RESUMEN
PDZ domain mediated interactions with voltage-gated calcium (CaV) channel C-termini play important roles in localizing membrane Ca2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and CaV2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to CaV2 chanels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and CaV2 channels from cnidarians and placozoans interact in vitro, and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis. Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly bound the divergent C-terminal ligands of cnidarian and placozoan CaV2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore CaV2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind CaV2 channels predates bilaterian animals, and that evolutionary changes in CaV2 channel C-terminal sequences resulted in altered binding modalities with Mint.
Asunto(s)
Dominios PDZ , Unión Proteica , Animales , Secuencia de Aminoácidos , Placozoa/metabolismo , Placozoa/genética , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Humanos , Canales de Calcio/metabolismo , Canales de Calcio/genéticaRESUMEN
Studies have demonstrated the therapeutic effects of Lindera plants. This study was undertaken to reveal the antihypertensive properties of Lindera erythrocarpa leaf ethanolic extract (LEL). Aorta segments of Sprague-Dawley rats were used to study the vasodilatory effect of LEL, and the mechanisms involved were evaluated by treating specific inhibitors or activators that affect the contractility of blood vessels. Our results revealed that LEL promotes a vasorelaxant effect through the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway, blocking the Ca2+ channels, opening the K+ channels, and inhibiting the vasoconstrictive action of angiotensin II. In addition, the effects of LEL on blood pressure were investigated in spontaneously hypertensive rats by the tail-cuff method. LEL (300 or 1000 mg/kg) was orally administered to the rats, and 1000 mg/kg of LEL significantly lowered the blood pressure. Systolic blood pressure decreased by -20.06 ± 4.87%, and diastolic blood pressure also lowered by -30.58 ± 5.92% at 4 h in the 1000 mg/kg LEL group. Overall, our results suggest that LEL may be useful to treat hypertensive diseases, considering its vasorelaxing and hypotensive effects.
Asunto(s)
Antihipertensivos , Presión Sanguínea , GMP Cíclico , Hipertensión , Lindera , Óxido Nítrico , Extractos Vegetales , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Animales , Antihipertensivos/farmacología , Extractos Vegetales/farmacología , Óxido Nítrico/metabolismo , Presión Sanguínea/efectos de los fármacos , GMP Cíclico/metabolismo , Masculino , Hipertensión/tratamiento farmacológico , Ratas , Lindera/química , Canales de Potasio/metabolismo , Canales de Potasio/efectos de los fármacos , Canales de Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Hojas de la Planta/química , Vasodilatación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Activating Ca2+-sensitive enzymes of oxidative metabolism while preventing calcium overload that leads to mitochondrial and cellular injury requires dynamic control of mitochondrial Ca2+ uptake. This is ensured by the mitochondrial calcium uptake (MICU)1/2 proteins that gate the pore of the mitochondrial calcium uniporter (mtCU). MICU1 is relatively sparse in the heart, and recent studies claimed the mammalian heart lacks MICU1 gating of mtCU. However, genetic models have not been tested. We find that MICU1 is present in a complex with MCU in nonfailing human hearts. Furthermore, using murine genetic models and pharmacology, we show that MICU1 and MICU2 control cardiac mitochondrial Ca2+ influx, and that MICU1 deletion alters cardiomyocyte mitochondrial calcium signaling and energy metabolism. MICU1 loss causes substantial compensatory changes in the mtCU composition and abundance, increased turnover of essential MCU regulator (EMRE) early on and, later, of MCU, that limit mitochondrial Ca2+ uptake and allow cell survival. Thus, both the primary consequences of MICU1 loss and the ensuing robust compensation highlight MICU1's relevance in the beating heart.
