Systematic Review of Calcium Channels and Intracellular Calcium Signaling: Relevance to Pesticide Neurotoxicity

Pesticides of different chemical classes exert their toxic effects on the nervous system by acting on the different regulatory mechanisms of calcium (Ca²⁺) homeostasis. Pesticides have been shown to alter Ca²⁺ homeostasis, mainly by increasing its intracellular concentration above physiological levels. The pesticide-induced Ca²⁺ overload occurs through two main mechanisms: the entry of Ca²⁺ from the extracellular medium through the different types of Ca²⁺ channels present in the plasma membrane or its release into the cytoplasm from intracellular stocks, mainly from the endoplasmic reticulum. It has also been observed that intracellular increases in the Ca²⁺ concentrations are maintained over time, because pesticides inhibit the enzymes involved in reducing its levels. Thus, the alteration of Ca²⁺ levels can lead to the activation of various signaling pathways that generate oxidative stress, neuroinflammation and, finally, neuronal death. In this review, we also discuss some proposed strategies to counteract the detrimental effects of pesticides on Ca²⁺ homeostasis.     

Two-Dimensional Interfacial Exchange Diffusion Has the Potential to Augment Spatiotemporal Precision of Ca2+ Signaling

Nano-junctions between the endoplasmic reticulum and cytoplasmic surfaces of the plasma membrane and other organelles shape the spatiotemporal features of biological Ca²⁺ signals. Herein, we propose that 2D Ca²⁺ exchange diffusion on the negatively charged phospholipid surface lining nano-junctions participates in guiding Ca²⁺ from its source (channel or carrier) to its target (transport protein or enzyme). Evidence provided by in vitro Ca²⁺ flux experiments using an artificial phospholipid membrane is presented in support of the above proposed concept, and results from stochastic simulations of Ca²⁺ trajectories within nano-junctions are discussed in order to substantiate its possible requirements. Finally, we analyze recent literature on Ca²⁺ lipid interactions, which suggests that 2D interfacial Ca²⁺ diffusion may represent an important mechanism of signal transduction in biological systems characterized by high phospholipid surface to aqueous volume ratios.     

Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution

The GTP-binding protein-coupled receptors (GPCRs) play important roles in physiology and neuronal signaling. More than a thousand genes, excluding the olfactory receptors, have been identified that encode these integral membrane proteins. Their pharmacological and functional properties make them fascinating targets for drug development, since various disease states can be treated and overcome by pharmacologically addressing these receptors and/or their downstream interacting partners. The activation of the GPCRs typically causes transient changes in the intracellular second messenger concentrations as well as in membrane conductance. In contrast to ion channel-mediated electrical signaling which results in spontaneous cellular responses, the GPCR-mediated metabotropic signals operate at a different time scale. Here we have studied the kinetics of two common GPCR-induced signaling pathways: (a) Ca²⁺ release from intracellular stores and (b) cyclic adenosine monophosphate (cAMP) production. The latter was monitored via the activation of cyclic nucleotide-gated (CNG) ion channels causing Ca²⁺ influx into the cell. Genetically modified and stably transfected cell lines were established and used in stopped-flow experiments to uncover the individual steps of the reaction cascades. Using two homologous biogenic amine receptors, either coupling to G_o/q or G_s proteins, allowed us to determine the time between receptor activation and signal output. With ~350 ms, the release of Ca²⁺ from intracellular stores was much faster than cAMP-mediated Ca²⁺ entry through CNG channels (~6 s). The measurements with caged compounds suggest that this difference is due to turnover numbers of the GPCR downstream effectors rather than the different reaction cascades, per se.     

Crosstalk among Calcium ATPases: PMCA,SERCA and SPCA in Mental Diseases

Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca²⁺ signal is well-known to trigger a cascade of events leading to altered response to variety of stimuli and persistent accumulation of pathological changes at the molecular level. To counterbalance potentially detrimental consequences of Ca²⁺, neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca²⁺-ATPase (PMCA), sarco/endoplasmic Ca²⁺-ATPase (SERCA) and secretory pathway Ca²⁺-ATPase (SPCA) are considered efficient line of defense against abnormal Ca²⁺ rises. However, their role is not limited only to Ca²⁺ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein—calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca²⁺-ATPases to neuropathology, with a special emphasis on mental diseases.     

Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca²⁺ imaging, and computational modeling, we evaluated the effects of S1P on the Ca²⁺-activated K⁺ currents (I_K(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BK_Ca channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca²⁺ concentration (Ca_i) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BK_Ca was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed I_K(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of I_K(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced I_K(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BK_Ca conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited I_K(Ca) and the permissive role of S1P on the BK_Ca activity was also effectively observed in the PC12 cell system. The S1P-mediated I_K(Ca) stimulation may result from the elevated Ca_i, whereas the inhibition of BK_Ca activity by S1P appears to be direct. By the differentiated tailoring BK_Ca channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.     

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKβ/AMPK Signaling Pathway in Human Endothelial Cells

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca²⁺ concentration and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and Ca²⁺/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca²⁺ concentration and phosphorylation of CaMKII, CaMKKβ, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKβ inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca²⁺/CaMKII and CaM/CaMKKβ/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.     

Intracellular Ca2+-Mediated AE2 Is Involved in the Vectorial Movement of HaCaT Keratinocyte

Keratinocyte migration is initiated toward the wound skin barrier as a crucial process in wound healing. However, the migratory machinery used by keratinocytes is relatively unknown. Histamine signaling, including an increase in the Ca²⁺ signal, mediated the enhanced protein expression and chloride/bicarbonate exchange activity of anion exchanger AE2 in keratinocytes. In this study, we applied an agarose spot assay to induce a vectorial motion. The vectorial stimulation of the histamine-containing agarose spot enhanced the HaCaT keratinocyte migration, compared to non-directional stimulation. AE2 is associated with the vectorial movement of HaCaT keratinocytes. Enhanced expression of AE2 was mainly associated with an increase in Ca²⁺ and was abolished by the treatment with the Ca²⁺ chelating agent BAPTA-AM. These findings revealed that the directionality of Ca²⁺-exerted stimulation can play a prominent role in facilitating migration through the involvement of AE2 as a migratory machinery in HaCaT keratinocytes.     

Signaling Pathways Used by the Specialized Pro-Resolving Mediator Maresin 2 Regulate Goblet Cell Function: Comparison with Maresin 1

Specialized pro-resolving mediators (SPMs), including Maresins (MaR)-1 and 2, contribute to tear film homeostasis and resolve conjunctival inflammation. We investigated MaR2′s signaling pathways in goblet cells (GC) from rat conjunctiva. Agonist-induced [Ca²⁺]_i and high-molecular weight glycoconjugate secretion were measured. MaR2 increased [Ca²⁺]_i and stimulated secretion. MaR2 and MaR1 stimulate conjunctival goblet cell function, especially secretion, by activating different but overlapping GPCR and signaling pathways, and furthermore counter-regulate histamine stimulated increase in [Ca²⁺]_i. Thus, MaR2 and MaR1 play a role in maintaining the ocular surface and tear film homeostasis in health and disease. As MaR2 and MaR1 modulate conjunctival goblet cell function, they each may have potential as novel, but differing, options for the treatment of ocular surface inflammatory diseases including allergic conjunctivitis and dry eye disease. We conclude that in conjunctival GC MaR2 and MaR1, both increase the [Ca²⁺]_i and stimulate secretion to maintain homeostasis by using one set of different, but overlapping, signaling pathways to increase [Ca²⁺]_i and another set to stimulate secretion. MaR2 also resolves ocular allergy.     

The p53 and Calcium Regulated Actin Rearrangement in Model Cells

Long-term cellular stress maintains high intracellular Ca²⁺ concentrations which ultimately initiates apoptosis. Our interest is focused on how the gelsolin (GSN) and junctional mediating and regulating Y protein (JMY) play important roles in stress response. Both of these proteins can bind p53 and actin. We investigated using in vitro fluorescence spectroscopy and found that the p53 competes with actin in GSN to inhibit p53–JMY complex formation. A high Ca²⁺ level initializes p53 dimerization; the dimer competes with actin on JMY, which can lead to p53–JMY cotransport into the nucleus. Here we investigated how the motility and division rate of HeLa cells changes due to low-voltage electroporation of GSN or JMY in scratching assays. We revealed that JMY inhibits their motion, but that it can accelerate the cell division. GSN treatment slows down cell division but does not affect cell motility. HeLa cells fully recovered the gap 20 h after the electroporation with JMY and then started to release from the glass slides. Taken together, our in vitro results indicate that GSN and JMY may play an important role in the cellular stress response.     

