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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，是理解所有电压门控离子通道结构-功能原理的模型系统。这些通道是神经中兴奋传播的基础，并且通道突变引起各种心脏、神经元和神经肌肉疾病。已知这些离子通道通过跨膜电压的变化而打开和关闭（即改变它们对离子流的电导率）。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”，但它如何移动以打开和关闭通道尚不清楚。最近，晶体学数据（KvAP通道）导致了一个新的和非常不同的电压门控模型，这是非常有争议的，似乎与生物物理和生物化学数据不一致。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中最大的问题：所提出的KvAP模型对于膜中的功能通道是否准确？LRET能够以亚埃的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，我们已经开发了一种新的配置LRET，测量从电压传感器上的网站到蝎子毒素绑定到外部口的离子孔的距离。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模型中所提出的。我们将使用LRET和常规FRET更准确地定义通道开放和关闭的基础构象变化。通过将LRET扩展到以前没有测试过的其他位点，我们将极大地限制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来研究称为ILT的突变Shaker的电压传感器，这使我们能够分别测量与多步骤通道开放过程中的几个步骤沿着相关的构象变化。在新的发光探针的设计和合成的潜在发展，使螯合物更适合作为LRET捐助者（和其他研究），提出。