Asunto(s)
Señalización del Calcio , Proteínas de Unión al Calcio , Calcio , Proteínas de Transporte de Catión , Proteínas de Transporte de Membrana Mitocondrial , Miocitos Cardíacos , Animales , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Ratones , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Humanos , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/genética , Miocitos Cardíacos/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Mitocondrias Cardíacas/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio/genética , Ratones Noqueados , Miocardio/metabolismo , MasculinoRESUMEN
The mitochondrial calcium (mCa2+) uniporter channel (mtCU) resides at the inner mitochondrial membrane and is required for Ca2+ to enter the mitochondrial matrix. The mtCU is essential for cellular function, as mCa2+ regulates metabolism, bioenergetics, signaling pathways and cell death. mCa2+ uptake is primarily regulated by the MICU family (MICU1, MICU2, MICU3), EF-hand-containing Ca2+-sensing proteins, which respond to cytosolic Ca2+ concentrations to modulate mtCU activity. Considering that mitochondrial function and Ca2+ signaling are ubiquitously disrupted in cardiovascular disease, mtCU function has been a hot area of investigation for the last decade. Here we provide an in-depth review of MICU-mediated regulation of mtCU structure and function, as well as potential mtCU-independent functions of these proteins. We detail their role in cardiac physiology and cardiovascular disease by highlighting the phenotypes of different mutant animal models, with an emphasis on therapeutic potential and targets of interest in this pathway.
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Canales de Calcio , Enfermedades Cardiovasculares , Humanos , Animales , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Canales de Calcio/metabolismo , Canales de Calcio/genética , Mitocondrias Cardíacas/metabolismo , Señalización del Calcio , Calcio/metabolismo , Activación del Canal Iónico , Relación Estructura-ActividadRESUMEN
Endothelial cells (ECs) are highly plastic, capable of differentiating into various cell types. Endothelial-to-mesenchymal transition (EndMT) is crucial during embryonic development and contributes substantially to vascular dysfunction in many cardiovascular diseases (CVDs). While targeting EndMT holds therapeutic promise, understanding its mechanisms and modulating its pathways remain challenging. Using single-cell RNA sequencing on three in vitro EndMT models, we identified conserved gene signatures. We validated original regulators in vitro and in vivo during embryonic heart development and peripheral artery disease. EndMT induction led to global expression changes in all EC subtypes rather than in mesenchymal clusters. We identified mitochondrial calcium uptake as a key driver of EndMT; inhibiting mitochondrial calcium uniporter (MCU) prevented EndMT in vitro, and conditional Mcu deletion in ECs blocked mesenchymal activation in a hind limb ischemia model. Tissues from patients with critical limb ischemia with EndMT features exhibited significantly elevated endothelial MCU. These findings highlight MCU as a regulator of EndMT and a potential therapeutic target.
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Señalización del Calcio , Células Endoteliales , Transición Epitelial-Mesenquimal , Mitocondrias , RNA-Seq , Análisis de la Célula Individual , Animales , Humanos , Mitocondrias/metabolismo , RNA-Seq/métodos , Ratones , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Canales de Calcio/metabolismo , Canales de Calcio/genética , Isquemia/metabolismo , Isquemia/patología , Calcio/metabolismo , Análisis de Expresión Génica de una Sola CélulaRESUMEN
In this issue of Structure, Chi et al.1 report structural and functional studies that reveal the inhibition mechanism of the lysosomal two-pore channel TPC2 by the antagonist SG-094, which is of interest for drug development. Antagonist binding induces the downward displacement of the voltage-sensor domain II (VSD II), which is accompanied by asymmetric conformational rearrangements of the entire channel.
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Canales de Calcio , Humanos , Canales de Calcio/metabolismo , Canales de Calcio/química , Dominios Proteicos , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/farmacologíaRESUMEN
Action potentials trigger neurotransmitter release at the presynaptic active zone with spatiotemporal precision. This is supported by protein machinery that mediates synaptic vesicle priming and clustering of CaV2 Ca2+ channels nearby. One model posits that scaffolding proteins directly tether vesicles to CaV2s; however, here we find that at mouse hippocampal synapses, CaV2 clustering and vesicle priming are executed by separate machineries. CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins but distinct interaction motifs independently execute these functions. In transfected cells, Liprin-α and RIM form co-assemblies that are separate from CaV2-organizing complexes. At synapses, Liprin-α1-Liprin-α4 knockout impairs vesicle priming but not CaV2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co-clustering CaV2s. We conclude that active zones consist of distinct machineries to organize CaV2s and prime vesicles, and Liprin-α and PTPσ specifically support priming site assembly.
