Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal subunit regulation of the cardiac voltage-gated sodium channel.

Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory ß-subunits. The ß-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous ß-subunits. To better reveal ß-subunit functions, we engineered a human cell line devoid of endogenous NaV ß-subunits and their immediate phylogenetic relatives. This new cell line, ß-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the ß-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac NaV α-subunit, NaV1.5, was highly sensitive to ß-subunit modulation and revealed that each ß-subunit and even MPZ imparted unique gating properties. Furthermore, combining ß1 and ß2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of ß-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other α/ß NaV complexes.

Resurgent Na+ currents promote ultrafast spiking in projection neurons that drive fine motor control.

The underlying mechanisms that promote precise spiking in upper motor neurons controlling fine motor skills are not well understood. Here we report that projection neurons in the adult zebra finch song nucleus RA display robust high-frequency firing, ultra-narrow spike waveforms, superfast Na+ current inactivation kinetics, and large resurgent Na+ currents (INaR). These properties of songbird pallial motor neurons closely resemble those of specialized large pyramidal neurons in mammalian primary motor cortex. They emerge during the early phases of song development in males, but not females, coinciding with a complete switch of Na+ channel subunit expression from Navß3 to Navß4. Dynamic clamping and dialysis of Navß4's C-terminal peptide into juvenile RA neurons provide evidence that Navß4, and its associated INaR, promote neuronal excitability. We thus propose that INaR modulates the excitability of upper motor neurons that are required for the execution of fine motor skills.

Cell-Adhesion Properties of ß-Subunits in the Regulation of Cardiomyocyte Sodium Channels.

Voltage-gated sodium (Nav) channels drive the rising phase of the action potential, essential for electrical signalling in nerves and muscles. The Nav channel α-subunit contains the ion-selective pore. In the cardiomyocyte, Nav1.5 is the main Nav channel α-subunit isoform, with a smaller expression of neuronal Nav channels. Four distinct regulatory ß-subunits (ß1-4) bind to the Nav channel α-subunits. Previous work has emphasised the ß-subunits as direct Nav channel gating modulators. However, there is now increasing appreciation of additional roles played by these subunits. In this review, we focus on ß-subunits as homophilic and heterophilic cell-adhesion molecules and the implications for cardiomyocyte function. Based on recent cryogenic electron microscopy (cryo-EM) data, we suggest that the ß-subunits interact with Nav1.5 in a different way from their binding to other Nav channel isoforms. We believe this feature may facilitate trans-cell-adhesion between ß1-associated Nav1.5 subunits on the intercalated disc and promote ephaptic conduction between cardiomyocytes.

A gain-of-function sodium channel ß2-subunit mutation in painful diabetic neuropathy.

Diabetes mellitus is a global challenge with many diverse health sequelae, of which diabetic peripheral neuropathy is one of the most common. A substantial number of patients with diabetic peripheral neuropathy develop chronic pain, but the genetic and epigenetic factors that predispose diabetic peripheral neuropathy patients to develop neuropathic pain are poorly understood. Recent targeted genetic studies have identified mutations in α-subunits of voltage-gated sodium channels (Navs) in patients with painful diabetic peripheral neuropathy. Mutations in proteins that regulate trafficking or functional properties of Navs could expand the spectrum of patients with Nav-related peripheral neuropathies. The auxiliary sodium channel ß-subunits (ß1-4) have been reported to increase current density, alter inactivation kinetics, and modulate subcellular localization of Nav. Mutations in ß-subunits have been associated with several diseases, including epilepsy, cancer, and diseases of the cardiac conducting system. However, mutations in ß-subunits have never been shown previously to contribute to neuropathic pain. We report here a patient with painful diabetic peripheral neuropathy and negative genetic screening for mutations in SCN9A, SCN10A, and SCN11A-genes encoding sodium channel α-subunit that have been previously linked to the development of neuropathic pain. Genetic analysis revealed an aspartic acid to asparagine mutation, D109N, in the ß2-subunit. Functional analysis using current-clamp revealed that the ß2-D109N rendered dorsal root ganglion neurons hyperexcitable, especially in response to repetitive stimulation. Underlying the hyperexcitability induced by the ß2-subunit mutation, as evidenced by voltage-clamp analysis, we found a depolarizing shift in the voltage dependence of Nav1.7 fast inactivation and reduced use-dependent inhibition of the Nav1.7 channel.

