Regulation of CRAC channels by protein interactions and post-translational modification

Store-operated Ca²⁺ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca²⁺ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca²⁺ store depletion that subsequently activates Ca²⁺-release-activated-Ca²⁺ (CRAC) channels, a prototype of store-operated Ca²⁺ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca²⁺ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function.     

钙离子通道蛋白的研究进展

钙离子是最广泛存在的细胞内信使,调控着几乎所有生命过程。最近的结构生物学研究解析了很多不同种类的钙离子通道在不同开放-关闭状态下的近原子分辨率结构。有关进展揭示了这些通道的分子组成、动态活动、生理功能、调控修饰的分子基础,为阐明钙信号转导和相关疾病的微观机制提供了理论基础.     

Ca signaling and STIM1

An increase in the intracellular calcium ion concentration ([Ca²⁺]) impacts a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Elevation of intracellular calcium ion (Ca²⁺) regulates various cellular events after the stimulation of cells. Initial increase in Ca²⁺ comes from the endoplasmic reticulum (ER), intracellular storage space. However, the continuous influx of extracellular Ca²⁺ is required to maintain the increased level of Ca²⁺ inside cells. Store-operated Ca²⁺ entry (SOCE) manages this process, and STIM1, a newly discovered molecule, has a unique and essential role in SOCE. STIM1 can sense the exhaustion of Ca²⁺ in the ER, and activate the SOC channel in the plasma membrane, leading to the continuous influx of extracellular Ca²⁺. STIM1 senses the status of the intracellular Ca²⁺ stores via a luminal N-terminal Ca²⁺-binding EF-hand domain. Dissociation of Ca²⁺ from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca²⁺ channels/entry (calcium-release-activated calcium channels/entry). In this review, we summarize the mechanism by which STIM1 regulates SOCE, and also its role in the control of mast cell functions and allergic responses.     

Ca microdomains in smooth muscle

In smooth muscle, Ca²⁺ controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca²⁺ to perform these multiple functions is the cell's ability to localize Ca²⁺ signals to certain regions by creating high local concentrations of Ca²⁺ (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca²⁺ influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca²⁺ store. A single Ca²⁺ channel can create a microdomain of several micromolar near (200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca²⁺] and the rapid rates of decline target Ca²⁺ signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca²⁺ by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca²⁺. In this review, the generation of microdomains arising from Ca²⁺ influx across the plasma membrane and the release of the ion from the SR Ca²⁺ store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

Perspective: The List of Potential Volume-sensitive Chloride Currents Continues to Swell (and Shrink)

The channel of the glutamate N-methyl-d-aspartate receptor (NMDAR) transports Ca²⁺ approximately four times more efficiently than that of Ca²⁺-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs⁺ efflux and Ca²⁺ influx in recombinant NMDAR and Ca²⁺-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca²⁺ using whole-cell currents and fluorometric techniques (dye overload) with Cs⁺ internally and Ca²⁺ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was ∼2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca²⁺ and Cs⁺ as the reference ions, Ca²⁺ permeability (P_Ca/P_Cs) was concentration dependent, being highest around physiological concentrations (1–1.8 mM; P_Ca/P_Cs ≈ 6.1) and reduced at both higher (110 mM; P_Ca/P_Cs ≈ 2.6) and lower (0.18 mM; P_Ca/P_Cs ≈ 2.2) concentrations. P_Ca/P_Cs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3–110 mM Ca²⁺. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca²⁺ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca²⁺ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca²⁺ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca²⁺, whereas that of AMPAR channels only a single site.     

