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Voltage-gated sodium channels initiate action potentials in brain neurons. In the 1970s, much was known about the function of sodium channels from measurements of ionic currents using the voltage clamp method, but there was no information about the sodium channel molecules themselves. As a postdoctoral fellow and staff scientist at the National Institutes of Health, I developed neurotoxins as molecular probes of sodium channels in cultured neuroblastoma cells. During those years, Bruce Ransom and I crossed paths as members of the laboratories of Marshall Nirenberg and Philip Nelson and shared insights about sodium channels in neuroblastoma cells from my work and electrical excitability and synaptic transmission in cultured spinal cord neurons from Bruce’s pioneering electrophysiological studies. When I established my laboratory at the University of Washington in 1977, my colleagues and I used those neurotoxins to identify the protein subunits of sodium channels, purify them, and reconstitute their ion conductance activity in pure form. Subsequent studies identified the molecular basis for the main functions of sodium channels—voltage-dependent activation, rapid and selective ion conductance, and fast inactivation. Bruce Ransom and I re-connected in the 1990s, as ski buddies at the Winter Conference on Brain Research and as faculty colleagues at the University of Washington when Bruce became our founding Chair of Neurology and provided visionary leadership of that department. In the past decade my work on sodium channels has evolved into structural biology. Molecular modeling and X-ray crystallographic studies have given new views of sodium channel function at atomic resolution. Sodium channels are also the molecular targets for genetic diseases, including Dravet Syndrome, an intractable pediatric epilepsy disorder with major co-morbidities of cognitive deficit, autistic-like behaviors, and premature death that is caused by loss-of-function mutations in the brain sodium channel Na V 1.1. Our work on a mouse genetic model of this disease has shown that its multi-faceted pathophysiology and co-morbidities derive from selective loss of electrical excitability and action potential firing in GABAergic inhibitory neurons, which disinhibits neural circuits throughout the brain and leads directly to the epilepsy, premature death and complex co-morbidities of this disease. It has been rewarding for me to use our developing knowledge of sodium channels to help understand the pathophysiology and to suggest potential therapeutic approaches for this devastating childhood disease.

A highlight of the knowledge derived in large part from structural work on physical motions and chemical interactions involved in voltage sensing, pore opening, ion conductance and selectivity, and voltage-dependent inactivation mechanisms of the voltage-gated channels Na V and Ca V . Electrical signals generated by minute currents of ions moving across cell membranes are central to all rapid processes in biology. Initiation and propagation of electrical signals requires voltage-gated sodium (Na V ) and calcium (Ca V ) channels. These channels contain a tetramer of membrane-bound subunits or domains comprising a voltage sensor and a pore module. Voltage-dependent activation occurs as membrane depolarization drives outward movements of positive gating changes in the voltage sensor via a sliding-helix mechanism, which leads to a conformational change in the pore module that opens its intracellular activation gate. A unique negatively charged site in the selectivity filter conducts hydrated Na + or Ca 2+ rapidly and selectively. Ion conductance is terminated by voltage-dependent inactivation, which causes asymmetric pore collapse. This Review focuses on recent advances in structure and function of Na V and Ca V channels that expand our current understanding of the chemical basis for electrical signaling mechanisms conserved from bacteria to humans.

Worldwide medicinal use of cannabis is rapidly escalating, despite limited evidence of its efficacy from preclinical and clinical studies. Here we show that cannabidiol (CBD) effectively reduced seizures and autistic-like social deficits in a well-validated mouse genetic model of Dravet syndrome (DS), a severe childhood epilepsy disorder caused by loss-of-function mutations in the brain voltage-gated sodium channel NaV1.1. The duration and severity of thermally induced seizures and the frequency of spontaneous seizures were substantially decreased. Treatment with lower doses of CBD also improved autistic-like social interaction deficits in DS mice. Phenotypic rescue was associated with restoration of the excitability of inhibitory interneurons in the hippocampal dentate gyrus, an important area for seizure propagation. Reduced excitability of dentate granule neurons in response to strong depolarizing stimuli was also observed. The beneficial effects of CBD on inhibitory neurotransmission were mimicked and occluded by an antagonist of GPR55, suggesting that therapeutic effects of CBD are mediated through this lipid-activated G protein-coupled receptor. Our results provide critical preclinical evidence supporting treatment of epilepsy and autistic-like behaviors linked to DS with CBD. We also introduce antagonism of GPR55 as a potential therapeutic approach by illustrating its beneficial effects in DS mice. Our study provides essential preclinical evidence needed to build a sound scientific basis for increased medicinal use of CBD.