Asunto(s)
Hipocampo , Sinapsis , Vesículas Sinápticas , Animales , Hipocampo/metabolismo , Hipocampo/citología , Vesículas Sinápticas/metabolismo , Ratones , Sinapsis/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ratones Noqueados , Caveolina 2/metabolismo , Canales de Calcio/metabolismo , Ratones Endogámicos C57BLRESUMEN
NVA1309 is a non-brain penetrant next-generation gabapentinoid shown to bind Cavα2δ at R243 within a triple Arginine motif forming the binding site for gabapentin and pregabalin. In this study we have compared the effects of NVA1309 with Mirogabalin, a gabapentinoid drug with higher affinity for the voltage-gated calcium channel subunit Cavα2δ-1 than pregabalin which is approved for post-herpetic neuralgia in Japan, Korea and Taiwan. Both NVA1309 and mirogabalin inhibit Cav2.2 currents in vitro and decrease Cav2.2 plasma membrane expression with higher efficacy than pregabalin. Mutagenesis of the classical binding residue arginine R243 and the newly identified binding residue lysine K615 reverse the effect of mirogabalin on Cav2.2 current, but not that of NVA1309.
Asunto(s)
Gabapentina , Humanos , Gabapentina/farmacología , Animales , Unión Proteica , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Células HEK293 , Ácido gamma-Aminobutírico/metabolismo , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Pregabalina/farmacología , Canales de Calcio/metabolismo , Compuestos Bicíclicos con PuentesRESUMEN
Orai1 is a plasma membrane Ca2+ channel involved in store operated calcium entry (SOCE). SOCE can regulate cell growth, exocytosis, gene expression and inflammation. We previously found that short palate lung and nasal epithelial clone 1's (SPLUNC1) sixth α-helix (α6) bound Orai1 to inhibit SOCE. SPLUNC1 was not proteolytically stable, so we developed ELD607, an 11 amino acid peptide based on SPLUNC1's α6 region which was more stable and more potent than SPLUNC1/α6. Here, we studied ELD607's mechanism of action. We overexpressed either Orai1-HA or Orai1-YFP in HEK293T cells to probe ELD607-Orai1 interactions by confocal microscopy. We also measured changes in Fluo-4 fluorescence in a multiplate reader as a marker of cytoplasmic Ca2+ levels. ELD607 internalized Orai1 independently of STIM1. Both 15 min and 3 h exposure to ELD607 similarly depleted Orai1 in the plasma membrane. However, 3 h exposure to ELD607 yielded greater inhibition of SOCE. ELD607 continued to colocalize with Orai1 after internalization and this process was dependent on the presence of the ubiquitin ligase NEDD4.2. Similarly, ELD607 increased the colocalization between Orai1 and ubiquitin. ELD607 also increased the colocalization between Orai1 and Rab5 and 7, but not Rab11, suggesting that Orai1 trafficked through early and late but not recycling endosomes. Finally, ELD607 caused Orai1, but not Orai2, Orai3, or STIM1 to traffic to lysosomes. We conclude that ELD607 rapidly binds to Orai1 and works in an identical fashion as full length SPLUNC1 by internalizing Orai1 and sending it to lysosomes, leading to a decrease in SOCE.
Asunto(s)
Calcio , Lisosomas , Proteína ORAI1 , Humanos , Proteína ORAI1/metabolismo , Células HEK293 , Calcio/metabolismo , Lisosomas/metabolismo , Canales de Calcio/metabolismo , Transporte de Proteínas , Membrana Celular/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.
Asunto(s)
Calcio , Epitelio Corneal , Hidrazonas , Mitocondrias , Humanos , Epitelio Corneal/metabolismo , Epitelio Corneal/efectos de los fármacos , Calcio/metabolismo , Mitocondrias/metabolismo , Hidrazonas/farmacología , Retículo Endoplásmico/metabolismo , Tapsigargina/farmacología , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Células Cultivadas , terc-Butilhidroperóxido/farmacología , Homeostasis/fisiologíaRESUMEN
Most neurons are not replaced after injury and thus possess robust intrinsic mechanisms for repair after damage. Axon injury triggers a calcium wave, and calcium and cAMP can augment axon regeneration. In comparison to axon regeneration, dendrite regeneration is poorly understood. To test whether calcium and cAMP might also be involved in dendrite injury signaling, we tracked the responses of Drosophila dendritic arborization neurons to laser severing of axons and dendrites. We found that calcium and subsequently cAMP accumulate in the cell body after both dendrite and axon injury. Two voltage-gated calcium channels (VGCCs), L-Type and T-Type, are required for the calcium influx in response to dendrite injury and play a role in rapid initiation of dendrite regeneration. The AC8 family adenylyl cyclase, Ac78C, is required for cAMP production after dendrite injury and timely initiation of regeneration. Injury-induced cAMP production is sensitive to VGCC reduction, placing calcium upstream of cAMP generation. We propose that two VGCCs initiate global calcium influx in response to dendrite injury followed by production of cAMP by Ac78C. This signaling pathway promotes timely initiation of dendrite regrowth several hours after dendrite damage.
