It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.

Scn2a encodes the voltage-gated sodium channel NaV1.2, a main mediator of neuronal action potential firing. The current paradigm suggests that NaV1.2 gain-of-function variants enhance neuronal excitability, resulting in epilepsy, whereas NaV1.2 deficiency impairs neuronal excitability, contributing to autism. However, this paradigm does not explain why ∼20%-30% of individuals with NaV1.2 deficiency still develop seizures. Here, we report the counterintuitive finding that severe NaV1.2 deficiency results in increased neuronal excitability. Using a NaV1.2-deficient mouse model, we show enhanced intrinsic excitability of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in Scn2a-related seizures. This increased excitability is autonomous and reversible by genetic restoration of Scn2a expression in adult mice. RNA sequencing reveals downregulation of multiple potassium channels, including KV1.1. Correspondingly, KV channel openers alleviate the hyperexcitability of NaV1.2-deficient neurons. This unexpected neuronal hyperexcitability may serve as a cellular basis underlying NaV1.2 deficiency-related seizures.

Neuronal ankyrin-B is an intracellular scaffolding protein that plays multiple roles in the axon. By contrast, relatively little is known about the function of ankyrin-B in dendrites, where ankyrin-B is also localized in mature neurons. Recently, we showed that ankyrin-B acts as a scaffold for the voltage-gated sodium channel, NaV1.2, in dendrites of neocortical pyramidal neurons. How ankyrin-B is itself targeted to the dendritic membrane is not well understood. Here, we report that ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic localization of NaV1.2. We identify the palmitoyl acyl transferase zDHHC17 as a key mediator of ankyrin-B palmitoylation in heterologous cells and in neurons. Additionally, we find that zDHHC17 regulates ankyrin-B protein levels independently of its S-acylation function through a conserved binding mechanism between the ANK repeat domain of zDHHC17 and the zDHHC ankyrin-repeat binding motif of ankyrin-B. We subsequently identify five cysteines in the N-terminal ankyrin repeat domain of ankyrin-B that are necessary for ankyrin-B palmitoylation. Mutation of these five cysteines to alanines not only abolishes ankyrin-B palmitoylation, but also prevents ankyrin-B from scaffolding NaV1.2 at dendritic membranes of neurons due to ankyrin-B's inability to localize properly at dendrites. Thus, we show palmitoylation is critical for localization and function of ankyrin-B at dendrites. Strikingly, loss of ankyrin-B palmitoylation does not affect ankyrin-B-mediated axonal cargo transport of synaptic vesicle synaptotagmin-1 in neurons. This is the first demonstration of S-palmitoylation of ankyrin-B as an underlying mechanism required for ankyrin-B localization and function in scaffolding NaV1.2 at dendrites.

Voltage-gated sodium channels are targets for many analgesic and antiepileptic drugs whose therapeutic mechanisms and binding sites have been well characterized. We describe the identification of a previously unidentified receptor site within the NavMs voltage-gated sodium channel. Tamoxifen, an estrogen receptor modulator, and its primary and secondary metabolic products bind at the intracellular exit of the channel, which is a site that is distinct from other previously characterized sodium channel drug sites. These compounds inhibit NavMs and human sodium channels with similar potencies and prevent sodium conductance by delaying channel recovery from the inactivated state. This study therefore not only describes the structure and pharmacology of a site that could be leveraged for the development of new drugs for the treatment of sodium channelopathies but may also have important implications for off-target health effects of this widely used therapeutic drug.

Epilepsy is a disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels (NaVs). The C121W mutation of the β1 subunit, in particular, gives rise to the thermosensitive generalized epilepsy with febrile seizures plus (GEFS+) phenotype. Lacosamide is used to treat epileptic seizures and is distinct from other anti-seizure drugs by targeting NaV slow-inactivation. We studied the effects of a physiologically relevant concentration of lacosamide on the biophysical properties of NaV1.2 channels associated with either WT-β1 or the mutant C121W-β1 subunit. Biophysical parameters were measured at both normal (22°C) and elevated (34°C) temperatures to elicit the differential temperature-sensitivity of C121W. Lacosamide was more effective in NaV1.2 associated with the WT-β1 than with C121W-β1 at either temperature. There is also a more potent effect by lacosamide on slow inactivation at elevated temperatures. Our data suggest a modulatory role is imparted by the β1 subunit in the interaction between the drug and the channel.