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.

Electrical signaling was a dramatic development in evolution, allowing complex single-cell organisms like Paramecium to coordinate movement and early metazoans like worms and jellyfish to send regulatory signals rapidly over increasing distances. But how are electrical signals generated in biology? In fact, voltage-gated sodium channels conduct sodium currents that initiate electrical signals in all kingdoms of life, from bacteria to man. They are responsible for generating the action potential in vertebrate nerve and muscle, neuroendocrine cells, and other cell types 1 , 2 . Because of the high level of conservation of their core structure, it is likely that their fundamental mechanisms of action are conserved as well. Here we describe the complete cycle of conformational changes that a bacterial sodium channel undergoes as it transitions from resting to activated/open and inactivated/closed states, based on high-resolution structural studies of a single sodium channel. We further relate this conformational cycle of the ancestral sodium channel to the function of its vertebrate orthologs. The strong conservation of amino acid sequence and three-dimensional structure suggests that this model, at a fundamental level, is relevant for both prokaryotic and eukaryotic sodium channels, as well as voltage-gated calcium and potassium channels. The conformational cycle of a bacterial voltage-gated sodium channel as it transitions from resting to activated open to inactivated closed states can be constructed from various crystal structure snapshots.

Haploinsufficiency of the voltage-gated sodium channel Na V1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of Na V1.1 in forebrain GABAergic-inhibitory neurons is sufficient to cause all the manifestations of Dravet syndrome in mice, but the physiological roles of specific subtypes of GABAergic interneurons in the cerebral cortex in this disease are unknown. Voltage-clamp studies of dissociated interneurons from cerebral cortex did not detect a significant effect of the Dravet syndrome mutation on sodium currents in cell bodies. However, current-clamp recordings of intact interneurons in layer V of neocortical slices from mice with haploinsufficiency in the gene encoding the Na V1.1 sodium channel, Scn1a , revealed substantial reduction of excitability in fast-spiking, parvalbumin-expressing interneurons and somatostatin-expressing interneurons. The threshold and rheobase for action potential generation were increased, the frequency of action potentials within trains was decreased, and action-potential firing within trains failed more frequently. Furthermore, the deficit in excitability of somatostatin-expressing interneurons caused significant reduction in frequency-dependent disynaptic inhibition between neighboring layer V pyramidal neurons mediated by somatostatin-expressing Martinotti cells, which would lead to substantial disinhibition of the output of cortical circuits. In contrast to these deficits in interneurons, pyramidal cells showed no differences in excitability. These results reveal that the two major subtypes of interneurons in layer V of the neocortex, parvalbumin-expressing and somatostatin-expressing, both have impaired excitability, resulting in disinhibition of the cortical network. These major functional deficits are likely to contribute synergistically to the pathophysiology of Dravet syndrome.

Calcium channel blockers are widely used to treat cardiovascular diseases; new structural studies uncover how two different types of calcium-channel blockers bind to the channel; the drugs bind in different locations, revealing different mechanisms of drug action. Calcium channel blocker structures This paper reports the structural basis for the actions of two different classes of calcium channel blocking drugs: the dihydropyridines and the phenylalkylamines, used to treat high blood pressure and control cardiac arrhythmias, respectively. Lin Tang et al . present crystallographic and functional analyses of how the two drug types bind to the bacterial homotetrameric model Ca V channel Ca V Ab, which is inhibited by dihydropyridines and phenylalkylamines with nanomolar affinity in a state-dependent manner. They find that the binding site for dihydropyridines is located on the external, lipid-facing surface of the pore and that for phenylalkylamines is in the central cavity of the pore on the intracellular side of the selectivity filter. These results define the mechanisms of action of these two classes of calcium channel blockers and could facilitate the design and development of new efficacious antagonists with fewer side effects. Ca 2+ antagonist drugs are widely used in therapy of cardiovascular disorders 1 , 2 . Three chemical classes of drugs bind to three separate, but allosterically interacting, receptor sites on Ca V 1.2 channels, the most prominent voltage-gated Ca 2+ (Ca V ) channel type in myocytes in cardiac and vascular smooth muscle 3 , 4 , 5 , 6 , 7 , 8 , 9 . The 1,4-dihydropyridines are used primarily for treatment of hypertension and angina pectoris and are thought to act as allosteric modulators of voltage-dependent Ca 2+ channel activation, whereas phenylalkylamines and benzothiazepines are used primarily for treatment of cardiac arrhythmias and are thought to physically block the pore 1 , 2 . The structural basis for the different binding, action, and therapeutic uses of these drugs remains unknown. Here we present crystallographic and functional analyses of drug binding to the bacterial homotetrameric model Ca V channel Ca V Ab, which is inhibited by dihydropyridines and phenylalkylamines with nanomolar affinity in a state-dependent manner. The binding site for amlodipine and other dihydropyridines is located on the external, lipid-facing surface of the pore module, positioned at the interface of two subunits. Dihydropyridine binding allosterically induces an asymmetric conformation of the selectivity filter, in which partially dehydrated Ca 2+ interacts directly with one subunit and blocks the pore. In contrast, the phenylalkylamine Br-verapamil binds in the central cavity of the pore on the intracellular side of the selectivity filter, physically blocking the ion-conducting pathway. Structure-based mutations of key amino-acid residues confirm drug binding at both sites. Our results define the structural basis for binding of dihydropyridines and phenylalkylamines at their distinct receptor sites on Ca V channels and offer key insights into their fundamental mechanisms of action and differential therapeutic uses in cardiovascular diseases.