Asunto(s)
Adenilil Ciclasas , Canales de Calcio Tipo L , Calcio , AMP Cíclico , Dendritas , Animales , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/genética , Axones/metabolismo , Axones/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Señalización del Calcio/genética , AMP Cíclico/metabolismo , Dendritas/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regeneración Nerviosa/fisiología , Regeneración Nerviosa/genética , Neuronas/metabolismo , Regeneración/genética , Regeneración/fisiología , Transducción de SeñalRESUMEN
The regulation of the cell membrane potential plays a crucial role in governing the transmembrane transport of various ions and cellular life processes. However, in situ and on-demand modulation of cell membrane potential for ion channel regulation is challenging. Herein, we have constructed a supramolecular assembly system based on water-soluble cationic oligo(phenylenevinylene) (OPV) and cucurbit[7]uril (CB[7]). The controllable disassembly of OPV/4CB[7] combined with the subsequent click reaction provides a step-by-step adjustable surface positive potential. These processes can be employed in situ on the plasma membrane to modulate the membrane potential on-demand for precisely controlling the activation of the transient receptor potential vanilloid 1 (TRPV1) ion channel and up-regulating exogenous calcium-responsive gene expression. Compared with typical optogenetics, electrogenetics, and mechanogenetics, our strategy provides a perspective supramolecular genetics toolbox for the regulation of membrane potential and downstream intracellular gene regulation events.
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Imidazoles , Potenciales de la Membrana , Imidazoles/química , Humanos , Hidrocarburos Aromáticos con Puentes/química , Polivinilos/química , Membrana Celular/metabolismo , Membrana Celular/química , Canales Catiónicos TRPV/metabolismo , Células HEK293 , Calcio/metabolismo , Calcio/química , Canales de Calcio/metabolismo , Canales de Calcio/química , Compuestos Heterocíclicos con 2 Anillos , Compuestos Macrocíclicos , ImidazolidinasRESUMEN
Vasodilation in response to low oxygen (O2) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O2 supply to tissues according to demand. However, how blood vessels react to O2 deficiency is not well understood. A common belief is that arterial myocytes are O2-sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Ca2+channels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O2 modulation of Ca2+ channels and hypoxic vasodilation. Mitochondria function as O2 sensors and effectors that signal myocyte Ca2+ channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O2-sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia , Mitocondrias , Oxígeno , Vasodilatación , Animales , Mitocondrias/metabolismo , Oxígeno/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Transducción de Señal , Masculino , Hipoxia/metabolismo , Ratones Endogámicos C57BL , Arterias/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Ratones Noqueados , Transporte de Electrón , Canales de Calcio/metabolismo , Canales de Calcio/genéticaRESUMEN
The abundance of CaV2 voltage-gated calcium channels is linked to presynaptic homeostatic plasticity (PHP), a process that recalibrates synaptic strength to maintain the stability of neural circuits. However, the molecular and cellular mechanisms governing PHP and CaV2 channels are not completely understood. Here, we uncover a previously not described form of PHP in Caenorhabditis elegans, revealing an inverse regulatory relationship between the efficiency of neurotransmitter release and the abundance of UNC-2/CaV2 channels. Gain-of-function unc-2SL(S240L) mutants, which carry a mutation analogous to the one causing familial hemiplegic migraine type 1 in humans, showed markedly reduced channel abundance despite increased channel functionality. Reducing synaptic release in these unc-2SL(S240L) mutants restored channel levels to those observed in wild-type animals. Conversely, loss-of-function unc-2DA(D726A) mutants, which harbor the D726A mutation in the channel pore, exhibited a marked increase in channel abundance. Enhancing synaptic release in unc-2DA mutants reversed this increase in channel levels. Importantly, this homeostatic regulation of UNC-2 channel levels is accompanied by the structural remodeling of the active zone (AZ); specifically, unc-2DA mutants, which exhibit increased channel abundance, showed parallel increases in select AZ proteins. Finally, our forward genetic screen revealed that WWP-1, a HECT family E3 ubiquitin ligase, is a key homeostatic mediator that removes UNC-2 from synapses. These findings highlight a self-tuning PHP regulating UNC-2/CaV2 channel abundance along with AZ reorganization, ensuring synaptic strength and stability.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Neurotransmisores , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio/genética , Transmisión Sináptica/fisiología , Plasticidad Neuronal , Mutación , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Neuronas/metabolismo , Proteínas de la MembranaRESUMEN
