Intracellular Calcium Signaling Research Articles

Inspiratory and sigh breathing rhythms depend on distinct cellular signalling mechanisms in the preBötzinger complex.

Breathing behaviour involves the generation of normal breaths (eupnoea) on a timescale of seconds and sigh breaths on the order of minutes. Both rhythms emerge in tandem from a single brainstem site, but whether and how a single cell population can generate two disparate rhythms remains unclear. We posit that recurrent synaptic excitation in concert with synaptic depression and cellular refractoriness gives rise to the eupnoea rhythm, whereas an intracellular calcium oscillation that is slower by orders of magnitude gives rise to the sigh rhythm. A mathematical model capturing these dynamics simultaneously generates eupnoea and sigh rhythms with disparate frequencies, which can be separately regulated by physiological parameters. We experimentally validated key model predictions regarding intracellular calcium signalling. All vertebrate brains feature a network oscillator that drives the breathing pump for regular respiration. However, in air-breathing mammals with compliant lungs susceptible to collapse, the breathing rhythmogenic network may have refashioned ubiquitous intracellular signalling systems to produce a second slower rhythm (for sighs) that prevents atelectasis without impeding eupnoea. KEY POINTS: A simplified activity-based model of the preBötC generates inspiratory and sigh rhythms from a single neuron population. Inspiration is attributable to a canonical excitatory network oscillator mechanism. Sigh emerges from intracellular calcium signalling. The model predicts that perturbations of calcium uptake and release across the endoplasmic reticulum counterintuitively accelerate and decelerate sigh rhythmicity, respectively, which was experimentally validated. Vertebrate evolution may have adapted existing intracellular signalling mechanisms to produce slow oscillations needed to optimize pulmonary function in mammals.

Modulation of Type 5 Metabotropic Glutamate Receptor-Mediated Intracellular Calcium Mobilization by Regulator of G Protein Signaling 4 (RGS4) in Cultured Astrocytes.

Acting as GTPase activating proteins promoting the silencing of activated G-proteins, regulators of G protein signaling (RGSs) are generally considered negative modulators of cell signaling. In the CNS, the expression of RGS4 is altered in diverse pathologies and its upregulation was reported in astrocytes exposed to an inflammatory environment. In a model of cultured cortical astrocytes, we herein investigate the influence of RGS4 on intracellular calcium signaling mediated by type 5 metabotropic glutamate receptor (mGluR5), which is known to support the bidirectional communication between neurons and glial cells. RGS4 activity was manipulated by exposure to the inhibitor CCG 63802 or by infecting the cells with lentiviruses designed to achieve the silencing or overexpression of RGS4. The pharmacological inhibition or silencing of RGS4 resulted in a decrease in the percentage of cells responding to the mGluR5 agonist DHPG and in the proportion of cells showing typical calcium oscillations. Conversely, RGS4-lentivirus infection increased the percentage of cells showing calcium oscillations. While the physiological implication of cytosolic calcium oscillations in astrocytes is still under investigation, the fine-tuning of calcium signaling likely determines the coding of diverse biological events. Indirect signaling modulators such as RGS4 inhibitors, used in combination with receptor ligands, could pave the way for new therapeutic approaches for diverse neurological disorders with improved efficacy and selectivity.

CAMTA3 repressor destabilization triggers TIR domain protein TN2-mediated autoimmunity in the Arabidopsis exo70B1 mutant.

Calcium-dependent protein kinases (CPKs) can decode and translate intracellular calcium signals to induce plant immunity. Mutation of the exocyst subunit gene EXO70B1 causes autoimmunity that depends on CPK5 and the Toll/interleukin-1 receptor (TIR) domain resistance protein TIR-NBS2 (TN2), where direct interaction with TN2 stabilizes CPK5 kinase activity. However, how the CPK5-TN2 interaction initiates downstream immune responses remains unclear. Here, we show that, besides CPK5 activity, the physical interaction between CPK5 and functional TN2 triggers immune activation in exo70B1 and may represent reciprocal regulation between CPK5 and the TIR domain functions of TN2 in Arabidopsis (Arabidopsis thaliana). Moreover, we detected differential phosphorylation of the calmodulin-binding transcription activator 3 (CAMTA3) in the cpk5 background. CPK5 directly phosphorylates CAMTA3 at S964, contributing to its destabilization. The gain-of-function CAMTA3A855V variant that resists CPK5-induced degradation rescues immunity activated through CPK5 overexpression or exo70B1 mutation. Thus, CPK5-mediated immunity is executed through CAMTA3 repressor degradation via phosphorylation-induced and/or calmodulin-regulated processes. Conversely, autoimmunity in camta3 also partially requires functional CPK5. While the TIR domain activity of TN2 remains to be tested, our study uncovers a TN2-CPK5-CAMTA3 signaling module for exo70B1-mediated autoimmunity, highlighting the direct embedding of a calcium-sensing decoder element within resistance signalosomes.