A Novel Model for Studying Voltage-Gated Ion Channel Gene Expression during Reversible Ischemic Stroke.

The dysfunction of voltage-gated ion channels contributes to the pathology of ischemic stroke. In this study, we developed rat models of transient ischemic attack (TIA) and reversible ischemic neurological deficit (RIND) that was induced via the injection of artificial embolic particles during full consciousness, that allow us to monitor the neurologic deficit and positron emission tomography (PET) scans in real-time. We then evaluated the infarction volume of brain tissue was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining, and gene expressions were evaluated by quantitative real-time PCR (qPCR). We found that rats with TIA or RIND exhibited neurological deficits as determined by negative TTC and PET findings. However, the expression of voltage-gated sodium channels in the hippocampus was significantly up-regulated in the qPCR array study. Furthermore, an altered expression of sodium channel ß-subunits and potassium channels, were observed in RIND compared to TIA groups. In conclusion, to our knowledge, this is the first report of the successful evaluation of voltage-gated ion channel gene expression in TIA and RIND animal models. This model will aid future studies in investigating pathophysiological mechanisms, and in developing new therapeutic compounds for the treatment of TIA and RIND.

Mining Protein Evolution for Insights into Mechanisms of Voltage-Dependent Sodium Channel Auxiliary Subunits.

Voltage-gated sodium channel (VGSC) beta (ß) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC ß-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC ß1-ß4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC ß-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern ß-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and ß eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for ß-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

Voltage-Gated Sodium Channel ß Subunits and Their Related Diseases.

Voltage-gated sodium channels are protein complexes comprised of one pore forming α subunit and two, non-pore forming, ß subunits. The voltage-gated sodium channel ß subunits were originally identified to function as auxiliary subunits, which modulate the gating, kinetics, and localization of the ion channel pore. Since that time, the five ß subunits have been shown to play crucial roles as multifunctional signaling molecules involved in cell adhesion, cell migration, neuronal pathfinding, fasciculation, and neurite outgrowth. Here, we provide an overview of the evidence implicating the ß subunits in their conducting and non-conducting roles. Mutations in the ß subunit genes (SCN1B-SCN4B) have been linked to a variety of diseases. These include cancer, epilepsy, cardiac arrhythmias, sudden infant death syndrome/sudden unexpected death in epilepsy, neuropathic pain, and multiple neurodegenerative disorders. ß subunits thus provide novel therapeutic targets for future drug discovery.

Voltage-gated sodium channel ß subunits: The power outside the pore in brain development and disease.

Voltage gated sodium channels (VGSCs) were first identified in terms of their role in the upstroke of the action potential. The underlying proteins were later identified as saxitoxin and scorpion toxin receptors consisting of α and ß subunits. We now know that VGSCs are heterotrimeric complexes consisting of a single pore forming α subunit joined by two ß subunits; a noncovalently linked ß1 or ß3 and a covalently linked ß2 or ß4 subunit. VGSC α subunits contain all the machinery necessary for channel cell surface expression, ion conduction, voltage sensing, gating, and inactivation, in one central, polytopic, transmembrane protein. VGSC ß subunits are more than simple accessories to α subunits. In the more than two decades since the original cloning of ß1, our knowledge of their roles in physiology and pathophysiology has expanded immensely. VGSC ß subunits are multifunctional. They confer unique gating mechanisms, regulate cellular excitability, affect brain development, confer distinct channel pharmacology, and have functions that are independent of the α subunits. The vast array of functions of these proteins stems from their special station in the channelome: being the only known constituents that are cell adhesion and intra/extracellular signaling molecules in addition to being part of channel complexes. This functional trifecta and how it goes awry demonstrates the power outside the pore in ion channel signaling complexes, broadening the term channelopathy beyond defects in ion conduction. This article is part of the Special Issue entitled 'Channelopathies.'

Contribution of Cardiac Sodium Channel ß-Subunit Variants to Brugada Syndrome.

