The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked to the induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for the regulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to the understanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

A recurrent missense variant in ITPR3 causes demyelinating Charcot-Marie-Tooth with variable severity.

Charcot-Marie-Tooth (CMT) disease is a neuromuscular disorder affecting the peripheral nervous system. The diagnostic yield in demyelinating CMT (CMT1) is typically ∼80-95%, of which at least 60% is due to the PMP22 gene duplication. The remainder of CMT1 is more genetically heterogeneous. We used whole exome and whole genome sequencing data included in the GENESIS database to investigate novel causal genes and mutations in a cohort of ∼2,670 individuals with CMT neuropathy. A recurrent heterozygous missense variant p.Thr1424Met in the recently described CMT gene ITPR3, encoding IP3R3 (inositol 1,4,5-trisphosphate receptor 3) was identified. This previously reported p.Thr1424Met change was present in 33 affected individuals from nine unrelated families from multiple populations, representing an unusual recurrence rate at a mutational hotspot, strengthening the gene-disease relationship (GnomADv4 allele frequency 1.76e-6). Sanger sequencing confirmed the co-segregation of the CMT phenotype with the presence of the mutation in autosomal dominant and de novo inheritance patterns, including a four-generation family with multiple affected second-degree cousins. Probands from all families presented with slow nerve conduction velocities, matching the diagnostic category of CMT1. Remarkably, we observed a uniquely variable clinical phenotype for age at onset and phenotype severity in p.Thr1424Met carrying patients, even within families. Finally, we present data supportive of a dominant-negative effect of the p.Thr1424Met mutation with associated changes in protein expression in patient-derived cells.

Effects of temperature on action potentials and ion conductances in type II taste-bud cells.

Temperature strongly influences the intensity of taste, but it remains understudied despite its physiological, hedonic, and commercial implications. The relative roles of the peripheral gustatory and somatosensory systems innervating the oral cavity in mediating thermal effects on taste sensation and perception are poorly understood. Type II taste-bud cells, responsible for sensing sweet, bitter umami, and appetitive NaCl, release neurotransmitters to gustatory neurons by the generation of action potentials, but the effects of temperature on action potentials and the underlying voltage-gated conductances are unknown. Here, we used patch-clamp electrophysiology to explore the effects of temperature on acutely isolated type II taste-bud cell electrical excitability and whole cell conductances. Our data reveal that temperature strongly affects action potential generation, properties, and frequency and suggest that thermal sensitivities of underlying voltage-gated Na+ and K+ channel conductances provide a mechanism for how and whether voltage-gated Na+ and K+ channels in the peripheral gustatory system contribute to the influence of temperature on taste sensitivity and perception.NEW & NOTEWORTHY The temperature of food affects how it tastes. Nevertheless, the mechanisms involved are not well understood, particularly whether the physiology of taste-bud cells in the mouth is involved. Here we show that the electrical activity of type II taste-bud cells that sense sweet, bitter, and umami substances is strongly influenced by temperature. These results suggest a mechanism for the influence of temperature on the intensity of taste perception that resides in taste buds themselves.

Coupled transmembrane mechanisms control MCU-mediated mitochondrial Ca2+ uptake.

Ca2+ uptake by mitochondria regulates bioenergetics, apoptosis, and Ca2+ signaling. The primary pathway for mitochondrial Ca2+ uptake is the mitochondrial calcium uniporter (MCU), a Ca2+-selective ion channel in the inner mitochondrial membrane. MCU-mediated Ca2+ uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca2+ uptake is tightly regulated to maintain low matrix [Ca2+] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca2+ regulation of intermembrane space-localized MICU1/2 is controlled by Ca2+-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca2+ that permeates through the channel pore regulates Ca2+ affinities of coupled inhibitory and activating sensors in the matrix. Ca2+ binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca2+ binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca2+ regulation of channel activity. Our results demonstrate how Ca2+ influx into mitochondria is tuned by coupled Ca2+-regulatory mechanisms on both sides of the inner mitochondrial membrane.

