Cloning of LVA Ca 2＋ channel and its primary structure features / 细胞与分子免疫学杂志

ABSTRACT

Aim To clone LVA calcium channel (also known as T-type calcium channel) from INS-1 cell line derived from rat pancreatic β cells. Methods RT-PCR, 3′ -RACE and 5′ -RACE were used to clone coding sequence of the whole gene. DNA sequencing and genomic DNA walking were used to identify the primary structure features and exons. Results The cloned gene, named as α 1 G-INS, was composed of 6 864 bp, encoding 2 288 amino acids, which shares 96.3％ identity to α 1G, the neuronal T-type calcium channel. Compared to α 1G, the amino acids in membrane-spanning regions of four transmembrane domains and intracellular loop LI-II of α 1G-INS were highly conserved, but there are three distinct regions. This discrepancy was due to alternative mRNA splicing. Scattering on other regions of the molecule there were 10 single amino acid substitutions, which could be explained as site-mutations of the gene. Conclusion A new isoform,α 1G-INS, of T-type calcium channel is successfully cloned from rat insulin-secreting cell line INS-1, which is significant to further understanding many Ca2＋ -involved biologically essential processes.