BACKGROUND: Brugada syndrome (BrS) is an inheritable cardiac disease associated with syncope, malignant ventricular arrhythmias and sudden cardiac death. The largest proportion of mutations in BrS is found in the SCN5A gene encoding the α-subunit of cardiac sodium channels (Nav1.5). Causal SCN5A mutations are present in 18-30% of BrS patients. The additional genetic diagnostic yield of variants in cardiac sodium channel ß-subunits in BrS patients was explored and functional studies on 3 novel candidate variants were performed. METHODS AND RESULTS: TheSCN1B-SCN4B genes were screened, which encode the 5 sodium channel ß-subunits, in a SCN5A negative BrS population (n=74). Five novel variants were detected; in silico pathogenicity prediction classified 4 variants as possibly disease causing. Three variants were selected for functional study. These variants caused only limited alterations of Nav1.5 function. Next generation sequencing of a panel of 88 arrhythmia genes could not identify other major causal mutations. CONCLUSIONS: It was hypothesized that the studied variants are not the primary cause of BrS in these patients. However, because small functional effects of these ß-subunit variants can be discriminated, they might contribute to the BrS phenotype and be considered a risk factor. The existence of these risk factors can give an explanation to the reduced penetrance and variable expressivity seen in this syndrome. We therefore recommend including the SCN1-4B genes in a next generation sequencing-based gene panel.

Sodium channel ß subunits: emerging targets in channelopathies.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and ß subunits. Although ß subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. ß subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding ß subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC ß subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target.

A new look at sodium channel ß subunits.

Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated ß subunits. The ß subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human ß3 and ß4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that ß3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some ß subunits.

The role of non-pore-forming ß subunits in physiology and pathophysiology of voltage-gated sodium channels.

Voltage-gated sodium channel ß1 and ß2 subunits were discovered as auxiliary proteins that co-purify with pore-forming α subunits in brain. The other family members, ß1B, ß3, and ß4, were identified by homology and shown to modulate sodium current in heterologous systems. Work over the past 2 decades, however, has provided strong evidence that these proteins are not simply ancillary ion channel subunits, but are multifunctional signaling proteins in their own right, playing both conducting (channel modulatory) and nonconducting roles in cell signaling. Here, we discuss evidence that sodium channel ß subunits not only regulate sodium channel function and localization but also modulate voltage-gated potassium channels. In their nonconducting roles, VGSC ß subunits function as immunoglobulin superfamily cell adhesion molecules that modulate brain development by influencing cell proliferation and migration, axon outgrowth, axonal fasciculation, and neuronal pathfinding. Mutations in genes encoding ß subunits are linked to paroxysmal diseases including epilepsy, cardiac arrhythmia, and sudden infant death syndrome. Finally, ß subunits may be targets for the future development of novel therapeutics.

Is sudden unexplained nocturnal death syndrome in Southern China a cardiac sodium channel dysfunction disorder?

Sudden unexplained nocturnal death syndrome (SUNDS) remains an enigma to both forensic pathologists and physicians. Previous epidemiological, clinical, and pilot genetic studies have implicated that SUNDS is most likely a disease allelic to Brugada syndrome (BrS). We have performed postmortem genetic testing to address the spectrum and role of genetic abnormalities in the SCN5A-encoded cardiac sodium channel and its several associated proteins in SUNDS victims from Southern China. Genomic DNA extracted from the blood samples of 123 medico-legal autopsy-negative SUNDS cases and 104 sex-, age- and ethnic-matched controls from Southern China underwent comprehensive amino acid coding region mutational analysis for the BrS associated genes SCN5A, SCN1B, SCN2B, SCN3B, SCN4B, MOG1, and GPD1-L using PCR and direct sequencing. We identified a total of 7 unique (4 novel) putative pathogenic mutations (all in SCN5A; V95I, R121Q [2 cases], R367H, R513H, D870H, V1764D, and S1937F) in 8/123 (6.5%) SUNDS cases. Three SCN5A mutations (V95I, R121Q, and R367H) have been previously implicated in BrS. An additional 8 cases hosted rare variants of uncertain clinical significance (SCN5A: V1098L, V1202M, R1512W; SCN1B: V138I [3 cases], T189M [2 cases]; SCN3B: A195T). There were no non-synonymous mutations found in SCN2B, SCN4B, MOG1, or GPD1-L. This first comprehensive genotyping for SCN5A and related genes in the Chinese Han population with SUNDS discovered 13 mutations, 4 of them novel, in 16 cases, which suggests cardiac sodium channel dysfunction might account for the pathogenesis of 7-13% of SUNDS in Southern China.

