CHARACTERISTICS OF POTASSIUM CHANNEL GENES

电压依赖性钾通道对正常的 神经系统和肌肉中可兴奋细胞的功能。 不同功能家族的钾通道参与 对某些可激发性质进行修饰和微调。vt.给出 这些渠道的广泛多样性及其在其中发挥的关键作用 兴奋性组织的正常功能，对 了解钾通道多样性和功能的分子基础。 一个家族的几个成员，A型钾通道，一直是 以cDNA克隆的形式分离并在非洲爪哇卵母细胞中表达。鲜为人知 关于钾的生物化学，特别是分子生物学 来自其他家庭的频道。钾离子通道的纯化 速度慢，且没有序列数据可用于合成的设计 可用于常规cdna分离的寡核苷酸探针。 因此，我们设计了一种序列依赖的分离方法 编码不同钾通道家族成员的cDNA。至 为了做到这一点，我们在转录中使用了不同的cdna克隆库- 体外合成信使核糖核酸的有效载体。离子通道的表达 在非洲爪哇卵母细胞中形成转录本并将cDNA库细分为 越来越小的鸡尾酒导致了一个孤立的 编码一种新的钾通道的单个cDNA克隆。使用此策略在 结合常规的克隆程序，我们计划分离钾 属于不同家族的频道克隆。我们计划在功能上 通过在非洲爪哇卵母细胞中的表达来鉴定这些分离物。 不同家族成员氨基酸序列的比较 揭示对一般结构和结构可能重要的保守区域 功能，可能涉及特定功能的不同区域 一些钾离子通道。这些地区将被选为 定点突变。氨基酸残基可能是功能上的 重要的内容将会被更改。我们将在以下方面引入保守的改革 不得扰乱蛋白质的包装和构象。突变的cDNA将 被转录和体外制备的mRNA将被注射并在 非洲爪哇卵母细胞。这样的结构-功能分析将使我们能够 更好地理解离子通道和分子事件的生物物理学 这是兴奋性现象的根本原因。

Voltage-dependent potassium channels are important for the proper functioning of excitable cells in the nervous system and in muscle. Different functional families of potassium channels are involved in modification and fine tuning of some of the excitable properties. Given the wide diversity of these channels and the crucial role they play in proper functioning of the excitable tissues, it is of great importance to understand the molecular basis of potassium channel diversity and function. Several members of one family, the A-type potassium channels, have been isolated as cDNA clones and expressed in Xenopus oocytes. Little is known about the biochemistry and especially the molecular biology of potassium channels from other families. Purification of potassium channels has been slow, and no sequence data is available for the design of synthetic oligonucleotides probes that could be used for routine cDNA isolation. Therefore, we have designed a sequence-dependent approach for the isolation of cDNAs encoding members of different potassium channel families. To achieve this, we use pools of different cDNA clones in a transcription- competent vector to synthesize mRNA in vitro. Expression of ion channels form transcripts in Xenopus oocytes and subdividing cDNA pools into "cocktails" of smaller and smaller sizes has led to the isolation of a single cDNA clone encoding a new potassium channel. Using this strategy in combination with routine cloning procedures, we plan to isolate potassium channel clones that belong to different families. We plan to functionally characterize these isolates through expression in Xenopus oocytes. Comparison of amino acid sequences of members of different families will reveal conserved regions that may be important for general structure and function, diverged regions that may be involved in specific functions of some of the potassium channels. Such regions will be chosen as targets for site directed mutagenesis. Amino acid residues that may be functionally important will be altered. We shall introduce conservative changes in order not to perturb protein packing and conformation. Mutated cDNAs will be transcribed and in vitro prepared mRNA will be injected and expressed in Xenopus oocytes. Such a structure-function analysis will enable us to better understand the biophysics of ion channels and the molecular events underlying the phenomenon of excitability.