Alterations in the expression, molecular composition, and localization of voltage-gated sodium channels play major roles in a broad range of neurological disorders. Recent evidence identifies sodium channel proteolysis as a key early event after ischemia and traumatic brain injury, further expanding the role of the sodium channel in neurological diseases. In this study, we investigate the protease responsible for proteolytic cleavage of voltage-gated sodium channels (NaChs). NaCh proteolysis occurs after protease activation in rat brain homogenates, pharmacological disruption of ionic homeostasis in cortical cultures, and mechanical injury using an in vitro model of traumatic brain injury. Proteolysis requires Ca(2+) and calpain activation but is not influenced by caspase-3 or cathepsin inhibition. Proteolysis results in loss of the full-length alpha-subunits, and the creation of fragments comprising all domains of the channel that retain interaction even after proteolysis. Cell surface biotinylation after mechanical injury indicates that proteolyzed NaChs remain in the membrane before noticeable evidence of neuronal death, providing a mechanism for altered action potential initiation, propagation, and downstream signaling events after Ca(2+) elevation.

Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage-gated sodium or Nav channels underlie action potential initiation and propagation, dendritic excitability, and many other aspects of neuronal excitability. Various protein kinases have been suggested to phosphorylate the primary or alpha subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav alpha subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in mediating diverse aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav alpha subunits affinity-purified from rat brain with two distinct monoclonal antibodies to identify 15 phosphorylation sites on Nav1.2, 12 of which have not been previously reported. We also found 3 novel phosphorylation sites on Nav1.1. In general, commonly used phosphorylation site prediction algorithms did not accurately predict these novel in vivo phosphorylation sites. Our results demonstrate that specific Nav alpha subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nav channel activity in mammalian brain. Identification of phosphorylation sites using monoclonal antibody-based immunopurification and mass spectrometry is an effective approach to define the phosphorylation status of Nav channels and other important membrane proteins in mammalian brain.

Voltage-gated sodium channels (Nav1.1-Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein-protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.

Proteins that interact with voltage-gated sodium (Na(v)) channels are important in channel sorting and modulation. In this study, we identified the transcriptional regulator, Sin3B, as a novel binding partner of Na(v) channels in a yeast two-hybrid screen and confirmed the interaction using pull-down assays, co-immunoprecipitation, and immunofluorescence-colocalization. Because both long (~1100-residue) and short (N-terminal 293 residues) Sin3B variants interacted with Na(v) channels, binding occurred within the N-terminal region containing two paired-amphipathic helix domains. In Na(v) channels, Sin3B bound to a 132-residue portion of the cytoplasmic C-terminus. Expression of the short Sin3B variant strongly reduced native sodium current and Na(v)-channel gating charge in the neuronal cell line N1E-115, without affecting the voltage-dependence of activation. Because the total amount of channel protein was unchanged by Sin3B, binding of Sin3B likely decreases the number of channels in the plasma membrane, suggesting that interaction with Sin3B influences Na(v)-channel trafficking or stability in the membrane.