Na+ channel-dependent recruitment of Navß4 to axon initial segments and nodes of Ranvier.

The axon initial segment (AIS) and nodes of Ranvier are the sites of action potential initiation and regeneration in axons. Although the basic molecular architectures of AIS and nodes, characterized by dense clusters of Na(+) and K(+) channels, are similar, firing patterns vary among cell types. Neuronal firing patterns are established by the collective activity of voltage-gated ion channels and can be modulated through interaction with auxiliary subunits. Here, we report the neuronal expression pattern and subcellular localization of Navß4, the modulatory Na(+) channel subunit thought to underlie resurgent Na(+) current. Immunostaining of rat tissues revealed that Navß4 is strongly enriched at the AIS of a select set of neuron types, including many characterized by high-frequency firing, and at nodes of Ranvier in the PNS and some nodes in the CNS. By introducing full-length and mutant GFP-tagged Navß4 into cultured neurons, we determined that the AIS and nodal localization of Navß4 depends on its direct interaction with Na(+) channel α subunits through an extracellular disulfide bond. Based on these results, we propose that differences in the specific composition of the Na(+) channel complexes enriched at the AIS and nodes contribute to the diverse physiologies observed among cell types.

Co-expression of Na(V)ß subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking µ-conotoxins.

BACKGROUND AND PURPOSE: Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing α-subunit (NaV 1) and one or two accessory ß-subunits (NaV ßs). Neurons in mammals can express one or more of seven isoforms of NaV 1 and one or more of four isoforms of NaV ß. The peptide µ-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in NaV 1. Hitherto, the effects of NaV ß-subunit co-expression on the activity of these toxins have not been comprehensively assessed. EXPERIMENTAL APPROACH: Four µ-conotoxins (µ-TIIIA, µ-PIIIA, µ-SmIIIA and µ-KIIIA), TTX and STX were tested against NaV 1.1, 1.2, 1.6 or 1.7, each co-expressed in Xenopus laevis oocytes with one of NaV ß1, ß2, ß3 or ß4 and, for NaV 1.7, binary combinations of thereof. KEY RESULTS: Co-expression of NaV ß-subunits modifies the block by µ-conotoxins: in general, NaV ß1 or ß3 co-expression tended to increase kon (in the most extreme instance by ninefold), whereas NaV ß2 or ß4 co-expression decreased kon (in the most extreme instance by 240-fold). In contrast, the block by TTX and STX was only minimally, if at all, affected by NaV ß-subunit co-expression. Tests of NaV ß1 : ß2 chimeras co-expressed with NaV 1.7 suggest that the extracellular portion of the NaV ß subunit is largely responsible for altering µ-conotoxin kinetics. CONCLUSIONS AND IMPLICATIONS: These results are the first indication that NaV ß subunit co-expression can markedly influence µ-conotoxin binding and, by extension, the outer vestibule of the pore of VGSCs. µ-Conotoxins could, in principle, be used to pharmacologically probe the NaV ß subunit composition of endogenously expressed VGSCs.

Field-collected permethrin-resistant Aedes aegypti from central Thailand contain point mutations in the domain IIS6 of the sodium channel gene (KDR).

One of the mechanisms responsible for pyrethroid resistance in mosquitoes is mutations in domain IIS6 of voltage-gated sodium channel gene (kdr). Aedes aegypti larvae were collected from the central provinces of Thailand (Bangkok, Prachin Buri and Ratchaburi) and colonized until they became adults. Partial fragment of kdr of permethrin-resistant mosquitoes were amplified by RT-PCR and sequenced. Among the four nucleotide mutations detected, two mutations resulted in two amino acid substitutions, S(TCC) 989 P(CCC) and V(GTA)1016 G(GGA). Among 94 permethrin-resistant mosquitoes, the SS genotype (SS/VV) was found to predominate (n = 74), followed by SR (SP/VG) (n = 15) and RR (PP/ GG) genotypes (n = 5), with the resistant allele frequency ranging from 0.03 to 0.17. As pyrethroid insecticides are currently being advocated for use in Thailand, investigations of pyrethroid resistance in other regions of the country are needed to prevent potential cross-resistance among different types of insecticides.