HERG Channel in Acquired and Inherited Long-QT Syndrome

描述(申请人提供)：HERG钾通道是药物诱导的长QT综合征的主要原因。电生理学和关键位置的非自然氨基酸突变将被用来确定药物与哺乳动物细胞表达的Berg通道的结合。要研究的药物选自不同的结构类别：西沙必利、特非那定、多非利特、阿司咪唑、MK-499、奎尼丁、维斯利酮和氯喹。被纳入的非天然氨基酸包括操纵阳离子-pi、疏水、氢键和主链相互作用的系列。最初的实验将产生关于额外位置的进一步假设，导致关于每种药物与通道之间独特相互作用的接触点性质的一系列结论。此外，突变的Berg钾通道的缺陷运输是遗传性长QT综合征的主要原因。荧光非天然氨基酸侧链将被用来实时研究野生型和突变型HERG通道在活哺乳动物细胞中的运输。HERG亚基之间的荧光共振能量转移(FRET)将监测通道的组装和化学计量以及细胞内运输的细节；HERG与其他蛋白质之间的FRET将定义进一步的相互作用。电穿孔技术、新的正交化tRNA抑制子种类以及延伸和释放因子的水平将被优化，以通过无义抑制在哺乳动物细胞中掺入非天然氨基酸。这些正在进行的方法学发展将加强研究，并将为研究哺乳动物细胞中含有非天然氨基酸残基的膜蛋白提供通用工具。该项目有助于美国国立卫生研究院路线图目标“分子图书馆和成像”的进展。

DESCRIPTION (provided by applicant): The hERG potassium channel is a major cause of drug-induced Iong-QT syndrome. Electrophysiology and unnatural amino-acid mutagenesis at key positions will be employed to define binding of drugs to bERG channels expressed in mammalian cells. The drugs to be investigated are chosen from various structural classes: cisapride, terfenadine, dofetilide, astemizole, MK-499, quinidine, vesnarinone, and chloroquine. The unnatural amino acids to be incorporated include series that manipulate cation-pi, hydrophobic, hydrogen-bond, and main-chain interactions. The initial experiments will generate further hypotheses about additional positions, leading to a series of conclusions about the nature of contact points for the unique interaction between each drug and the channel. Furthermore, defective trafficking of mutant bERG potassium channels is a major cause of inherited Iong-QT syndrome. Fluorescent unnatural amino-acid side chains will be used to investigate trafficking of wild type and mutant hERG channels in real time in living mammalian cells. Fluorescence resonance energy transfer (FRET) between hERG subunits will monitor assembly and stoichiometry of the channel and the details of intracellular trafficking; and FRET between hERG and other proteins will define further interactions. Electroporation techniques, novel orthogonal tRNA suppressor species, and levels of elongation and release factors will be optimized for unnatural amino-acid incorporation by nonsense suppression in mammalian cells. These ongoing methodological developments will enhance the studies and will also provide general tools for investigating membrane proteins that contain unnatural amino-acid residues in mammalian cells. This project contributes to progress on the NIH roadmap goals, "Molecular Libraries and Imaging".