"There's plenty of room at the bottom": deep brain imaging with holographic endo-microscopy.

Over more than 300 years, microscopic imaging keeps providing fundamental insights into the mechanisms of living organisms. Seeing microscopic structures beyond the reach of free-space light-based microscopy, however, requires dissection of the tissue-an intervention seriously disturbing its physiological functions. The hunt for low-invasiveness tools has led a growing community of physicists and engineers into the realm of complex media photonics. One of its activities represents exploiting multimode optical fibers (MMFs) as ultra-thin endoscopic probes. Employing wavefront shaping, these tools only recently facilitated the first peeks at cells and their sub-cellular compartments at the bottom of the mouse brain with the impact of micro-scale tissue damage. Here, we aim to highlight advances in MMF-based holographic endo-microscopy facilitating microscopic imaging throughout the whole depth of the mouse brain. We summarize the important technical and methodological prerequisites for stabile high-resolution imaging in vivo. We showcase images of the microscopic building blocks of brain tissue, including neurons, neuronal processes, vessels, intracellular calcium signaling, and red blood cell velocity in individual vessels. This perspective article helps to understand the complexity behind the technology of holographic endo-microscopy, summarizes its recent advances and challenges, and stimulates the mind of the reader for further exploitation of this tool in the neuroscience research.

Calcium transferring from ER to mitochondria via miR-129/ITPR2 axis controls cellular senescence in vitro and in vivo

Senescent cells are known to be accumulated in aged organisms. Although the two main characteristics, cell cycle arrest (for dividing cells) and secretion of senescence-associated secretory phenotype (SASP) factors, have been well described, the lack of sufficient senescent markers and incomplete understanding of mechanisms have limited the progress of the anti-senescence field. Calcium transferred from the endoplasmic reticulum (ER) via inositol 1, 4, 5-trisphosphate receptor type 2 (ITPR2) to mitochondria has emerged as a key player during cellular senescence and aging. However, the internal regulatory mechanisms, particularly those of endogenous molecules, remain only partially understood. Here we identified miRNA-129 (miR-129) as a direct repressor of ITPR2. Interestingly, miR-129 controlled a cascade of intracellular calcium signaling, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA damage, and consequently cellular senescence through ITPR2 and mitochondrial calcium uniporter (MCU). In addition, miR-129 was repressed in different senescence models and delayed bleomycin-induced cellular senescence. Importantly, intraperitoneal injection of miR-129 partly postponed bleomycin-accelerated lung aging and natural aging markers as well as reduced immunosenescence markers in mice. Altogether, these findings demonstrated that miR-129 regulated cellular senescence and aging markers via intracellular calcium signaling by directly targeting ITPR2.

Calcium chelation independent effects of BAPTA on endogenous ANO6 channels in HEK293T cells

Selective calcium chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) is a common tool to investigate calcium signaling. However, BAPTA express various effects on intracellular calcium signaling, which are not related to its ability to bind Ca2+. In patch clamp experiments, we investigated calcium chelation independent effects of BAPTA on endogenous calcium-activated chloride channels ANO6 (TMEM16F) in HEK293T cells. We have found that application of BAPTA to intracellular solution led to two distinct effects on channels properties. On the one hand, application of BAPTA acutely reduced amplitude of endogenous ANO6 channels induced by 10 μM Ca2+ in single channel recordings. On the other hand, BAPTA application by itself induced ANO6 channel activity in the absence of the intracellular calcium elevation. Open channel probability was enhanced by increasing the intracellular BAPTA concentration from 0.1 to 1 and 10 mM. Another calcium chelator EGTA did not demonstrate chelation independent effects on the ANO6 activity in the same conditions. Due to off target effects BAPTA should be used with caution when studying calcium-activated ANO6 channels.

