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The correlation between the loss of Profilin 1 (Pfn1) with tumor progression indicated that Pfn1 is a tumor suppressor in human carcinoma. The molecular mechanisms underlying Pfn1 tumor suppression has yet to be elucidated. In this study, we showed that Pfn1 overexpression sensitizes cancer cells to apoptosis through the typical intrinsic apoptotic pathway. Mechanistically, the increased Pfn1 expression mediated the upregulation of p53R273H, one of the most common tumor-associated hotspot mutations of p53, with transactivation deletion in tumorigenesis and increased localization of p53R273H in cytoplasm. Further studies showed that mutant p53R273H was involved in apoptosis induced by Staurosporine (STS) via transcription-independent mitochondrial functions. We observed (i) the increased cytosolic localization of p53R273H, (ii) the activation of phosphorylation at Ser15, (iii) its mitochondrial localization; Pfn1 acted as a positive regulator of these processes. We also found that Pfn1 interacted with p53R273H and thus facilitated its exertion over the transcription-independent activity in the cytoplasm during drug action. Our results define a new function and mechanism of Pfn1 demonstrating that the combined effect with apoptotic agents led to a synergistic increase in apoptosis. In addition, p53R273H abrogating DNA binding was found to play a major role in the Pfn1- sensitized apoptosis through a transactivation-independent and cytosolic activity.

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TRP proteins form ion channels that are activated following receptor stimulation. Several members of the TRP family are likely to be expressed in lymphocytes. However, in many studies, messenger RNA (mRNA) but not protein expression was analyzed and cell lines but not primary human or murine lymphocytes were used. Among the expressed TRP mRNAs are TRPC1, TRPC3, TRPM2, TRPM4, TRPM7, TRPV1, and TRPV2. Regulation of Ca2+ entry is a key process for lymphocyte activation, and TRP channels may both increase Ca2+ influx (such as TRPC3) or decrease Ca2+ influx through membrane depolarization (such as TRPM4). In the future, linking endogenous Ca2+/cation channels in lymphocytes with TRP proteins should lead to a better molecular understanding of lymphocyte activation.

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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.

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In skeletal muscle the oligomeric alpha(1S), alpha(2)/delta-1 or alpha(2)/delta-2, beta1, and gamma1 L-type Ca(2+) channel or dihydropyridine receptor functions as a voltage sensor for excitation contraction coupling and is responsible for the L-type Ca(2+) current. The gamma1 subunit, which is tightly associated with this Ca(2+) channel, is a membrane-spanning protein exclusively expressed in skeletal muscle. Previously, heterologous expression studies revealed that gamma1 might modulate Ca(2+) currents expressed by the pore subunit found in heart, alpha(1C), shifting steady state inactivation, and increasing current amplitude. To determine the role of gamma1 assembled with the skeletal subunit composition in vivo, we used gene targeting to establish a mouse model, in which gamma1 expression is eliminated. Comparing litter-matched mice with control mice, we found that, in contrast to heterologous expression studies, the loss of gamma1 significantly increased the amplitude of peak dihydropyridine-sensitive I(Ca) in isolated myotubes. Whereas the activation kinetics of the current remained unchanged, inactivation of the current was slowed in gamma1-deficient myotubes and, correspondingly, steady state inactivation of I(Ca) was shifted to more positive membrane potentials. These results indicate that gamma1 decreases the amount of Ca(2+) entry during stimulation of skeletal muscle.

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Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca(2+) entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry that TRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only other TRP transcript found at considerable levels was TRP1, whereas TRP2, TRP3, TRP5(CCE2), and TRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca(2+) (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

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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.

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Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca2+ release channel (in the membrane of internal Ca2+ stores) and the dihydropyridine receptor (DHPR)-Ca2+ channel (in the plasma membrane). To ensure expression of functional L-type Ca+ channels, we expressed alpha2, beta, and gamma DHPR subunits and a chimeric DHPR alpha(i) subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca2+ transient (CaT) in the absence of extracellular Ca2+ ([Ca2+]o). However, in the presence of [Ca2+]o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca2+ concentration ([Ca2+]i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca2+]i which lasted for 5-10 s (the second component). Our results suggest that the first component primarily reflected Ca2+ influx through Ca2+ channels, whereas the second component resulted from Ca2+ release through the RyR expressed in the membrane of internal Ca2+ stores. However, the onset and the rate of Ca2+ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca2+ current. These results suggest that the skeletal muscle RyR isoform supports Ca2+-induced Ca2+ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional "close coupling" like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.

