Role of a novel MCU variant in Ca2+ handling in human platelets
Authors: Polina Iuliia , Chaput Isabel , Landherr Maria , Cypress Michael , Jhun Bong Sook , O-Uchi Jin 
Published: May 2023

Introduction: Discovery of the molecular identity of the mitochondrial Ca2+ (mtCa2+) uniporter complex (mtCUC) provides rapid progress in characterizing mtCa2+ handling and its roles in regulating cellular functions. MCU, a pore-forming subunit of mtCUC, which resides at the inner mitochondrial membrane, forms the major mtCa2+ influx pathway. Human MCU gene is predicted to produce mRNA that codes a shorter transcript variant (termed MCU-S) in addition to canonical full-length MCU transcript (renamed as MCU-L). However, its functional relevance in human cells is not fully investigated. Hypothesis: MCU-S variant can form Ca2+-permeable channels in addition to canonical MCU-L. Methods: Human platelets from adult healthy donors, human megakaryoblastic leukemia cell line MEG-01, and HEK293T cells were used for biomechanical, cell biological, and physiological assays. Ca2+ permeability via MCU-S was assessed by patch clamp and Ca2+ imaging under confocal microscope. Results: The existence of the mRNA and protein of MCU-S was confirmed in various human cells and tissues. Specifically, highest MCU-S mRNA levels was observed in human platelets compared to other cells and tissues including HEK293T, MEG-01 cell lines and the heart tissues. Expression of MCU and several other mtCUC components in the plasma membrane (PM) was confirmed by cell-surface protein biotinylation. Immunostaining of the human platelets exhibited extra-mitochondrial expression of MCU protein in addition to mitochondria. Overexpressed MCU-S is localized at the PM rather than in the mitochondria, while overexpressed MCU-L is exclusively expressed in the mitochondria, as assessed by live cell imaging and cell-surface protein biotinylation. Conventional whole-cell patch clamping showed that MCU-S expression HEK293T cells (but not control cells) exhibit an inward Ca2+ current sensitive to Ru360, the conventional MCU blocker. MEG-01 overexpressing MCU-S (but not the cells expressing GFP as a control or dominant-negative pore-forming mutant of MCU-S, termed MCU-S-DN) showed Ru360-sensitive PM Ca2+ permeabilization after switching the extracellular Ca2+ concentration from 0 mM to physiological range in the presence of BTP2, a selective inhibitor for the store-operated Ca2+ entry (SOCE). There is no significant difference in the magnitude of SOCE between cells expressing MCU-S, MCU-S-DN, and GFP. Lastly, we confirmed that human platelets also possess the Ru360-sensitive, but BTP-insensitive Ca2+ permeabilization at the PM, which can significantly alter the cytosolic Ca2+ concentration. Conclusion: MCU-S variant is capable of forming Ca2+-permeable channels at the PM. Elucidation of the role of MCU transcript variants may provide new insights into the molecular basis of mitochondrial and cellular Ca2+ handling in human platelet activation under physiological and pathological conditions. NIH/NHLBI R01HL136757 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.