Changes of Trigeminal Ganglion Neurons Innervating the Temporomandibular Joint in Chronic Pain Rat Model.

Background: Phenotype alterations of nociceptive neurons have been shown to be a key step in the pathogenesis of many pain-related diseases. However, it is unclear if the characteristic changes of temporomandibular joint (TMJ) primary afferent neurons are related to the pathogenesis of temporomandibular joint osteoarthritis (TMJOA) chronic pain. This study aimed to determine the morphological and neurochemical changes in trigeminal ganglion (TG) neurons innervating the TMJ in TMJOA chronic pain rats. Materials and Methods: Monosodium iodoacetate (MIA)-induced TMJOA chronic pain rat model was established (n = 6), and saline was injected in rats of the control group (n = 6). TMJ primary afferent neurons were labeled with retrograde tracing (Dil). The spatial distribution and the expression of calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), and neurofilament 200 (NF200) of TMJ primary afferent neurons in TG were investigated using immunofluorescence. Intracellular calcium signaling was recorded by calcium imaging (n = 20). Results: TMJ primary afferent neurons were located only in the V3 region of the TG from both saline- and MIA-injected rats. The number of TG neurons innervating the TMJ was increased in MIA-injected rats. Elevated number and intracellular calcium concentration of small- and medium-sized instead of large-sized Dil+ TG neurons were found in MIA-injected rats. The upregulated expression of CGRP and IB4, but not NF200, in TG neurons innervating the rat TMJs was accompanied by TMJOA chronic pain. Conclusion: This study suggests that sensitization of small- to medium-sized Dil+ TG neurons and CGRP- and IB4-positive Dil+ TG neurons might contribute to the development of TMJOA chronic pain in rats. This will provide valuable information for more efficient control of TMJOA chronic pain.

Ca2+-PP2B-PSD-95 axis: A novel regulatory mechanism of the phosphorylation state of Serine 295 of PSD-95.

The phosphorylation state of PSD-95 at Serine 295 (Ser295) is important for the regulation of synaptic plasticity. Although the activation of NMDA receptors (NMDARs), which initiates an intracellular calcium signaling cascade, decreases phosphorylated Ser295 (pS295) of PSD-95, the molecular mechanisms are not fully understood. We found that the calcium-activated protein phosphatase PP2B dephosphorylated pS295 not only in basal conditions but also in NMDAR-activated conditions in cultured neurons. The biochemical assay also revealed the dephosphorylation of pS295 by PP2B, consistently supporting the results obtained using neurons. The newly identified calcium signaling cascade "Ca2+-PP2B-PSD-95 axis" would play an important role in the molecular mechanism for NMDA receptor-dependent plasticity.

Coffee and mental disorders: How caffeine affects anxiety and depression.

Caffeine, the main psychoactive component in coffee, has garnered significant attention for its potential impact on the most prevalent mental health conditions like anxiety and depression. This chapter comprehensively examines the neurobiological effects of caffeine, its influence on anxiety and depression, and relevant clinical studies. Caffeine exerts its psychostimulant effects primarily through antagonizing adenosine receptors, modulating neurotransmitter systems, and influencing intracellular calcium signaling in the brain. Caffeine exhibits dose-dependent effects. While moderate caffeine consumption is safe in healthy adults and may offer benefits for mental health, excessive intake is linked to adverse effects on neurological and psychiatric health and can aggravate symptoms, highlighting the importance of adjusting consumption patterns. High caffeine intake correlates with elevated anxiety levels, especially in individuals predisposed to anxiety disorders. However, the relationship between caffeine consumption and the risk of depression is intricate, with some studies suggesting a potential protective effect of moderate intake, while others find no significant association. Individual variations in caffeine metabolism, sensitivity, and genetic factors considerably impact responses to caffeine. The chapter also explores the therapeutic potential of caffeine as an adjunct treatment and outlines challenges and future research directions in elucidating caffeine's multifaceted role in mental health.

