SINGLE MOLECULE DETECTION OF ION CHANNELS IN NEURONS

描述(申请人提供)：我们将研究调节高电导、钙和电压门控BK通道的亚细胞分布的因素，这是一种调节健康和疾病中神经元放电输出的关键离子通道。尽管它在调节兴奋性方面发挥着强大的作用，但实验证据表明，它稀疏地分布在质膜上，这一现象是通过与大脑特定的附属亚单位相互作用来调节的，4.传统的研究膜蛋白定位的方法严重依赖于标记蛋白的过度表达，这种方法可以通过改变靶标的化学计量比及其调节因子来显著改变蛋白质的分布。为了准确地确定BK通道是如何在细胞内分布的，重要的是能够确定单个分子在内源表达水平上的位置，以保持与调控伙伴之间关键的浓度依赖的相互作用。我们开发了一种具有高荧光发射的新型蛋白质/染料标签，可以对高丰度和低丰度蛋白质进行单分子检测。为了保持正常的通道表达水平，我们将产生一只转基因小鼠，其中该标签已插入内源性BK通道基因。将评估该通道在来自这些动物的初级神经元中的定位，并确定其附属亚单位和活动调节的表面分布。

DESCRIPTION (provided by applicant): We will investigate the factors regulating the subcellular distribution of the high conductance, calcium- and voltage-gated BK channel, a critical ion channel that regulates neuronal firing output in health and disease. Despite its powerful role in modulating excitability, experimental evidence indicates that it is sparsely distributed at the plasma membrane, a phenomenon that is regulated via interactions with the brain-specific accessory subunit, ?4. Conventional methods to study membrane protein localization have relied heavily upon overexpression of tagged proteins, a method that can significantly alter protein distribution by changing the stoichiometry of the target with its regulatory factors. To accurately determine how BK channels are distributed across the cell, it is important to be able to determine the location of individual molecules at endogenous expression levels to preserve critical concentration-dependent interactions with regulatory partners. We have developed a novel protein/dye tag with high-fluorescence emission that enables single-molecule detection, for both high- and low-abundance proteins. To preserve normal channel expression levels, we will generate a transgenic mouse where this tag has been inserted into the endogenous BK channel gene. The localization of this channel in primary neurons derived from these animals will be evaluated, and its accessory subunit and activity-regulated surface distribution will be determined.