RESUMO
The endomembrane system consists of organellar membranes in the biosynthetic pathway [endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles] as well as those in the degradative pathway (early endosomes, macropinosomes, phagosomes, autophagosomes, late endosomes, and lysosomes). These endomembrane organelles/vesicles work together to synthesize, modify, package, transport, and degrade proteins, carbohydrates, and lipids, regulating the balance between cellular anabolism and catabolism. Large ion concentration gradients exist across endomembranes: Ca2+ gradients for most endomembrane organelles and H+ gradients for the acidic compartments. Ion (Na+, K+, H+, Ca2+, and Cl-) channels on the organellar membranes control ion flux in response to cellular cues, allowing rapid informational exchange between the cytosol and organelle lumen. Recent advances in organelle proteomics, organellar electrophysiology, and luminal and juxtaorganellar ion imaging have led to molecular identification and functional characterization of about two dozen endomembrane ion channels. For example, whereas IP3R1-3 channels mediate Ca2+ release from the ER in response to neurotransmitter and hormone stimulation, TRPML1-3 and TMEM175 channels mediate lysosomal Ca2+ and H+ release, respectively, in response to nutritional and trafficking cues. This review aims to summarize the current understanding of these endomembrane channels, with a focus on their subcellular localizations, ion permeation properties, gating mechanisms, cell biological functions, and disease relevance.
Assuntos
Canais Iônicos , Humanos , Animais , Canais Iônicos/metabolismo , Membranas Intracelulares/metabolismo , Organelas/metabolismo , Organelas/fisiologiaRESUMO
Among the infectious diseases caused by pathogenic microorganisms such as bacteria, viruses, parasites, or fungi, the most prevalent ones today are malaria, tuberculosis, influenza, HIV/AIDS, Ebola, dengue fever, and methicillin-resistant Staphylococcus aureus (MRSA) infection, and most recently Covid-19 (SARS-CoV2). Others with a rather devastating history and high fatality rates such as plague, cholera, or typhus seem less threatening today but have not been eradicated, and with a declining efficacy of current antibiotics they ought to be watched carefully. Another emerging issue in this context is health-care associated infection. About 100,000 hospitalized patients in the USA ( www.cdc.gov ) and 33,000 in Europe ( https://www.ecdc.europa.eu ) die each year as a direct consequence of an infection caused by bacteria resistant to antibiotics. Among viral infections, influenza is responsible for about 3-5 million cases of severe illness, and about 250,000 to 500,000 deaths annually ( www.who.int ). About 37 million people are currently living with HIV infection and about one million die from it each year. Coronaviruses such as MERS-CoV, SARS-CoV, but in particular the recent outbreak of Covid-19 (caused by SARS-CoV2) have resulted in large numbers of infections worldwide with an estimated several hundred thousand deaths (anticipated fatality rate: <5%). With a comparatively low mortality rate dengue virus causes between 50 and 100 million infections every year, leading to 50,000 deaths. In contrast, Ebola virus is the causative agent for one of the deadliest viral diseases. The Ebola outbreak in West Africa in 2014 is considered the largest outbreak in history with more than 11,000 deaths. Many of the deadliest pathogens such as Ebola virus, influenza virus, mycobacterium tuberculosis, dengue virus, and cholera exploit the endo-lysosomal trafficking system of host cells for penetration into the cytosol and replication. Defects in endo-lysosomal maturation, trafficking, fusion, or pH homeostasis can efficiently reduce the cytotoxicity caused by these pathogens. Most of these functions critically depend on endo-lysosomal membrane proteins such as transporters and ion channels. In particular, cation channels such as the mucolipins (TRPMLs) or the two-pore channels (TPCs) are involved in all of these aspects of endo-lysosomal integrity. In this review we will discuss the correlations between pathogen toxicity and endo-lysosomal cation channel function, and their potential as drug targets for infectious disease therapy.