A piezoelectric micromachined ultrasound transducer combined with recording electrodes for acute brain preparations in vitro

BackgroundUltrasound stimulation is used to noninvasively stimulate the local and deep areas of the brain. However, the detailed cellular mechanisms of neural activation are still unclear because studies on micro-stimulation at the cellular level are lacking. New methodTo modulate neural activity at the cellular level, we developed a piezoelectric micromachined ultrasound transducer (PMUT), having circular diaphragms for application on acute brain slice preparations. To monitor neural activities, additionally, we fabricated recording microelectrodes onto the same PMUT device for closed-loop application. ResultsTo examine the PMUT-driven cellular responses of a brain slice, intracellular calcium signals in individual cells were measured using two calcium indicators. We successfully observed the intracellular responses triggered by the ultrasound of our novel PMUT. In addition, we performed recordings of local field potentials in a brain slice, demonstrating its usefulness as a simultaneous recording interface. Comparison with existing method(s)Conventional ultrasound stimulators are open-loop systems that risk inducing excessive neural activity because of the absence of neural activity monitoring. In contrast, our PMUT is packaged in a single device with both stimulation and sensor interface for neuromodulation. Further, there are no published reports on in vitro microdevices that can be used for ultrasound stimulation in rodent cortical slices that are several hundred micrometers thick, which maintain the cortical laminar structure and intrinsic neural networks. ConclusionsOur findings suggest that this novel PMUT device has the potential for being a powerful tool for in vitro brain slice applications and effective closed loop ultrasound stimulation.

Involvement of CATSPER 2 mutation in a familial context of unexplained infertility and fertilization failure associated with hearing loss: a case report

To explore the functional implications of a homozygous CATSPER 2 (cation channel for sperm) deletion within the acrosome reaction pathway during fertilization in 2 brothers, who have unexplained infertility and hearing loss. Case report. Two twin brothers aged 30 years with hearing loss and unexplained infertility. Molecular genetic diagnosis of deafness. Evaluation of the acrosome reaction and calcium mobilization assays after induction by progesterone and ionomycin on spermatozoa of the CATSPER 2-mutated patient and on fertile controls. Fertilization rate during conventional in vitro fertilization. Molecular genetic test. Percentage of acrosome-reacted spermatozoa with peanut agglutinin lectin staining. Recording of progesterone and ionomycin-induced intracellular calcium signals with a fluorescent probe. Mr. S and his brother have normal, conventional sperm parameters. Both brothers have had repeated intrauterine insemination failures and one fertilization failure after conventional in vitro fertilization. Mr. S obtained 2 healthy babies after intracytoplasmic sperm injection. Genetic analysis found a homozygote deletion of the STRC (stereocilin) gene (NM 153700: c.1-? 5328+?del) that removes the CATSPER 2 gene. Mutation of the STRC gene is known to be associated with hearing loss. Sperm functional tests revealed an inability of progesterone to activate intracellular calcium signaling and to induce acrosome reaction. We demonstrate the absence of a calcium signal and acrosome reaction after progesterone in our patient with a CATSPER 2 mutation. We emphasize the importance of the male medical interview and of the genetic investigation of hearing loss. We show that in vitro fertilization-intracytoplasmic sperm injection is necessary, even where normal sperm parameters are present.

The Relationship between Trop-2, Chemotherapeutic Drugs, and Chemoresistance.

Trop-2 is a highly conserved one-pass transmembrane mammalian glycoprotein that is normally expressed in tissues such as the lung, intestines, and kidney during embryonic development. It is overexpressed in many epithelial cancers but is absent in non-epithelial tumors. Trop-2 is an intracellular calcium signal transducer that participates in the promotion of cell proliferation, migration, invasion, metastasis, and probably stemness. It also has some tumor suppressor effects. The pro-tumoral actions have been thoroughly investigated and reported. However, Trop-2's activity in chemoresistance is less well known. We review a possible relationship between Trop-2, chemotherapy, and chemoresistance. We conclude that there is a clear role for Trop-2 in some specific chemoresistance events. On the other hand, there is no clear evidence for its participation in multidrug resistance through direct drug transport. The development of antibody conjugate drugs (ACD) centered on anti-Trop-2 monoclonal antibodies opened the gates for the treatment of some tumors resistant to classic chemotherapies. Advanced urothelial tumors and breast cancer were among the first malignancies for which these ACDs have been employed. However, there is a wide group of other tumors that may benefit from anti-Trop-2 therapy as soon as clinical trials are completed.

