IP3Rs and nSOCE-Tied Together at Two Ends.

With a concentration of 55,000 mM, water is by far the most prevalent molecule in biological systems. For many years it was assumed that biological membranes are freely water permeable, so that the existence of a family of water channels would not have been predicted. However, biophysical studies in erythrocytes and kidney tubules in the 1960s through the 1980s revealed that some membranes are more water permeable than others and have certain biophysical properties, such as weakly temperature-dependent water transport and an enhanced osmotic compared with diffusional water permeability, which suggested water movement through a pore-like pathway.The molecular identity of the first water channel, aquaporin 1 (AQP1), was reported in 1992 (Preston et al., 1992). AQP1 had little similarity to other known proteins, except for the major integral protein (MIP) of lens fiber, which was subsequently renamed AQP0. Soon thereafter, additional AQP family members were identified from animal, yeast, bacterial and plant sources, with at least 13 AQPs in humans.This Cell Science at a Glance article aims to highlight the biological roles of mammalian AQPs. Although some roles of AQPs, such as their function in the concentration of urine and the secretion of gland fluid, were predictable, other roles, such as those in cell migration and neural signaling, were unexpected. Following a brief review of AQP structure, function and regulation, this article will focus on the physiological roles of AQPs, with an emphasis on current knowledge of the cellular mechanisms. In addition, aquaporinopathies – AQP-related human diseases – and possibilities for AQP-based therapeutics, will be discussed.Compared with ion channels and solute transporters, AQPs are relatively simple proteins with regard to their structure and function. High-resolution X-ray crystal structures have been determined for several mammalian AQPs. Each ~30-kDa AQP monomer is made up of six membrane-spanning helical domains (H1–H6) and two short helical segments (HB and HE) that surround cytoplasmic and extracellular vestibules, respectively. These vestibules are connected by a narrow aqueous pore of ~25 Å (1 Å=0.1 nm) in length (reviewed by Fujiyoshi et al., 2002; Walz et al., 2009) (poster panel 1). Four AQP monomers assemble to form tetramers in the plasma membrane. Tetramers of one of the AQPs, AQP4, further assemble into supramolecular square arrays – called orthogonal arrays of particles – which are maintained by inter-tetrameric N-terminal interactions involving specific residues (Crane and Verkman, 2009).Structural data on AQPs, together with mutagenesis and molecular dynamics simulations, have indicated that single-file transport occurs through a narrow pore in each monomer, where water selectivity is conferred by electrostatic and steric factors (Hub et al., 2009; Khalali-Araghi et al., 2009). Glycerol-transporting AQPs, called aquaglyceroporins, have a less-constricted pore compared with that of water-selective AQPs (diameter of 3.4 Å compared with 2.8 Å, respectively), with relatively more hydrophobic residues lining the pore.Many mammalian AQPs, including AQP1, AQP2, AQP4, AQP5 and AQP8, function primarily as bidirectional water-selective transporters. Cells expressing AQPs on their plasma membrane have an ~5- to 50-fold higher osmotic water permeability than membranes that do not (Verkman and Mitra, 2000). Water transport through single-file pores poses a biophysical limitation on the efficiency with which AQPs can transport water, so that AQPs must be present in the membrane at a high density to increase membrane water permeability substantially (Yang and Verkman, 1997). AQP-expressing cells generally contain several thousands, or more, AQPs per μm2 of membrane, as compared with ten or fewer ion channels per μm2 of membrane.A subset of the AQPs, the aquaglyceroporins AQP3, AQP7 and AQP9, transport both water and glycerol, and studies have suggested that AQP9 can also transport other small polar solutes, including amino acids, sugars and even arsenite (Tsukaguchi et al., 1998; Wu and Beitz, 2007; Carbrey et al., 2009). AQP6 has been proposed to function as an intracellular chloride channel (Yasui et al., 1999). The functions and cellular localization of AQPs 10–13 are not clear (Ishibashi, 2009). In addition to their well-established water- and glycerol-transporting functions, AQPs have also been proposed to transport other small molecules and gases, including carbon dioxide, ammonia, nitric oxide and hydrogen peroxide (Musa-Aziz et al., 2009; Miller et al., 2010; Wang and Tajkhorshid, 2010). Although some gases are theoretically small enough to pass through the aqueous pore of the AQP, compelling evidence is lacking for physiologically relevant gas transport, partly because the intrinsic lipid-mediated membrane permeability to most gases is high (Yang et al., 2000; Missner et al., 2008; Tornroth-Horsefield et al., 2010). Current evidence suggests that most, if not all, significant biological functions of the mammalian AQPs, including those described here, can be attributed to AQP-facilitated water and/or glycerol transport.Because most AQPs are constitutively expressed at the cell plasma membrane, regulation of their function occurs mainly at the level of transcription. There are numerous descriptive studies of up- and down-regulation by various AQPs in response to various stresses, such as AQP4 upregulation in brain trauma and inflammation (Aoki-Yoshino et al., 2005; Tomura et al., 2011). With the exception of AQP2, the biological significance of transcriptional regulation of AQP expression is unclear. Transcription and post-translational regulation of AQP2 expression are particularly important for the mechanism of urine concentration (Nedvetsky et al., 2009; Noda et al., 2010). Similar to the process of GLUT4 translocation to the membrane in response to insulin, targeting of AQP2 to the apical plasma membrane in the collecting duct of the kidney is controlled by the antidiuretic hormone vasopressin through a mechanism involving regulated vesicular trafficking of