KCNN4 mediated potassium ion efflux maintains mitochondrial functions leading to platelet biogenesis

Background: While potassium ion (K+) is known to be vital to platelet functions, we have limited knowledge about K+ channels involved in thrombopoiesis from imMKCLs, the iPSC-derived megakaryocyte progenitor cell lines that we have established for ex vivo manufacturing of platelet products for clinical use. Objective: We aimed to elucidate how K+ channels contribute to platelet biogenesis and focused on the role of Ca2+-activated K+ channel KCNN4 (also known as KCa3.1). Methods: Using imMKCLs and human cord blood hematopoietic stem cell-derived megakaryocytes (CB-megakaryocytes), we examined the dynamic changes of intracellular cations during platelet biogenesis. Along with RNA-seq profiling of K+ channels, we investigated the role of KCNN4 by using inhibitors or by direct gene knockdown in proplatelet formation or platelet production. We further examined its relationship with tubulin reorganization, mitochondrial functions, and reactive oxygen species (ROS) levels. Results: Continuous reduction of intracellular K+ levels ([K+]i) was observed during the 6-day of maturing imMKCLs. KCNN4 was expressed at the initiation of platelet generation in megakaryocytes and KCNN4 inhibition resulted in impaired proplatelet formation and reduced platelet productivity in imMKCLs and CB-megakaryocytes, accompanied by decreased [K+]i, diminished mitochondrial membrane potential and an elevated level of ROS. Conclusions: Our findings suggest that the decline of [K+]i via KCNN4 is a key mechanism linking tubulin regulation, ROS, and mitochondrial functions to proplatelet formation and intact thrombopoiesis. This study sheds new light on thrombopoiesis mechanism that contributes to improved ex vivo platelet manufacturing.