In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.

SUMMARY1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca21]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells.2. Calcium influx through an inward calcium release-activated calcium current (ICRAC) was induced by three independent mechanisms that result in store depletion: 6. The selectivity of 'CRAC for Ca2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the calcium-sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na' and K+ by N-methyl-D-glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of 'CRAC by only 9% further suggesting a high specificity for Ca2+ ions. M1S 1625M. HOTH AND -R. PENNER 7. The current amplitude was not greatly affected by variations of external Mg2" in the range of 0-12 mm. Even at 12 mm Mg2+ the current amplitude was reduced by only 23 %.8. 1CRAC was dose-dependently inhibited by Cd2+. The concentration-response relationship for Cd21 could be described by a Michaelis-Menten function with an apparent KD of 0 24 mm and a Hill coefficient of 1.9. All other tested divalent ions also dose-dependently and reversibly inhibited ICRAC The order of potency was determined by the relative blocking efficacy of 1 mm of the respective ions: Ba2+ % Sr2+ < Ni2 < Mn2+Co2-" Be2 < Cd2 < Zn2rThe trivalent ion La3+ was the most potent blocker of 1CRAC' 10. 1CRAc excluded monovalent ions in the presence of divalent ions. Complete removal of divalent ions typically resulted in a triphasic conductance change: an initial decrease in the calcium current, an abrupt increase in inward current with modest inward rectification due to passage of monovalent ions, and a subsequent decrease in total current with a linear current-voltage relationship. At the same time, these changes were accompanied by a shift in the reversal potential from > +50 to 0 mV.11. While all the features of 1CRAC are compatible with an ion channel mechanism, there was no significant increase in current noise associated with its activation. 12. Our results suggest that the calcium current activated by depletion of intracellular calcium stores is a highly selective pathway for calcium entry into mast cells and may constitute one of the mechanisms underlying the plateau phase of elevated cytosolic calcium concentration following receptor-mediated release of intracellular calcium.

Summary Despite success with BRAFV600E–inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multi-drug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (OXPHOS) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1Bhigh subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anti-cancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.

Mitochondria act as potent buffers of intracellular Ca2+ in many cells, but a more active role in modulating the generation of Ca2+ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or “capacitative” Ca2+ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca2+ store with thapsigargin (TG) activates Ca2+ release-activated Ca2+ (CRAC) channels in T cells, and the ensuing influx of Ca2+ loads a TG- insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca2+ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na+ depletion, which inhibits Na+-dependent Ca2+ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca2+ levels that are consistent with its localization in the TG-insensitive store. Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca2+ uptake is sensitive to extracellular [Ca2+], indicating that mitochondria sense Ca2+ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na+ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca2+ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

T helper (Th) cell activation is required for the adaptive immune response. Formation of the immunological synapse (IS) between Th cells and antigen-presenting cells is essential for Th cell activation. IS formation induces the polarization and redistribution of many signaling molecules; however, very little is known about organelle redistribution during IS formation in Th cells. We show that formation of the IS induced cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. Using total internal reflection microscopy, we found that upon stimulation, the distance between the IS and mitochondria was decreased to values <200 nm. Consequently, mitochondria close to the IS took up more Ca 2؉ than the ones farther away from the IS. The redistribution of mitochondria to the IS was necessary to maintain Ca 2؉ influx across the plasma membrane and Ca 2؉ -dependent Th cell activation. Our results suggest that mitochondria are part of the signaling complex at the IS and that their localization close to the IS is required for Th cell activation.calcium ͉ lymphocyte ͉ mitochondria

Mitochondrial reactive oxygen species (ROS) play a central role in most aging-related diseases. ROS are produced at the respiratory chain that demands NADH for electron transport and are eliminated by enzymes that require NADPH. The nicotinamide nucleotide transhydrogenase (Nnt) is considered a key antioxidative enzyme based on its ability to regenerate NADPH from NADH. Here, we show that pathological metabolic demand reverses the direction of the Nnt, consuming NADPH to support NADH and ATP production, but at the cost of NADPH-linked antioxidative capacity. In heart, reverse-mode Nnt is the dominant source for ROS during pressure overload. Due to a mutation of the Nnt gene, the inbred mouse strain C57BL/6J is protected from oxidative stress, heart failure, and death, making its use in cardiovascular research problematic. Targeting Nnt-mediated ROS with the tetrapeptide SS-31 rescued mortality in pressure overload-induced heart failure and could therefore have therapeutic potential in patients with this syndrome.

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In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca 2+ signals. In many cells, mitochondria act as Ca 2+ buffers by taking up and releasing Ca 2+ , but this simple buffering action by itself often cannot explain the organelle's effects on Ca 2+ signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca 2+ channels in T lymphocytes as a mechanism of mitochondrial Ca 2+ signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca 2+ stores causes sustained activation of the store-operated Ca 2+ current, I CRAC (CRAC, Ca 2+ release-activated Ca 2+ ). Inhibition of mitochondrial Ca 2+ uptake by compounds that dissipate the intramitochondrial potential unmasks Ca 2+ -dependent inactivation of I CRAC . Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca 2+ accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca 2+ uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca 2+ ] i elevation, indicating a functional link between mitochondrial regulation of I CRAC and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca 2+ channel activity and signal transmission from the plasma membrane to the nucleus.