Fluorescence Changes in Shaker Potassium lon Channel

说明(申请人提供)：Shaker是一种电压门控钾离子通道，是一个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、神经和神经肌肉疾病。众所周知，这些离子通道是通过膜上电压的变化来开启和关闭的(即将它们的导电性改变为离子流)。但这是如何实现的呢？具体地说，离子通道的一个部分是已知的“电压传感器”，但它如何运动以控制通道的开启和关闭尚不清楚。最近，(KvAP通道的)结晶学数据导致了一种新的、非常不同的电压门控模型，该模型极具争议性，似乎与生物物理和生化数据不一致。 我们正在应用一种名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模型对于膜上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置之间的距离和距离变化。我们已经证明，振动器电压传感器上的LRET信号与电生理测量有很强的相关性[1]。现在，我们已经为LRET开发了一种新的配置，它可以测量从电压传感器上的位置到结合到离子孔外部嘴的蝎子毒素的距离。通过这种布置，我们可以严格地测试电压传感器是否如KvAP模型中所建议的那样具有大的跨膜运动。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扩展到以前没有测试过的其他位置，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结晶学数据。我们还在使用LRET来研究一种名为ILT的突变振荡器的电压传感器，它允许我们分别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。提出了在设计和合成新的发光探针方面的潜在进展，使其更适合作为LRET供体(以及用于其他研究)。

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.