LVA钙通道的克隆鉴定及一级结构特征

Cloning of LVA Ca^(2+) channel and its primary structure features

目的从大鼠胰腺β细胞中，克隆ＬＶＡ钙通道（即Ｔ－型钙通道）基因，并鉴定其一级结构的特征。方法用ＲＴ－ＰＣＲ，３’－ＲＡＣＥ及５’－ＲＡＣＥ法，从总ＲＮＡ中克隆Ｔ－型钙通道全长基因；用ＤＮＡ序列测定与ＤＮＡ步移实验，分析基因一级结构及外显子剪接。结果克隆了Ｔ－型钙通道的全长结构基因，命名为α１Ｇ－ＩＮＳ。该基因由６８６４个碱基组成，编码２２８８个氨基酸，与从神经组织中克隆的钙通道α１Ｇ相比较：在氨基酸水平上有９６．３％的同源性，在４个结构域的跨膜区内的氨基酸及Ⅰ、Ⅱ结构域间的胞内环链部分的氨基酸具有高度保守性；两者间具有３个明显不同的结构区段，基因组ＤＮＡ步移法分析表明，这种差异是由于ｍＲＮＡ前体的不同剪接所致。此外，尚有１０个氨基酸替代散在于分子内其它区域，这种替代是由于基因突变造成的。结论成功地从大鼠胰腺β细胞中，克隆到ＬＶＡ钙通道基因中的１个新亚型（ｉｓｏｆｏｒｍ）α１Ｇ－ＩＮＳ，对深入研究钙离子所涉及的许多重要的基本生命过程有着重要的意义。

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 α1 G-INS, was composed of 6864 bp, encoding 2 288 amino acids, which shares 96. 3% identity to α1G, the neuronal T-type calcium channel. Compared to α1G, the amino acids in membrane-spanning regions of four transmembrane domains and intracellular loop LⅠ-Ⅱ of α1G-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, α1G-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.