Fluorescence Changes in Shaker Potassium lon Channel

描述（由申请人提供）：Shaker是一个电压门控钾离子通道和一个模型系统，用于理解所有电压门控离子通道的结构-功能原理。这些通道是神经兴奋传播的基础，通道突变可引起各种心脏、神经元和神经肌肉疾病。众所周知，这些离子通道是通过膜上电压的变化来打开和关闭的（即改变它们的导电性）。但这是如何实现的呢？具体来说，已知离子通道的一部分是“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，这是高度争议的，似乎与生物物理和生化数据不协调。

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.