1. We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Nabeta1.1 and Nabeta2.1 and the novel, recently cloned subunit, beta3. 2. In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and beta3 mRNA at embryonic stages whilst Nabeta1.1 and Nabeta2.1 mRNA was absent throughout this period. 3. Nabeta1.1 and Nabeta2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. beta3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum. 4. Emulsion-dipped slides showed co-localisation of beta3 with Nav1.3 mRNA in areas of the CNS suggesting that these subunits may be capable of functional interaction. 5. Co-expression in Xenopus oocytes revealed that beta3 could modify the properties of Nav1.3; beta3 changed the equilibrium of Nav1.3 between the fast and slow gating modes and caused a negative shift in the voltage dependence of activation and inactivation. 6. In conclusion, beta3 is shown to be the predominant beta subunit expressed during development and is capable of modulating the kinetic properties of the embryonic Nav1.3 subunit. These findings provide new information regarding the nature and properties of voltage-gated sodium channels during development.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in neurons. The human genome includes ten human VGSC α-subunit genes, SCN(X)A, encoding Nav1.1-1.9 plus Nax. To understand the unique role that each VGSC plays in normal and pathophysiological function in neural networks, compounds with high affinity and selectivity for specific VGSC subtypes are required. Toward that goal, a structural analog of the VGSC pore blocker tetrodotoxin, 4,9-anhydrotetrodotoxin (4,9-ah-TTX), has been reported to be more selective in blocking Na+ current mediated by Nav1.6 than other TTX-sensitive VGSCs, including Nav1.2, Nav1.3, Nav1.4, and Nav1.7. While SCN1A, encoding Nav1.1, has been implicated in several neurological diseases, the effects of 4,9-ah-TTX on Nav1.1-mediated Na+ current have not been tested. Here, we compared the binding of 4,9-ah-TTX for human and mouse brain preparations, and the effects of 4,9-ah-TTX on human Nav1.1-, Nav1.3- and Nav1.6-mediated Na+ currents using the whole-cell patch clamp technique in heterologous cells. We show that, while 4,9-ah-TTX administration results in significant blockade of Nav1.6-mediated Na+ current in the nanomolar range, it also has significant effects on Nav1.1-mediated Na+ current. Thus, 4,9-ah-TTX is not a useful tool in identifying Nav1.6-specific effects in human brain networks.

Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs), this AP back-propagation is supported by dendritic voltage-gated Na+ (Nav) channels, whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we revealed the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here, the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability.

Many developmental and epileptic encephalopathies (DEEs) result from variants in cation channel genes. Using mRNA transfection, we generated and characterised an induced pluripotent stem cell (iPSC) line from the fibroblasts of a male late-onset DEE patient carrying a heterozygous missense variant (E1211K) in Nav1.2(SCN2A) protein. The iPSC line displays features characteristic of the human iPSCs, colony morphology and expression of pluripotency-associated marker genes, ability to produce derivatives of all three embryonic germ layers, and normal karyotype without SNP array-detectable abnormalities. We anticipate that this iPSC line will aid in the modelling and development of precision therapies for this debilitating condition.

Keratinocytes are implicated in sensory transduction and can influence nociception, but whether these contribute to chronic pain is not known. In neurons, voltage-gated sodium channels (Na(v)) are involved in neuropathic pain and are activated by depolarization. Since keratinocytes can also show changes in membrane potential, we used RT-PCR, in situ hybridization, and immunohistochemistry to investigate the expression of sodium channels in these cells. Na(v)1.1, Na(v)1.6, and Na(v)1.8 were localized within keratinocytes in rat epidermis. In addition, sodium channels contribute to the release of ATP from rat keratinocytes in response to increased [K(+)](o), implicating sodium channels in keratinocyte ligand release and nociception. To examine whether keratinocytes may contribute to human pain states, we analyzed sodium channel expression in human skin biopsies from subjects with complex regional pain syndrome Type 1 (CRPS) and post-herpetic neuralgia (PHN) using immunohistochemistry. Control skin exhibited immunolabeling for Na(v)1.5, Na(v)1.6 and Na(v)1.7. In contrast, painful skin from CRPS and PHN subjects displayed Na(v)1.1, Na(v)1.2, and Na(v)1.8 immunolabeling, in addition to substantially increased signal for Na(v)1.5, Na(v)1.6, Na(v)1.7. These observations lead us to propose that pathological increases in keratinocyte sodium channel expression may contribute to pain by increasing epidermal ATP release, resulting in excessive activation of P2X receptors on primary sensory axons. Consistent with this hypothesis, animal models of neuropathic pain exhibit increases in subcutaneous ATP release and activity of primary sensory neurons, and peripheral administration of P2X antagonists has been shown to reduce neuropathic pain in humans.

