Voltage-gated sodium channels ß3 subunit promotes tumorigenesis in hepatocellular carcinoma by facilitating p53 degradation.

The voltage-gated sodium channels (VGSCs) are aberrantly expressed in a variety of tumors and play an important role in tumor growth and metastasis. Here, we show that VGSCs auxiliary ß3 subunit, encoded by the SCN3B gene, promotes proliferation and suppresses apoptosis in HepG2 cells by promoting p53 degradation. ß3 significantly increases HepG2 cell proliferation, promotes tumor growth in mouse xenograft models, and suppresses senescence and apoptosis. We found that ß3 knockdown stabilizes p53 protein, leading to potentiation of p53-induced cell cycle arrest, senescence, and apoptosis. Mechanistic studies revealed that ß3 could bind to p53, promoting p53 ubiquitination and degradation by stabilizing the p53/MDM2 complex. Our results suggest that ß3 is a novel negative regulator of p53 and a potential oncogenic factor.

An Analysis of the Association between Epilepsy-Related Genes and Vertigo in the Polish Population.

Considering the possibility of a common genetic background of vertigo and epilepsy, we genotyped an affected group of individuals with vertigo and an unaffected group, by studying 26 single-nucleotide polymorphisms (SNPs) in 14 genes which were previously reported to be of particular importance for epilepsy. Significant differences were found between the patients and the control group (χ2 = 38.3, df = 3, p = 1.6 × 10-7) for the frequencies of haplotypes consist ing of 2 SNPs located in chromosome 11 (rs1939012 and rs1783901 within genes MMP8 and SCN3B, respectively). The haplotype rs1939012:C-rs1783901:A, consisting of the minor-frequency alleles was found to be associated with a higher risk of vertigo (OR = 5.0143, 95% CI = 1.6991-14.7980, p = 0.0035). In contrast, the haplotype rs1939012:T-rs1783901:A showed a significant association with a decreased risk of the disease (OR = 0.0597, 95% CI = 0.0136-0.2620, p = 0.0002). Our results suggest that the SNPs rs1939012 and rs1783901 may play a potential role of gene regulation and/or epistasis in a complex etiology of vertigo.

Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice.

The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. We generated an epitope-tagged NaV1.7 mouse that showed normal pain behaviours to identify channel-interacting proteins. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo The sodium channel ß3 (Scn3b), rather than the ß1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing-response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein interactors including membrane-trafficking protein synaptotagmin-2 (Syt2), L-type amino acid transporter 1 (Lat1) and transmembrane P24-trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co-immunoprecipitation (Co-IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein-regulated inducer of neurite outgrowth (Gprin1), an opioid receptor-binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope-tagged mouse should provide useful insights into the many functions now associated with the NaV1.7 channel.

Neuronal Nav1.8 Channels as a Novel Therapeutic Target of Acute Atrial Fibrillation Prevention.

BACKGROUND: Ganglionated plexus have been developed as additional ablation targets to improve the outcome of atrial fibrillation (AF) besides pulmonary vein isolation. Recent studies implicated an intimate relationship between neuronal sodium channel Nav1.8 (encoded by SCN10A) and AF. The underlying mechanism between Nav1.8 and AF remains unclear. This study aimed to determine the role of Nav1.8 in cardiac electrophysiology in an acute AF model and explore possible therapeutic targets. METHODS AND RESULTS: Immunohistochemical study was used on canine cardiac ganglionated plexus. Both Nav1.5 and Nav1.8 were expressed in ganglionated plexus with canonical neuronal markers. Sixteen canines were randomly administered either saline or the Nav1.8 blocker A-803467. Electrophysiological study was compared between the 2 groups before and after 6-hour rapid atrial pacing. Compared with the control group, administration of A-803467 decreased the incidence of AF (87.5% versus 25.0%, P<0.05), shortened AF duration, and prolonged AF cycle length. A-803467 also significantly suppressed the decrease in the effective refractory period and the increase in effective refractory period dispersion and cumulative window of vulnerability caused by rapid atrial pacing in all recording sites. Patch clamp study was performed under 100 nmol/L A-803467 in TSA201 cells cotransfected with SCN10A-WT, SCN5A-WT, and SCN3B-WT. INa,P was reduced by 45.34% at -35 mV, and INa,L by 68.57% at -20 mV. Evident fast inactivation, slow recovery, and use-dependent block were also discovered after applying the drug. CONCLUSIONS: Our study demonstrates that Nav1.8 could exert its effect on electrophysiological characteristics through cardiac ganglionated plexus. It indicates that Nav1.8 is a novel target in understanding cardiac electrophysiology and SCN10A-related arrhythmias.

