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）：振动筛是一个电压门控钾离子通道和一个模型系统，用于了解所有电压门控离子通道的结构功能原理。这些通道是神经激发传播的基础，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，这些离子通道通过整个膜的电压变化打开和关闭（即将其导电率更改为离子流）。但是如何实现？具体而言，已知离子通道的一部分是“电压传感器”，但是如何移动以使通道开关的通道尚不清楚。最近，晶体学数据（KVAP通道的）导致了一种新的且非常不同的电压门控模型，该模型引起了极大争议，并且似乎与生物物理和生化数据不符。我们正在应用一种称为发光共振能量传递（LRET）的技术来回答现场最大的问题：拟议的KVAP模型是否准确地用于膜中功能通道？ LRET能够测量具有子角精度的蛋白质上两个位点之间的距离和距离变化。我们已经表明，振动振压传感器上的LRET信号与电生理测量值密切相关[1]。现在，我们已经开发了一种针对LRET的新配置，该配置可以测量从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。通过这种布置，我们可以严格测试电压传感器是否具有较大的跨膜运动，如KVAP模型中所提出的那样。我们将使用LRET和常规FRET来更准确地定义基于渠道打开和关闭的构象变化。通过将LRET扩展到以前未测试的其他站点，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的突变振动器的电压传感器，这使我们能够测量沿多步通道打开过程的几个步骤相关的构象变化。提出了新发光探针设计和合成的潜在发展，这使螯合物更适合作为LRET供体（以及其他研究）。