AQP2-containing endosomes. In addition to the wealth of information described above about AQP structure, function and regulation, there is an abundance of information about the roles of AQPs in cell and organ physiology, which will be discussed in the following sections.A major role of epithelial cells is the transport of fluid across tissue barriers. Therefore, the involvement of AQPs in epithelial fluid transport is not unexpected. Certain epithelia carry out what has been called 'active near-isosmolar' fluid transport, such as fluid absorption by kidney proximal tubule and fluid secretion by salivary gland acinar epithelium. Active transepithelial fluid transport involves the generation of an osmotic gradient by active solute transport, which drives osmotic water transport (poster panel 2). The high transepithelial water permeability conferred by AQPs, increases net fluid transport.Mice that lack AQP1, which is expressed in the kidney proximal tubule, show defective fluid absorption (Schnermann et al., 1998). Mice lacking AQP5, which is expressed in salivary and airway submucosal glands, show defective secretion of saliva (Ma et al., 1999) and airway mucus (Song and Verkman, 2001). AQP-facilitated fluid secretion has also been found in the ocular ciliary epithelium (Zhang et al., 2002; Verkman et al., 2008b), which produces aqueous fluid, and in the brain choroid plexus (Oshio et al., 2005), which produces cerebrospinal fluid (CSF). There is a caveat, however: AQPs appear to be required for active transepithelial fluid transport only when fluid transport rate, normalized to epithelial surface area, is very high, as it is in the proximal tubule and salivary gland. By contrast, in the lung alveolus, deletion of AQP5 in mice does not impair active fluid absorption, although it is expressed in alveolar epithelial cells and is responsible for ~90% of osmotically driven water transport (Ma et al., 2000a). This is because the area-normalized rate of fluid absorption by the alveolus is more than 100-fold lower than that in the proximal tubule. The basal (AQP independent) water permeability of the alveolar epithelium is sufficient to support relatively slow fluid absorption.In addition to their role in active near-isosmolar fluid absorption and secretion, AQPs also facilitate passive transepithelial water transport in response to sustained large osmotic gradients, as exemplified in the thin descending limb of Henle and the collecting duct in the kidney (poster panel 3). Osmotic water transport by the thin descending limb of Henle, where AQP1 is highly expressed, is required for the countercurrent multiplication mechanism to generate concentrated urine (Chou et al., 1999). Water transport across the collecting duct epithelium involves vasopressin-regulated AQP2 trafficking to the apical plasma membrane, as mentioned above, with constitutively high water permeability of the basolateral membrane conferred by the constitutive expression of AQP3 and AQP4 (Verkman, 2008; Noda et al., 2010).To concentrate the urine, the high water permeability of the kidney collecting duct facilitates osmotic water transport from the tubule lumen to the hypertonic renal interstitium (generated by countercurrent multiplication). Mice lacking one of the AQPs 1–4 manifest defects in urinary concentrating ability (Ma et al., 1997; Ma et al., 1998; Ma et al., 2000b; Yang et al., 2009). In humans, loss-of-function mutations in AQP2 result in the rare genetic disease nephrogenic diabetes insipidus (NDI) (Deen et al., 1994), which is characterized by polyuria and urinary hypo-osmolality. Mice lacking AQP11 manifest an interesting polycystic kidney disease phenotype (Morishita et al., 2005), although it remains unclear how loss of AQP11 produces renal cysts. AQP-facilitated water transport in various epithelia is thus crucial to a wide range of physiological processes.Water movement across barriers also occurs in the brain, but it is the astrocytes rather than epithelial cells that are involved. The AQP4 water channel is expressed in astrocytes throughout the central nervous system, particularly at brain–fluid interfaces at the blood–brain and ependymal–CSF barriers (poster panel 4). In cytotoxic (cellular) brain edema, water moves into the brain through an intact blood–brain barrier in response to osmotic driving forces. For example, the acute serum hyponatremia that occurs during water intoxication causes brain swelling by a simple osmotic mechanism. Mice lacking AQP4 show improved outcome and reduced brain water accumulation compared with that in wild-type mice in models of cytotoxic brain edema, such as water intoxication, ischemic stroke and bacterial meningitis (Manley et al., 2000; Papadopoulos and Verkman, 2005).In contrast to cytotoxic edema, vasogenic (leaky-vessel) brain edema involves water movement into the brain by a bulk-fluid flow mechanism, through a leaky blood–brain barrier, and exit from the brain through the AQP4-rich glia limitans, which lines the ventricles and the surface of the brain (poster panel 4). When these water exit routes are blocked, such as in obstructive hydrocephalus, water also moves out of the brain through microvessels at the blood–brain barrier. Mice lacking AQP4 have a worse clinical outcome and greater brain water accumulation in models of vasogenic brain edema, including intra-parenchymal fluid infusion, cortical-freeze injury, brain tumor and brain abscess (Papadopouolos et al., 2004; Bloch et al., 2005), and in a model of obstructive hydrocephalus (Bloch et al., 2006). Therefore, as a bidirectional water channel, AQP4 facilitates brain water accumulation in cytotoxic edema and the clearance of excess brain water in vasogenic edema. AQP4 appears to have a similar role in the spinal cord, reducing cytotoxic swelling and improving the clinical outcome following spinal cord compression injury (Saadoun et al., 2008), whereas increasing vasogenic swelling following spinal cord contusion injury (Kimura et al., 2010).AQPs are expressed in electrically excitable tissues in supportive cells adjacent to excitable cells (e.g. in astrocytes but not neurons in the