Molecular identity of exosomal BK channels

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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.