Assuntos
COVID-19 , Cólera , Ebolavirus , Infecções por HIV , Doença pelo Vírus Ebola , Influenza Humana , Staphylococcus aureus Resistente à Meticilina , Humanos , COVID-19/metabolismo , Doença pelo Vírus Ebola/metabolismo , Influenza Humana/metabolismo , Cólera/metabolismo , Infecções por HIV/metabolismo , RNA Viral/metabolismo , SARS-CoV-2 , Lisossomos/metabolismo , Cátions/metabolismoRESUMO
BACKGROUND INFORMATION: Two pore channels (TPCs) are voltage-gated ion channel superfamily members that release Ca2+ from acidic intracellular stores and are ubiquitously present in both animals and plants. Starvation initiates multicellular development in Dictyostelium discoideum. Increased intracellular calcium levels bias Dictyostelium cells towards the stalk pathway and thus we decided to analyze the role of TPC2 in development, differentiation, and autophagy. RESULTS: We showed TPC2 protein localizes in lysosome-like acidic vesicles and the in situ data showed stalk cell biasness. Deletion of tpc2 showed defective and delayed development with formation of multi-tipped structures attached to a common base, while tpc2OE cells showed faster development with numerous small-sized aggregates and wiry fruiting bodies. The tpc2OE cells showed higher intracellular cAMP levels as compared to the tpc2- cells while pinocytosis was found to be higher in the tpc2- cells. Also, TPC2 regulates cell-substrate adhesion and cellular morphology. Under nutrient starvation, deletion of tpc2 reduced autophagic flux as compared to Ax2. During chimera formation, tpc2- cells showed a bias towards the prestalk/stalk region while tpc2OE cells showed a bias towards the prespore/spore region. tpc2 deficient strain exhibits aberrant cell-type patterning and loss of distinct boundary between the prestalk/prespore regions. CONCLUSION: TPC2 is required for effective development and differentiation in Dictyostelium and supports autophagic cell death and cell-type patterning. SIGNIFICANCE: Decreased calcium due to deletion of tpc2 inhibit autophagic flux.
Assuntos
Autofagia , Dictyostelium , Proteínas de Protozoários , Dictyostelium/genética , Dictyostelium/metabolismo , Dictyostelium/citologia , Dictyostelium/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Deleção de Genes , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Cálcio/metabolismo , Diferenciação CelularRESUMO
Two-pore channels and TRP mucolipins are ubiquitous endo-lysosomal cation channels of pathophysiological relevance. Both are Ca2+-permeable and regulated by phosphoinositides, principally PI(3,5)P2. Accumulating evidence has uncovered synergistic channel activation by PI(3,5)P2 and endogenous metabolites such as the Ca2+ mobilizing messenger NAADP, synthetic agonists including approved drugs and physical cues such as voltage and osmotic pressure. Here, we provide an overview of this coordination.
Assuntos
Canais de Cálcio , Canais de Potencial de Receptor Transitório , Canais de Cálcio/metabolismo , Canais de Dois Poros , Cálcio/metabolismo , Lisossomos/metabolismo , NADP/metabolismo , Pressão Osmótica , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
The ion channel two-pore channel 2 (TPC2), localised on the membranes of acidic organelles such as endo-lysosomes and melanosomes, has been shown to play a role in pathologies including cancer, and it is differently expressed in primary versus metastatic melanoma cells. Whether TPC2 plays a pro- or anti-oncogenic role in different tumour conditions is a relevant open question which we have explored in melanoma at different stages of tumour progression. The behaviour of primary melanoma cell line B16F0 and its metastatic subline B16F10 were compared in response to TPC2 modulation by silencing (by small interfering RNA), knock-out (by CRISPR/Cas9) and overexpression (by mCherry-TPC2 transfected plasmid). TPC2 silencing increased cell migration, epithelial-to-mesenchymal transition and autophagy in the metastatic samples, but abated them in the silenced primary ones. Interestingly, while TPC2 inactivation failed to affect markers of proliferation in both samples, it strongly enhanced the migratory behaviour of the metastatic cells, again suggesting that in the more aggressive phenotype TPC2 plays a specific antimetastatic role. In line with this, overexpression of TPC2 in B16F10 cells resulted in phenotype rescue, that is, a decrease in migratory ability, thus collectively resuming traits of the B16F0 primary cell line. Our research shows a novel role of TPC2 in melanoma cells that is intriguingly different in initial versus late stages of cancer progression.
