Fluorescence Changes in Shaker Potassium lon Channel

摇床钾离子通道的荧光变化

基本信息

批准号：
7476560
负责人：
PAUL R SELVIN
金额：
$ 32.3万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-01 至 2009-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7476560
关键词：
Aeropyrum Binding Biochemical Biological Biological Models Cardiac Charge Cleaved cell Condition Cysteine Data Development Docking Energy Transfer Fluorescence Fluorescence Resonance Energy Transfer Gated Ion Channel Helix (Snails)Ion Channel Ions Label Lanthanoid Series Elements Measurement Measures Membrane Membrane Lipids Membrane Proteins Modeling Motion Movement Mutation Nerve Neuromuscular Diseases Neurons Oocytes Oral cavity Pathway interactions Photobleaching Physiological Positioning Attribute Potassium Potassium Channel Process Protein Region Proteins Quantum Dots Rate Relative (related person)Reporting Research Personnel Scheme Scorpions Semiconductors Signal Transduction Site Solvents Specificity Structure Sulfhydryl Compounds Surface Techniques Testing Time Toxin Toxin Conjugates Vertebral column Voltage-Gated Potassium Channel Water conformational conversion design ear helix fluorophore improved interest luminescence luminescence resonance energy transfer molecular dynamics mutant programs protein expression research study sensor single molecule voltage

项目摘要

DESCRIPTION (provided by applicant): Shaker is a voltage-gated potassium ion channel and a model system for understanding the structure-function principles underlying all voltage-gated ion channels. These channels underlie excitation propagation in nerves, and channel mutations cause various cardiac, neuronal, and neuromuscular diseases. It is known that these ion channels are turned on and off (i.e. change their conductivity to ion flow) by changes in voltage across the membrane. But how is this achieved? Specifically, one part of the ion channel is known to be the "voltage-sensor," but how this moves in order to gate the channel on and off is not known. Recently, crystallographic data (of the KvAP channel) has led to a new and very different model of voltage-gating which is highly controversial and seems incongruous with biophysical and biochemical data. We are applying a technique called Luminescence Resonance Energy Transfer (LRET) to answer the biggest question in the field: Is the proposed KvAP model accurate for functional channels in a membrane? LRET is capable of measuring distances and distance changes between two sites on a protein with subangstrom precision. We have shown that LRET signals on the Shaker voltage-sensor strongly correlate with electrophysiological measurements [1]. Now we have developed a new configuration for LRET that measures the distance from sites on the voltage-sensor to a scorpion toxin bound to the external mouth of the ion pore. With this arrangement we can test rigorously whether the voltage-sensor has a large transmembrane movement, as proposed in the KvAP model. We will use LRET and conventional FRET to define more exactly the conformational changes that underlie channel opening and closing. By extending LRET to other sites not previously tested, we will greatly constrain models of the voltage-sensor structure, which will assist in interpreting the recent crystallographic data. We also are using LRET to study the voltage-sensor of a mutant Shaker called ILT, which allows us to measure separately conformational changes associated with several steps along the multi-step channel opening process. Potential developments in the design and synthesis of new luminescent probes, making the chelates more suitable as LRET donors (and for other studies), are presented.

描述（由申请人提供）：Shaker 是一种电压门控钾离子通道，也是一种用于理解所有电压门控离子通道的结构功能原理的模型系统。这些通道是神经兴奋传播的基础，通道突变会导致各种心脏、神经元和神经肌肉疾病。众所周知，这些离子通道通过膜上电压的变化而打开和关闭（即改变其对离子流的电导率）。但这是如何实现的呢？具体来说，离子通道的一部分被称为“电压传感器”，但其如何移动以控制通道的打开和关闭尚不清楚。最近，（KvAP 通道的）晶体学数据产生了一种新的、非常不同的电压门控模型，该模型备受争议，并且似乎与生物物理和生化数据不一致。我们正在应用一种称为发光共振能量转移 (LRET) 的技术来回答该领域最大的问题：所提出的 KvAP 模型对于膜中的功能通道是否准确？ LRET 能够以亚埃精度测量蛋白质上两个位点之间的距离和距离变化。我们已经证明 Shaker 电压传感器上的 LRET 信号与电生理测量密切相关 [1]。现在，我们开发了一种新的 LRET 配置，可以测量电压传感器上的位点到与离子孔外口结合的蝎子毒素的距离。通过这种安排，我们可以严格测试电压传感器是否具有大的跨膜运动，如 KvAP 模型中提出的那样。我们将使用 LRET 和传统的 FRET 来更准确地定义通道打开和关闭背后的构象变化。通过将 LRET 扩展到以前未测试过的其他位置，我们将极大地约束电压传感器结构的模型，这将有助于解释最近的晶体学数据。我们还使用 LRET 研究称为 ILT 的突变 Shaker 的电压传感器，它使我们能够单独测量与多步骤通道开放过程中的几个步骤相关的构象变化。介绍了新型发光探针设计和合成的潜在进展，使螯合物更适合作为 LRET 供体（以及其他研究）。