Osteoporosis, a systemic skeletal disorder marked by diminished bone mass and compromised bone microarchitecture, is becoming increasingly prevalent due to an aging population. The underlying pathophysiology of osteoporosis is attributed to an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Osteoclasts play a crucial role in the development of osteoporosis through various molecular pathways, including the RANK/RANKL/OPG signaling axis, cytokines, and integrins. Notably, the calcium signaling pathway is pivotal in regulating osteoclast activation and function, influencing bone resorption activity. Disruption in calcium signaling can lead to increased osteoclast-mediated bone resorption, contributing to the progression of osteoporosis. Emerging research indicates that calcium-permeable channels on the cellular membrane play a critical role in bone metabolism by modulating these intracellular calcium pathways. Here, we provide an overview of current literature on the regulation of plasma membrane calcium channels in relation to bone metabolism with particular emphasis on their dysregulation during the progression of osteoporosis. Targeting these calcium channels may represent a potential therapeutic strategy for treating osteoporosis.
Asunto(s)
Canales de Calcio , Osteoporosis , Humanos , Osteoporosis/metabolismo , Canales de Calcio/metabolismo , Animales , Resorción Ósea/metabolismo , Osteoclastos/metabolismo , Señalización del Calcio/fisiologíaRESUMEN
Voltage-gated sodium (Nav) and calcium (Cav) channels are responsible for the initiation of electrical signals. They have long been targeted for the treatment of various diseases. The mounting number of cryoelectron microscopy (cryo-EM) structures for diverse subtypes of Nav and Cav channels from multiple organisms necessitates a generic residue numbering system to establish the structure-function relationship and to aid rational drug design or optimization. Here we suggest a structure-based residue numbering scheme, centering around the most conserved residues on each of the functional segments. We elaborate the generic numbers through illustrative examples, focusing on representative drug-binding sites of eukaryotic Nav and Cav channels. We also extend the numbering scheme to compare common disease mutations among different Nav subtypes. Application of the generic residue numbering scheme affords immediate insights into hotspots for pathogenic mutations and critical loci for drug binding and will facilitate drug discovery targeting Nav and Cav channels.
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Canales de Calcio , Humanos , Canales de Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio/genética , Canales de Sodio Activados por Voltaje/química , Canales de Sodio Activados por Voltaje/metabolismo , Canales de Sodio Activados por Voltaje/genética , Animales , Sitios de Unión , Mutación , Microscopía por Crioelectrón , Modelos Moleculares , Secuencia de AminoácidosRESUMEN
BACKGROUND: One of major challenges in breast tumor therapy is the existence of breast cancer stem cells (BCSCs). BCSCs are a small subpopulation of tumor cells that exhibit characteristics of stem cells. BCSCs are responsible for progression, recurrence, chemoresistance and metastasis of breast cancer. Ca2+ signalling plays an important role in diverse processes in cancer development. However, the role of Ca2+ signalling in BCSCs is still poorly understood. METHODS: A highly effective 3D soft fibrin gel system was used to enrich BCSC-like cells from ER+ breast cancer lines MCF7 and MDA-MB-415. We then investigated the role of two Ca2+-permeable ion channels Orai1 and Orai3 in the growth and stemness of BCSC-like cells in vitro, and tumorigenicity in female NOD/SCID mice in vivo. RESULTS: Orai1 RNA silencing and pharmacological inhibition reduced the growth of BCSC-like cells in tumor spheroids, decreased the expression levels of BCSC markers, and reduced the growth of tumor xenografts in NOD/SCID mice. Orai3 RNA silencing also had similar inhibitory effect on the growth and stemness of BCSC-like cells in vitro, and tumor xenograft growth in vivo. Mechanistically, Orai1 and SPCA2 mediate store-operated Ca2+ entry. Knockdown of Orai1 or SPCA2 inhibited glycolysis pathway, whereas knockdown of Orai3 or STIM1 had no effect on glycolysis. CONCLUSION: We found that Orai1 interacts with SPCA2 to mediate store-independent Ca2+ entry, subsequently promoting the growth and tumorigenicity of BCSC-like cells via glycolysis pathway. In contrast, Orai3 and STIM1 mediate store-operated Ca2+ entry, promoting the growth and tumorigenicity of BCSC-like cells via a glycolysis-independent pathway. Together, our study uncovered a well-orchestrated mechanism through which two Ca2+ entry pathways act through distinct signalling axes to finely control the growth and tumorigenicity of BCSCs.