Regulation of SCN3B/scn3b by Interleukin 2 (IL-2): IL-2 modulates SCN3B/scn3b transcript expression and increases sodium current in myocardial cells.

BACKGROUND: In the initiation and maintenance of arrhythmia, inflammatory processes play an important role. IL-2 is a pro-inflammatory factor which is associated with the morbidity of arrhythmias, however, how IL-2 affects the cardiac electrophysiology is still unknown. METHODS: In the present study, we observed the effect of IL-2 by qRT-PCR on the transcription of ion channel genes including SCN2A, SCN3A, SCN4A, SCN5A, SCN9A, SCN10A, SCN1B, SCN2B, SCN3B, KCNN1, KCNJ5, KCNE1, KCNE2, KCNE3, KCND3, KCNQ1, KCNA5, KCNH2 and CACNA1C. Western blot assays and electrophysiological studies were performed to demonstrate the effect of IL-2 on the translation of SCN3B/scn3b and sodium currents. RESULTS: The results showed that transcriptional level of SCN3B was up-regulated significantly in Hela cells (3.28-fold, p = 0.022 compared with the control group). Consistent results were verified in HL-1 cells (3.73-fold, p = 0.012 compared with the control group). The result of electrophysiological studies showed that sodium current density increased significantly in cells which treated by IL-2 and the effect of IL-2 on sodium currents was independent of SCN3B (1.4 folds, p = 0.023). Western blot analysis showed IL-2 lead to the significantly increasing of p53 and scn3b (2.1 folds, p = 0.021 for p53; 3.1 folds, p = 0.023 for scn3b) in HL-1 cells. Consistent results were showed in HEK293 cells using qRT-PCR analysis (1.43 folds for P53, p = 0.022; 1.57 folds for SCN3B, p = 0.05). CONCLUSIONS: The present study suggested that IL-2, may play role in the arrhythmia by regulating the expression of SCN3B and sodium current density.

Deletion of FoxO1 leads to shortening of QRS by increasing Na(+) channel activity through enhanced expression of both cardiac NaV1.5 and ß3 subunit.

Our in vitro studies revealed that a transcription factor, Forkhead box protein O1 (FoxO1), negatively regulates the expression of NaV1.5, a main α subunit of the cardiac Na(+) channel, by altering the promoter activity of SCN5a in HL-1 cardiomyocytes. The in vivo role of FoxO1 in the regulation of cardiac NaV1.5 expression remains unknown. The present study aimed to define the role of FoxO1 in the regulation of NaV1.5 expression and cardiac Na(+) channel activity in mouse ventricular cardiomyocytes and assess the cardiac electrophysiological phenotype of mice with cardiac FoxO1 deletion. Tamoxifen-induced and cardiac-specific FoxO1 deletion was confirmed by polymerase chain reaction (PCR). Cardiac FoxO1 deletion failed to result in either cardiac functional changes or hypertrophy as assessed by echocardiography and individual ventricular cell capacitances, respectively. Western blotting showed that FoxO1 was significantly decreased while NaV1.5 protein level was significantly increased in mouse hearts with FoxO1 deletion. Reverse transcription-PCR (RT-PCR) revealed that FoxO1 deletion led to an increase in NaV1.5 and Na(+) channel subunit ß3 mRNA, but not ß1, 2, and 4, or connexin 43. Whole patch-clamp recordings demonstrated that cardiac Na(+) currents were significantly augmented by FoxO1 deletion without affecting the steady-state activation and inactivation, leading to accelerated depolarization of action potentials in mouse ventricular cardiomyocytes. Electrocardiogram recordings showed that the QRS complex was significantly shortened and the P wave amplitude was significantly increased in conscious and unrestrained mice with cardiac FoxO1 deletion. NaV1.5 expression was decreased in the peri-infarct (border-zone) of mice with myocardial infarction and FoxO1 accumulated in the cardiomyocyte nuclei of chronic ischemic human hearts. Our findings indicate that FoxO1 plays an important role in the regulation of NaV1.5 and ß3 subunit expressions as well as Na(+) channel activity in the heart and that FoxO1 is involved in the modulation of NaV1.5 expression in ischemic heart disease.

