Ion Regulation of Kv Channel Gating and Permeation

描述（由申请人提供）：细胞中的钾通道活性是正常细胞功能的关键决定因素。这种活性取决于通道特性的集合，包括渗透和门控。渗透离子本身调节这些特性，从而表明一种潜在的自动调节通道活性的手段，这可能是重要的稳态电活动。我们的长期目标是了解渗透离子调节和协同孔特性，门控（特别是慢失活），和电压传感器的运动的自动调节机制。这项建议分为两个目标。第一个目的是研究渗透离子对钾通道门控和渗透的调节机制。通过利用一系列具有不同缓慢失活动力学的Shaker突变体，将考虑几种假设。具体来说，我们将测试如何突变体，提高缓慢失活的速率大幅降低宏观电导Cs +离子，这是否是由于改变渗透性/选择性，较低的值的单通道电导，改变离子占有的孔，和/或降低的概率开放。在其他电生理学方法中，我们将使用HEK细胞中表达的电流的非平稳噪声分析。第二个目标将测试选择性过滤器中的离子是否调制电压传感器的运动。这些研究将需要测量HEK细胞中门控电流的动力学和电压依赖性。

DESCRIPTION (provided by applicant): Potassium channel activity in a cell is a critical determinant of normal cell function. This activity depends on an ensemble of channel properties including permeation and gating. Permeant ions themselves modulate these properties, thereby suggesting a potential means of autoregulation of channel activity, which could be important for homeostatic electrical activity. Our long term goal is to understand the autoregulatory mechanisms by which permeant ions modulate and synergize pore properties, gating (specifically slow inactivation), and movement of the voltage sensor. This proposal is divided into two aims. The first aim investigates the mechanisms of permeant ion modulation of gating and permeation in potassium channels. Several hypotheses will be considered by exploiting a series of Shaker mutants having different kinetics of slow inactivation. Specifically, we will test how mutants that enhance the rate of slow inactivation have drastically reduced macroscopic conductances to Cs + ions, whether this is due to altered permeabilities/selectivity, lower values of single channel conductance, altered ion occupancy of the pore, and/or decreased probabilities of opening. Among other electrophysiological approaches, we will use nonstationary noise analysis of currents expressed in HEK cells. The second aim will test whether ions in the selectivity filter modulate the movement of voltage sensors. These studies will entail measurements of kinetics and voltage dependence of gating currents in HEK cells.