Lipid rafts/caveolae as microdomains of calcium signaling

Ca²⁺ is a major signaling molecule in both excitable and non-excitable cells, where it serves critical functions ranging from cell growth to differentiation to cell death. The physiological functions of these cells are tightly regulated in response to changes in cytosolic Ca²⁺ that is achieved by the activation of several plasma membrane (PM) Ca²⁺ channels as well as release of Ca²⁺ from the internal stores. One such channel is referred to as store-operated Ca²⁺ channel that is activated by the release of endoplasmic reticulum (ER) Ca²⁺ which initiates store-operated Ca²⁺ entry (SOCE). Recent advances in the field suggest that some members of TRPCs and Orai channels function as SOCE channels. However, the molecular mechanisms that regulate channel activity and the exact nature of where these channels are assembled and regulated remain elusive. Research from several laboratories has demonstrated that key proteins involved in Ca²⁺ signaling are localized in discrete PM lipid rafts/caveolar microdomains. Lipid rafts are cholesterol and sphingolipid-enriched microdomains that function as unique signal transduction platforms. In addition lipid rafts are dynamic in nature which tends to scaffold certain signaling molecules while excluding others. By such spatial segregation, lipid rafts not only provide a favorable environment for intra-molecular cross-talk but also aid to expedite the signal relay. Importantly, Ca²⁺ signaling is shown to initiate from these lipid raft microdomains. Clustering of Ca²⁺ channels and their regulators in such microdomains can provide an exquisite spatiotemporal regulation of Ca²⁺-mediated cellular function. Thus in this review we discuss PM lipid rafts and caveolae as Ca²⁺-signaling microdomains and highlight their importance in organizing and regulating SOCE channels.     

ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance

Ca²⁺ homeostasis controls a diversity of cellular processes including proliferation and apoptosis. A very important aspect of Ca²⁺ signaling is how different Ca²⁺ signals are translated into specific cell functions. In T cells, Ca²⁺ signals are induced following the recognition of antigen by the T cell receptor and depend mainly on Ca²⁺ influx through store-operated CRAC channels, which are mediated by ORAI proteins following their activation by STIM proteins. The complete absence of Ca²⁺ influx caused by mutations in Stim1 and Orai1 leads to severe immunodeficiency. Here we summarize how Ca²⁺ signals are tuned to regulate important T cell functions as proliferation, apoptosis and tolerance, the latter one being a special state of immune cells in which they can no longer respond properly to an otherwise activating stimulus. Perturbations of Ca²⁺ signaling may be linked to immune suppressive diseases and autoimmune diseases.     

Longitudinal smooth muscle of the mammalian intestine

Ca²⁺ mobilization in muscle cells from the circular muscle layer of the mammalian intestine is mediated by IP₃-dependent Ca²⁺ release. Ca²⁺ mobilization in muscle from the adjacent longitudinal muscle layer involves a distinct, phosphoinositide-independent pathway. Receptors for contractile agonists in longitudinal muscle cells are coupled via a pertussis toxinsensitive G protein to activation of PLA₂ and formation of arachidonic acid (AA). The latter activates Cl⁻ channels resulting in depolarization of the plasma membrane and opening of voltage-sensitive Ca²⁺ channels. Ca²⁺ influx via these channels induces Ca²⁺ release by activating sarcoplasmic ryanodine receptor/Ca²⁺ channels. The increase in [Ca²⁺]_i activates membrane-bound ADP ribosyl cyclase, and the resultant formation of cADPR enhances Ca²⁺-induced Ca²⁺ release.     

Ion channels in cancer: future perspectives and clinical potential

Ion transport across the cell membrane mediated by channels and carriers participate in the regulation of tumour cell survival, death and motility. Moreover, the altered regulation of channels and carriers is part of neoplastic transformation. Experimental modification of channel and transporter activity impacts tumour cell survival, proliferation, malignant progression, invasive behaviour or therapy resistance of tumour cells. A wide variety of distinct Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels have been implicated in tumour growth and metastasis. Further experimental information is, however, needed to define the specific role of individual channel isoforms critically important for malignancy. Compelling experimental evidence supports the assumption that the pharmacological inhibition of ion channels or their regulators may be attractive targets to counteract tumour growth, prevent metastasis and overcome therapy resistance of tumour cells. This short review discusses the role of Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels in tumour growth and metastasis and the therapeutic potential of respective inhibitors.     

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca²⁺-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd³⁺ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca²⁺]_i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca²⁺ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca²⁺ influx to a small Ca²⁺-activated K⁺ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca²⁺ signaling that may play a role in tumor progression.     

Overexpression of SERCA2 ATPaase in vascular smooth muscle cells treated with oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca²⁺ _i homeostasis and altered two Ca²⁺ release mechanisms in these cells (IP₃ and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca²⁺ pump. One of the primary Ca²⁺ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP₃ and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca²⁺ homeostasis in VSMC chronically exposed to atherogenic oxLDL.     

