Regulation of ion channels by methionine oxidation

描述(申请人提供)：许多生理和病理生理现象，包括衰老、缺血/缺氧、糖尿病和一些神经退行性疾病，促进细胞成分的氧化。蛋白质中的蛋氨酸和半胱氨酸残基很容易被氧化；经常导致蛋白质功能的显着变化。半胱氨酸和蛋氨酸的可逆氧化被认为是调节蛋白质功能的一种生理调节机制。为了验证这一假设，这里提出的研究计划将使用电生理测试结合分子突变来检测半胱氨酸和蛋氨酸的氧化如何改变人类大电导钙依赖钾通道(hSIO通道)的门控行为。拟议的项目将研究蛋氨酸和半胱氨酸氧化如何改变hSIO通道门控。半胱氨酸和蛋氨酸氧化的生物物理和分子靶标将被确定。该研究项目还将研究血红素、一氧化氮和低氧对hSIO通道的调节。假设这些生理相关的变量部分通过半胱氨酸和/或蛋氨酸氧化改变了hSIO通道的功能。许多实验将在异源表达系统中进行，以便更好地控制潜在的混杂变量，如通道亚基组成。这些结果将通过使用海马神经元和大脑皮层神经元的自然通道得到证实。电生理结果被定量分析，以阐明半胱氨酸/蛋氨酸的氧化和血红素/一氧化氮的应用改变了哪些特定的门控转换。以前使用天然钙依赖钾通道的研究经常产生相互矛盾的结果。这项研究计划的预期结果将澄清提出的许多重要问题，并为离子通道的氧化调节提供分子和生物物理方面的见解。

DESCRIPTION (provided by applicant): Many physiological and pathophysiological phenomena, including aging, ischemia/hypoxia, diabetes and some neurodegenerative diseases, promote oxidation of cellular constituents. Methionine and cysteine residues in proteins are readily oxidized; often inducing marked changes in protein function. It is hypothesized that reversible oxidation of cysteine and methionine serves as a physiological modulatory mechanism to regulate protein function. To test this hypothesis, the research program proposed here will examine how oxidation of cysteine and methionine alters gating behavior of human large-conductance calcium-dependent potassium channels (hSIo channels) using electrophysiological assays in combination with molecular mutagenesis. The proposed project will examine how hSIo channel gating is altered by methionine and cysteine oxidation. The biophysical and molecular targets of cysteine and methionine oxidation will be identified. The research program will also study regulation of the hSIo channel by heme, nitric oxide and hypoxia. It is hypothesized that these physiologically relevant variables alter the hSIo channel function in part by cysteine and/or methionine oxidation. Many of the experiments will be conducted in heterologous expression systems so that potential confounding variables, such as the channel subunit composition, are better controlled. These results will be confirmed by using native channels in hippocampal and cortical neurons. The electrophysiological results are quantitatively analyzed to elucidate which specific gating transitions are altered by oxidation of cysteine/methionine and by application of heme/nitric oxide. Previous studies using native calcium-dependent potassium channels often produced conflicting results. The results expected from this research program will clarify many of the important issues raised and provide molecular and biophysical insights into oxidative regulation of ion channels.