Signaling Transduction of ABA,ROS, and Ca2+ in Plant Stomatal Closure in Response to Drought

Drought is a global threat that affects agricultural production. Plants have evolved several adaptive strategies to cope with drought. Stomata are essential structures for plants to control water status and photosynthesis rate. Stomatal closure is an efficient way for plants to reduce water loss and improve survivability under drought conditions. The opening and closure of stomata depend on the turgor pressure in guard cells. Three key signaling molecules, including abscisic acid (ABA), reactive oxygen species (ROS), and calcium ion (Ca²⁺), play pivotal roles in controlling stomatal closure. Plants sense the water-deficit signal mainly via leaves and roots. On the one hand, ABA is actively synthesized in root and leaf vascular tissues and transported to guard cells. On the other hand, the roots sense the water-deficit signal and synthesize CLAVATA3/EMBRYO-SURROUNDING REGION RELATED 25 (CLE25) peptide, which is transported to the guard cells to promote ABA synthesis. ABA is perceived by pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) receptors, which inactivate PP2C, resulting in activating the protein kinases SnRK2s. Many proteins regulating stomatal closure are activated by SnRK2s via protein phosphorylation. ABA-activated SnRK2s promote apoplastic ROS production outside of guard cells and transportation into the guard cells. The apoplastic H₂O₂ can be directly sensed by a receptor kinase, HYDROGEN PEROXIDE-INDUCED CA²⁺ INCREASES1 (HPCA1), which induces activation of Ca²⁺ channels in the cytomembrane of guard cells, and triggers an increase in Ca²⁺ in the cytoplasm of guard cells, resulting in stomatal closure. In this review, we focused on discussing the signaling transduction of ABA, ROS, and Ca²⁺ in controlling stomatal closure in response to drought. Many critical genes are identified to have a function in stomatal closure under drought conditions. The identified genes in the process can serve as candidate genes for genetic engineering to improve drought resistance in crops. The review summarizes the recent advances and provides new insights into the signaling regulation of stomatal closure in response to water-deficit stress and new clues on the improvement of drought resistance in crops.     

Oxidative Stress and Immune Response in Melanoma: Ion Channels as Targets of Therapy

Oxidative stress and immune response play an important role in the development of several cancers, including melanoma. Ion channels are aberrantly expressed in tumour cells and regulate neoplastic transformation, malignant progression, and resistance to therapy. Ion channels are localized in the plasma membrane or other cellular membranes and are targets of oxidative stress, which is particularly elevated in melanoma. At the same time, ion channels are crucial for normal and cancer cell physiology and are subject to multiple layers of regulation, and therefore represent promising targets for therapeutic intervention. In this review, we analyzed the effects of oxidative stress on ion channels on a molecular and cellular level and in the context of melanoma progression and immune evasion. The possible role of ion channels as targets of alternative therapeutic strategies in melanoma was discussed.     

Effects of dissolved calcium and magnesium ions on lead-induced stress corrosion cracking susceptibility of nuclear steam generator tubing alloy in high temperature crevice solutions

The effects of Ca²⁺ and Mg²⁺ ions on the stress corrosion cracking (SCC) susceptibility of UNS N08800 are investigated using constant extension rate tensile (CERT) tests at 300 °C in simulated crevice chemistries. The presence of lead contamination in the crevice chemistries increases significantly the SCC susceptibility of the alloy. The lead-assisted SCC (PbSCC) susceptibility is reduced markedly by the addition of Ca²⁺ and Mg²⁺ ions into the solution and this mitigating effect is enhanced by increasing the total concentration of Ca²⁺ + Mg²⁺. The CERT test results are consistent with the types of fracture surfaces shown by Scanning Electron Microscopy (SEM). There is a reasonable correlation between the SCC susceptibility and the donor densities in the anodic films in accord with the role of lead-induced passivity degradation in PbSCC.     

The Role of CaMKII and ERK Signaling in Addiction

Nicotine is the predominant addictive compound of tobacco and causes the acquisition of dependence through its interactions with nicotinic acetylcholine receptors and various neurotransmitter releases in the central nervous system. The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) play a pivotal role in synaptic plasticity in the hippocampus. CaMKII is involved in long-term potentiation induction, which underlies the consolidation of learning and memory; however, the roles of CaMKII in nicotine and other psychostimulant-induced addiction still require further investigation. This article reviews the molecular mechanisms and crucial roles of CaMKII and ERK in nicotine and other stimulant drug-induced addiction. We also discuss dopamine (DA) receptor signaling involved in nicotine-induced addiction in the brain reward circuitry. In the last section, we introduce the association of polyunsaturated fatty acids and cellular chaperones of fatty acid-binding protein 3 in the context of nicotine-induced addiction in the mouse nucleus accumbens and provide a novel target for the treatment of drug abuse affecting dopaminergic systems.     