Heterogeneous organization of the locus coeruleus projections to prefrontal and motor cortices.

The brainstem nucleus locus coeruleus (LC) is the primary source of norepinephrine (NE) to the mammalian neocortex. It is believed to operate as a homogeneous syncytium of transmitter-specific cells that regulate brain function and behavior via an extensive network of axonal projections and global transmitter-mediated modulatory influences on a diverse assembly of neural targets within the CNS. The data presented here challenge this longstanding notion and argue instead for segregated operation of the LC-NE system with respect to the functions of the circuits within its efferent domain. Anatomical, molecular, and electrophysiological approaches were used in conjunction with a rat model to show that LC cells innervating discrete cortical regions are biochemically and electrophysiologically distinct from one another so as to elicit greater release of norepinephrine in prefrontal versus motor cortex. These findings challenge the consensus view of LC as a relatively homogeneous modulator of forebrain activity and have important implications for understanding the impact of the system on the generation and maintenance of adaptive and maladaptive behaviors.

Study of the residues involved in the binding of ß1 to ß3 subunits in the sodium channel.

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one ß subunit. Up to now, five ß subunits are known: ß1/ß1A, ß1B, ß2, ß3, and ß4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between ß1 and ß3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of ß1 subunit for homology modelling, which shares higher similarity to ß3. Docking studies of the ß1 and ß3 homology model were conducted, and likely ß1 and ß3 binding loci were investigated. The results revealed that ß1-ß3 is more likely to form a di-polymer than ß1-ß1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of ß1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of ß3 were predicted to play a functional role.

Crystal structure and molecular imaging of the Nav channel ß3 subunit indicates a trimeric assembly.

The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated ß subunits. Here, we report the crystal structure of the human ß3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the ß3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length ß3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length ß3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that ß3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the ß3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations.

The immunoglobulin domain of the sodium channel ß3 subunit contains a surface-localized disulfide bond that is required for homophilic binding.

The ß subunits of voltage-gated sodium (Na(v)) channels possess an extracellular immunoglobulin (Ig) domain that is related to the L1 family of cell-adhesion molecules (CAMs). Here we show that in HEK293 cells, secretion of the free Ig domain of the ß3 subunit is reduced significantly when it is coexpressed with the full-length ß3 and ß1 subunits but not with the ß2 subunit. Using immunoprecipitation, we show that the ß3 subunit can mediate trans homophilic-binding via its Ig domain and that the ß3-Ig domain can associate heterophilically with the ß1 subunit. Evolutionary tracing analysis and structural modeling identified a cluster of surface-localized amino acids fully conserved between the Ig domains of all known ß3 and ß1 sequences. A notable feature of this conserved surface cluster is the presence of two adjacent cysteine residues that previously we have suggested may form a disulfide bond. We now confirm the presence of the disulfide bond in ß3 using mass spectrometry, and we show that its integrity is essential for the association of the full-length, membrane-anchored ß3 subunit with itself. However, selective reduction of this surface disulfide bond did not inhibit homophilic binding of the purified ß3-Ig domain in free solution. Hence, the disulfide bond itself is unlikely to be part of the homophilic binding site. Rather, we suggest that its integrity ensures the Ig domain of the membrane-tethered ß3 subunit adopts the correct orientation for productive association to occur in vivo.