brain, in Müller cells but not bipolar cells in the retina, in supportive cells but not hair cells in the inner ear, and in support cells but not olfactory receptor neurons in the olfactory epithelium). Electrophysiological measurements show impaired vision (Li et al., 2002), hearing (Li and Verkman, 2001) and olfaction (Lu et al., 2008) in Aqp4-null mice. In the brain, the seizure threshold is reduced and seizure duration prolonged by an AQP4 deficiency (Binder et al., 2006). Possible mechanisms for altered neuroexcitation in AQP4 deficiency include slower K+ re-uptake into astrocytes following neuroexcitation (Padmawar et al., 2005; Binder et al., 2006) and mild expansion of the extracellular space (ECS) surrounding these cells (Yao et al., 2008; Zhang and Verkman, 2010b).How AQP4 alters K+ re-uptake following neuroexcitation is the subject of ongoing investigation. It had been postulated that the interaction between AQP4 and the inwardly rectifying K+ channel Kir4.1 was responsible. However, patch-clamp analysis has indicated that AQP4 deficiency does not affect Kir4.1 K+ channel function in brain astrocytes or retinal Müller cells (Ruiz-Ederra et al., 2007). We propose that AQP4-dependent water permeability enhances K+ transport by a 'pseudo-solvent drag' mechanism (poster panel 5). Neuroexcitation involves ion buffering in the ECS, which is the small aqueous volume surrounding cells (~20% of the volume in brain). Excess K+ released by neurons during excitation is taken up and 'siphoned' largely by astrocytes. We postulate that AQP4-facilitated water transport in astrocytes in the brain is an important determinant of both water and ion movement between cells and the ECS during neuroexcitation. Re-uptake of K+ following neuroexcitation results in osmotic water influx into AQP4-expressing astrocytes and consequent ECS shrinkage, which maintains the electrochemical driving force for K+ re-uptake (by maintaining elevated K+ concentrations in the ECS). The diminished astrocyte water permeability that occurs in AQP4 deficiency would reduce ECS contraction and hence slow K+ re-uptake. This hypothesis, although unproven as yet, is attractive because it relates neuroexcitation phenotypes to the water-transporting role of AQP4.The involvement of AQPs in cell migration was discovered following the observation of impaired tumor angiogenesis in Aqp1-null mice and the characterization of endothelial cell cultures derived from wild-type and Aqp1-null mice (Saadoun et al., 2005a). Motivated by the high expression of AQP1 in tumor microvessels (Endo et al., 1999), we found that AQP1 deletion in mice reduces the growth and vascularity of implanted tumors (Saadoun et al., 2005a). Cultured aortic endothelial cells from Aqp1-null mice migrated slower than cells from wild-type mice in response to a chemotactic stimulus, and cell migration was increased when various cells not normally expressing AQPs were transfected with AQPs. Further evidence, including AQP polarization to the leading edge of migrating cells, increased lamellipodial dynamics in AQP-expressing cells and AQP-dependent cell migration in different cells types and with different AQPs, suggested a mechanism for AQP-facilitated migration (Papadopoulos et al., 2008; Loitto et al., 2009) (poster panel 6).On the basis of these results, we have proposed that actin depolymerization and ion influx increase cytoplasmic osmolality at the leading edge of a migrating cell, driving water influx through the plasma membrane (Saadoun et al., 2005a). This idea of water flow into and out of migrating cells is supported by data showing that migration can be modulated by changes in extracellular osmolality and transcellular osmotic gradients (Saadoun et al., 2005b). Our model postulates that water influx causes expansion of the adjacent plasma membrane by increased hydrostatic pressure, which is followed by actin repolymerization to stabilize the cell membrane protrusion. In support of this idea is the observation that regional hydrostatic pressure changes within cells do not equilibrate throughout the cytoplasm, on a distance scale of 10 μm and a time scale of 10 seconds, and could thus contribute to the formation of localized cell membrane protrusions (Charras et al., 2005). However, this mechanism remains unproven and other mechanisms, such as AQP-dependent changes in cell volume during migration and interaction of AQPs with other proteins, are potential alternatives.Regardless of the exact biophysical mechanism, AQP-facilitated cell migration appears to be a general phenomenon relevant not only in angiogenesis but also in tumor spread, wound healing and immune cell chemotaxis. AQP expression in tumor cells increases their ability to extravasate across blood vessels and to invade locally (Hu and Verkman, 2006), which might account for the high level of AQP expression in many tumor types and the correlation between AQP expression and tumor grade in some tumors, such as glioblastomas (Verkman et al., 2008a). Expression of AQP4 in brain astrocytes increases their migration towards a chemotaxic stimulus and increases glial scarring (Saadoun et al., 2005b; Auguste et al., 2007), and expression of AQP3 in skin and cornea facilitates wound healing (Levin et al., 2006; Hara-Chikuma and Verkman, 2008b).Although the evidence remains preliminary, AQPs might also be involved in intracellular vesicular transport processes, such as pancreatic granule exocytosis and astrocyte cytokine secretion (Cho et al., 2002; Li et al., 2011), perhaps by a mechanism involving rapid AQP-dependent changes in cell volume, which might accompany vesicle fusion.The functional significance of glycerol transport by the aquaglyceroporins, such as AQP3 in skin and AQP7 in adipocytes was, for many years, unclear. However, more recent studies have indicated roles for these channels in skin hydration and fat metabolism. AQP3-facilitated glycerol transport in skin is an important determinant of epidermal and stratum corneum hydration (Ma et al., 2002) (poster panel 7). Mice lacking AQP3, which is normally expressed in the basal layer of proliferating keratinocytes in