CHARACTERISTICS OF POTASSIUM CHANNEL GENES

电压依赖性钾离子通道对于正常的 神经系统和肌肉中可兴奋细胞的功能。 钾通道的不同功能家族参与了 修改和微调的一些可激发的属性。 给定 这些渠道的广泛多样性以及它们在 正常运作的兴奋组织，这是非常重要的， 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型钾离子通道，已经被 作为cDNA克隆分离并在非洲爪蟾卵母细胞中表达。 知之甚少 关于钾的生物化学，尤其是分子生物学 其他家庭的频道。 钾离子通道的纯化 缓慢，并且没有序列数据可用于合成的设计。 这些寡核苷酸探针可用于常规cDNA分离。 因此，我们设计了一种序列依赖的方法来分离 不同钾通道家族成员的cDNA编码。 到 为了实现这一点，我们在转录中使用不同cDNA克隆的库- 在体外合成mRNA的感受态载体。 离子通道的表达 在非洲爪蟾卵母细胞中形成转录本， 越来越小的“鸡尾酒”导致了一个 编码一种新钾通道的cDNA克隆。使用这种策略， 结合常规克隆程序，我们计划分离钾 属于不同家族的通道克隆。 我们计划在功能上 通过在非洲爪蟾卵母细胞中的表达来表征这些分离物。 不同家族成员的氨基酸序列的比较将 揭示可能对一般结构重要的保守区域， 功能，可能涉及特定功能的分叉区域， 一些钾离子通道 这些地区将被选为目标， 定点诱变 可能在功能上 重要的是要改变。 我们将在以下方面引入保守的变化： 为了不扰乱蛋白质包装和构象。 突变的cDNA将 将转录并在体外制备的mRNA注射并表达于 非洲爪蟾卵母细胞。 这种结构-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 这是兴奋性现象的基础。

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.