Assuntos
Melanoma , Humanos , Melanoma/metabolismo , Canais de Dois Poros , Lisossomos/metabolismo , Linhagem Celular , Autofagia/fisiologia , Cálcio/metabolismoRESUMO
Lysosomes, the degradation center of the cell, are filled with acidic hydrolases. Lysosomes generate nutrient-sensitive signals to regulate the import of H+, hydrolases, and endocytic and autophagic cargos, as well as the export of their degradation products (catabolites). In response to environmental and cellular signals, lysosomes change their positioning, number, morphology, size, composition, and activity within minutes to hours to meet the changing cellular needs. Ion channels in the lysosome are essential transducers that mediate signal-initiated Ca2+/Fe2+/Zn2+ release and H+/Na+/K+-dependent changes of membrane potential across the perimeter membrane. Dysregulation of lysosomal ion flux impairs lysosome movement, membrane trafficking, nutrient sensing, membrane repair, organelle membrane contact, and lysosome biogenesis and adaptation. Hence, activation and inhibition of lysosomal channels by synthetic modulators may tune lysosome function to maintain cellular health and promote cellular clearance in lysosome storage disorders.
Assuntos
Canais Iônicos/metabolismo , Lisossomos/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Humanos , Canais Iônicos/química , Lisossomos/química , Modelos Moleculares , Tamanho da PartículaRESUMO
Lysosomal function and organellar Ca2+ homeostasis become dysfunctional in Stroke causing disturbances in autophagy, the major process for the degradation of abnormal protein aggregates and dysfunctional organelles. However, the role of autophagy in Stroke is controversial since excessive or prolonged autophagy activation exacerbates ischemic brain injury. Of note, glutamate evokes NAADP-dependent Ca2+ release via lysosomal TPC2 channels thus controlling basal autophagy. Considering the massive release of excitotoxins in Stroke, autophagic flux becomes uncontrolled with abnormal formation of autophagosomes causing, in turn, disruption of excitotoxins clearance and neurodegeneration. Here, a fine regulation of autophagy via a proper pharmacological modulation of lysosomal TPC2 channel has been tested in preclinical Stroke models. Primary cortical neurons were subjected to oxygen and glucose deprivation+reoxygenation to reproduce in vitro brain ischemia. Focal brain ischemia was induced in rats by transient middle cerebral artery occlusion (tMCAO). Under these conditions, TPC2 protein expression as well as autophagy and endoplasmic reticulum (ER) stress markers were studied by Western blotting, while TPC2 localization and activity were measured by immunocytochemistry and single-cell video-imaging, respectively. TPC2 protein expression and immunosignal were highly modulated in primary cortical neurons exposed to extreme hypoxic conditions causing dysfunction in organellar Ca2+ homeostasis, ER stress and autophagy-induced cell death. TPC2 knocking down and pharmacological inhibition by Ned-19 during hypoxia induced neuroprotection. The effect of Ned-19 was reversed by the permeable form of TPC2 endogenous agonist, NAADP-AM. Of note, Ned-19 prevented ER stress, as measured by GRP78 (78 kDa glucose-regulated protein) protein reduction and caspase 9 downregulation. In this way Ned-19 restored organellar Ca2+ level. Interestingly, Ned-19 reduced the infarct volume and neurological deficits in rats subjected to tMCAO and prevented hypoxia-induced cell death by blocking autophagic flux. Collectively, the pharmacological inhibition of TPC2 lysosomal channel by Ned-19 protects from focal ischemia by hampering a hyperfunctional autophagy.