the epidermis, manifest reduced stratum corneum hydration and skin elasticity. This is caused by reduced epidermal cell glycerol permeability, resulting in reduced glycerol content in the stratum corneum and epidermis, where it acts as a 'humectant', or water-retaining osmolyte (Hara et al., 2002). Topical or systemic administration of glycerol normalized stratum corneum glycerol content and corrected the skin-hydration defect in AQP3 deficiency (Hara and Verkman, 2003). These findings provided a rationale for the common use of glycerol in cosmetics and various skin medical formulations and generated interest in the involvement of AQP3 in skin diseases (reviewed by Hara-Chikuma and Verkman, 2008c).An unanticipated role of AQP3 in cell proliferation was observed in several AQP3-expressing tissues, including skin, colon and cornea. AQP3-deficient mice manifest impaired cutaneous and corneal wound healing (Hara-Chikuma and Verkman, 2008b; Levin and Verkman, 2006) and colonic epithelial cell regeneration (Thiagarajah et al., 2007). In addition, a remarkable tumor phenotype was found in Aqp3-null mice, which showed complete resistance to the formation of skin tumors in response to a tumor initiator-promoter protocol that produces multiple tumors in wild-type mice (Hara-Chikuma and Verkman, 2008a). Biochemical studies in epidermal cells of these AQP3-deficient mice showed impaired cellular glycerol metabolism and biosynthesis, with reduced ATP content and impaired MAPK signaling (poster panel 8). Thus, we proposed that AQP3-facilitated glycerol transport is a key determinant of cell proliferation through a mechanism involving reduced epidermal glycerol concentration in AQP3 deficiency, impaired lipid biosynthesis, reduced glycerol metabolism and ATP, impaired MAPK signaling (particularly p38 kinase) and, finally, reduced cell proliferation. Further investigation of glycerol metabolism in AQP3-expressing cells is needed to verify this proposed mechanism. In any case, the possibility of AQP3 inhibition being used to prevent or treat certain tumors is intriguing because AQP3 inhibition is predicted to reduce both migration and proliferation in tumor cells.A quite unexpected role of an aquaporin in obesity has been discovered. AQP7 is expressed in the plasma membrane of adipocytes. Aqp7-null mice manifest a remarkable progressive increase in fat mass and adipocyte hypertrophy, accumulating glycerol and triglycerides in their adipocytes, as they age (Hara-Chikuma et al., 2005; Hibuse et al., 2005). Biochemical studies suggested that this adipocyte hypertrophy is the consequence of reduced plasma membrane glycerol permeability, resulting in cellular glycerol and triacylglycerol accumulation, and glycerol kinase upregulation (poster panel 9). These results focus attention on adipocyte glycerol permeability as a novel regulator of adipocyte size and whole-body fat mass, suggesting that the modulation of adipocyte AQP7 expression and/or function can alter fat mass. AQP9 has been suggested as an important route for hepatic glycerol uptake (Carbrey et al., 2003), and AQP7 and AQP9 as key metabolic regulators in diabetes and obesity (Maeda et al., 2009). However, additional work is needed to validate these ideas.In humans, loss-of-function mutations in AQPs exist but are rare. As mentioned above, mutations in AQP2 produce non-X-linked NDI by a recessive mechanism involving defective AQP2 protein folding and retention in the endoplasmic reticulum (ER), as well as by a dominant mechanism, which results from interactions between wild-type and mutant AQP2 in the ER and/or Golgi that prevent plasma membrane targeting of wild-type AQP2 (Bichet, 2006). The incidence of NDI caused by AQP2 mutations is fewer than one in 20 million births.For other AQPs, only a handful of subjects have been identified with loss-of-function mutations. A few subjects that lack functional AQP1, which were identified by blood-group screening, are phenotypically but manifest defective urinary concentrating function when of water et al., 2001). of the of as well as a few subjects that lack functional AQP3 or AQP7 et al., 2002), and because of wide phenotype in humans, little information is about the roles of these AQPs in in (MIP) in lens by a mechanism that is to defective rather than impaired water transport et al., 2000; et al., 2008; 2009). mutations of other AQPs in between AQP and disease have so far been disease is an interesting that has It is a relatively rare of multiple that primarily the and the spinal cord, and and it has a even with A of is the of serum extracellular on AQP4 et al., 2005). are to their by to AQP4 on which and and et al., (poster panel It is not known what the of how they the or are largely in brain and in AQP4-expressing Active is being out to models of et al., 2009; et al., and to such as or that the of to AQP4.The AQP has the molecular of the first AQP two By we have a of knowledge on AQP structure, cellular expression and biological However, in and to AQP to the work is needed to cellular mechanisms for the many interesting phenotypes by AQP deficiency, such as impaired cell cell neuroexcitation and fat metabolism. phenotypes will to be discovered and will about water transport physiology, such as the impaired in AQP1 deficiency (Zhang and Verkman, and the reduced in AQP4 deficiency (Li et al., studies in mice that AQP-based for human diseases might be AQP or are predicted to reduce urine a water greater than for of such as AQP4 are predicted to reduce brain swelling in cytotoxic edema, following brain injury and and reducing in meningitis and various of AQPs in tumor cells and microvessels are predicted to reduce tumor and tumor of AQP4-facilitated glial cell migration is predicted to glial formation following brain and spinal cord injury, regeneration and improving Topical of AQP3 in the skin might reduce skin and increasing adipocyte glycerol permeability, perhaps by of AQP7 might a novel for There is work the possibility of AQP5 for salivary gland et al., 2009). of the of will the of which is the subject of ongoing in numerous and Thus, many to AQP from the into clinical

