The structure and function of voltage-independent Ca^<2+> channels involved in vascular contractions

迄今为止，我们发现ET-1激活了大鼠胸主动脉血管平滑肌细胞中三种类型的Ca^<2+> -可渗透通道：两种类型的非选择性阳离子通道（指定为NSCC-1和NSCC-2）和储存型Ca^<2+>通道（SOCC）。本研究的目的是阐明在血管收缩剂如内皮素-1 （ET-1）和去甲肾上腺素诱导的血管收缩过程中，电压无关的Ca^<2+>-可渗透通道的结构、功能和功能意义，并开发特异性作用于这些通道的药物。为了分离编码这些通道的cdna，我们决定使用爪蟾卵母细胞的表达克隆策略。为此，我们开发了一种使用^<45>Ca^<2+>摄取来特异性检测Ca^<2+>进入的筛选方法。首先，我们筛选了不同组织和细胞系中三种通道的高表达水平，发现三种通道的表达水平在CHO细胞中最高。作为表达克隆的第一步，我们以重组ET A型受体cDNA （ET_AR）为模板，制备了体外转录的cRNA，并从CHO细胞中提取mrna，根据其大小用蔗糖梯度超离心分离。在第一次筛选中，我们向爪蟾卵母细胞微注射了ET_AR和任意mRNA片段的cRNA，并检测了^<45>Ca^<2+>进入卵母细胞的情况。从阳性片段中提取cDNA文库，利用ZAP表达载体进行cDNA文库的分离。在第二次和接下来的筛选中，卵母细胞被微注射了用于ET_AR的cRNA和从cDNA文库中任一部分制备的cRNA，并检测^<45>Ca<2+>的摄取。通过重复这种筛选，我们最终获得了16个阳性克隆。我们现在正在确定每个克隆的序列和功能。之后，我们计划开发专门作用于这些通道的药物。

We have so far found that ET-1 activated three types of Ca^<+2>-permeable channel in vascular smooth muscle cells derived from rat thoracic aortae : two types of nonselective cation channel (designated NSCC-1 and NSCC-2) and store-onerated Ca^<2+> channel (SOCC). The purpose of the present study is to clarify the structure, function, and functional significance of voltage-independent Ca^<2+>-permeable channels involved in vascular contractions induced by vasoconstricting agents such as endothelin-1 (ET-1) and noradrenaline, and also to develop drugs specifically acting on these channels. To isolate cDNAs encoding these channels, we decided to use expression cloning strategy with Xenopus oocytes. For this purpose, we developed a screening method using ^<45>Ca^<2+> uptake to specifically detect Ca^<2+> entry. First we screened varying tissues and cell lines for high expression levels of the three channels, and found that expression levels of the three channels were highest in CHO cells. For the first step toward expression cloning, we prepared cRNA which was trascribed in vitro using recombinant cDNA for ET type A receptor (ET_AR) as a template : mRNAs were extracted from CHO cells and fractionated according to their sizes with sucrose-gradient ultracentrifugation. For the first screening, we microinjected cRNA for ET_AR and either of the mRNA fractions into Xenopus oocytes, and assayed for ^<45>Ca^<2+> uptake into the oocytes. From thepositive fraction, cDNA library was prepared using ZAP Express Vector and separated into 10 parts. For the second and the following screening, the oocytes were microinjected with cRNA for ET_AR and cRNA prepared from either of the cDNA library parts, and assayed for ^<45>Ca<2+> uptake. By repeating this type of screening, we finally obtained 16 positive clones. We are now determining sequences and function of each clone. After that, we plan to to develop drugs specifically acting on these channels.