Assuntos
Isquemia Encefálica , Acidente Vascular Cerebral , Animais , Ratos , Autofagia , Isquemia Encefálica/metabolismo , Chaperona BiP do Retículo Endoplasmático , Hipóxia/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Lisossomos/metabolismo , Neuroproteção , Neurotoxinas , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismoRESUMO
Two-pore channels (TPCs) are novel intracellular cation channels, which play a key role in numerous (patho-)physiological and immunological processes. In this chapter, we focus on their function in immune cells and immune reactions. Therefore, we first give an overview of the cellular immune response and the partaking immune cells. Second, we concentrate on ion channels which in the past have been shown to play an important role in the regulation of immune cells. The main focus is then directed to TPCs, which are primarily located in the membranes of acidic organelles, such as lysosomes or endolysosomes but also certain other vesicles. They regulate Ca2+ homeostasis and thus Ca2+ signaling in immune cells. Due to this important functional role, TPCs are enjoying increasing attention within the field of immunology in the last few decades but are also becoming more pertinent as pharmacological targets for the treatment of pro-inflammatory diseases such as allergic hypersensitivity. However, to uncover the precise molecular mechanism of TPCs in immune cell responses, further molecular, genetic, and ultrastructural investigations on TPCs are necessary, which then may pave the way to develop novel therapeutic strategies to treat diseases such as anaphylaxis more specifically.
Assuntos
Canais de Cálcio , Lisossomos , Humanos , Canais de Cálcio/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Sistema Imunitário/metabolismo , Endossomos/metabolismo , Cálcio/metabolismo , Sinalização do CálcioRESUMO
Two-pore channels, TPC1 and TPC2, are Ca2+- and Na+-permeable cation channels expressed on the membranes of endosomes and lysosomes in nearly all mammalian cells. These channels have been implicated in Ca2+ signaling initiated from the endolysosomes, vesicular trafficking, cellular metabolism, macropinocytosis, and viral infection. Although TPCs have been shown to mediate Ca2+ release from acidic organelles in response to NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca2+ mobilizing messenger, questions remain whether NAADP is a direct ligand of these channels. In whole-endolysosomal patch clamp recordings, it has been difficult to detect NAADP-evoked currents in vacuoles that expressed TPC1 or TPC2, while PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate) activated a highly Na+-selective current under the same experimental configuration. In this chapter, we summarize recent progress in this area and provide our observations on NAADP-elicited TPC2 currents from enlarged endolysosomes as well as their possible relationships with the currents evoked by PI(3,5)P2. We propose that TPCs are channels dually regulated by PI(3,5)P2 and NAADP in an interdependent manner and the two endogenous ligands may have both distinguished and cooperative roles.
Assuntos
Canais de Cálcio , Transdução de Sinais , Animais , Humanos , Canais de Cálcio/metabolismo , NADP/metabolismo , Lisossomos/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Mamíferos/metabolismoRESUMO
Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.
Assuntos
Autofagia/efeitos dos fármacos , Melanoma Experimental/patologia , Melanossomas/patologia , Pigmentação da Pele/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Linhagem Celular Tumoral , Melaninas/biossíntese , Melanossomas/efeitos dos fármacos , Camundongos , Triterpenos/química , alfa-MSH/farmacologia , Ácido UrsólicoRESUMO
Two-pore channels (TPCs) are endolysosomal cation channels. Two members exist in humans, TPC1 and TPC2. Functional roles associated with the ubiquitously expressed TPCs include VEGF-induced neoangiogenesis, LDL-cholesterol trafficking and degradation, physical endurance under fasting conditions, autophagy regulation, the acrosome reaction in sperm, cancer cell migration, and intracellular trafficking of pathogens such as Ebola virus or bacterial toxins (e.g., cholera toxin). In a genome-wide association study for variants associated with human pigmentation characteristics two coding variants of TPC2, rs35264875 (encoding M484L) and rs3829241 (encoding G734E), have been found to be associated with a shift from brown to blond hair color. In two recent follow-up studies a role for TPC2 in pigmentation has been further confirmed. However, these human polymorphic variants have not been functionally characterized until now. The development of endolysosomal patch-clamp techniques has made it possible to investigate directly ion channel activities and characteristics in isolated endolysosomal organelles. We applied this technique here to scrutinize channel characteristics of the polymorphic TPC2 variants in direct comparison with WT. We found that both polymorphisms lead to a gain of channel function by independent mechanisms. We next conducted a clinical study with more than 100 blond- and brown/black-haired individuals. We performed a genotype/phenotype analysis and subsequently isolated fibroblasts from WT and polymorphic variant carriers for endolysosomal patch-clamp experimentation to confirm key in vitro findings.