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Highly Ca2+ selective Ca2+ channels activated by store depletion have been recently described in several cell types and have been termed CRAC channels (for calcium release-activated calcium). The present study shows that following store depletion in mast and RBL-1 cells, monovalent outward currents could be recorded if the internal solution contained K+ but not Cs+. The activation of the outward K+ current correlated with the activation of ICRAC, in both time and amplitude, suggesting that the K+ current might be carried by CRAC channels. The amplitude of the outward current was increased if external Ca2+ was reduced or replaced by external Ba2+. The outward K+ conductance might have a physiological role in maintaining the driving force for Ca2+ entry during the activation of CRAC channels.

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Prolonged Ca2+ influx is an essential signal for the activation of T lymphocytes by antigen. This influx is thought to occur through highly selective Ca2+ release-activated Ca2+ (CRAC) channels that are activated by the depletion of intracellular Ca2+ stores. We have isolated mutants of the Jurkat human T cell line NZdipA to explore the molecular mechanisms that underlie capacitative Ca2+ entry and to allow a genetic test of the functions of CRAC channels in T cells. Five mutant cell lines (CJ-1 through CJ-5) were selected based on their failure to express a lethal diphtheria toxin A chain gene and a lacZ reporter gene driven by NF-AT, a Ca(2+)- and protein kinase C-dependent transcription factor. The rate of Ca2+ influx evoked by thapsigargin was reduced to varying degrees in the mutant cells whereas the dependence of NF-AT/lacZ gene transcription on [Ca2+]i was unaltered, suggesting that the transcriptional defect in these cells is caused by a reduced level of capacitative Ca2+ entry. We examined several factors that determine the rate of Ca2+ entry, including CRAC channel activity, K(+)-channel activity, and Ca2+ clearance mechanisms. The only parameter found to be dramatically altered in most of the mutant lines was the amplitude of the Ca2+ current (ICRAC), which ranged from 1 to 41% of that seen in parental control cells. In each case, the severity of the ICRAC defect was closely correlated with deficits in Ca2+ influx rate and Ca(2-)-dependent gene transcription. Behavior of the mutant cells provides genetic evidence for several roles of ICRAC in T cells. First, mitogenic doses of ionomycin appear to elevate [Ca2+]i primarily by activating CRAC channels. Second, ICRAC promotes the refilling of empty Ca2+ stores. Finally, CRAC channels are solely responsible for the Ca2+ influx that underlies antigen-mediated T cell activation. These mutant cell lines may provide a useful system for isolating, expressing, and exploring the functions of genes involved in capacitative Ca2+ entry.

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A Ca2+ current activated by store depletion has been described recently in several cell types and has been termed ICRAC (for Ca2+ release-activated Ca2+ current). In this paper, the Ca2+ and Ba2+ permeability of CRAC channels is investigated in mast cells, rat basophilic leukaemia cells (RBL) and human T-lymphocytes (Jurkat). The selectivity of CRAC channels for Ca2+ over monovalent cations is identical in all three cell types and is at least as high as that of voltage-operated Ca2+ (VOC) channels in the various tissues tested. The amplitude of Ba2+ currents relative to Ca2+ currents (IBa/ICa) through CRAC channels was found to be strongly dependent on the membrane potential and was much smaller in Jurkat cells compared to mast and RBL cells. An anomalous mole-fraction behavior was observed at very negative membrane potentials in all three cell types when using different mixtures of external Ca2+ and Ba2+. In contrast to VOC channels, the anomalous mole-fraction effect was not observed at potentials positive to -20 mV.

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Calcium entry in non-excitable cells occurs through calcium-selective currents activated secondarily to store depletion and/or through non-selective cation channels (e.g., receptor- or second-messenger-activated channels). The driving force for calcium influx can be modified by chloride or potassium channels, which set the membrane potential of cells. Together, these conductances determine the extent of calcium entry. Mast cells are an excellent model system for studying calcium influx, because calcium-release-activated calcium currents (ICRAC), second-messenger-activated non-selective currents and chloride currents are present in these cells. Whole-cell patch-clamp recordings were used to test the effects of the commonly used calcium entry blockers econazole and SK&F 96365, as well as the antiallergic and anti-inflammatory drugs tenidap, ketotifen and cromolyn on these channels. All tested drugs blocked the three different channel types with a similar order of magnitude (IC50 values ranging from micromolar to millimolar). Hence, these drugs cannot be used to discriminate between different calcium entry mechanisms.