Epigallocatechin-3-gallate elicits Ca spike in MCF-7 breast cancer cells: Essential role of Cav3.2 channels

We used MCF-7 human breast cancer cells that endogenously express Cav3.1 and Cav3.2 T-type Ca²⁺ channels toward a mechanistic study on the effect of EGCG on [Ca²⁺]_i. Confocal Ca²⁺ imaging showed that EGCG induces a [Ca²⁺]_i spike which is due to extracellular Ca²⁺ entry and is sensitive to catalase and to low-specificity (mibefradil) and high-specificity (Z944) T-type Ca²⁺channel blockers. siRNA knockdown of T-type Ca²⁺ channels indicated the involvement of Cav3.2 but not Cav3.1. Application of EGCG to HEK cells expressing either Cav3.2 or Cav3.1 induced enhancement of Cav3.2 and inhibition of Cav3.1 channel activity. Measurements of K⁺ currents in MCF-7 cells showed a reversible, catalase-sensitive inhibitory effect of EGCG, while siRNA for the Kv1.1 K⁺ channel induced a reduction of the EGCG [Ca²⁺]_i spike. siRNA for Cav3.2 reduced EGCG cytotoxicity to MCF-7 cells, as measured by calcein viability assay. Together, data suggest that EGCG promotes the activation of Cav3.2 channels through K⁺ current inhibition leading to membrane depolarization, and in addition increases Cav3.2 currents. Cav3.2 channels are in part responsible for EGCG inhibition of MCF-7 viability, suggesting that deregulation of [Ca²⁺]_i by EGCG may be relevant in breast cancer treatment.     

Activation of Nicotinic Acetylcholine Receptors Augments Calcium Channel-mediated Exocytosis in Rat Pheochromocytoma (PC12) Cells

The functional effect of activating Ca²⁺-permeable neuronal nicotinic acetylcholine receptors (nAChRs) on vesicle secretion was studied in PC12 cells. Single cells were patch-clamped in the whole-cell configuration and stimulated with either brief pulses of nicotine to activate the Ca²⁺-permeable nAChRs or with voltage steps to activate voltage-dependent Ca²⁺ channels. Membrane capacitance was used as a measure of vesicle secretion. Activation of nAChRs by nicotine application to cells voltage clamped at −80 mV evoked secretion. This secretion was completely abolished by nicotinic antagonists. When the cells were voltage clamped at +20 mV in the presence of Cd²⁺ to block voltage-activated Ca²⁺ channels, nicotine elicited a small amount of secretion. Most interestingly, when the nAChRs were activated coincidentally with voltage-dependent Ca²⁺ channels, secretion was augmented approximately twofold over the secretion elicited with voltage-dependent Ca²⁺ channels alone. Our data suggest that Ca²⁺ influx via nAChRs affects Ca²⁺-dependent cellular functions, including vesicle secretion. In addition to the secretion evoked by nAChR activation at hyperpolarized potentials, we demonstrate that even at depolarized potentials, nAChRs provide an important Ca²⁺ entry pathway underlying Ca²⁺-dependent cellular processes such as exocytosis.     

Calcium current kinetics in young and aged human cultured myotubes

There is evidence that the complex process of sarcopenia in human aged skeletal muscle is linked to the modification of mechanisms controlling Ca²⁺ homeostasis. To further clarify this issue, we assessed the changes in the kinetics of activation and inactivation of T- and L-type Ca²⁺ currents in in vitro differentiated human myotubes, derived from satellite cells of healthy donors aged 2, 12, 76 and 86 years. The results showed an age-related decrease in the occurrence of T- and L-type currents. Moreover, significant age-dependent alterations were found in L-(but not T) type current density, and activation and inactivation kinetics, although an interesting alteration in the kinetics of T-current inactivation was observed. The T- and L-type Ca²⁺ currents play a crucial role in regulating Ca²⁺ entry during satellite cells differentiation and fusion into myotubes. Also, the L-type Ca²⁺ channels underlie the skeletal muscle excitation–contraction coupling mechanism. Thus, our results support the hypothesis that the aging process could negatively affect the Ca²⁺ homeostasis of these cells, by altering Ca²⁺ entry through T- and L-type Ca²⁺ channels, thereby putting a strain on the ability of human satellite cells to regenerate skeletal muscle in elderly people.     

