Chemical Basis for Potassium Channel Modulation

描述（由申请人提供）：离子通道是一类蛋白质，通过介导细胞的兴奋性在细胞信号传导中起关键作用。离子通道的这种活性不是静态的，而是随着细胞内和细胞外的各种信号而不断变化的，近年来，许多直接调节离子通道活性的因素已经开始被发现。了解这些蛋白质的活性如何被调节的化学基础是现代科学中最重要的领域之一，因为它影响了我们对神经系统如何工作的理解，以及我们在药理学上操纵它的能力。提出的研究旨在描述人类以太-a-go-go相关基因（HERG）离子通道的几种潜在调制模式，HERG离子通道是大脑和心脏中发现的电压门控钾通道。在体外，HERG通道的活性受氧（02）和活性氧（ROS）的调节，并且已经提出HERG通道在体内作为O2/ROS的直接传感器。HERG通道的O2/ROS感知是由通道内蛋氨酸残基的可逆氧化介导的这一假设将通过表征特定位点将蛋氨酸亚砜掺入通道对通道生物物理的生物物理效应来评估。HERG通道的活性也受体内各种小分子的调节。然而，除了少数情况外，这些化合物的结合位点都没有被确定。小分子通过结合n端（PAS）结构域来调节HERG通道活性的假设将通过核磁共振波谱（检测与配体结合相关的结构扰动）和电生理学（定义与配体结合相关的生物物理变化）进行评估。最后，HERG通道活性似乎也受到各种蛋白质-蛋白质相互作用的调节。为了确定HERG中与其他蛋白质相互作用的位点，能够形成蛋白质交联的光反应性氨基酸将被特异性地结合到HERG中。

DESCRIPTION (provided by applicant): Ion channels are a class of proteins that play critical role in cellular signaling by mediating the excitability of cells. This activity of ion channels is not static, but constantly changes in response to various intracellular and extracellular signals and, in recent years, many of the factors that directly modulate the activity of ion channels have begun to be identified. Understanding the chemical basis for how the activity of these proteins is modulated represents one of the most important areas of modern science as it impacts our understanding of how the nervous system works as well as our ability to pharmacologically manipulate it. The proposed research seeks to characterize several potential modes of modulation of the human ether-a-go-go related gene (HERG) ion channel, a voltage-gated potassium channel found in brain and heart. The activity of HERG channels is modulated by oxygen (02) and reactive oxygen species (ROS) in vitro and it has been proposed that HERG channels function as direct sensors of O2/ROS in vivo. The hypothesis that O2/ROS sensing by HERG channels is mediated by the reversible oxidation of methionine residues within the channel will be evaluated by characterizing the biophysical effects of site-specifically incorporating methionine sulfoxide into the channel on channel biophysics. HERG channel activity is also modulated by various classes of small molecules in vivo. In all but a few cases, the binding site(s) for these compounds have not been identified, however. The hypothesis that small molecules modulate the activity of HERG channels by binding to the N-terminal (PAS) domain will be evaluated using NMR spectroscopy (to detect structural perturbations associated with ligand binding) and electrophysiology (to define biophysical changes associated with ligand binding). Finally, HERG channel activity also appears to be modulated by various protein-protein interactions. To determine the site(s) in HERG that interact with other proteins, photoreactive amino acids capable of forming protein cross-links will be site-specifically incorporated into the HERG.