Cloning and expression of the two new variants of Nav1.5/SCN5A in rat brain

Abstract

The α-subunit of tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel (VSGC, Nav1.5/SCN5A) has been found from the rat heart and human neuroblastoma cell line NB-1, but its expression in rat brain has not been identified radically. In this study, a reverse transcriptase-polymerase chain reaction was used to clone the full sequence of Nav1.5 (designated as rN1) α-subunit in rat brain and compared the distribution in different lobe of brain in different developmental stages. The open reading frame of rN1 encodes 2,016 amino acid residues and sequence analysis indicated that rN1 is highly homologous with 96.53% amino acids identity to rat cardiac Nav1.5 (rH1) and 96.13% amino acids identity to human neuroblastoma Nav1.5 (hNbR1). It has all the structural features of a VSGC and the presence of a cysteine residue (C373) in the pore loop region of domain I suggests that this channel is resistant to TTX. A new exon (exon6A) that is distinct from rH1 was found in DI-S3-S4, meanwhile an isomer of alternative splicing that deleted 53 amino acids (exon18) was found for the first time in domain DII-III in rN1. (designated as rN1-2). Distribution results demonstrated that rN1 expressed discrepancy in different ages and lobe in brain. The expression level of rN1 was gradually more stable in adult than in neonatal; these results suggest that rN1 has a newly identified exon for alternative splicing that is differentfrom rat heart and is more widely expressed in rat brain than previously thought.