Molecular identity of exosomal BK channels

摘要 自1983年发现细胞外小泡以来，细胞外小泡作为一种重要的细胞外信号受到了广泛的关注。 细胞间通讯的介体、潜在的疾病标志物、治疗靶点和药物 送货车辆。尽管人们普遍认为电动汽车是在细胞内包装的，但通过 细胞外环境，并将货物运送到目标细胞。然而，即使在37年后，它也不是 已确定，1)EVS如何处理不同的离子环境(细胞质与细胞外)，2)EVS是否 是否拥有任何功能离子通道，以及3)这些通道中是否有任何起到生理作用。我们 重点回答了这些问题，并专注于具有最大梯度的离子，即钾。vbl.使用 在电子计算机方法中，我们发现了几个离子通道，其中最突出的离子通道，我们 在外体中发现的是BK。我们采用了一种新的电生理学方法，近场 电生理学，因为经典的膜片钳方法是不可行的，因为外体的大小。我们 发现功能BK通道存在于外切体中，并决定外切体的完整性。我们的 初步数据还表明，胞外BK对心脏缺血再灌注损伤具有保护作用。我们 现在将测试含有BK的外切体决定外切体含量的假设， 它们在多变的离子环境中存活，并保护心脏免受IR损伤。总体而言，数据支持 上述假设将使用多种方法进行检验，并追求以下具体目标， 1.在外体中建立BK的存在、分子鉴定和生物物理性质，2. 确定BK在外切体中的生理作用；3.阐明外切体的作用机制 BK通道在心脏保护中的作用在我们的提案中，我们纳入了遗传小鼠模型，以及 以创新和新颖的技术来理解一个非常基本和广泛的生物学问题。 该计划的结果将为研究包括BK在内的胞外离子通道提供机会 通道，并通过确定外切体如何生存变量来推进外切体领域 渗透压，建立外体离子通道的分子同一性，了解货物含量 受胞外体离子通道的调节，以及胞外体离子通道的作用和机制 心脏保护。在未来，我们的研究将为探索外体中的其他离子通道奠定基础 不同的生物以及器官系统。

Abstract Extracellular vesicles (EVs) have gained significant attention since their discovery in 1983 as important mediators of intercellular communications, potential disease markers, therapeutic targets, and drug delivery vehicles. Though it is widely accepted that EVs get packaged inside the cell, pass through the extracellular environment, and deliver the cargo to the target cells. However, even after 37 yrs it is not determined, 1) how EVs handle the differential ionic environment (cytoplasm vs extracellular), 2) whether EVs possess any functional ion channels, and 3) whether any of these channels play a physiological role. We focused on answering these questions and focused on an ion with the largest gradient, i.e., potassium. Using the in silico approach, we discovered several ion channels, and the most prominent ion channels, we discovered in exosomes is BK. We incorporated a novel electrophysiology approach, near field electrophysiology, as canonical patch-clamp methods are not feasible due to the size of exosomes. We discovered that functional BK channels exist in exosomes, and decide the integrity of exosomes. Our preliminary data also indicate that exosomal BK can protect the heart from ischemia-reperfusion injury. We will now test the hypothesis that exosomes containing BK determine the content of exosomes, facilitate their survival in variable ionic environments, and protect the heart from IR injury. Overall the data supports the above hypothesis which will be tested using multiple approaches and pursuing the following specific aims to, 1. establish a presence, molecular identity, and biophysical properties of BK in exosomes, 2. determine the physiological role of BK in exosomes., and 3. elucidate the mechanistic role of exosomal BK channels in cardioprotection. In our proposal, we have incorporated genetic mice models, and innovative as well as a novel technology to understand a very basic and broad biological question. The outcome of this program will open an opportunity to study exosomal ion channels including BK channels, and advance the exosome field by determining how exosome survive variable osmolarities, establishing the molecular identity of exosomal ion channels, understand how cargo content is regulated by exosomal ion channels, and the role and mechanism of exosomal ion channels in cardioprotection. In the future, our study will set the ground for exploring other ion channels in exosomes from different living beings as well as organ systems.