Joint Study on the Molecular Biology of SA Channels

拉伸激活离子通道表达于多种类型的细胞中，在细胞生长、分裂、形态发生和体积调节等基本细胞功能中发挥重要作用。然而，由于缺乏关于其分子结构的信息，进一步探索SA通道面临着困难。最近从大肠杆菌中分离到一个编码SA通道的基因，然而，它显示了一个特殊的序列，与真核细胞中已知的离子通道序列没有任何同源性。本研究的目的是从真核细胞中寻找SA通道基因，并开发SA通道的特异性阻断剂，以探索SA通道的结构、功能和生理作用。我们在MDCK细胞中发现了一个候选基因，其序列与mec基因有显著的同源性，该基因可能是SA通道基因。我们还在果蝇中发现了一种编码新型cGMP门控离子通道的基因，这可能有助于该物种的机械转导。这些基因的表达和功能分析正在进行中。已发现一种蜘蛛毒液中含有专门阻断SA通道的多肽。我们成功地纯化了其中一些对SA通道具有不同亲和力的多肽。成功地确定了低α亲和力分子的结构。目前正在确定高亲和力的一种。我们正计划利用该多肽来纯化SA通道蛋白，从而制备一种亲和柱。我们还准备制备一种荧光偶联肽，用它来研究SA通道在细胞表面的分布。

Stretch activated (SA) ion channels are expressed in many types of cells and supposed to play an important role in fundamental cell functions including cell growth, division, morphogenesis, and volume regulation. However, because of the lack of information on their molecular structure, further exploration of SA channels is confronted with difficulties. Very recently a gene encoding an SA channel has been isolated from E.coli, however, its shows a peculiar sequence devoid of any homology with known sequences of ion channels in eucaryotic cels. The aim of the present study was to find SA channel genes from eucaryotic cells and to develop a specific blockers to the SA channel, by which we could explore structure-function and physiological roles of SA channels. We found a candidate gene in MDCK cells of which sequence has a significant homology with mec gene that is supposed to be a putative SA channel gene. We also found, in Drosophila, a gene encoding new type of cGMP gated ion channel that may contribute to mechanotransduction in this species. Expression and functional assay of these genes are in progress. A certain spider venom has been found to include peptides that specifically block the SA channel. We succeeded in purifying some of these peptides with defferent degree of affinity to the SA channel. The structure of a low a affinity one has been sccessfully determined. Determination of a high affinity one is now in progress. We are planning to prepare an affinity column by using the peptide to purify SA channel proteins. We are also going to prepare a fluorescent-conjugated peptide by which the distribution of SA channels on the cell surface could be studied.