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Neoplasias de la Mama , Canales de Calcio , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas , Proteína ORAI1 , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Humanos , Animales , Femenino , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Ratones , Canales de Calcio/metabolismo , Canales de Calcio/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Transducción de Señal , Señalización del Calcio , Células MCF-7RESUMEN
At chemical synapses, voltage-gated Ca2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition (E-I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation-inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 ß-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Unión Neuromuscular , Transmisión Sináptica , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Proteínas Portadoras , Proteínas de la Membrana , Mutación , Red Nerviosa/fisiología , Red Nerviosa/metabolismo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Sinapsis/metabolismo , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismoRESUMEN
BACKGROUND: Tight control of cytoplasmic Ca2+ concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavß3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier. METHODS: We investigated the function of Cavß3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavß3-/- (Cavß3-deficient) mice as controls. RESULTS: We identified Cavß3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavß3. After induction of experimental autoimmune encephalomyelitis, Cavß3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavß3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavß3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavß3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavß3 with inositol 1,4,5-trisphosphate receptor proteins. CONCLUSIONS: Independent of its function as a subunit of Cav channels, Cavß3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ concentration and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavß3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.
Asunto(s)
Barrera Hematoencefálica , Señalización del Calcio , Encefalomielitis Autoinmune Experimental , Células Endoteliales , Animales , Femenino , Masculino , Ratones , Barrera Hematoencefálica/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio/genética , Permeabilidad Capilar , Células Cultivadas , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Células Endoteliales/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , FosforilaciónRESUMEN
As the uncontrolled entry of calcium ions (Ca2+) through plasmalemmal calcium channels is a cell death trigger, the conjecture is here raised that mitigating such an excess of Ca2+ entry should rescue from death the vulnerable neurons in neurodegenerative diseases (NDDs). However, this supposition has failed in some clinical trials (CTs). Thus, a recent CT tested whether isradipine, a blocker of the Cav1 subtype of voltage-operated calcium channels (VOCCs), exerted a benefit in patients with Parkinson's disease (PD); however, outcomes were negative. This is one more of the hundreds of CTs done under the principle of one-drug-one-target, that have failed in Alzheimer's disease (AD) and other NDDs during the last three decades. As there are myriad calcium channels to let Ca2+ ions gain the cell cytosol, it seems reasonable to predict that blockade of Ca2+ entry through a single channel may not be capable of preventing the Ca2+ flood of cells by the uncontrolled Ca2+ entry. Furthermore, as Ca2+ signaling is involved in the regulation of myriad functions in different cell types, it seems also reasonable to guess that a therapy should be more efficient by targeting different cells with various drugs. Here, we propose to mitigate Ca2+ entry by the simultaneous partial blockade of three quite different subtypes of plasmalemmal calcium channels that is, the Cav1 subtype of VOCCs, the Orai1 store-operated calcium channel (SOCC), and the purinergic P2X7 calcium channel. All three channels are expressed in both microglia and neurons. Thus, by targeting the three channels with a combination of three drug blockers we expect favorable changes in some of the pathogenic features of NDDs, namely (i) to mitigate Ca2+ entry into microglia; (ii) to decrease the Ca2+-dependent microglia activation; (iii) to decrease the sustained neuroinflammation; (iv) to decrease the uncontrolled Ca2+ entry into neurons; (v) to rescue vulnerable neurons from death; and (vi) to delay disease progression. In this review we discuss the arguments underlying our triad hypothesis in the sense that the combination of three repositioned medicines targeting Cav1, Orai1, and P2X7 calcium channels could boost neuroprotection and delay the progression of AD and other NDDs.