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 α 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 IC 50 for the TTX block was ≈ 8 µ m in both variants. These results suggest that SCN5A has a newly identified exon for alternative splicing and is more widely expressed than previously thought.