CLONING OF T-TYPE CALCIUM CHANNEL GENE

将功能性表达T型Ca通道但不表达L型Ca通道的小鼠骨髓瘤细胞系派用作克隆T型Ca通道基因的源材料。根据L型钙通道基因序列设计了几对简并寡聚DNA引物，利用聚合酶链反应从细胞中制备cDNA，以扩增可能的T型钙通道基因。我们获得了一个独特的序列，它与已知的L型钙通道基因具有同源部分，但与迄今报道的任何DNA序列不同。我们也建立了非洲爪蟾卵母细胞表达系统来鉴定我们克隆的基因的功能特性。我们从大鼠脑和胰腺中分离了L型钙通道的2个α亚基（4A和4B）和3个β亚基的基因。亚基基因以不同的组合在青蛙卵母细胞和中国仓鼠卵巢细胞中表达，并对通道的电生理特性进行了研究。由4A和β 2基因诱导的钙通道电流显示出比其他基因组合的失活时间更长。通过对小鼠胰岛β细胞克隆细胞株MIN6的cDNA文库进行低严格筛选，获得了一个封闭的GTP结合蛋白偶联膜受体蛋白基因。利用青蛙卵母细胞表达系统鉴定该基因编码第3型PACAP受体。

A mouse myeloma cell line, PAI, which functionally expresses T-type Ca channel, but no L-type Ca channels, was used as a source material for cloning a T-type Ca channel gene. cDNA was made from the cells using several pairs of degenerate oligo DNA primers, which were designed based on L-type Ca channel gene sequences, were used for the polymerase chain reactions to amplify a possibly T-type Ca channel gene. We obtained a unique sequence which has homologous parts to already known L-type Ca channel genes but is different from any DNA sequences reported to date. Work of sequensing full length of the gene is now undertaken.We have also made a Xenopus o oocyte-expression system to identify functional properties of the genes we cloned. We have isolated genes of 2 alpha-subunits (4A and 4B) and 3 beta-subunits of L-type Ca channel from rat brain and pancreas. The subunit genes were expressed in different combinations in frog g oocytes and Chinese hamster ovary cells, and properties of the channels were investigated electrophysiologically. Ca channels currents induced by 4A and beta2 genes showed extremely longer inactivation time than those of the other combinations of the genes. The injection of an alpha-subunit gene alone did not induce any detectable Ca channel current.A gene of GTP-binding protein coupling membrane receptor protein has been closed by low-stringency screening of cDNA library of a mouse clonal cell line of a pancreatic beta-cell MIN6. The frog oocyte expression system was used to identify that the gene encodes the 3rd type of PACAP (pituitary adenylate cyclase-activating polypeptide) receptor.