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Voltage-gated sodium (Na v ) channels are targeted by a number of widely used and investigational drugs for the treatment of epilepsy, arrhythmia, pain, and other disorders. Despite recent advances in structural elucidation of Na v channels, the binding mode of most Na v -targeting drugs remains unknown. Here we report high-resolution cryo-EM structures of human Na v 1.7 treated with drugs and lead compounds with representative chemical backbones at resolutions of 2.6-3.2 Å. A binding site beneath the intracellular gate (site BIG) accommodates carbamazepine, bupivacaine, and lacosamide. Unexpectedly, a second molecule of lacosamide plugs into the selectivity filter from the central cavity. Fenestrations are popular sites for various state-dependent drugs. We show that vinpocetine, a synthetic derivative of a vinca alkaloid, and hardwickiic acid, a natural product with antinociceptive effect, bind to the III-IV fenestration, while vixotrigine, an analgesic candidate, penetrates the IV-I fenestration of the pore domain. Our results permit building a 3D structural map for known drug-binding sites on Na v channels summarized from the present and previous structures. Voltage-gated sodium (Nav) channels are targeted by various clinically applied and investigational drugs. Here cryo-EM structures of Nav1.7 bound to 7 compounds with diverse chemical skeletons reveal the structural basis of action of these drugs and provide a 3D structural map for drug binding sites on Nav channels.

Cannabidiol (CBD), a major non-psychoactive phytocannabinoid in cannabis, is an effective treatment for some forms of epilepsy and pain. At high concentrations, CBD interacts with a huge variety of proteins, but which targets are most relevant for clinical actions is still unclear. Here we show that CBD interacts with Na v 1.7 channels at sub-micromolar concentrations in a state-dependent manner. Electrophysiological experiments show that CBD binds to the inactivated state of Na v 1.7 channels with a dissociation constant of about 50 nM. The cryo-EM structure of CBD bound to Na v 1.7 channels reveals two distinct binding sites. One is in the IV-I fenestration near the upper pore. The other binding site is directly next to the inactivated “wedged” position of the Ile/Phe/Met (IFM) motif on the short linker between repeats III and IV, which mediates fast inactivation. Consistent with producing a direct stabilization of the inactivated state, mutating residues in this binding site greatly reduced state-dependent binding of CBD. The identification of this binding site may enable design of compounds with improved properties compared to CBD itself. Cannabidiol (CBD), the nonpsychoactive component in cannabis, is an effective treatment for epilepsy and pain. Here, authors explored the mode of action of CBD on hNa v 1.7 channels through two distinct binding sites, suggesting a direct stabilization of the inactivated state of channels.

Voltage-gated sodium channels (Navs) play essential roles in excitable tissues, with their activation and opening resulting in the initial phase of the action potential. The cycling of Navs through open, closed and inactivated states, and their closely choreographed relationships with the activities of other ion channels lead to exquisite control of intracellular ion concentrations in both prokaryotes and eukaryotes. Here we present the 2.45 Å resolution crystal structure of the complete NavMs prokaryotic sodium channel in a fully open conformation. A canonical activated conformation of the voltage sensor S4 helix, an open selectivity filter leading to an open activation gate at the intracellular membrane surface and the intracellular C-terminal domain are visible in the structure. It includes a heretofore unseen interaction motif between W77 of S3, the S4–S5 interdomain linker, and the C-terminus, which is associated with regulation of opening and closing of the intracellular gate. Voltage-gated sodium (Nav) channels are crucial for action potential initiation in excitable cells. Here the authors present the complete structure of prokaryotic NavMs in a fully open state, providing structural insight into the opening and closure of the channel's intracellular gate.

Voltage-gated sodium (Na V ) channels play fundamental roles in initiating and propagating action potentials. Na V 1.3 is involved in numerous physiological processes including neuronal development, hormone secretion and pain perception. Here we report structures of human Na V 1.3/β1/β2 in complex with clinically-used drug bulleyaconitine A and selective antagonist ICA121431. Bulleyaconitine A is located around domain I-II fenestration, providing the detailed view of the site-2 neurotoxin binding site. It partially blocks ion path and expands the pore-lining helices, elucidating how the bulleyaconitine A reduces peak amplitude but improves channel open probability. In contrast, ICA121431 preferentially binds to activated domain IV voltage-sensor, consequently strengthens the Ile-Phe-Met motif binding to its receptor site, stabilizes the channel in inactivated state, revealing an allosterically inhibitory mechanism of Na V channels. Our results provide structural details of distinct small-molecular modulators binding sites, elucidate molecular mechanisms of their action on Na V channels and pave a way for subtype-selective therapeutic development. Na V 1.3 is involved in neuronal development, hormone secretion and pain perception. Here, the authors elucidate the molecular mechanism for modulation of Na V 1.3 by a site-2 neurotoxin bulleyaconitine A and a subtype selective antagonist ICA121431.