Intracellular free Ca 2+ in dissociated cells of the chick pineal gland: regulation by membrane depolarization, second messengers and neuromodulators, and evidence for release of intracellular Ca 2+ stores

The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.

The present study was aimed at localization of plasma membrane (PMCA) and intracellular (SERCA) Ca2+ pumps and characterizing their role in initiation and propagation of Ca2+ waves. Specific and polarized expression of Ca2+ pumps was observed in all epithelial cells examined. Immunolocalization revealed expression of PMCA in both the basolateral and luminal membranes of all cell types. SERCA2a appeared to be expressed in the luminal pole, whereas SERCA2b was expressed in the basal pole and the nuclear envelope of pancreatic acini. Interestingly, SERCA2b was found in the luminal pole of submandibular salivary gland acinar and duct cells. These cells expressed SERCA3 in the basal pole. To examine the significance of the polarized expression of SERCA and perhaps PMCA pumps in secretory cells, we compared the effect of inhibition of SERCA pumps with thapsigargine and partial Ca2+ release with ionomycin on Ca2+ release evoked by agonists and Ca2+ uptake induced by antagonists. Despite their polarized expression, Ca2+ uptake by SERCA pumps and Ca2+ efflux by PMCA resulted in uniform reduction in [Ca2+]i. Surprisingly, inhibition of the SERCA pumps, but not Ca2+ release by ionomycin, eliminated the distinct initiation sites and propagated Ca2+ waves, leading to a uniform increase in [Ca2+]i. In addition, inhibition of SERCA pumps reduced the rate of Ca2+ release from internal stores. The implication of these findings to rates of Ca2+ diffusion in the cytosol, compartmentalization of Ca2+ signaling complexes, and mechanism of Ca2+ wave propagation are discussed.

