SINGLE MOLECULE DETECTION OF ION CHANNELS IN NEURONS

描述（由申请人提供）：我们将研究调节高电导，钙和电压门控BK通道亚细胞分布的因素，BK通道是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，它在质膜上分布稀疏，这一现象是通过与脑特异性附属亚基的相互作用来调节的。研究膜蛋白定位的传统方法严重依赖于标记蛋白的过表达，这种方法可以通过改变靶标及其调节因子的化学计量来显著改变蛋白质的分布。为了准确地确定BK通道如何在细胞中分布，重要的是能够确定内源性表达水平下单个分子的位置，以保持与调节伙伴的关键浓度依赖性相互作用。我们开发了一种具有高荧光发射的新型蛋白质/染料标签，可用于高丰度和低丰度蛋白质的单分子检测。为了保持正常通道的表达水平，我们将在内源性BK通道基因中插入该标签，产生转基因小鼠。该通道在源自这些动物的初级神经元中的定位将被评估，其附属亚基和活动调节的表面分布将被确定。

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.