Multi-Ion, Free Energy Study of Effect of Ba2+ Blockage on KcsA Ion Selectivity

描述（由申请人提供）：拟研究的目的是阐明钡阻断钾通道选择性过滤器的机制，并确定钡阻断对KcsA钾通道E71A突变体离子选择性的影响。由于钾离子通道的选择性过滤器是高度保守的，因此从这些研究中获得的所有知识都可以立即应用于广泛的钾离子通道。电压活化钾（K+）通道是在神经冲动传播中起关键作用的膜蛋白。对它们的功能至关重要的是，它们保持非常高的K+ o Na+选择性，但也以接近扩散极限的速率传导离子。目前，钡阻断电生理实验是测定高选择性钾离子通道中离子选择性最可靠的方法之一。然而，在这些实验中，关于钡和钾在通道中的定位或钡阻断对通道中结合位点选择性的影响的细节知之甚少。最近，首次进行了实验研究，研究了已知结构的钾通道，即KcsA的E71A突变体中的钡阻断。KcsA的E71A突变体抑制了缓慢失活，因此获得的关于离子选择性的所有信息都属于选择性过滤器的导电形式。KcsA的许多结构研究以及KcsA中离子选择性的一些研究使其非常适合基于显式极化全原子分子动力学模拟的详细计算研究。平衡和非平衡分子动力学方法（自由能摄动）将用于确定KcsA钾通道E71A突变体中钡阻断的机制以及钡阻断对离子选择性的影响。

DESCRIPTION (provided by applicant): The goal of the proposed research is to elucidate the mechanism of barium blockade of the selectivity filter of potassium channels and to determine the effects of barium blockade upon ion selectivity in the E71A mutant of the KcsA potassium channel. Since the selectivity filter of potassium channels is highly conserved, all knowledge gained from these studies is immediately applicable to a wide range of potassium channels. Voltage-activated potassium (K+) channels are membrane proteins that play a key role in the propagation of the nerve impulse. It is critical to their function that they maintain very high K+ o Na+ selectivity, but also conduct ions at a rate that approaches the diffusion limit. Currently, barium blockade electrophysiological experiments are one of the most reliable methods to determine ion selectivity in highly-selective potassium channels. However, little is known in detail about the positioning of the barium and potassium in the channels during these experiments or the effect of a barium blockade on the selectivity of the binding sites in the channel. Recently, and for the first time, experimental studies took place that investigated barium blockade in a potassium channel of known structure, the E71A mutant of KcsA. Slow inactivation is suppressed in the E71A mutant of KcsA, therefore that all information obtained about ion selectivity pertains to the conductive form of the selectivity filter. The existence of many structural studies of KcsA and additionally several studies of ion selectivity in KcsA make it uniquely suited for a detailed computational studies based on explicitly polarizable all-atom molecular dynamics simulations. Equilibrium and non-equilibrium molecular dynamics methods (free energy perturbation) will be used to determine the mechanism of barium blockade in the E71A mutant of the KcsA potassium channel and the effects of barium blockade on ion selectivity.