The mitochondrial Ca 2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

The mitochondrial uniporter (MCU) Ca 2+ ion channel represents the primary means for Ca 2+ uptake into mitochondria. Here we employed in vitro and in vivo models with MCU genetically eliminated to understand how MCU contributes to tumor formation and progression. Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced mitochondrial Ca 2+ uptake, suppression of inactivating-phosphorylation of pyruvate dehydrogenase, a modest increase of basal mitochondrial respiration and a significant increase of acute Ca 2+ -dependent stimulation of mitochondrial respiration. Inhibition of mitochondrial Ca 2+ uptake by genetic deletion of MCU markedly inhibited growth of HEK293T cells and of transformed fibroblasts in mouse xenograft models. Reduced tumor growth was primarily a result of substantially reduced proliferation and fewer mitotic cells in vivo , and slower cell proliferation in vitro associated with delayed progression through S-phase of the cell cycle. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro , both predictors of metastatic potential. Surprisingly, mitochondrial matrix Ca 2+ concentration, membrane potential, global dehydrogenase activity, respiration and ROS production were unchanged by genetic deletion of MCU in transformed cells. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca 2+ signals. Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on mitochondrial Ca 2+ uptake for cell metabolism and Ca 2+ dynamics necessary for cell-cycle progression and cell proliferation.

The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals. Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation.

Davallialactone from mushroom reduced premature senescence and inflammation on glucose oxidative stress in human diploid fibroblast cells.

Mushrooms are both food and a source of natural compounds of biopharmaceutical interest. The purpose of this study was to clarify whether davallialactone from mushroom extract affected the pathogenesis of hyperglycemia oxidative stress and the aging process in human diploid fibroblast (HDF) cells. The high-glucose state with glucose oxidase resulted in glucose oxidative stress, induction of inflammatory molecules, dysfunction of antioxidant molecules, and activation of mitogen-activated protein kinase (MAPKs) and its downstream signaling in old HDF cells. The exposure of glucose oxidative stress in middle-stage cells led to stress-induced premature senescence (SIPS) via senescence-associated ß-galactosidase (SA ß-gal) activity and displayed replicative senescence phenomena. However, davallialactone reduces the pathogenesis of glucose oxidative stress and the aging process through down-regulation of SA ß-gal activity. These results strongly suggest that natural compounds, especially mushroom extract davallialactone, improve the pathogenesis of glucose oxidative stress and the aging process. Hence, davallialactone has potential in the treatment of diabetes mellitus or age-related disease complications.

Davallialactone reduces inflammation and repairs dentinogenesis on glucose oxidase-induced stress in dental pulp cells.

INTRODUCTION: The chronic nature of diabetes mellitus (DM) raises the risk of oral complication diseases. In general, DM causes oxidative stress to organs. This study aimed to evaluate the cellular change of dental pulp cells against glucose oxidative stress by glucose oxidase with a high glucose state. The purpose of this study was to test the antioxidant character of davallialactone and to reduce the pathogenesis of dental pulp cells against glucose oxidative stress. METHODS: The glucose oxidase with a high glucose concentration was tested for hydroxy peroxide (H2O2) production, cellular toxicity, reactive oxygen species (ROS) formation, induction of inflammatory molecules and disturbance of dentin mineralization in human dental pulp cells. The anti-oxidant effect of Davallilactone was investigated to restore dental pulp cells' vitality and dentin mineralization via reduction of H2O2 production, cellular toxicity, ROS formation and inflammatory molecules. RESULTS: The treatment of glucose oxidase with a high glucose concentration increased H2O2 production, cellular toxicity, and inflammatory molecules and disturbed dentin mineralization by reducing pulp cell activity. However, davallialactone reduced H2O2 production, cellular toxicity, ROS formation, inflammatory molecules, and dentin mineralization disturbances even with a long-term glucose oxidative stress state. CONCLUSIONS: The results of this study imply that the development of oral complications is related to the irreversible damage of dental pulp cells by DM-induced oxidative stress. Davallialactone, a natural antioxidant, may be useful to treat complicated oral disease, representing an improvement for pulp vital therapy.