Assuntos
Canais de Cálcio/genética , Cabelo/química , Pigmentação/genética , Polimorfismo Genético , Canais de Cálcio/fisiologia , Estudo de Associação Genômica Ampla , Células HEK293 , Cabelo/metabolismo , Humanos , Técnicas de Patch-Clamp , FenótipoRESUMO
Transient receptor potential (TRP) or transient receptor potential channels are a highly diverse family of mostly non-selective cation channels. In the mammalian genome, 28 members can be identified, most of them being expressed predominantly in the plasma membrane with the exception of the mucolipins or TRPMLs which are expressed in the endo-lysosomal system. In mammalian organisms, TRPMLs have been associated with a number of critical endo-lysosomal functions such as autophagy, endo-lysosomal fusion/fission and trafficking, lysosomal exocytosis, pH regulation, or lysosomal motility and positioning. The related non-selective two-pore cation channels (TPCs), likewise expressed in endosomes and lysosomes, have also been found to be associated with endo-lysosomal trafficking, autophagy, pH regulation, or lysosomal exocytosis, raising the question why these two channel families have evolved independently. We followed TRP/TRPML channels and TPCs through evolution and describe here in which species TRP/TRPMLs and/or TPCs are found, which functions they have in different species, and how this compares to the functions of mammalian orthologs.
Assuntos
Canais de Cálcio/fisiologia , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Evolução Molecular , Proteínas Fúngicas/fisiologia , Humanos , Proteínas de Plantas/fisiologia , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
During the development of the early spinal circuitry in zebrafish, spontaneous Ca2+ transients in the primary motor neurons (PMNs) are reported to transform from being slow and uncorrelated, to being rapid, synchronized and patterned. In this study, we demonstrated that in intact zebrafish, Ca2+ release via two-pore channel type 2 (TPC2) from acidic stores/endolysosomes is required for the establishment of synchronized activity in the PMNs. Using the SAIGFF213A;UAS:GCaMP7a double-transgenic zebrafish line, Ca2+ transients were visualized in the caudal PMNs (CaPs). TPC2 inhibition via molecular, genetic or pharmacological means attenuated the CaP Ca2+ transients, and decreased the normal ipsilateral correlation and contralateral anti-correlation, indicating a disruption in normal spinal circuitry maturation. Furthermore, treatment with MS-222 resulted in a complete (but reversible) inhibition of the CaP Ca2+ transients, as well as a significant decrease in the concentration of the Ca2+ mobilizing messenger, nicotinic acid adenine diphosphate (NAADP) in whole embryo extract. Together, our new data suggest a novel function for NAADP/TPC2-mediated Ca2+ signaling in the development, coordination, and maturation of the spinal network in zebrafish embryos.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Neurônios Motores/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Técnicas de Cultura de Células , Imuno-Histoquímica , NADP/análogos & derivados , NADP/metabolismo , Peixe-Zebra/metabolismoRESUMO
Autophagy is an evolutionarily conserved lysosomal degradation process, and is involved in various cellular processes. Here we studied the role of two pore channel 2 (TPC2), a lysosomal non-selective Na+/Ca2+ channel, in autophagy progression. We found that TPC overexpression in 4T1 mouse breast cancer cell line or in HeLa human cervical cancer cell line inhibited the fusion between autophagosome and lysosome, resulting in the accumulation of autophagosomes accompanied with increased lysosomal pH and TFEB nuclear localization. Interestingly, we also found that extracellular vesicle (EV) secretion was markedly decreased in TPC2 overexpressing cells but was induced in TPC2 knockdown cells. In addition, migration of TPC2 knockdown cells, not TPC2 overexpressing cells, was inhibited. Taken together, these results support a role of TPC2 in autophagy progression and EV trafficking in cancer cells.