Voltage-gated sodium (Na v ) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Na v channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider ( Heteroscodra maculata ) toxins that selectively activate the Na v 1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Na v 1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Na v 1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Na v 1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain. Two spider toxins are shown to target the Na v 1.1 subtype of sodium channel specifically, shedding light on the role of these channels in mechanical pain signalling. Na v 1.1 channels mediate mechanical pain Mutations affecting several Na v 1 subtype voltage-gated sodium channels have been shown to be associated with insensitivity to pain or persistent pain syndromes. Na v 1.1 is expressed by somatosensory neurons, but no direct link has been established between this subtype and nociception. Further studies have been hampered by a paucity of pharmacological agents that discriminate between the closely related members of the Na v 1 family. Now David Julius and colleagues have identified two spider toxins specifically targeting Na v 1.1, and use them to show that this channel is key to the specific transduction of mechanical but not thermal pain by myelinated Aδ sensory fibres. Previous genetic studies of Na v 1.1 indicate that such selective agents may open therapeutic avenues in disorders associated with the central nervous system, such as epilepsy, autism and Alzheimer disease. The involvement of Na v 1.1 channels in mediating mechanical pain reported here was unexpected.

Voltage-gated sodium channel Na V 1.7 plays essential roles in pain and odor perception. Na V 1.7 variants cause pain disorders. Accordingly, Na V 1.7 has elicited extensive attention in developing new analgesics. Here we present cryo-EM structures of human Na V 1.7/β1/β2 complexed with inhibitors XEN907, TC-N1752 and Na V 1.7-IN2, explaining specific binding sites and modulation mechanism for the pore blockers. These inhibitors bind in the central cavity blocking ion permeation, but engage different parts of the cavity wall. XEN907 directly causes α- to π-helix transition of DIV-S6 helix, which tightens the fast inactivation gate. TC-N1752 induces π-helix transition of DII-S6 helix mediated by a conserved asparagine on DIII-S6, which closes the activation gate. Na V 1.7-IN2 serves as a pore blocker without causing conformational change. Electrophysiological results demonstrate that XEN907 and TC-N1752 stabilize Na V 1.7 in inactivated state and delay the recovery from inactivation. Our results provide structural framework for Na V 1.7 modulation by pore blockers, and important implications for developing subtype-selective analgesics. The voltage-gated sodium channel Na V 1.7 plays essential roles in pain sensation. The authors report cryo-EM structures of Na V 1.7 in complexes with three pore blockers, elucidating distinct mechanisms of action of their modulation on Na V 1.7.

Voltage-gated sodium (Na V ) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na + channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7 Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4–S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ∼4.6 Å wide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na + selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level. Mechanism of Na+ channel action The X-ray crystal structure of a voltage-gated sodium channel from Arcobacter butzleri has been determined, with the channel in the closed-pore conformation. Channels of this type initiate electrical signalling in excitable cells and are the molecular targets for many drugs, but the structural basis for their voltage-dependent activation and ion selectivity is not known. The selectivity filter in this sodium channel is found to be quite short, compared with those in open-pore potassium channels, and the voltage-sensor domains and linkers between segments S4 and S5 seem to dilate the central pore by pivoting together.