Novel SCN3B mutation associated with brugada syndrome affects intracellular trafficking and function of Nav1.5.

BACKGROUND: Brugada syndrome (BrS) is characterized by specific alterations on ECG in the right precordial leads and associated with ventricular arrhythmia that may manifest as syncope or sudden cardiac death. The major causes of BrS are mutations in SCN5A for a large subunit of the sodium channel, Nav1.5, but a mutation in SCN3B for a small subunit of sodium channel, Navß3, has been recently reported in an American patient. METHODS AND RESULTS: A total of 181 unrelated BrS patients, 178 Japanese and 3 Koreans, who had no mutations in SCN5A, were examined for mutations in SCN3B by direct sequencing of all exons and adjacent introns. A mutation, Val110Ile, was identified in 3 of 178 (1.7%) Japanese patients, but was not found in 480 Japanese controls. The SCN3B mutation impaired the cytoplasmic trafficking of Nav1.5, the cell surface expression of which was decreased in transfected cells. Whole-cell patch clamp recordings of the transfected cells revealed that the sodium currents were significantly reduced by the SCN3B mutation. CONCLUSIONS: The Val110Ile mutation of SCN3B is a relatively common cause of SCN5A-negative BrS in Japan, which has a reduced sodium current because of the loss of cell surface expression of Nav1.5.

Increased expression of the beta3 subunit of voltage-gated Na+ channels in the spinal cord of the SOD1G93A mouse.

Amyotrophic lateral sclerosis (ALS) is an adult-onset disease characterized by the progressive degeneration of motoneurons (MNs). Altered electrical properties have been described in familial and sporadic ALS patients. Cortical and spinal neurons cultured from the mutant Cu,Zn superoxide dismutase 1 (SOD1G93A) mouse, a murine model of ALS, exhibit a marked increase in the persistent Na+ currents. Here, we investigated the effects of the SOD1G93A mutation on the expression of the voltage-gated Na+ channel alpha subunit SCN8A (Nav1.6) and the beta subunits SCN1B (beta1), SCN2B (beta2), and SCN3B (beta3) in MNs of the spinal cord in presymptomatic (P75) and symptomatic (P120) mice. We observed a significant increase, within lamina IX, of the beta3 transcript and protein expression. On the other hand, the beta1 transcript was significantly decreased, in the same area, at the symptomatic stage, while the beta2 transcript levels were unaltered. The SCN8A transcript was significantly decreased at P120 in the whole spinal cord. These data suggest that the SOD1G93A mutation alters voltage-gated Na+ channel subunit expression. Moreover, the increased expression of the beta3 subunit support the hypothesis that altered persistent Na+ currents contribute to the hyperexcitability observed in the ALS-affected MNs.

Mutations in sodium channel ß-subunit SCN3B are associated with early-onset lone atrial fibrillation.

AIMS: Atrial fibrillation (AF) is the most frequent arrhythmia. Screening of SCN5A-the gene encoding the α-subunit of the cardiac sodium channel-has indicated that disturbances of the sodium current may play a central role in the mechanism of lone AF. We tested the hypothesis that lone AF in young patients is associated with genetic mutations in SCN3B and SCN4B, the genes encoding the two ß-subunits of the cardiac sodium channel. METHODS AND RESULTS: In 192 unrelated lone AF patients, the entire coding sequence and splice junctions of SCN3B and SCN4B were bidirectionally sequenced. Three non-synonymous mutations were found in SCN3B (R6K, L10P, and M161T). Two mutations were novel (R6K and M161T). None of the mutations were present in the control group (n = 432 alleles), nor have any been previously reported in conjunction with AF. All SCN3B mutations affected residues that are evolutionarily conserved across species. Electrophysiological studies on the SCN3B mutation were carried out and all three SCN3B mutations caused a functionally reduced sodium channel current. One synonymous variant was found in SCN4B. CONCLUSION: In 192 young lone AF patients, we found three patients with suspected disease-causing non-synonymous mutations in SCN3B, indicating that mutations in this gene contribute to the mechanism of lone AF. The three mutations in SCN3B were investigated electrophysiologically and all led to loss of function in the sodium current, supporting the hypothesis that decreased sodium current enhances AF susceptibility.