Apoptosis and survival pathways are intimately connected signal transduction cascades that determine the fate of cells suffering from insults such as DNA damage, oxidative stress, intracellular Ca2+ overload, mild excitotoxicity, accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), and many others, or induced to autoelimination by extracellular signals. The apoptotic pathways, respectively, intrinsic or extrinsic, are executed through molecular signals travelling inside the cells. If the initial studies considered the nucleus, and specifically DNA, as both primary sensor and executioner of apoptosis, active roles of other intracellular organelles have been pointed out in the last two decades, identifying plasma membrane as the initiator of the extrinsic pathway and mitochondria as the pivot of the intrinsic pathway. In apoptosing cells, the outer membrane of mitochondria is permeabilized by the proapoptotic members of the Bcl-2 family, promoting the release of proapoptotic factors such as the primary and secondary activators of caspases (cytochrome c and SMAC/diablo, resp.). Stress signals, typically inducing the intrinsic apoptotic pathway, must be perceived by the damaged cells and translated into signals that promote mitochondria permeabilization, after which caspases are activated. This implies that other organelles participate in apoptotic signaling upstream to mitochondria involvement, acting as sensors of damage or signal transducers. Recently, a key role of the endoplasmic reticulum (ER) is emerging, consisting of the ability of amplifying or dampening the apoptotic signal prior to mitochondria involvement, through the establishment of active Ca2+ fluxes that control the physical and functional interaction between mitochondria and ER. Also lysosomes participate to the intrinsic apoptotic signaling, and their dialogs with mitochondria have been described. Other important examples of organelles cross-talk in apoptosis include nucleus-mitochondria exchange of proteins such as p53, which is main controller of the cell response to DNA damage. To date, the role of organelles other than mitochondria in perceiving and signaling cell damage, and the interrelation of the organelles during apoptotic signaling, are still poorly explored matters. In this special issue dedicated to Organelle Cross-talk in Apoptotic and Survival Pathways, original research papers and review articles are collected with the aim of giving an impulse to this promising field of research on apoptosis and stimulating discussion between interdisciplinary readers. The paper written by Y.-B. Ouyang and R. G. Giffard describes ER-mitochondria cross-talk during cerebral ischemia. The authors precisely describe the chaperone machineries at both the ER and mitochondrion. Moreover, they contribute the new view that the chaperones organise not only the regulation of Ca2+ signaling between these two organelles and control bioenergetics, cell survival, but also cell death decision. In the paper entitled “Mitochondrial dynamics: functional link with apoptosis”, written by H. Otera and K. Mihara, the readers are introduced to “intrinsic” apoptotic pathway in the context of regulation and physiologic significance of mitochondrial fusion and fission. The authors of this review underline also that the ER-mitochondria contact is involved in the regulation of mitochondrial energy, lipid metabolism, calcium signaling, and even in mitochondrial fission. An important issue arising is the cross-talk of apoptosis with autophagy, a stress response pathway controlled by key intracellular regulators such as m-TOR, induced by insults or conditions ranging from nutrient depletion, protein malfolding, or cell ageing. The paper written by H. Rikiishi focusing on the recent advances regarding autophagy in cancer and the techniques currently available to manipulate autophagy. The author of this paper provides a complete overview of the literature on molecular mechanism of autophagy and its cross-talk with apoptosis. Additionally, the author indicates the technical problems in the study of autophagy and suggest that new methods need to be developed to ensure progress in this area of investigation. Moreover, they highlight that the multifaceted nature of autophagy and its diverse cross-talk with other biological processes must be carefully considered when the autophagic system is targeted for anticancer benefits. The paper entitled “Effects of cisplatin in neuroblastoma rat cells: damage to cellular organelles” written by G. Santin et al. provides the practical analysis of organelle cross-talk during cell death. The authors indicate that B50 neuroblastoma cells treated with cisplatin undergo apoptosis through organelles injury, and not only as a consequent of DNA damage, but also that cisplatin does not induce any autophagic process as alternative response to the treatment. In conclusion, the studies collected in this issue effectively contribute to the general problem of organelles as target of cell damage versus transducer of the apoptotic signal. Lina Ghibelli Alina Grzanka

Truly endothelial progenitor cells (EPCs) can be mobilized from bone marrow to support the vascular network of growing tumors, thereby sustaining the metastatic switch. Endothelial colony forming cells (ECFCs) are the only EPC subtype belonging to the endothelial phenotype and capable of incorporating within neovessels. The intracellular Ca(2+) machinery plays a key role in ECFC activation and is remodeled in renal cellular carcinoma-derived ECFCs (RCC-ECFCs). Particularly, RCC-ECFCs seems to undergo a drop in endoplasmic reticulum (ER) Ca(2+) concentration ([Ca(2+) ]ER ). This feature is remarkable when considering that inositol-1,4,5-trisphosphate (InsP3 )-dependent ER-to-mitochondria Ca(2+) transfer regulates the intrinsic apoptosis pathway. Herein, we sought to assess whether: (1) the [Ca(2+) ]ER and the InsP3 -induced ER-mitochondria Ca(2+) shuttle are reduced in RCC-ECFCs; and (2) the dysregulation of ER Ca(2+) handling leads to apoptosis resistance in tumor-derived cells. RCC-ECFCs displayed a reduction both in [Ca(2+) ]ER and in the InsP3 -dependent mitochondrial Ca(2+) uptake, while they expressed normal levels of Bcl-2 and Bak. The decrease in [Ca(2+) ]ER was associated to a remarkable ER expansion in RCC-ECFCs, which is a hallmark of ER stress, and did not depend on the remodeling of the Ca(2+) -transporting and the ER Ca(2+) -storing systems. As expected, RCC-ECFCs were less sensitive to rapamycin- and thapsigargin-induced apoptosis; however, buffering intracellular Ca(2+) levels with BAPTA dampened apoptosis in both cell types. Finally, store-operated Ca(2+) entry was seemingly uncoupled from the apoptotic machinery in RCC-ECFCs. Thus, the chronic underfilling of the ER Ca(2+) pool could confer a survival advantage to RCC-ECFCs and underpin RCC resistance to pharmacological treatment. J. Cell. Biochem. 117: 2260-2271, 2016. © 2016 Wiley Periodicals, Inc.

