Tetrodotoxin-resistant Na+ channels in human neuroblastoma cells are encoded by new variants of Nav1.5/SCN5A.
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.
Ou
Shao-Wu
SW
Department of Physiology, Graduate School of Medical & Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
Kameyama
Asako
A
Hao
Li-Ying
LY
Horiuchi
Masahisa
M
Minobe
Etsuko
E
Wang
Wu-Yang
WY
Makita
Naomasa
N
Kameyama
Masaki
M
D000595:Amino Acid Sequence; D015152:Blotting, Northern / Q000379:methods; D045744:Cell Line, Tumor; D002893:Chromosomes, Human, Pair 3; D003001:Cloning, Molecular / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004558:Electric Stimulation; D005091:Exons; D014644:Genetic Variation; D006801:Humans; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics* / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel; D009447:Neuroblastoma; D018408:Patch-Clamp Techniques / Q000379:methods; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000235:genetics* / Q000378:metabolism; D013779:Tetrodotoxin / Q000494:pharmacology*; D014162:Transfection / Q000379:methods
D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C549756:NBR1 protein, human; D011506:Proteins; D012333:RNA, Messenger; C568320:SCN5A protein, human; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013779:Tetrodotoxin