(Questions) on phloem biology. 1. Electropotential waves, Ca fluxes and cellular cascades along the propagation pathway

This review explores the relationships between electrical long-distance signalling, Ca²⁺ influx coincident with propagation of electropotential waves, and cellular responses to Ca²⁺ influx including the consequences for sieve-tube conductivity and mass flow. Ca²⁺ influx is inherent to electropotential waves and appears to constitute the key link between rapid physical signals and resultant chemical cascades in sieve tubes and adjacent cells. Members of several channel groups are likely involved the regulation of Ca²⁺ levels in sieve elements. Among them are hyperpolarization-activated, depolarization-activated, and mechanosensitive Ca²⁺ channels located in the plasma membrane and Ca²⁺ dependent Ca²⁺ channels that reside in ER-membranes of sieve elements. These channels collectively determine intracellular Ca²⁺ levels in sieve elements and their neighbour cells. The latter cells react to Ca²⁺ elevation by inducing diverse functional responses dependent on the cell type. If the Ca²⁺ concentration in sieve elements surpasses a threshold level, dual sieve-plate occlusion by proteins and callose deposition is triggered. Occlusion is reversed when Ca²⁺ levels subside. Electrical messages may regulate the degree of sieve plate hydraulic conductivity in intact plants by partial sieve-plate occlusion that has a major impact on volume flow through sieve tubes. Furthermore, complete but temporary occlusion of sieve tubes may modify mass flow patterns in intact plants.     

Effect of thapsigargin on Ca2+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca²⁺]_i levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Thapsigargin at concentrations between 10?nM and 10 µM increased [Ca²⁺]_i in a concentration-dependent fashion. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺ indicating that Ca²⁺ entry and release both contributed to the [Ca²⁺]_i rise. This Ca²⁺ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca²⁺ channels or by modulation of protein kinase C activity. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca²⁺ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca²⁺]_i rise, suggesting that thapsigargin released Ca²⁺ from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca²⁺]_i rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca²⁺ with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca²⁺]_i rises by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A2-sensitive Ca²⁺ channels. Thapsigargin also induced cell death via Ca²⁺-dependent pathways and Ca²⁺-independent apoptotic pathways.     

Effects of amyloid-β peptides on voltage-gated L-type Ca(V)1.2 and Ca(V)1.3 Ca(2+) channels

Overload of intracellular Ca²⁺ has been implicated in the pathogenesis of neuronal disorders, such as Alzheimer’s disease. Various mechanisms produce abnormalities in intracellular Ca²⁺ homeostasis systems. L-type Ca²⁺ channels have been known to be closely involved in the mechanisms underlying the neurodegenerative properties of amyloid-β (Aβ) peptides. However, most studies of L-type Ca²⁺ channels in Aβ-related mechanisms have been limited to Ca_V1.2, and surprisingly little is known about the involvement of Ca_V1.3 in Aβ-induced neuronal toxicity. In the present study, we examined the expression patterns of Ca_V1.3 after Aβ_25–35 exposure for 24 h and compared them with the expression patterns of Ca_V1.2. The expression levels of Ca_V1.3 were not significantly changed by Aβ_25–35 at both the mRNA levels and the total protein level in cultured hippocampal neurons. However, surface protein levels of Ca_V1.3 were significantly increased by Aβ_25–35, but not by Aβ_35–25. We next found that acute treatment with Aβ_25–35 increased Ca_V1.3 channel activities in HEK293 cells using whole-cell patch-clamp recordings. Furthermore, using GTP pulldown and co-immunoprecipitation assays in HEK293 cell lysates, we found that amyloid precursor protein interacts with β₃ subunits of Ca²⁺ channels instead of Ca_V1.2 or Ca_V1.3 α₁ subunits. These results show that Aβ_25–35 chronically or acutely upregulates Ca_V1.3 in the rat hippocampal and human kidney cells (HEK293). This suggests that Ca_V1.3 has a potential role along with Ca_V1.2 in the pathogenesis of Alzheimer’s disease.     

Mitochondrial ryanodine receptors and other mitochondrial Ca permeable channels

Ca²⁺ channels that underlie mitochondrial Ca²⁺ transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca²⁺ uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca²⁺ transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca²⁺ overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca²⁺ channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca²⁺ channels.