Ca2+-specific minielectrodes were used to monitor changes in the ambient free Ca2+ concentration [( Ca2+]a) maintained by the intracellular organelles of permeabilized GH3 cells. Mitochondria maintained a [Ca2+]a steady state of around 500 nM and displayed a very high capacity for Ca2+ uptake. A nonmitochondrial pool, tentatively identified as the endoplasmic reticulum (ER), displayed higher affinity for Ca2+ by maintaining a steady state of approximately 170 nM. The capacity of this pool was around 10 nmol/mg cell protein. Inositol 1,4,5-trisphosphate (InsP3) released Ca2+ specifically from the ER, with an EC50 of approximately 2 microM, and gave maximal release of around 4 nmol Ca2+/mg of cell protein. Repeated InsR3 additions under conditions allowing for functional mitochondrial transport resulted in successively attenuated peaks, leading eventually to the depletion of the InsP3 sensitive portion of the ER. However, Ca2+ could still be released from the total ER pool with the ATPase inhibitor, vanadate. This InsP3-insensitive store did not reaccumulate InsP3 releasable Ca2+ nor could it directly refill the sensitive pool. However, the attenuation of the InsP3 responses could be overcome by repleting the sensitive pool with exogenous Ca2+ or by inhibiting Ca2+ uptake into the mitochondria. The results suggest: 1) the ER is the major intracellular organelle buffering Ca2+ in nonstimulated GH3 cells; 2) InsP3 releases Ca2+ from only a portion of the ER; 3) the InsP3-sensitive and -insensitive ER pools are functionally distinct; 4) InsP3 addition results in a transfer of Ca2+ from the ER to the mitochondria.

The activity of BiP, the major chaperone of the endoplasmic reticulum (ER) lumen, is known to be Ca2+-regulated; however, the participation of this protein in the ER storage of the cation has not yet been investigated. Here such a role is demonstrated in human epithelial (HeLa) cells transiently transfected with the hamster BiP cDNA and incubated in Ca2+-free medium, as revealed by two different techniques. In the first, co-transfected aequorin was employed as a probe for assaying either the cytosolic of the mitochondrial free Ca2+ concentration. By this approach higher Ca2+ release responses were revealed in BiP-transfected cells by experiments in which extensive store depletion was induced either by repetitive stimulation with inositol 1,4,5-trisphosphate-generating agonists or by treatment with the Ca2+ ionophore, A23187. In the second technique the cells were loaded at the equilibrium with 45Ca, and the release of the tracer observed upon treatment with thapsigargin, a blocker of the ER Ca2+ ATPases, was larger in BiP-transfected than in control cells. The latter results were obtained also when BiP was overexpressed not via transfection but as a response to ER stress by tunicamycin. These results are sustained by increases of the ER Ca2+ storage capacity rather than by artifacts or indirect readjustments induced in the cells by the overexpression of the chaperone since (a) the exogenous and endogenous BiP were both confined to the ER, (b) the expression levels of other proteins active in the ER Ca2+ storage were not changed, and (c) effects similar to those of wild type BiP were obtained with a deletion mutant devoid of chaperone activity. The specificity of the results was confirmed by parallel 45Ca experiments carried out in HeLa cells transfected with two other Ca2+-binding proteins, calreticulin and CaBP2(ERp72), only the first of which induced increases of Ca2+ capacity. We conclude that BiP has a dual function, in addition to its chaperone role it is a bona fide ER lumenal Ca2+ storage protein contributing, under resting cell conditions, to around 25% of the store, with a stoichiometry of 1-2 moles of calcium/mole of BiP.

Numerous cellular processes and signaling mechanisms have been identified that contribute to Alzheimer's disease (AD) pathology; however, a comprehensive or unifying pathway that binds together the major disease features remains elusive. As an upstream mechanism, altered calcium (Ca2+) signaling is a common driving force for many pathophysiological events that emerge during normal aging and development of neurodegenerative disease. Recent Advances: Over the previous three decades, accumulated evidence has validated the concept that intracellular Ca2+ dysregulation is centrally involved in AD pathogenesis, including the aggregation of pathogenic β-amyloid (Aβ) and phospho-τ species, synapse loss and dysfunction, cognitive impairment, and neurotoxicity. Although neuronal Ca2+ signaling within the cytosol and endoplasmic reticulum (ER) has been well studied, other critical central nervous system-resident cell types affected by aberrant Ca2+ signaling, such as astrocytes and microglia, have not been considered as thoroughly. In addition, certain intracellular Ca2+-harboring organelles have been well studied, such as the ER and mitochondria; however other critical Ca2+-regulated organelles, such as lysosomes and autophagosomes, have only more recently been investigated. In this review, we examine Ca2+ dysregulation in microglia and astrocytes, as well as key intracellular organelles important for cellular maintenance and protein handling. Ca2+ dysregulation within these non-neuronal cells and organelles is hypothesized to disrupt the effective clearance of misaggregated proteins and cellular signaling pathways needed for memory networks. Overall, we aim to explore how these disrupted mechanisms could be involved in AD pathology and consider their role as potential therapeutic targets. Antioxid. Redox Signal. 29, 1158-1175.

