FUNCTIONAL EFFECTS OF RNA EDITING ON A KV1 K+ CHANNEL

了解离子通道的调节对于理解细胞兴奋性的基本机制至关重要。离子通道基因缺陷及其基因产物的错误处理，已被认为与迅速扩大的疾病清单有关，包括囊性纤维化、心律失常和某些形式的癫痫。RNA编辑是突触离子通道的重要调节机制。这一建议广泛借鉴了电压依赖性K+挑战GO的生物物理分析，回答了一个基本的生物学重要性问题：RNA编辑在动作电位水平上也很重要吗？这些研究的重点是鱿鱼巨轴突(SqKv1a)的延迟整流钾通道。到目前为止，已经确定了12个编辑过的密码子。这些位点分散在各种功能重要的通道结构域中，初步数据表明它们可以改变生理特性。这项工作的目的是定义编辑在频道的得分和四聚化(T1)领域的功能效果。5个核心编辑位点的未知组合会影响失活和稳态电压敏感性。将确定相关地点，并将在单一渠道一级审查其行动的生物物理基础。T1域的第87位从精氨酸编辑为甘氨酸(R87G)，对非洲爪哇卵母细胞的功能表达具有巨大的调节作用。R87G同源异构体的表达水平比编辑后的同源异构体低100倍。因为这个位置在巨大的轴突中被部分编辑，所以它对异源四聚体表达的影响将被检测。为了实现这一点，将开发单体特定的标签。今后，这些研究将扩展到本地系统。出于这个原因，巨型轴突是理想的：它是可识别的，经过广泛研究，其尺寸允许在宏观、门控和单通道水平上进行高分辨率电压钳。

Understanding the regulation ion channel is critical to understanding basic mechanisms of cellular excitability. Defective ion channel loci, and misprocessing of their gene products, have been linked to a rapidly expanding list of disorders, including cystic fibrosis, cardiac arrhythmias, and certain forms of epilepsy. RNA editing is an important regulatory mechanism for ion channels at the synapse. This proposal draws extensively from biophysical analysis of voltage-dependent K+ challenge go answer a question of fundamental biological importance: is RNA editing also important at the level of the action potential? The focus of these studies is the delayed rectifier K+ channel of the squid giant axon (SqKv1A). Thus far, 12 codons which are edited have been identified. These sites are scattered in a variety of functionally important channel domains, and preliminary data suggest that they modify physiological properties. The aim of this work is to define the functional effects of editing in the channel's score and tetramerization (T1) domain. An unknown combination of 5 core editing sites effect deactivation and steady-state voltage sensitivity. The relevant sites will be identified, and the biophysical basis for their actions will be examined on a single channel level. Position 87 in the T1 domain, which is edited from arginine to glycine (R87G), has a tremendous effect on regulating functional expression in Xenopus oocytes. R87 G homotetramers express at 100 fold lower levels than their edited counterparts. Because this position is partially edited in the giant axon, it's effect on heterotetramer expression will be examined. To accomplish this, monomer specific tags will be developed. In the future it is intended that these studies will be extended to the native system. For this reason the giant axon is ideal: it is identifiable, extensively studied, and its dimensions permit high resolution voltage clamp on the macroscopic, gating, and single channel levels.