Studies on neural calcium signaling using knock-out mice

我们计划引入有针对性地破坏P/Q型和N型电压门控钙通道基因，但未能达到目的。为了制备钙通道敲除小鼠，我们不仅在建立同源重组胚胎干细胞系方面存在困难，而且在通过注射胚胎干细胞制造嵌合小鼠方面也存在困难。不知道为什么我们有这么多困难。但如果我们考虑到目前还没有人成功敲除钙通道基因的情况，这些基因可能存在一些特定的调控机制。同时，我们继续表征离子传导和钙通道激活的功能特性。此外，我们对编码一种新的钙通道实体的cdna进行了分子克隆，这种钙通道实体不依赖电压，但通过细胞内未识别的信使被细胞表面受体激活。我们获得了几个cdna，我们现在正在使用重组表达系统表征它们的功能特性。一个摧毁这个频道家族的项目正在进行中。我们已经建立了同源重组的胚胎干细胞系，并期望在不久的将来有嵌合小鼠。由于P/Q型钙通道基因突变可引起小鼠和人类小脑共济失调和其他神经系统症状，我们对小脑浦肯野细胞进行了电生理研究，并与重组系统中获得的功能特性进行了比较。这些结果将使我们对钙通道缺陷的病理机制有更深入的了解。

We planned to introduce targetted disruption of the genes of P/Q type and N type voltage-gated calcium channels, but we failed to attain the goal. For producing calcium channel-knock-out mice, we had difficulties not only in establishing homologously recombinated ES cell lines but also in making chimeric mice by injecting the ES cells. It is not known why we had so many difficulties. But if we consider the current situation that no one has succeeded in knocking out calcium channel genes, there may be some specific regulatory mechanism for those genes.In meantime, we continued to characterize functional properties in ion conduction and activation of calcium channels. Also we made molecular cloning of cDNAs encoding a new entity of calcium channels that are not voltage-dependent but activated by cell surface receptors through unidentified intracellular messengers. We obtained several cDNAs, and we are now characterizing their functional properties using a recombinant expresion system. A project to knock out one of this channel family is in progress. We have already established homologously recombinated ES cell lines, and expect to have chimeric mice in near future.Because mutations in the P/Q type calcium channel gene can cause cerebellar ataxia and other neurological symptoms in mice and human, we studied cerebellar Purkinje cells electrophysiologically, and compared the functional properties with those we obtained in recombinant system. These results will give us an insight into the pathological mechanism of calcium channel defects.