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、钙门控和电压门控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。尽管其强大的作用，在调节兴奋性，实验证据表明，它是稀疏分布在质膜，一种现象，是通过与大脑特定的辅助亚基，？4.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蛋白与其调节因子的化学计量来显著改变蛋白质分布。为了准确地确定BK通道如何分布在整个细胞中，重要的是能够确定内源性表达水平下单个分子的位置，以保持与调控伙伴的关键浓度依赖性相互作用。我们开发了一种新型的蛋白质/染料标签，具有高荧光发射，能够对高丰度和低丰度蛋白质进行单分子检测。为了保持正常通道表达水平，我们将产生转基因小鼠，其中该标签已插入内源BK通道基因。将评估该通道在这些动物的原代神经元中的定位，并确定其辅助亚基和活性调节的表面分布。

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.