The structure and function of Ca^<2+>-permeable nonselective cation channels

本研究旨在阐明内皮素-1 （ET-1）在血管平滑肌细胞（VSMCs）中激活的Ca^<2+>通道的结构、功能、药理及功能意义。ET-1激活了来自大鼠胸主动脉平滑肌细胞的A7r5细胞中的三种Ca^<2+>-可渗透通道：两种非选择性阳离子通道（指定为NSCC-1和NSCC-2）和储存操作Ca^<2+>通道（SOCC）。重要的是，这些通道被SK&F 96365和LOE 908这两种药物区分开来，这两种药物属于“所谓的”受体操作的Ca^<2+>通道的阻滞剂。利用这些阻滞，发现ET-1在VSMCs中诱导的细胞内游离Ca^<2+>浓度的收缩和增加实际上与Ca^<2+>通过NSCC-1、NSCC-2和SOCC进入有关。为了分离编码这些通道的cdna，我们采用了两种策略：一种是基于pcr的克隆，另一种是用爪蟾卵母细胞进行表达克隆。在前一种策略中，Ca^<2+>通道的保守区域称为瞬时受体电位（trp）作为探针。在后一种策略中，开发了一种使用^<45>Ca^<2+>摄取的筛选方法，以特异性检测Ca^<2+>的进入。在基于pcr的方法中，分离了两种形式的cDNA （trp4及其推定的剪接变体trp 4Δ）。trp4的序列与从其他组织中分离出来的序列基本相似，但trp 4Δ的独特之处在于它缺少与第一和第二膜跨越区相对应的部分序列。当在卵母细胞和HEK 293细胞中表达时，这两种cdna在功能上是完整的。它们被储存耗尽激活，并表现出与SOCC相同的药理特性。值得注意的是，trp 4Δ比trp 4表现出更大的反应。在表达克隆策略中，筛选VSMCs cDNA文库，获得5个阳性克隆。这些克隆体的结构和功能正在被确定。

The purpose of the present study is to clarify the structure, function, pharmacology and functional significance of Ca^<2+> channels activated bv endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). ET-1 activated three types of Ca^<2+>-permeable channel in A7r5 cells derived from rat thoracic aortic smooth muscle cells : two types of nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca^<2+> channel (SOCC). Importantly, these channels were discriminated by two drugs like SK＆F 96365 and LOE 908 belonging to blockers of the "so-called" receptor-operated Ca^<2+> channel. Using these blockes, contractions and inreases in the intracellular free Ca^<2+> concentration induced by ET-1 in VSMCs were found to actually involve Ca^<2+> entry through NSCC-1, NSCC-2 and SOCC.To isolate cDNAs encoding these channels, we used two strategies : one is PCR-based cloning and the other is expression cloning with Xenopus oocytes. In the former strategy, the conserved region of Ca^<2+> channels called transient receptor potentials (trp) was used as probe. In the latter strategy, a screening method using ^<45>Ca^<2+> uptake was developed to specifically detect Ca^<2+> entry. In the PCR-based method, two forms of cDNA (trp 4 and its putative splice variant designated trp 4Δ) were isolated. The sequence of trp 4 was essentially similar to that isolated from other tissues, but trp 4Δ was unique in that it lacks part of the sequence corresponding to the first and second membrane spanning region. When expressed in the oocytes and HEK 293 cells, both cDNAs were functionally intact. They were activated by store depletion, and showed the same pharmacological properties as those of SOCC.Notably, trp 4Δ showed larger responses than trp 4. In the expression cloning strategy, cDNA library from VSMCs was screened and five positive clones were obtained. The structure and function of these clones are being determined.