Tetrodotoxin‐resistant Na⁺ channels in human neuroblastoma cells are encoded by new variants of Nav1.5/SCN5A

Abstract

Both tetrodotoxin-sensitive (TTX-S) and TTX-resistant (TTX-R) voltage-dependent Na+ channels are expressed in the human neuroblastoma cell line NB-1, but a gene encoding the TTX-R Na+ channel has not been identified. In this study, we have cloned cDNA encoding the alpha subunit of the TTX-R Na+ channel in NB-1 cells and designated it hNbR1. The longest open reading frame of hNbR1 (accession no. AB158469) encodes 2016 amino acid residues. Sequence analysis has indicated that hNbR1 is highly homologous with human cardiac Nav1.5/SCN5A with > 99% amino acid identity. The presence of a cysteine residue (Cys373) in the pore-loop region of domain I is consistent with the supposition that hNbR1 is resistant to TTX. Analysis of the genomic sequence of SCN5A revealed a new exon encoding S3 and S4 of domain I (exon 6A). In addition, an alternative splicing variant, lacking exon 18, that encodes 54 amino acids in the intracellular loop between domains II and III was found (hNbR1-2; accession no. AB158470). Na+ currents in human embryonic kidney cells (HEK293) transfected with hNbR1 or hNbR1-2 showed electrophysiological properties similar to those for TTX-R I(Na) in NB-1 cells. The IC50 for the TTX block was approximately 8 microM in both variants. These results suggest that SCN5A has a newly identified exon for alternative splicing and is more widely expressed than previously thought.

Expression of neonatal Nav1.5 in human brain astrocytoma and its effect on proliferation, invasion and apoptosis of astrocytoma cells

“…The former is a neonatal isoform (6,7,9,(28)(29)(30)(31), namely nNav1.5 (neonatal Nav1.5), while the latter is a mature isoform. Our and various other studies suggest that the Nav1.5 subtype of VGSCs encoded by the SCN5A gene could potentially serve as a marker of tumor metastasis and as a therapeutic target (8,(10)(11)(12)26,(30)(31)(32)(33)(34)(35)(36). Recent studies have found that the functions of VGSCs may vary with changes in mRNA splicing (2).…”

“…These channels not only play important roles in various physiological activities such as growth and metabolism in glial cells (19), but also in cell cycle regulation, proliferation, metastasis, invasion and apoptosis of astrocytoma cells (8,(19)(20)(21)(22)(23)(24)(25). VGSCs are mainly expressed in tumors of epithelial origin, and can participate in the biological behaviors of tumor cells, including direct spread, lateral movement, galvanotaxis, invasion and the secretory activity of the membrane (8,26,27). In the present study, we proposed that various VGSC subtypes may be related to the occurrence and development of astrocytoma, and we carried out a series of assays to confirm this hypothesis.…”

“…Our preliminary findings confirmed that Nav1.5 is widely distributed in the CNS. Therefore, we first cloned the complete sequence of the Nav1.5 gene expressed in human cortical neurons and identified its function (8). The results showed that the SCN5A gene which encodes Nav1.5 in brain tissue is different than that in human cardiomyocytes, and they belong to two different subtypes.…”

“…Thus, Na + channels are expected to become a new target for gene therapy for astrocytoma (3)(4)(5)(6). Our previous studies found that a new neonatal isoform of the Nav1.5 Na + channel, neonatal Nav1.5 (nNav1.5), was functionally expressed in human brain neuroblastoma NB-1 cells (7)(8)(9). This neonatal isoform is considered to be the re-expression of an embryonic gene or oncogene, and its expression is intimately related to the occurrence and development of tumors, which has been confirmed in lymphoma and breast cancer (10)(11)(12).…”

Mechanisms of voltage-gated ion channel regulation: from gene expression to localization

“…For example, two splice variants of Na v 1.5, hNbR1 and hNbR1 -2, differ in the presence or absence of exon 18 as a result of exon skipping. hNbR1, which includes exon 18, has a lower I Na activation, along with lower peak current and average range of I Na than hNbR1 -2, which does not include exon 18 [46]. The functional significance of the presence of these splice variants in cells has yet to be investigated, but these results suggest that hNbR1 may be more easily activated and deactivated, leading to increased excitability.…”