Decoupling the roles of mechanical stretching and piezoelectrical stimulation in calcium signal mediated osteogenic differentiation of MSCs on biomimetic nanofibers

Piezoelectric materials implanted in vivo will be affected by physiological stress, leading to the generation of dynamic piezoelectrical signals. The cells identify both piezoelectrical and mechanical stimuli, and the way they behave is regulated through an electromechanical coupling process. Understanding the specific functions of individual stimulation on the osteogenic differentiation of mesenchymal stem cells (MSCs) would aid the development of ideal biomaterials for bone tissue engineering. In this work, by utilizing the controllable piezoelectric properties of polylactic acid materials, we prepared biomimetic piezoelectric poly (L-lactic acid) nanofibers and its non-piezoelectric isomer poly (D, l-lactide) nanofibers using the electrospinning technology. The dynamic piezoelectric and mechanical stimuli on the nanofibers were theoretically and experimentally investigated. A home-made in-situ mechanical loading platform was used to apply predetermined tensile loading on the nanofibers under cell culture conditions. The results indicated that piezo-potential ranging from 11 to 103 μV could be produced by cell adhesion forces varied between 1 and 15 nN, while mechanical loading ranging from 1 % to 15 % strain resulted in piezoelectrical signals in the magnitude of several volts. These piezoelectric signals could influence cell adhesion through enhancing cell aspect ratio and cell area. During the process of cell contraction and re-spreading caused by the mechanical loading, the piezoelectric signals were capable of accelerating the recovery of spreading and adhesion. Furthermore, individual piezoelectric and mechanical signals could modulate intracellular calcium signals, and significantly promote osteogenic differentiation of MSCs. Specifically, the mechanical stimulation could activate PIEZO signaling pathway, resulting in a greater upregulation of early osteogenic differentiation markers of Collagen type I and Bone morphogenetic protein-2 compared to the piezoelectric signal. However, the combination of the two stimuli could obviously and effectively enhance osteogenic differentiation of MSCs in a synergistic manner. These findings offer fresh understandings regarding the electromechanical influences in the process of bone tissue regeneration, which afford additional possibilities for optimizing piezoelectric biomaterials for biomedical applications in a more reasonable manner.

Piezoelectric nanogenerators enabled neuromodulation rescued dopaminergic neuron loss in Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disease with dopaminergic neurons loss, which induces dopamine reduction and body motion disorder. Traditional deep brain stimulation (DBS), which involves the implanting metal electrodes into the subcortical nucleus of the brain, has a huge risk of invasive surgery and low resolution. Here we present that pomegranate-like piezoelectric nanogenerators are able to efficiently transduce ultrasonic wave into electric power generation under ultrasound stimulation, which modulates intracellular calcium signaling and tyrosine hydroxylase levels to activate the neuron activity and process of deep-brain tissue in vitro and in vivo. Strikingly, after one-week nanogenerator-mediated neuromodulation, the damaged dopaminergic neurons in PD-zebrafish and mice were reversed with a decrease in α-synuclein aggregates and an increase in tyrosine hydroxylase for neuronal recovery of PD. The behaviors of PD mice model also have a significant improvement, compared with untreated group. This approach provides a minimally invasive and high precision neurotechnology protect against neuronal death and loss of synapse in PD and other neurodegenerative diseases.

Platelet-activating factor and protease-activated receptor 2 cooperate to promote neutrophil recruitment and lung inflammation through nuclear factor-kappa B transactivation