Assuntos
Canais de Cálcio/metabolismo , Núcleo Celular/metabolismo , Vesículas Extracelulares/metabolismo , Lisossomos/metabolismo , Autofagossomos/fisiologia , Autofagia/fisiologia , Células HeLa , Humanos , Transdução de Sinais/fisiologiaRESUMO
Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases.
Assuntos
Exocitose/fisiologia , Canais Iônicos/fisiologia , Lisossomos/fisiologia , Animais , Homeostase/fisiologia , Humanos , Íons/metabolismo , Doenças por Armazenamento dos Lisossomos/fisiopatologia , Potenciais da Membrana/fisiologiaRESUMO
Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca(2+) signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca(2+) mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca(2+) stores, resulting in Ca(2+) release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2(-/-) mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca(2+) release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca(2+) release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2(-/-) mice, but was unaffected in Tpcn1(-/-) animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca(2+) signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Carbolinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Camundongos , Camundongos Knockout , NADP/análogos & derivados , NADP/antagonistas & inibidores , NADP/genética , NADP/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Piperazinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Pancreatic ß cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic ß cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the ß cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in ß cells.
Assuntos
Canais de Cálcio/metabolismo , Endossomos/metabolismo , Células Secretoras de Insulina/metabolismo , NADP/análogos & derivados , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Células Cultivadas , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Masculino , Potenciais da Membrana , Camundongos , Camundongos Knockout , NADP/metabolismoRESUMO
Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.
Assuntos
Álcalis/metabolismo , Autofagia , Canais de Cálcio/metabolismo , Lisossomos/metabolismo , Fusão de Membrana , Fagossomos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/ultraestrutura , Células HeLa , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Fusão de Membrana/efeitos dos fármacos , Camundongos , NADP/análogos & derivados , NADP/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fagossomos/efeitos dos fármacos , Fagossomos/ultraestrutura , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab de Ligação ao GTP/efeitos dos fármacos , proteínas de unión al GTP Rab7RESUMO
Calcium (Ca2+) plays a pivotal role in cellular signal transmission by triggering downstream signaling in response to an increase in the cytosolic Ca2+ concentration. Intracellular organelles serve as Ca2+ stores that induce differently shaped Ca2+ signals. We discuss a study by Yuan et al. that investigated the interplay between the lysosomal two-pore channel 2 (TPC2) and endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptors (IP3Rs).
Assuntos
Canais de Cálcio , Sinalização do Cálcio , Receptores de Inositol 1,4,5-Trifosfato , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Humanos , Animais , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismoRESUMO
Besides controlling several organellar functions, lysosomal channels also guide the catabolic "self-eating" process named autophagy, which is mainly involved in protein and organelle quality control. Neuronal cells are particularly sensitive to the rate of autophagic flux either under physiological conditions or during the degenerative process. Accordingly, neurodegeneration occurring in Parkinson's (PD), Alzheimer's (AD), and Huntington's Diseases (HD), and Amyotrophic Lateral Sclerosis (ALS) as well as Lysosomal Storage Diseases (LSD) is partially due to defective autophagy and accumulation of toxic aggregates. In this regard, dysfunction of lysosomal ionic homeostasis has been identified as a putative cause of aberrant autophagy. From a therapeutic perspective, Transient Receptor Potential Channel Mucolipin 1 (TRPML1) and Two-Pore Channel isoform 2 (TPC2), regulating lysosomal homeostasis, are now considered promising druggable targets in neurodegenerative diseases. Compelling evidence suggests that pharmacological modulation of TRPML1 and TPC2 may rescue the pathological phenotype associated with autophagy dysfunction in AD, PD, HD, ALS, and LSD. Although pharmacological repurposing has identified several already used drugs with the ability to modulate TPC2, and several tools are already available for the modulation of TRPML1, many efforts are necessary to design and test new entities with much higher specificity in order to reduce dysfunctional autophagy during neurodegeneration.