Although enhanced calpain activity is well documented after traumatic brain injury (TBI), the pathways targeting specific substrate proteolysis are less defined. Our past work demonstrated that calpain cleaves voltage gated sodium channel (NaCh) α-subunits in an in vitro TBI model. In this study, we investigated the pathways leading to NaCh cleavage utilizing our previously characterized in vitro TBI model, and determined the location of calpain activation within neuronal regions following stretch injury to micropatterned cultures. Calpain specific breakdown products of α-spectrin appeared within axonal, dendritic, and somatic regions 6 h after injury, concurrent with the appearance of NaCh α-subunit proteolysis in both whole cell or enriched axonal preparations. Direct pharmacological activation of either NMDA receptors (NMDArs) or NaChs resulted in NaCh proteolysis. Likewise, a chronic (6 h) dual inhibition of NMDArs/NaChs but not L-type voltage gated calcium channels significantly reduced NaCh proteolysis 6 h after mechanical injury. Interestingly, an early, transient (30 min) inhibition of NMDArs alone significantly reduced NaCh proteolysis. Although a chronic inhibition of calpain significantly reduced proteolysis, a transient inhibition of calpain immediately after injury failed to significantly attenuate NaCh proteolysis. These data suggest that both NMDArs and NaChs are key contributors to calpain activation after mechanical injury, and that a larger temporal window of sustained calpain activation needs consideration in developing effective treatments for TBI.

Propranolol is a widely used, non-selective β-adrenergic receptor antagonist with proven efficacy in treating cardiovascular disorders and in the prevention of migraine headaches. At plasma concentrations exceeding those required for β-adrenergic receptor inhibition, propranolol also exhibits anti-arrhythmic ("membrane stabilizing") effects that are not fully explained by β-blockade. Previous in vitro studies suggested that propranolol may have local anesthetic effects. We directly tested the effects of propranolol on heterologously expressed recombinant human cardiac (NaV1.5) and brain (NaV1.1, NaV1.2, NaV1.3) sodium channels using whole-cell patch-clamp recording. We found that block was not stereospecific as we observed approximately equal IC50 values for tonic and use-dependent block by R-(+) and S-(-) propranolol (tonic block: R: 21.4 μM vs S: 23.6 μM; use-dependent block: R: 2.7 μM vs S: 2.6 μM). Metoprolol and nadolol did not block NaV1.5 indicating that sodium channel block is not a class effect of β-blockers. The biophysical effects of R-(+)-propranolol on NaV1.5 and NaV1.1 resembled that of the prototypical local anesthetic lidocaine including the requirement for a critical phenylalanine residue (F1760 in NaV1.5) in the domain 4 S6 segment. Finally, we observed that brain sodium channels exhibited less sensitivity to R-(+)-propranolol than NaV1.5 channels. Our findings establish sodium channels as targets for propranolol and may help explain some beneficial effects of the drug in treating cardiac arrhythmias, and may explain certain adverse central nervous system effects.

The voltage-gated sodium channel subtype 1.2 (NaV1.2) is instrumental in the initiation of action potentials in the nervous system, making it a natural drug target for neurological diseases. Therefore, there is much pharmacological interest in finding blockers of NaV1.2 and improving their affinity and selectivity properties. An extensive family of peptide toxins from cone snails (conotoxins) block NaV channels, thus they provide natural templates for the design of drugs targeting NaV channels. Unfortunately, progress was hampered due to the absence of any NaV structures. The recent determination of cryo-EM structures for NaV channels has finally broken this impasse. Here, we use the NaV1.2 structure in complex with μ-conotoxin KIIIA (KIIIA) in computational studies with the aim of improving KIIIA's affinity and blocking capacity for NaV1.2. Only three KIIIA amino acid residues are available for mutation (S5, S6, and S13). After performing molecular modeling and simulations on NaV1.2-KIIIA complex, we have identified the S5R, S6D, and S13K mutations as the most promising for additional contacts. We estimate these contacts to boost the affinity of KIIIA for NaV1.2 from nanomole to picomole domain. Moreover, the KIIIA[S5R, S6D, S13K] analogue makes contacts with all four channel domains, thus enabling the complete blocking of the channel (KIIIA partially blocks as it has contacts with three domains). The proposed KIIIA analogue, once confirmed experimentally, may lead to novel anti-epileptic drugs.