Mutational analysis of SCN2B, SCN3B and SCN4B in a large Chinese Han family with generalized tonic-clonic seizure.

Voltage-gated sodium channel genes are associated with idiopathic generalized epilepsy. Our group preciously identified a suggestive new locus on chromosome 11q22.1-23.3 in a five-generational Chinese epileptic family with generalized tonic-clonic seizure. SCN2B, SCN3B and SCN4B, which located at 11q22.1-23.3 locus, were chosen as candidate genes for this family. In the present study, genomic DNA was extracted in six affected family members. All exons of SCN2B, SCN3B and SCN4B were sequenced using direct DNA sequence analysis. The results showed that no mutation or polymorphism of coding regions of SCN2B, SCN3B and SCN4B was detected in the tested family members. Therefore, SCN2B, SCN3B and SCN4B are not major susceptibility genes contributed to our large family.

The sodium channel {beta}3-subunit induces multiphasic gating in NaV1.3 and affects fast inactivation via distinct intracellular regions.

Electrical excitability in neurons depends on the activity of membrane-bound voltage gated sodium channels (Na(v)) that are assembled from an ion conducting α-subunit and often auxiliary ß-subunits. The α-subunit isoform Na(v)1.3 occurs in peripheral neurons together with the Na(v) ß3-subunit, both of which are coordinately up-regulated in rat dorsal root ganglion neurons after nerve injury. Here we examine the effect of the ß3-subunit on the gating behavior of Na(v)1.3 using whole cell patch clamp electrophysiology in HEK-293 cells. We show that ß3 depolarizes the voltage sensitivity of Na(v)1.3 activation and inactivation and induces biphasic components of the inactivation curve. We detect both a fast and a novel slower component of inactivation, and we show that the ß3-subunit increases the fraction of channels inactivating by the slower component. Using CD and NMR spectroscopy, we report the first structural analysis of the intracellular domain of any Na(v) ß-subunit. We infer the presence of a region within the ß3-subunit intracellular domain that has a propensity to form a short amphipathic α-helix followed by a structurally disordered sequence, and we demonstrate a role for both of these regions in the selective stabilization of fast inactivation. The complex gating behavior induced by ß3 may contribute to the known hyperexcitability of peripheral neurons under those physiological conditions where expression of ß3 and Na(v)1.3 are both enhanced.

Functional dominant-negative mutation of sodium channel subunit gene SCN3B associated with atrial fibrillation in a Chinese GeneID population.

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the clinic, and accounts for more than 15% of strokes. Mutations in cardiac sodium channel alpha, beta1 and beta2 subunit genes (SCN5A, SCN1B, and SCN2B) have been identified in AF patients. We hypothesize that mutations in the sodium channel beta3 subunit gene SCN3B are also associated with AF. To test this hypothesis, we carried out a large scale sequencing analysis of all coding exons and exon-intron boundaries of SCN3B in 477 AF patients (28.5% lone AF) from the GeneID Chinese Han population. A novel A130V mutation was identified in a 46-year-old patient with lone AF, and the mutation was absent in 500 controls. Mutation A130V dramatically decreased the cardiac sodium current density when expressed in HEK293/Na(v)1.5 stable cell line, but did not have significant effect on kinetics of activation, inactivation, and channel recovery from inactivation. When co-expressed with wild type SCN3B, the A130V mutant SCN3B negated the function of wild type SCN3B, suggesting that A130V acts by a dominant negative mechanism. Western blot analysis with biotinylated plasma membrane protein extracts revealed that A130V did not affect cell surface expression of Na(v)1.5 or SCN3B, suggesting that mutant A130V SCN3B may not inhibit sodium channel trafficking, instead may affect conduction of sodium ions due to its malfunction as an integral component of the channel complex. This study identifies the first AF-associated mutation in SCN3B, and suggests that mutations in SCN3B may be a new pathogenic cause of AF.

