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.

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.

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.

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.