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Calcium influx in electrically non-excitable cells is regulated by the filling state of intracellular calcium stores. Depletion of stores activates plasma membrane channels that are voltage-independent and highly selective for Ca2+ ions. We report here that the activation of plasma membrane Ca2+ currents induced by depletion of Ca2+ stores requires a diffusible cytosolic factor that washes out with time when dialyzing cells in the whole-cell configuration of the patch-clamp technique. The activation of calcium release-activated calcium current (ICRAC) by ionomycin- or inositol 1,4,5-trisphosphate-induced store depletion is blocked by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and guanyl-5'-yl imidodiphosphate, non-hydrolyzable analogs of GTP, suggesting the involvement of a GTP-binding protein. The inhibition by GTP gamma S occurs at a step prior to the activation of ICRAC and is prevented by the addition of GTP. We conclude that the activation mechanism of depletion-induced Ca2+ influx encompasses a GTP-dependent step, possibly involving an as yet unidentified small GTP-binding protein.

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Changes in the concentration of intracellular Ca2+ are crucial for signal transduction in virtually every cell. In the past year, more of the diversity of receptor-mediated Ca2+ influx mechanisms has been shown, and it has been disclosed that one of the most effective Ca2+ influx pathways, known as 'capacitative Ca2+ entry', occurs via Ca(2+)-selective ion channels in the plasma membrane that are activated following depletion of intracellular Ca2+ stores. Although the exact activation mechanism of capacitative Ca2+ entry still remains a mystery, the identification of plasma membrane currents following store depletion and the characterization of their biophysical properties opens the possibility of unraveling the features and molecular components of the phenomenon of capacitative Ca2+ entry.

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1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]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: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to -100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to -200 mV. 6. The selectivity of ICRAC 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 ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 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. ICRAC was dose-dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)

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Whole-cell patch-clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study Mn2+ influx in rat peritoneal mast cells. The calcium-selective current, activated by depletion of intracellular calcium stores (ICRAC for calcium release-activated calcium current), supports a small but measurable Mn2+ current. In the presence of intracellular BAPTA, a Mn2+ current through ICRAC was recorded in isotonic MnCl2 (100 mM) without a significant quenching of fura-2 fluorescence. Its amplitude was 10% of that measured in physiological solution containing 10 mM Ca2+. However, following store depletion, a significant quenching of fura-2 fluorescence could be measured only when intracellular BAPTA was omitted, so that all the incoming Mn2+ could be captured by the fluorescent dye. Two other ionic currents activated by receptor stimulation also induced Mn2+ quenching of fura-2 fluorescence: a small current through non-specific cation channels of 50-pS unitary conductance and a distinct cationic current of large amplitude. In addition to these influx mechanisms, Mn2+ was taken up into calcium stores and was subsequently co-released with Ca2+ by Ca(2+)-mobilizing agonists.

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Whole-cell patch-clamp recordings of membrane currents and Fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study calcium influx through receptor-activated cation channels in rat peritoneal mast cells. Cation channels were activated by the secretagogue compound 48/80, whereas a possible concomitant Ca2+ entry through pathways activated by depletion of calcium stores was blocked by dialyzing cells with heparin. Heparin effectively suppressed the transient Ca2+ release induced by 48/80 and abrogated inositol 1,4,5-trisphosphate-induced calcium influx without affecting activation of 50-pS cation channels. There was a clear correlation between changes in [Ca2+]i and the activity of 50-pS channels. The changes in [Ca2+]i increased with elevation of extracellular Ca2+. At the same time, inward currents through 50-pS channels were diminished as more Ca2+ permeated. This effect was due to a decrease in slope conductance and a reduction in the open probability of the cation channels. In physiological solutions, 3.6% of the total current was carried by Ca2+. The cation channels were not only permeable to Ca2+ but also to Mn2+, as evidenced by the quench of Fura-2 fluorescence. Mn2+ current through 50-pS channels could not be resolved at the single-channel level. Our results suggest that 50-pS cation channels partially contribute to sustained increases of [Ca2+]i in mast cells following receptor activation.

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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.

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Functionally significant properties of domains in the amino acid sequence of potassium (K+) channel-forming proteins have been investigated by constructing chimeric K+ channels. The N-terminal domain of ShA2 channels was responsible for the fast inactivation (IKA) and also determined a shift in the threshold of activation whereas the membrane domain determined the timecourse of slow inactivation. The binding site for dendrotoxin (DTX), but not for mast cell degranulating peptide (MCDP), is completely located on the loop between the membrane spanning segments S5 and S6 in RCK1 channels. A certain part of this region which has recently been designated as a narrow part of the pore was found to be not responsible for the differences in the single-channel current amplitude between RCK4 and RCK2 K+ channels. Interchange of the C-terminal domain did not influence activation or inactivation of the channels.

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