Ion Regulation of Kv Channel Gating and Permeation

描述(申请人提供)：细胞中的钾通道活性是细胞正常功能的关键决定因素。这一活动取决于包括渗透和门控在内的一系列通道属性。离子本身调节这些属性，从而暗示了一种潜在的自动调节通道活动的方法，这可能对体内平衡的电活动很重要。我们的长期目标是了解自我调节机制，通过这些机制，离子可以调节和协同气孔特性、门控(特别是慢速失活)和电压传感器的移动。这项提议分为两个目标。第一个目的是研究离子对钾通道门控和通透的调节机制。通过利用一系列具有不同缓慢失活动力学的Shaker突变体，将考虑几个假设。具体地说，我们将测试提高缓慢失活速度的突变体如何大幅降低对Cs离子的宏观电导，无论这是由于渗透性/选择性的改变、单通道电导值的降低、孔的离子占有率的改变和/或开放概率的降低。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。第二个目标是测试选择性过滤器中的离子是否会调节电压传感器的移动。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.