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The mitochondrial calcium uniporter is a Ca 2+ channel that imports cytoplasmic Ca 2+ into the mitochondrial matrix to regulate cell bioenergetics, intracellular Ca 2+ signaling, and apoptosis. The uniporter contains the pore-forming MCU subunit, an auxiliary EMRE protein, and the regulatory MICU1/MICU2 subunits. Structural and biochemical studies have suggested that MICU1 gates MCU by blocking/unblocking the pore. However, mitoplast patch-clamp experiments argue that MICU1 does not block, but instead potentiates MCU via allosteric mechanisms. Here, we address this direct clash of the proposed MICU1 function. Supporting the MICU1-occlusion mechanism, patch-clamp demonstrates that purified MICU1 strongly suppresses MCU Ca 2+ currents, and this inhibition is abolished by mutating the MCU-interacting K126 residue. Moreover, a membrane-depolarization assay shows that MICU1 prevents MCU-mediated Na + flux into intact mitochondria under Ca 2+ -free conditions. Examining the observations underlying the potentiation model, we found that MICU1 occlusion was not detected in mitoplasts not because MICU1 cannot block, but because MICU1 dissociates from the uniporter complex. Furthermore, MICU1 depletion reduces uniporter transport not because MICU1 can potentiate MCU, but because EMRE is down-regulated. These results firmly establish the molecular mechanisms underlying the physiologically crucial process of uniporter regulation by MICU1.

Synovial sarcoma is a rare but aggressive soft-tissue sarcoma associated with translocation t(X;18). Metastasis occurs in approximately 50% of all patients, and curative outcomes are difficult to achieve in this group. Since the efficacies of current therapeutic approaches for metastatic synovial sarcoma remain limited, new therapeutic agents are urgently needed. Tilapia piscidin 4 (TP4), a marine antimicrobial peptide, is known to exhibit multiple biological functions, including anti-bacterial, wound-healing, immunomodulatory, and anticancer activities. In the present study, we assessed the anticancer activity of TP4 in human synovial sarcoma cells and determined the underlying mechanisms. We first demonstrated that TP4 can induce necrotic cell death in human synovial sarcoma AsKa-SS and SW982 cells lines. In addition, we saw that TP4 initiates reactive oxygen species (ROS) production and downregulates antioxidant proteins, such as uncoupling protein-2, superoxide dismutase (SOD)-1, and SOD-2. Moreover, TP4-induced mitochondrial hyperpolarization is followed by elevation of mitochondrial ROS. Calcium overload is also triggered by TP4, and cell death can be attenuated by a necrosis inhibitor, ROS scavenger or calcium chelator. In our experiments, TP4 displayed strong anticancer activity in human synovial sarcoma cells by disrupting oxidative status, promoting mitochondrial hyperpolarization and causing calcium overload.

NMDA receptor channels are characterized by high Ca 2+ permeability. It remains unclear whether extracellular Ca 2+ could directly modulate channel gating and control Ca 2+ influxes. We demonstrate a pore-blocking site external to the activation gate for extracellular Ca 2+ and Cd 2+ , which has the same charge and radius as Ca 2+ but is impermeable to the channel. The apparent affinity of Cd 2+ or Ca 2+ is higher toward the activated (a steady-state mixture of the open and desensitized, probably chiefly the latter) than the closed states. The blocking effect of Cd 2+ is well correlated with the number of charges in the DRPEER motif at the external pore mouth, with coupling coefficients close to 1 in double mutant cycle analyses. The effect of Ca 2+ and especially Cd 2+ could be allosterically affected by T647A mutation located just inside the activation gate. A prominent "hook" also develops after wash-off of Cd 2+ or Ca 2+ , suggesting faster unbinding rates of Cd 2+ and Ca 2+ with the mutation. We conclude that extracellular Ca 2+ or Cd 2+ directly binds to the DRPEER motif to modify NMDA channel activation (opening as well as desensitization), which seems to involve essential regional conformational changes centered at the bundle crossing point A652 (GluN1)/A651(GluN2).