FUNCTIONAL EFFECTS OF RNA EDITING ON A KV1 K+ CHANNEL

了解调控离子通道对于理解细胞兴奋性的基本机制至关重要。有缺陷的离子通道基因座及其基因产物的错误处理，已经与迅速扩大的疾病清单相关联，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控机制。该建议广泛借鉴了电压依赖性K+挑战的生物物理分析，以回答一个具有基本生物学重要性的问题：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K+通道。到目前为止，已鉴定出12个被编辑的密码子。这些位点分散在各种功能重要的通道域中，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通道的分数和四聚化（T1）域的功能影响。5个核心编辑位点的未知组合影响失活和稳态电压敏感性。将确定相关地点，并在单一通道水平上检查其作用的生物物理基础。T1结构域的87位（R87G）由精氨酸编辑为甘氨酸，对调节非洲爪蟾卵母细胞的功能表达有巨大作用。R87 G同型四聚体的表达水平比编辑后的低100倍。由于这个位置在巨大的轴突中被部分编辑，因此我们将研究它对异四聚体表达的影响。为了实现这一目标，将开发单体特定标签。在未来，我们打算将这些研究扩展到本地系统。由于这个原因，巨大的轴突是理想的：它是可识别的，被广泛研究，它的尺寸允许在宏观、门控和单通道水平上进行高分辨率电压箝位。

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.