The crystal structure of Na v Rh, a NaChBac orthologue from the marine Rickettsiales sp. HIMB114 , defines an ion binding site within the selectivity filter, and reveals several conformational rearrangements that may underlie the electromechanical coupling mechanism. High-resolution sodium channel structures There are many published structures for potassium channels, but structural information on voltage-gated sodium (Na v ) channels is much more scare, despite their importance in the initiation and propagation of action potentials in nerve cells, muscle cells and in the heart. Bacterial Na v channels provide a good model system for structure–function analyses, and here two groups report the X-ray crystal structure of bacterial Na v channels apparently in 'inactivated' conformations. Nieng Yan and colleagues determined the structure of Na v Rh from the marine bacterium known as alpha proteobacterium HIMB114 at 3.05-ångström resolution. William Catterall and colleagues report crystallographic snapshots of the Na v Ab channel from Arcobacter butzleri in two potentially inactivated states at 3.2-ångström resolution. Comparisons of these newly obtained structures with previously published data on Na v Ab in a 'pre-open' state reveal conformational rearrangements that may underlie the electromechanical coupling mechanism of these channels. This work is relevant to channelopathies and more widely to the design of neuroactive drugs. Voltage-gated sodium (Na v ) channels are essential for the rapid depolarization of nerve and muscle 1 , and are important drug targets 2 . Determination of the structures of Na v channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na v channels, exemplified by the Na + -selective channel of bacteria (NaChBac) 3 , provides a useful model system for structure–function analysis. Here we report the crystal structure of Na v Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 ( Rickettsiales sp. HIMB114 ; denoted Rh), at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr 178 and Leu 179 constitute an inner site within the selectivity filter where a hydrated Ca 2+ resides in the crystal structure. The outer mouth of the Na + selectivity filter, defined by Ser 181 and Glu 183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na v Rh is in an ‘inactivated’ conformation. Comparison of Na v Rh with Na v Ab 4 reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

X-ray crystal structures of a bacterial voltage-gated sodium channel in two ‘inactivated’ conformations are reported, revealing several conformational rearrangements that may underlie the electromechanical coupling of voltage sensor movement to inactivation of the pore. High-resolution sodium channel structures There are many published structures for potassium channels, but structural information on voltage-gated sodium (Na v ) channels is much more scare, despite their importance in the initiation and propagation of action potentials in nerve cells, muscle cells and in the heart. Bacterial Na v channels provide a good model system for structure–function analyses, and here two groups report the X-ray crystal structure of bacterial Na v channels apparently in 'inactivated' conformations. Nieng Yan and colleagues determined the structure of Na v Rh from the marine bacterium known as alpha proteobacterium HIMB114 at 3.05-ångström resolution. William Catterall and colleagues report crystallographic snapshots of the Na v Ab channel from Arcobacter butzleri in two potentially inactivated states at 3.2-ångström resolution. Comparisons of these newly obtained structures with previously published data on Na v Ab in a 'pre-open' state reveal conformational rearrangements that may underlie the electromechanical coupling mechanism of these channels. This work is relevant to channelopathies and more widely to the design of neuroactive drugs. In excitable cells, voltage-gated sodium (Na V ) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance 1 , 2 . Inactivation is a hallmark of Na V channel function and is critical for control of membrane excitability 3 , but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na V Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of Na V Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4 ), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na V Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na V Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na V channel gating and inactivation. These potential inactivated-state structures provide new insights into Na V channel gating and novel avenues to drug development and therapy for a range of debilitating Na V channelopathies.

The sodium channel Na V 1.6 is widely expressed in neurons of the central and peripheral nervous systems, which plays a critical role in regulating neuronal excitability. Dysfunction of Na V 1.6 has been linked to epileptic encephalopathy, intellectual disability and movement disorders. Here we present cryo-EM structures of human Na V 1.6/β1/β2 alone and complexed with a guanidinium neurotoxin 4,9-anhydro-tetrodotoxin (4,9-ah-TTX), revealing molecular mechanism of Na V 1.6 inhibition by the blocker. The apo-form structure reveals two potential Na + binding sites within the selectivity filter, suggesting a possible mechanism for Na + selectivity and conductance. In the 4,9-ah-TTX bound structure, 4,9-ah-TTX binds to a pocket similar to the tetrodotoxin (TTX) binding site, which occupies the Na + binding sites and completely blocks the channel. Molecular dynamics simulation results show that subtle conformational differences in the selectivity filter affect the affinity of TTX analogues. Taken together, our results provide important insights into Na V 1.6 structure, ion conductance, and inhibition. Na V 1.6 channel plays a critical role in neuronal excitability. Here, authors present human Na V 1.6 structures in apo and 4,9-anhydro-tetrodotoxin bound forms, which reveal molecular mechanisms of Na V 1.6 Na + conductance and inhibition by the blocker.