Development of a device to measure gap junction physiology

描述（由申请人提供）：间隙连接是传导离子电流并在细胞之间交换小分子的蛋白质通道。它们在身体的大多数组织如神经胶质、心脏、平滑肌和上皮细胞中产生功能性合胞体。缝隙连接功能障碍与许多疾病有关，但目前还没有特异性的试剂来激活或抑制缝隙连接。药物开发中的速率限制因素之一是当前测定的繁琐性质。本申请提出开发一种简单的工具，将当代微流体和电子技术应用于称为蔗糖间隙的已建立的电生理技术。将组织培养的细胞置于微流控芯片上，通过非导电溶液的窄流将其电分成两半。在音频频率下使用相位检测的四电极阵列测量从差距的一侧到另一侧的阻抗。我们还将跟踪荧光染料从差距的一侧到另一侧的扩散，同时进行电学测量。平均电流及其方差的分析将提供通道数量、单一电流以及打开和关闭动力学的估计。公共卫生相关性：缝隙连接是细胞之间的蛋白质孔，使它们能够与离子电流和化学信使通信。缝隙连接缺陷与许多疾病有关，包括心律失常、白内障和癌症。该提案将创建一种设备来筛选化学库中对间隙连接有活性的药物。

DESCRIPTION (provided by applicant): Gap junctions are protein channels that conduct ionic current and exchange small molecules between cells. They create functional syncitia in most tissues of the body such as glia, heart, smooth muscle and epithelia. Gap junction malfunction has been implicated in many diseases, but there are no specific reagents to activate or inactivate gap junctions. One of the rate limiting factors in drug development is the tedious nature of current assays. This application proposes to develop a simple tool that applies contemporary microfluidic and electronic technology to an established electrophysiological technique called the sucrose gap. Tissue cultured cells placed on a microfluidic chip are divided electrically in half by a narrow flow of non conducting solution. A four electrode array using phase detection at audio frequencies measures the impedance from one side of the gap to the other. We will also follow the diffusion of fluorescent dyes from one side of the gap to the other simultaneously with the electrical measurement. Analysis of mean current and its variance will provide an estimate of the number of channels, the unitary current and the kinetics of opening and closing. Public Health Relevance: Gap junctions are protein pores between cells that allow them to communicate with ionic currents and chemical messengers. Defects in gap junctions are associated with many diseases including cardiac arrhythmias, cataracts and cancer. This proposal will create a device to screen chemical libraries for drugs that are active on gap junctions.