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.

Bacterial voltage-gated sodium channels (BacNavs) serve as models of their vertebrate counterparts. BacNavs contain conserved voltage-sensing and pore-forming domains, but they are homotetramers of four identical subunits, rather than pseudotetramers of four homologous domains. Here, we present structures of two NaVAb mutants that capture tightly closed and open states at a resolution of 2.8–3.2 Å. Introduction of two humanizingmutations in the S6 segment (NaVAb/FY: T206F and V213Y) generates a persistently closed form of the activation gate in which the intracellular ends of the four S6 segments are drawn tightly together to block ion permeation completely. This construct also revealed the complete structure of the four-helix bundle that forms the C-terminal domain. In contrast, truncation of the C-terminal 40 residues in NavAb/1–226 captures the activation gate in an open conformation, revealing the open state of a BacNav with intact voltage sensors. Comparing these structures illustrates the full range of motion of the activation gate, from closed with its orifice fully occluded to open with an orifice of ∼10 Å. Molecular dynamics and free-energy simulations confirm designation of NaVAb/1–226 as an open state that allows permeation of hydrated Na⁺, and these results also support a hydrophobic gating mechanism for control of ion permeation. These two structures allow completion of a closed–open–inactivated conformational cycle in a single voltage-gated sodium channel and give insight into the structural basis for state-dependent binding of sodium channel-blocking drugs.

Voltage-gated calcium (Ca V ) channels catalyse rapid, highly selective influx of Ca 2+ into cells despite a 70-fold higher extracellular concentration of Na + . How Ca V channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial Na V channel Na V Ab. Our results reveal interactions of hydrated Ca 2+ with two high-affinity Ca 2+ -binding sites followed by a third lower-affinity site that would coordinate Ca 2+ as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side chains, which have a critical role in determining Ca 2+ selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations Cd 2+ and Mn 2+ , with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca 2+ ions bound in the selectivity filter and supports a ‘knock-off’ mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our Ca V Ab model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate Ca V channels. X-ray crystal structures of a voltage-gated Na + channel mutated to be highly Ca 2+ selective provide a framework for understanding the mechanisms of ion selectivity and conductance in vertebrate voltage-gated Ca 2+ channels. Super-selectivity of the calcium channel Voltage-gated calcium (Ca V ) channels show remarkable selectivity, facilitating the import of Ca 2+ from an extracellular medium where the Na + concentration is 70-fold higher than that of Ca 2+ . How Ca V channels solve this fundamental biophysical problem remains unclear. This study presents several X-ray crystal structures of the full-length tetrameric bacterial Na V channel engineered through mutations to act as a highly selective voltage-gated calcium channel. The structures reveal that there are three Ca 2+ -binding sites in the pore: two high-affinity Ca 2+ -binding sites, and a lower-affinity site that Ca 2+ occupies as it moves through the pore. The Ca 2+ ions remain nearly fully hydrated as they pass through the channel, which is completely contrary to the textbook picture of fully dehydrated metal ions binding to amino acid side chains or backbone carbonyl groups lining the selectivity filter.