Although it is well established that platelet-activated receptor (PAF) and protease-activated receptor 2 (PAR2) play a pivotal role in the pathophysiology of lung and airway inflammatory diseases, a role for a PAR2-PAFR cooperation in lung inflammation has not been investigated. Here, we investigated the role of PAR2 in PAF-induced lung inflammation and neutrophil recruitment in lungs of BALB/c mice. Mice were pretreated with the PAR2 antagonist ENMD1068, PAF receptor (PAFR) antagonist WEB2086, or aprotinin prior to intranasal instillation of carbamyl-PAF (C-PAF) or the PAR2 agonist peptide SLIGRL-NH2 (PAR2-AP). Leukocyte infiltration in bronchoalveolar lavage fluid (BALF), C-X-C motif ligand 1 (CXCL)1 and CXCL2 chemokines, myeloperoxidase (MPO), and N-acetyl-glycosaminidase (NAG) levels in BALF, or lung inflammation were evaluated. Intracellular calcium signaling, PAFR/PAR2 physical interaction, and the expression of PAR2 and nuclear factor-kappa B (NF-КB, p65) transcription factor were investigated in RAW 264.7 cells stimulated with C-PAF in the presence or absence of ENMD1068. C-PAF- or PAR2-AP-induced neutrophil recruitment into lungs was inhibited in mice pretreated with ENMD1068 and aprotinin or WEB2086, respectively. PAR2 blockade impaired C-PAF-induced neutrophil rolling and adhesion, lung inflammation, and production of MPO, NAG, CXCL1, and CXCL2 production in lungs of mice. PAFR activation reduced PAR2 expression and physical interaction of PAR2 and PAFR; co-activation is required for PAFR/PAR2 physical interaction. PAR2 blockade impaired C-PAF-induced calcium signal and NF-κB p65 translocation in RAW 264.7 murine macrophages. This study provides the first evidence for a cooperation between PAFR and PAR2 mediating neutrophil recruitment, lung inflammation, and macrophage activation.

Human neutrophil extracellular traps do not impair in vitro Toxoplasma gondii infection.

Toxoplasma gondii, responsible for causing toxoplasmosis, is a prevalent food and waterborne pathogen worldwide. It commonly infects warm-blooded animals and affects more than a third of the global human population. Once ingested, the parasite enters the host's small intestine and rapidly disseminates throughout the body via the bloodstream, infiltrating various tissues. Leukocyte-driven responses are vital against T. gondii, with neutrophils playing a dual role: swiftly recruited to infection sites, releasing inflammatory mediators, and serving as a replication hub and Trojan horses, aiding parasite spread. Neutrophils from various hosts release extracellular traps (NETs) against the protozoan. However, gaps persist regarding the mechanisms of NETs production to parasite and their significance in infection control. This study investigates the interplay between human neutrophils and T. gondii, exploring dynamics, key molecules, and signaling pathways involved in NETs production upon protozoan challenge. Using confocal and electron microscopy, live cell imaging, pharmacological inhibitors, and DNA quantification assays, we find that human neutrophils promptly release both classical and rapid NETs upon pathogen stimulation. The NETs structure exhibits diverse phenotypes over time and is consistently associated with microorganisms. Mechanisms involve neutrophil elastase and peptidylarginine deiminase, along with intracellular calcium signaling and the PI3K pathway. Unexpectedly, human traps do not diminish viability or infectivity, but potentially aid in capturing parasites for subsequent neutrophil phagocytosis and elimination. By revealing NETs formation mechanisms and their nuanced impact on T. gondii infection dynamics, our findings contribute to broader insights into host-pathogen relationships.

Effects of Acute and Chronic Gabapentin Treatment on Cardiovascular Function of Rats.

Gabapentin (GBP), a GABA analogue, is primarily used as an anticonvulsant for the treatment of partial seizures and neuropathic pain. Whereas a majority of the side effects are associated with the nervous system, emerging evidence suggests there is a high risk of heart diseases in patients taking GBP. In the present study, we first used a preclinical model of rats to investigate, firstly, the acute cardiovascular responses to GBP (bolus i.v. injection, 50 mg/kg) and secondly the effects of chronic GBP treatment (i.p. 100 mg/kg/day × 7 days) on cardiovascular function and the myocardial proteome. Under isoflurane anesthesia, rat blood pressure (BP), heart rate (HR), and left ventricular (LV) hemodynamics were measured using Millar pressure transducers. The LV myocardium and brain cortex were analyzed by proteomics, bioinformatics, and western blot to explore the molecular mechanisms underlying GBP-induced cardiac dysfunction. In the first experiment, we found that i.v. GBP significantly decreased BP, HR, maximal LV pressure, and maximal and minimal dP/dt, whereas it increased IRP-AdP/dt, Tau, systolic, diastolic, and cycle durations (* p < 0.05 and ** p < 0.01 vs. baseline; n = 4). In the second experiment, we found that chronic GBP treatment resulted in hypotension, bradycardia, and LV systolic dysfunction, with no change in plasma norepinephrine. In the myocardium, we identified 109 differentially expressed proteins involved in calcium pathways, cholesterol metabolism, and galactose metabolism. Notably, we found that calmodulin, a key protein of intracellular calcium signaling, was significantly upregulated by GBP in the heart but not in the brain. In summary, we found that acute and chronic GBP treatments suppressed cardiovascular function in rats, which is attributed to abnormal calcium signaling in cardiomyocytes. These data reveal a novel side effect of GBP independent of the nervous system, providing important translational evidence to suggest that GBP can evoke adverse cardiovascular events by depression of myocardial function.