Sudden infant death syndrome-associated mutations in the sodium channel beta subunits.

BACKGROUND: Approximately 10% of sudden infant death syndrome (SIDS) cases may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the Na(V)1.5 cardiac sodium channel. Recently, Na(V) beta subunits have been implicated in various cardiac arrhythmias. Thus, the 4 genes encoding Na(V) beta subunits represent plausible candidate genes for SIDS. OBJECTIVE: This study sought to determine the spectrum, prevalence, and functional consequences of sodium channel beta-subunit mutations in a SIDS cohort. METHODS: In this institutional review board-approved study, mutational analysis of the 4 beta-subunit genes, SCN1B to 4B, was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were coexpressed with SCN5A in HEK 293 cells and were whole-cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. RESULTS: Three rare (absent in 200 to 800 reference alleles) missense mutations (beta3-V36M, beta3-V54G, and beta4-S206L) were identified in 3 of 292 SIDS cases. Compared with SCN5A+beta3-WT, beta3-V36M significantly decreased peak I(Na) and increased late I(Na), whereas beta3-V54G resulted in a marked loss of function. beta4-S206L accentuated late I(Na) and positively shifted the midpoint of inactivation compared with SCN5A+beta4-WT. In native cardiomyocytes, beta4-S206L accentuated late I(Na) and increased the ventricular action potential duration compared with beta4-WT. CONCLUSION: This study provides the first molecular and functional evidence to implicate the Na(V) beta subunits in SIDS pathogenesis. Altered Na(V)1.5 sodium channel function due to beta-subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS cases.

Loss-of-function mutation of the SCN3B-encoded sodium channel {beta}3 subunit associated with a case of idiopathic ventricular fibrillation.

AIMS: Loss-of-function mutations in the SCN5A-encoded sodium channel SCN5A or Nav1.5 have been identified in idiopathic ventricular fibrillation (IVF) in the absence of Brugada syndrome phenotype. Nav1.5 is regulated by four sodium channel auxiliary beta subunits. Here, we report a case with IVF and a novel mutation in the SCN3B-encoded sodium channel beta subunit Navbeta3 that causes a loss of function of Nav1.5 channels in vitro. METHODS AND RESULTS: Comprehensive open reading frame mutational analysis of KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, GPD1L, four sodium channel beta subunit genes (SCN1-4B), and targeted scan of RYR2 was performed. A novel missense mutation, Navbeta3-V54G, was identified in a 20-year-old male following witnessed collapse and defibrillation from VF. The ECG exhibited epsilon waves, and imaging studies demonstrated a structurally normal heart. The mutated residue was highly conserved across species, localized to the Navbeta3 extracellular domain, and absent in 800 reference alleles. We found that HEK-293 cells had endogenous Navbeta3, but COS cells did not. Co-expression of Nav1.5 with Navbeta3-V54G (with or without co-expression of the Navbeta1 subunit) in both HEK-293 cells and COS cells revealed a significant decrease in peak sodium current and a positive shift of inactivation compared with WT. Co-immunoprecipitation experiments showed association of Navbeta3 with Nav1.5, and immunocytochemistry demonstrated a dramatic decrease in trafficking to the plasma membrane when co-expressed with mutant Navbeta3-V54G. CONCLUSION: This study provides molecular and cellular evidence implicating mutations in Navbeta3 as a cause of IVF.