The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca2+ ion channel in the plasma membrane. Ca2+ store depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners. Graphic abstractLegend: Upon intracellular Ca2+ store depletion of the endoplasmic reticulum (ER), Ca2+ dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca2+ influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca2+-selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca2+ store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca2+ store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right).

IP3 Receptors

Previous studies have demonstrated that myo-inositol 1,4,5-trisphosphate (IP3) mobilizes Ca2+ from the endoplasmic reticulum (ER) of digitonin-permeabilized islets and that an increase in intracellular free Ca2+ stimulates insulin release. Furthermore, glucose stimulates arachidonic acid metabolism in islets. In digitonin-permeabilized islets, exogenous arachidonic acid at concentrations between 1.25 to 10 microM elicited significant Ca2+ release from the ER at a free Ca2+ concentration of 0.1 microM. Arachidonic acid-induced Ca2+ release was not due to the metabolites of arachidonic acid. Arachidonic acid induced a rapid release of Ca2+ within 2 min. Comparison of arachidonic acid-induced Ca2+ release with IP3-induced Ca2+ release revealed a similar molar potency of arachidonic acid and IP3. The combination of both arachidonic acid and IP3 resulted in a greater effect on Ca2+ mobilization from the ER than either compound alone. The mass of endogenous arachidonic acid released by islets incubated with 28 mM glucose was measured by mass spectrometric methods and was found to be sufficient to achieve arachidonic acid concentrations equal to or exceeding those required to induce release of Ca2+ sequestered in the ER. These observations indicate that glucose-induced arachidonic acid release could participate in glucose-induced Ca2+ mobilization and insulin secretion by pancreatic islets, possibly in cooperation with IP3.

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

The proliferation and motility of vascular endothelial cells (ECs) are critical steps in angiogenesis and are strictly controlled by different extracellular signals. Among mitogens, peptides binding to tyrosine kinase receptors (i.e. VEGFs and FGFs) are well known and are released by several cell types, including ECs and tumor cells. The binding of mitogens to their specific receptors triggers intracellular signaling cascades, involving a number of messengers working in a sort of network. In particular, in this review we describe the increases of calcium levels in the cytosol, a universal, evolutionary conserved and highly versatile signal involved in the regulation of EC's proliferation and motility. Most mitogens, including angiogenic factors, generate cytosolic calcium rises through two mechanisms: entry from extracellular medium, through the opening of calcium permeable channels in the plasma membrane, or release from intracellular organelles (mainly endoplasmic reticulum, ER). Calcium entry, the main topic of this review, can be dependent on previously IP(3)-activated emptying of calcium stores (store-dependent or capacitative calcium entry--CCE), or independent on it (non capacitative calcium entry, NCCE). The intracellular pathways underlying calcium entry are under investigation and recently arachidonic acid (AA) and nitric oxide (NO) metabolism have been suggested to play a key role, at least in some cell types. Even if some calcium entry blockers are under clinical trial with encouraging results, a better knowledge about the molecular nature of calcium channels and their intracellular regulation, together with a more detailed description of spatiotemporal dynamics of intracellular calcium events, could lead to new and more specific strategies in therapeutical approach to cancer progression and angiogenesis.

It has been suggested that store-operated Ca(2+) entry (SOC) facilitates catecholamine secretion and synthesis in bovine adrenal medullary (AM) cells. However, there has been no experimental result clearly showing that cation channel activity is enhanced by store Ca(2+) depletion. Thus the present experiments were undertaken to address the issue of whether rat AM cells have SOC channels. Inhibition of the sarco(endo)plasmic reticulum Ca(2+) (SERCA) pump resulted in a sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat AM cells. This increase was completely suppressed by 2 mM Ni(2+) but not by 100 muM D600. A bath application of Ni(2+), but not D600, produced an outward current at -60 mV in rat AM cells, whereas exposure to a SERCA pump inhibitor did not affect either the whole cell current level or the Ni(2+)-induced outward current. The refilling of intracellular store sites was suppressed by the addition of Ni(2+) to the perfusate. RT-PCR revealed that transcripts for transient receptor potential channels 1 (TRPC1) and 5 (TRPC5) were present in rat adrenal medullas. Immunocytochemistry showed that TRPC1 channels, which have been implicated in SOC in certain types of cells, were mainly localized in the endoplasmic reticulum (ER) and not in the plasma membrane, and that STIM1, a Ca(2+) sensor in the ER, was not expressed in rat AM cells. On the basis of these results, we conclude that rat AM cells lack the SOC mechanism.