Mechanistic basis of ligand efficacy in the calcium-activated chloride channel TMEM16A.

Agonist binding in ligand-gated ion channels is coupled to structural rearrangements around the binding site, followed by the opening of the channel pore. In this process, agonist efficacy describes the equilibrium between open and closed conformations in a fully ligand-bound state. Calcium-activated chloride channels in the TMEM16 family are important sensors of intracellular calcium signals and are targets for pharmacological modulators, yet a mechanistic understanding of agonist efficacy has remained elusive. Using a combination of cryo-electron microscopy, electrophysiology, and autocorrelation analysis, we now show that agonist efficacy in the ligand-gated channel TMEM16A is dictated by the conformation of the pore-lining helix α6 around the Ca2+ -binding site. The closure of the binding site, which involves the formation of a π-helix below a hinge region in α6, appears to be coupled to the opening of the inner pore gate, thereby governing the channel's open probability and conductance. Our results provide a mechanism for agonist binding and efficacy and a structural basis for the design of potentiators and partial agonists in the TMEM16 family.

Targeting altered calcium homeostasis and uncoupling protein-2 promotes sensitivity in drug-resistant breast cancer cells.

Metastatic breast cancer has the highest mortality rate among women owing to its poor clinical outcomes. Metastatic tumors pose challenges for treatment through conventional surgery or radiotherapy because of their diverse organ localization and resistance to various cytotoxic agents. Chemoresistance is a significant obstacle to effective breast cancer treatment owing to cancer's heterogeneous nature. Abnormalities in intracellular calcium signaling, coupled with altered mitochondrial metabolism, play a significant role in facilitating drug resistance and contribute to therapy resistance. Uncoupling protein-2 (UCP2) is considered as a marker of chemoresistance and is believed to play a major role in promoting metabolic shifts and tumor metastasis. In this context, it is imperative to understand the roles of altered calcium signaling and metabolic switching in the development of chemotherapeutic resistance. This study investigates the roles of UCP2 and intracellular calcium signaling (Ca2+ ) in promoting chemoresistance against cisplatin. Additionally, we explored the effectiveness of combining genipin (GP, a compound that reverses UCP2-mediated chemoresistance) and thapsigargin (TG, a calcium signaling modulator) in treating highly metastatic breast cancers. Our findings indicate that both aberrant Ca2+ signaling and metabolic shifts in cancer cells contribute to developing drug-resistant phenotypes, and the combination treatment of GP and TG significantly enhances drug sensitivity in these cells. Collectively, our study underscores the potential of these drug combinations as an effective approach to overcome drug resistance in chemoresistant cancers.

Neuronal Calcium Sensor 1: a Zinc/Redox-Dependent Protein of Nervous System Signaling Pathways

Intracellular calcium signals play a key role in the regulation of nervous system structure and function. The control of neuronal excitability and plasticity by calcium ions underlies the mechanisms of higher nervous activity, and the mechanisms of this control are of particular interest to researchers. In recent decades, a family of highly specialized neuronal proteins that can translate the information contained in calcium signals into the regulation of channels, enzymes, receptors, and transcription factors has been described. The most abundant member of this family is neuronal calcium sensor-1 (NCS-1), which is intensively expressed in CNS neurons and controls such vital processes as neuronal growth and survival, reception, neurotransmission and synaptic plasticity. In addition to calcium ions, NCS-1 may bind intracellular ‘mobile’ zinc, increased concentration of which is a characteristic feature of cells under oxidative stress. Zinc coordination under these conditions stimulates NCS-1 oxidation to form a disulfide dimer (dNCS-1) with altered functional properties. The combined effect of mobile zinc and the increased redox potential of the medium can thus induce the aberrant NCS-1 activity involving signals for survival of neuronal cells or induction of their apoptosis and, as a consequence, the development of neurodegenerative processes. The review details the localization, regulation of expression, structure and molecular properties of NCS-1, as well as current data on its signaling activity in health and disease, including zinc-dependent redox-regulation cascades.