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.

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.

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.

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.