Extracellular vesicles (EVs or exosomes) are known for their role in cargo delivery to the target cell^1-3. They have emerged as important means for intercellular communication, and the transfer of biological and chemical messages between cells. Extensive discussions with scientists in the EV field and their potential role generated curiosity about how these small vesicles can handle ionic stress during their journey between organs. The motivation behind the manuscript originally came from the discussions about extracellular vesicles between Prof. Raj Kishore (Temple University)^4,5 and Prof. Harpreet Singh during the scientific sessions at the American Heart Association, identifying the need to understand better the mechanism of regulating ionic homeostasis in the vesicles. The very first experiment using a Nanion planar bilayer system by Shridhar Sanghvi (MS student) in the Singh lab at Drexel revealed specific currents in the EV membranes. Dr. Singh calculated all the Nernst potentials for different ions to study a specific ion channel, such as a potassium channel. This set the ground for a discussion between Prof. Singh and Prof. Vishnu Sundaresan at the Ohio State University (OSU, now at DARPA) to perform electrophysiological experiments in intact vesicles using ‘near field’ electrophysiology. The first challenge was to get pure EVs, and Prof. Mahmood Khan (OSU) was pioneering the purification of EVs. While Prof. Khan provided EVs, and Dr. Divya Sridharan (Khan lab) tested their physiological function⁶, Dr. Parker Evans (Sunderesan lab) fabricated actuators^7,8 to set up the first set of experiments in intact EVs. We ran low on null mutant mice plasma at one point, and Prof. Dan Halm from Wright State University promptly supplied us with samples. This combined multidisciplinary approach led to the very first discovery and characterization of a functional ion channel (BK) in the EVs, as reported in manuscript⁹.

Inspiration

The discovery of aquaporin and its role in handling osmotic stress in red blood cells^10-12 was the biggest inspiration for the hypothesis that functional ion channels and transporters are present in the EVs to handle osmotic shock during their transportation. Our work was driven by the need to identify these channels to understand the possible mechanism of ‘handling’ of osmotic stress. We subsequently calculated Nernst potential for all the ions in intracellular vs extracellular spaces and decided to chase K⁺ as it is a monovalent cation with a large intracellular concentration. In addition, our characterization of BK channels in the inner membrane of mitochondria¹³, neonatal cardiomyocytes¹⁴, and expression of BK in lysosomes^15,16 motivated us to start exploring the idea of BK channels in EVs.

Advancements and Scientific Significance

In this comprehensive study⁹, we revealed that functional BK channels are present in the extracellular vesicles. Though exocarta¹⁷ shows several other ion channels and transporters to be present in the extracellular vesicles, it was unclear whether any of them are functional or are just packaged in or associated with the vesicles. Here we also showed that BK channels are vital for maintaining the integrity, size, and number of EVs.  Along with Prof. Khan, we discovered that the packaging of microRNAs in EVs is also regulated by BK channels. It is an open question whether BK plays a role in selective packaging or whether EVs lacking BK disintegrate with the specific cargo. We further highlighted that similar to mitochondrial BK, EV BK is also important in protecting the heart from ischemia-reperfusion injury^13,14,18-20. These findings highlight the potential role of EV ion channels in therapeutic applications.

The Future

The study⁹ paves the way for future research on the characterization of ion channels and transporters in EVs. Though BK is the first functional ion channel to be reported⁹, there are several other ion channels and transporters present. For example, CLIC4 was recently reported to be present in EVs²¹ but whether the channel is functional or not is known. Future research can expand the EV ion channel and transporter field by combining near-field electrophysiology and traditional electrophysiology. Our manuscript⁹ also showed the presence of ATP in EVs and a Na-K ATPase which can provide a mechanism on how potassium ion concentration is regulated in EVs. Another important aspect is the possible application of loading/ packaging of EVs with charged particles. By understanding the mechanism of ionic homeostasis, the packaging can be better controlled for their therapeutic application.

Summary

We discovered the first functional ion channel in EVs which regulates their integrity and physiological properties⁹. The integrative approach identified a functional EV BK channel and established its role in regulating the content of EVs. Our study highlights the importance of ion channels and transporters for EV-centric innovative therapeutic solutions.

 References 

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