Dendrodendritic synaptic interactions between olfactory bulb mitral and granule cells represent a key neuronal mechanism of odor discrimination. Dendritic release of gamma-aminobutyric acid (GABA) from granule cells contributes to stimulus-dependent, rapid, and accurate odor discrimination, yet the physiological mechanisms governing this release and its behavioral relevance are unknown. Here, we show that granule cells express the voltage-gated sodium channel α-subunit NaV1.2 in clusters distributed throughout the cell surface including dendritic spines. Deletion of NaV1.2 in granule cells abolished spiking and GABA release as well as inhibition of synaptically connected mitral cells (MCs). As a consequence, mice required more time to discriminate highly similar odorant mixtures, while odor discrimination learning remained unaffected. In conclusion, we show that expression of NaV1.2 in granule cells is crucial for physiological dendritic GABA release and rapid discrimination of similar odorants with high accuracy. Hence, our data indicate that neurotransmitter-releasing dendritic spines function just like axon terminals.

The bacterial sodium channel NaChBac is the prokaryotic prototype for the eukaryotic NaV and CaV channels, which could be used as a relatively simple model to study their structure-function relationships. However, few modulators of NaChBac have been reported thus far, and the pharmacology of NaChBac remains to be investigated. In the present study, we show that the spider toxin κ-LhTx-1, an antagonist of the KV4 family potassium channels, potently inhibits NaChBac with an IC50 of 491.0 ± 61.7 nM. Kinetics analysis revealed that κ-LhTx-1 inhibits NaChBac by impeding the voltage-sensor activation. Site-directed mutagenesis confirmed that phenylalanine-103 (F103) in the S3-S4 extracellular loop of NaChBac was critical for interacting with κ-LhTx-1. Molecular docking predicts the binding interface between κ-LhTx-1 and NaChBac and highlights a dominant hydrophobic interaction between W27 in κ-LhTx-1 and F103 in NaChBac that stabilizes the interface. In contrast, κ-LhTx-1 showed weak activity on the mammalian NaV channels, with 10 µM toxin slightly inhibiting the peak currents of NaV1.2-1.9 subtypes. Taken together, our study shows that κ-LhTx-1 inhibits the bacterial sodium channel, NaChBac, using a voltage-sensor trapping mechanism similar to mammalian NaV site 4 toxins. κ-LhTx-1 could be used as a ligand to study the toxin-channel interactions in the native membrane environments, given that the NaChBac structure was successfully resolved in a nanodisc.

Brevetoxins are produced by dinoflagellates such as Karenia brevis in warm-water red tides and cause neurotoxic shellfish poisoning. They bind to voltage-gated sodium channels at neurotoxin receptor 5, making the channels more active by shifting the voltage-dependence of activation to more negative potentials and by slowing the inactivation process. Previous work using photoaffinity labeling identified binding to the IS6 and IVS5 transmembrane segments of the channel α subunit. We used alanine-scanning mutagenesis to identify molecular determinants for brevetoxin binding in these regions as well as adjacent regions IVS5-SS1 and IVS6. Most of the mutant channels containing single alanine substitutions expressed functional protein in tsA-201 cells and bound to the radioligand [42-3H]-PbTx3. Binding affinity for the great majority of mutant channels was indistinguishable from wild type. However, transmembrane segments IS6, IVS5 and IVS6 each contained 2 to 4 amino acid positions where alanine substitution resulted in a 2-3-fold reduction in brevetoxin affinity, and additional mutations caused a similar increase in brevetoxin affinity. These findings are consistent with a model in which brevetoxin binds to a protein cleft comprising transmembrane segments IS6, IVS5 and IVS6 and makes multiple distributed interactions with these α helices. Determination of brevetoxin affinity for Nav1.2, Nav1.4 and Nav1.5 channels showed that Nav1.5 channels had a characteristic 5-fold reduction in affinity for brevetoxin relative to the other channel isoforms, suggesting the interaction with sodium channels is specific despite the distributed binding determinants.