a framework for drug discovery. Voltage-gated sodium channel Na v 1.7 represents a promising target for pain relief. Here we report the cryo–electron microscopy structures of the human Na v 1.7-β1-β2 complex bound to two combinations of pore blockers and gating modifier toxins (GMTs), tetrodotoxin with protoxin-II and saxitoxin with huwentoxin-IV, both determined at overall resolutions of 3.2 angstroms. The two structures are nearly identical except for minor shifts of voltage-sensing domain II (VSD II ), whose S3-S4 linker accommodates the two GMTs in a similar manner. One additional protoxin-II sits on top of the S3-S4 linker in VSD IV . The structures may represent an inactivated state with all four VSDs “up” and the intracellular gate closed. The structures illuminate the path toward mechanistic understanding of the function and disease of Na v 1.7 and establish the foundation for structure-aided development of analgesics.

The human voltage-gated sodium channel, hNaV1.5, is responsible for the rapid upstroke of the cardiac action potential and is target for antiarrhythmic therapy. Despite the clinical relevance of hNaV1.5-targeting drugs, structure-based molecular mechanisms of promising or problematic drugs have not been investigated at atomic scale to inform drug design. Here, we used Rosetta structural modeling and docking as well as molecular dynamics simulations to study the interactions of antiarrhythmic and local anesthetic drugs with hNaV1.5. These calculations revealed several key drug binding sites formed within the pore lumen that can simultaneously accommodate up to two drug molecules. Molecular dynamics simulations identified a hydrophilic access pathway through the intracellular gate and a hydrophobic access pathway through a fenestration between DIII and DIV. Our results advance the understanding of molecular mechanisms of antiarrhythmic and local anesthetic drug interactions with hNaV1.5 and will be useful for rational design of novel therapeutics.

Bismuth(III) oxide is included as a radio-opacifier in dental materials, including hydraulic silicate cements, the material of choice for several endodontic procedures. It has been implicated in tooth discoloration after contact with endodontic irrigants, in particular NaOCl solution, To date, there has been no work on the chemistry: all reports have been of clinical findings only. The purpose now was to report the reactions leading to colour change from Bi 2 O 3 in contact with solutions used in routine endodontic practice. Ten-gram portions of Bi 2 O 3 were immersed in either water, NaOH, NaCl, NaOCl or HCl solution, either in the dark or exposed to visible light, and samples retrieved at 1, 4, 12 and 24 weeks. After washing, these were exposed to either added CO 2 or not, for 1 week while drying, and under the same dark or light conditions. Changes in appearance were monitored by photography and colour measurement, and chemically by X-ray diffraction and Fourier-transform infrared spectroscopy. 24-week material was studied using electron paramagnetic resonance and Raman spectroscopy; NaOCl-treated material was also examined by scanning electron microscopy. With water, NaCl and NaOH, bismuth subcarbonate was formed. With or without added carbon dioxide, discoloration occurred from pale yellow to light brown when exposed to light, and to a lesser extent in the dark, intensifying with time. In contrast, exposure to NaOCl rapidly formed a dark brown-black sodium bismuthate. With HCl, white BiOCl was formed. Bi 2 O 3 is not at all inert in this context as is commonly believed, denying its principle of use. Previously unreported solution-mediated reaction occurs readily even in water and NaCl solution, forming new compounds that discolour. In contact with NaOCl sodium bismuthate is formed; severe darkening occurs rapidly. The reactivity is such that Bi 2 O 3 is not indicated for dental materials and should be withdrawn from use.

Voltage-gated sodium （Nav） channels are essential for the rapid upstroke of action potentials and the propa- gation of electrical signals in nerves and muscles. Defects of Nav channels are associated with a variety of channelopathies. More than 1000 disease-related muta- tions have been identified in Nay channels, with Nay1.1 and Nay1.5 each harboring more than 400 mutations. Nay channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Nav chan- nels are required to understand their function and dis- ease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium （Car） channel Carl.1 provides a template for homology-based structural modeling of the evolutionarily related Nay channels. In this Resource article, we summarized all the reported disease-related mutations in human Nav channels, generated a homologous model of human Nay1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Nay channels, the analysis presented here serves as the base framework for mechanistic investi- gation of Nav channelopathies and for potential struc- ture-based drug discovery.

Membranes are widely used for separation processes in applications such as water desalination, batteries and dialysis, and are crucial in key sectors of our economy and society 1 . The majority of technologically exploited membranes are based on solid polymers and function as passive barriers, whose transport characteristics are governed by their chemical composition and nanostructure. Although such membranes are ubiquitous, it has proved challenging to maximize selectivity and permeability independently, leading to trade-offs between these pertinent characteristics 2 . Self-assembled biological membranes, in which barrier and transport functions are decoupled 3 , 4 , provide the inspiration to address this problem 5 , 6 . Here we introduce a self-assembly strategy that uses the interface of an aqueous two-phase system to template and stabilize molecularly thin (approximately 35 nm) biomimetic block copolymer bilayers of scalable area that can exceed 10 cm 2 without defects. These membranes are self-healing, and their barrier function against the passage of ions (specific resistance of approximately 1 MΩ cm 2 ) approaches that of phospholipid membranes. The fluidity of these membranes enables straightforward functionalization with molecular carriers that shuttle potassium ions down a concentration gradient with exquisite selectivity over sodium ions. This ion selectivity enables the generation of electric power from equimolar solutions of NaCl and KCl in devices that mimic the electric organ of electric rays. We introduce a self-assembly strategy that uses the interface of an aqueous two-phase system to template and stabilize molecularly thin biomimetic block copolymer bilayers of scalable area that can exceed 10 cm 2 without defects.