IRAG2 Interacts with IP3-Receptor Types 1, 2, and 3 and Regulates Intracellular Ca2+ in Murine Pancreatic Acinar Cells

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP₃ receptors /IP₃R) via its coiled-coil domain and modulates Ca²⁺ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca²⁺ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP₃ receptors and its impact on intracellular Ca²⁺ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP₃R and altered Ca²⁺ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca²⁺, suggesting that IRAG2 leads to activation of IP₃R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca²⁺ signaling, which regulates exocrine pancreatic function.     

Effect of Metformin on T2D-Induced MAM Ca2+ Uncoupling and Contractile Dysfunction in an Early Mouse Model of Diabetic HFpEF

Diabetic cardiomyopathy (DCM) is a leading complication in type 2 diabetes patients. Recently, we have shown that the reticulum-mitochondria Ca²⁺ uncoupling is an early and reversible trigger of the cardiac dysfunction in a diet-induced mouse model of DCM. Metformin is a first-line antidiabetic drug with recognized cardioprotective effect in myocardial infarction. Whether metformin could prevent the progression of DCM remains not well understood. We therefore investigated the effect of a chronic 6-week metformin treatment on the reticulum-mitochondria Ca²⁺ coupling and the cardiac function in our high-fat high-sucrose diet (HFHSD) mouse model of DCM. Although metformin rescued the glycemic regulation in the HFHSD mice, it did not preserve the reticulum-mitochondria Ca²⁺ coupling either structurally or functionally. Metformin also did not prevent the progression towards cardiac dysfunction, i.e., cardiac hypertrophy and strain dysfunction. In summary, despite its cardioprotective role, metformin is not sufficient to delay the progression to early DCM.     

Calmodulin and Its Binding Proteins in Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder that manifests with rest tremor, muscle rigidity and movement disturbances. At the microscopic level it is characterized by formation of specific intraneuronal inclusions, called Lewy bodies (LBs), and by a progressive loss of dopaminergic neurons in the striatum and substantia nigra. All living cells, among them neurons, rely on Ca²⁺ as a universal carrier of extracellular and intracellular signals that can initiate and control various cellular processes. Disturbances in Ca²⁺ homeostasis and dysfunction of Ca²⁺ signaling pathways may have serious consequences on cells and even result in cell death. Dopaminergic neurons are particularly sensitive to any changes in intracellular Ca²⁺ level. The best known and studied Ca²⁺ sensor in eukaryotic cells is calmodulin. Calmodulin binds Ca²⁺ with high affinity and regulates the activity of a plethora of proteins. In the brain, calmodulin and its binding proteins play a crucial role in regulation of the activity of synaptic proteins and in the maintenance of neuronal plasticity. Thus, any changes in activity of these proteins might be linked to the development and progression of neurodegenerative disorders including PD. This review aims to summarize published results regarding the role of calmodulin and its binding proteins in pathology and pathogenesis of PD.     

Spatial and Functional Crosstalk between the Mitochondrial Na+-Ca2+ Exchanger NCLX and the Sarcoplasmic Reticulum Ca2+ Pump SERCA in Cardiomyocytes

The mitochondrial Na⁺-Ca²⁺ exchanger, NCLX, was reported to supply Ca²⁺ to sarcoplasmic reticulum (SR)/endoplasmic reticulum, thereby modulating various cellular functions such as the rhythmicity of cardiomyocytes, and cellular Ca²⁺ signaling upon antigen receptor stimulation and chemotaxis in B lymphocytes; however, there is little information on the spatial relationships of NCLX with SR Ca²⁺ handling proteins, and their physiological impact. Here we examined the issue, focusing on the interaction of NCLX with an SR Ca²⁺ pump SERCA in cardiomyocytes. A bimolecular fluorescence complementation assay using HEK293 cells revealed that the exogenously expressed NCLX was localized in close proximity to four exogenously expressed SERCA isoforms. Immunofluorescence analyses of isolated ventricular myocytes showed that the NCLX was localized to the edges of the mitochondria, forming a striped pattern. The co-localization coefficients in the super-resolution images were higher for NCLX–SERCA2, than for NCLX–ryanodine receptor and NCLX–Na⁺/K⁺ ATPase α-1 subunit, confirming the close localization of endogenous NCLX and SERCA2 in cardiomyocytes. The mathematical model implemented with the spatial and functional coupling of NCLX and SERCA well reproduced the NCLX inhibition-mediated modulations of SR Ca²⁺ reuptake in HL-1 cardiomyocytes. Taken together, these results indicated that NCLX and SERCA are spatially and functionally coupled in cardiomyocytes.