The voltage-gated sodium channel isoform Na 1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function Na 1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate Na 1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting Na 1.7 have been advanced to clinical development, no Na 1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a Na 1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, Na 1.1-1.6 and Na 1.8. In contrast to other Na 1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting Na 1.7.

The epithelial sodium channel (ENaC), a member of the ENaC/DEG superfamily, regulates Na+ and water homeostasis. ENaCs assemble as heterotrimeric channels that harbor protease-sensitive domains critical for gating the channel. Here, we present the structure of human ENaC in the uncleaved state determined by single-particle cryo-electron microscopy. The ion channel is composed of a large extracellular domain and a narrow transmembrane domain. The structure reveals that ENaC assembles with a 1:1:1 stoichiometry of α:β:γ subunits arranged in a counter-clockwise manner. The shape of each subunit is reminiscent of a hand with key gating domains of a ‘finger’ and a ‘thumb.’ Wedged between these domains is the elusive protease-sensitive inhibitory domain poised to regulate conformational changes of the ‘finger’ and ‘thumb’; thus, the structure provides the first view of the architecture of inhibition of ENaC. The bodies of humans and other animals contain many different fluids that play vital roles in the body, such as blood, saliva and the fluids that surround cells in organs. These fluids all contain particles called ions, which can affect the flow of water into and out of cells and alter the activity of proteins. Therefore, in order to survive, an animal must tightly regulate the levels of ions in its body. Epithelial cells line the surface of organs, and the inside of the digestive system and other cavities in the human body. A channel known as ENaC is found on the surface of epithelial cells and controls the volume of the fluid surrounding cells, blood pressure and the volume of liquid in the airways. This channel spans the membrane surrounding each epithelial cell and allows sodium ions to pass into the cell. To promote the opening of the channel, enzymes remove portions of the ENaC called extracellular domain, which sits on the outside surface of an epithelial cell. Three components (or ‘subunits’) called alpha, beta and gamma are needed to form an ENaC, but it is not clear how they fit together to form a single working unit. Noreng et al. used a technique called cryo-electron microscopy to study the three-dimensional structure of the human ENaC. This revealed that a single channel contains one alpha, one beta and one gamma subunit, which sit next to each other to form a narrow tube through the membrane and a large extracellular domain. When viewed from the outside of the cell the subunits form a narrow ring in a counter-clockwise manner. Further analysis of the structure suggested that when enzymes remove pieces of the extracellular domain of ENaC, it becomes easier for the rest of the channel to adopt a shape that allows sodium ions to move through the pore. A next step will be to study the three-dimensional structure of ENaC when it takes on different shapes to better understand how it works.

Improper function of voltage-gated sodium channels (NaVs), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target NaVs hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of NaV subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of NaV through a systematic structure–activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human NaV1.7 (hNaV1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective NaV inhibitors.

NADH oxidation in the respiratory chain is coupled to ion translocation across the membrane to build up an electrochemical gradient. The sodium-translocating NADH:quinone oxidoreductase (Na + -NQR), a membrane protein complex widespread among pathogenic bacteria, consists of six subunits, NqrA, B, C, D, E and F. To our knowledge, no structural information on the Na + -NQR complex has been available until now. Here we present the crystal structure of the Na + -NQR complex at 3.5 Å resolution. The arrangement of cofactors both at the cytoplasmic and the periplasmic side of the complex, together with a hitherto unknown iron centre in the midst of the membrane-embedded part, reveals an electron transfer pathway from the NADH-oxidizing cytoplasmic NqrF subunit across the membrane to the periplasmic NqrC, and back to the quinone reduction site on NqrA located in the cytoplasm. A sodium channel was localized in subunit NqrB, which represents the largest membrane subunit of the Na + -NQR and is structurally related to urea and ammonia transporters. On the basis of the structure we propose a mechanism of redox-driven Na + translocation where the change in redox state of the flavin mononucleotide cofactor in NqrB triggers the transport of Na + through the observed channel. Here the structure of the membrane protein complex sodium-translocating NADH:quinone oxidoreductase (Na + -NQR) is described; as Na + -NQR is a component of the respiratory chain of various bacteria, including pathogenic ones, this structure may serve as the basis for the development of new antibiotics. A key bacterial respiratory-chain enzyme The sodium-translocating NADH: quinone oxidoreductase (Na + -NQR) is a membrane protein complex in the respiratory chain of various bacteria, including pathogens such as Vibrio cholerae . It is analogous to — but not homologous to — mitochondrial complex I. Julia Steuber et al . have solved the X-ray crystal structures of this enzyme from V. cholerae at 3.5 Å resolution, together with structures of its NqrA, NqrC and NqrF subunits at high resolution. Na + -NQR contains one FAD cofactor, a [2Fe-2S] cluster, two covalently bound flavin mononucleotide cofactors, a riboflavin cofactor and a ubiquinone cofactor. Analysis of the structure suggests that a change in redox state of the flavin mononucleotide cofactor in NqrB is critical for the transport of Na + through the channel of the NqrB subunit to occur. This structure may serve as a basis for the development of new antibiotics.

Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495-575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1-1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1-1.7 activator veratridine (EC50 10-29 μM) or the NaV1.1-1.8 inhibitor tetracaine (IC50's 6-66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.