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.     

Gentamicin Blocks the ACh-Induced BK Current in Guinea Pig Type II Vestibular Hair Cells by Competing with Ca2+ at the l-Type Calcium Channel

Type II vestibular hair cells (VHCs II) contain big-conductance Ca²⁺-dependent K⁺ channels (BK) and l-type calcium channels. Our previous studies in guinea pig VHCs II indicated that acetylcholine (ACh) evoked the BK current by triggering the influx of Ca²⁺ ions through l-type Ca²⁺ channels, which was mediated by M2 muscarinic ACh receptor (mAChRs). Aminoglycoside antibiotics, such as gentamicin (GM), are known to have vestibulotoxicity, including damaging effects on the efferent nerve endings on VHCs II. This study used the whole-cell patch clamp technique to determine whether GM affects the vestibular efferent system at postsynaptic M2-mAChRs or the membrane ion channels. We found that GM could block the ACh-induced BK current and that inhibition was reversible, voltage-independent, and dose-dependent with an IC₅₀ value of 36.3 ± 7.8 μM. Increasing the ACh concentration had little influence on GM blocking effect, but increasing the extracellular Ca²⁺ concentration ([Ca²⁺]_o) could antagonize it. Moreover, 50 μM GM potently blocked Ca²⁺ currents activated by (−)-Bay-K8644, but did not block BK currents induced by NS1619. These observations indicate that GM most likely blocks the M2 mAChR-mediated response by competing with Ca²⁺ at the l-type calcium channel. These results provide insights into the vestibulotoxicity of aminoglycoside antibiotics on mammalian VHCs II.     

NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD⁺ on: (1) the Ca²⁺-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca²⁺-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca²⁺-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca²⁺-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD⁺ increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca²⁺-uptake and the free Ca²⁺ concentration in the medium. NAD(H) suppressed the Ca²⁺-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca²⁺-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.     

Glutathionylation of the L-type Ca2+ Channel in Oxidative Stress-Induced Pathology of the Heart

There is mounting evidence to suggest that protein glutathionylation is a key process contributing to the development of pathology. Glutathionylation occurs as a result of posttranslational modification of a protein and involves the addition of a glutathione moiety at cysteine residues. Such modification can occur on a number of proteins, and exerts a variety of functional consequences. The L-type Ca²⁺ channel has been identified as a glutathionylation target that participates in the development of cardiac pathology. Ca²⁺ influx via the L-type Ca²⁺ channel increases production of mitochondrial reactive oxygen species (ROS) in cardiomyocytes during periods of oxidative stress. This induces a persistent increase in channel open probability, and the resulting constitutive increase in Ca²⁺ influx amplifies the cross-talk between the mitochondria and the channel. Novel strategies utilising targeted peptide delivery to uncouple mitochondrial ROS and Ca²⁺ flux via the L-type Ca²⁺ channel following ischemia-reperfusion have delivered promising results, and have proven capable of restoring appropriate mitochondrial function in myocytes and in vivo.     

Mitochondrial Protection and Anti-aging Activity of Astragalus Polysaccharides and Their Potential Mechanism

The current study was performed to investigate mitochondrial protection and anti-aging activity of Astragalus polysaccharides (APS) and the potential underlying mechanism. Lipid peroxidation of liver and brain mitochondria was induced by Fe²⁺–Vit C in vitro. Thiobarbituric acid (TBA) colorimetry was used to measure the content of thiobarbituric acid reactive substances (TBARS). Mouse liver mitochondrial permeability transition (PT) was induced by calcium overload in vitro and spectrophotometry was used to measure it. The scavenging activities of APS on superoxide anion (O₂^•−) and hydroxyl radical (•OH), which were produced by reduced nicotinamide adenine dinucleotide (NADH)—N-Methylphenazonium methyl sulfate (PMS) and hydrogen peroxide (H₂O₂)–Fe²⁺ system respectively, were measured by 4-nitrobluetetrazolium chloride (NBT) reduction and Fenton reaction colorimetry respectively. The Na₂S₂O₃ titration method was used to measure the scavenging activities of APS on H₂O₂. APS could inhibit TBARS production, protect mitochondria from PT, and scavenge O₂^•−, •OH and H₂O₂ significantly in a concentration-dependent manner respectively. The back of the neck of mice was injected subcutaneously with D-galactose to induce aging at a dose of 100 mg/kg/d for seven weeks. Moreover, the activities of catalase (CAT), surperoxide dismutase (SOD) and glutathione peroxidase (GPx) and anti-hydroxyl radical which were assayed by using commercial monitoring kits were increased significantly in vivo by APS. According to this research, APS protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting mitochondrial PT and increasing the activities of antioxidases. Therefore, APS has the effect of promoting health.     

Dynamin-Related Protein 1 Inhibitors Protect against Ischemic Toxicity through Attenuating Mitochondrial Ca2+ Uptake from Endoplasmic Reticulum Store in PC12 Cells

Intracellular calcium homeostasis disorder and mitochondrial dysfunction are involved in many acute and chronic brain diseases, including ischemic brain injury. An imbalance in mitochondrial fission and fusion is one of the most important structural abnormalities found in a large number of mitochondrial dysfunction related diseases. Here, we investigated the effects of mitochondrial division inhibitor A (mdivi A) and mdivi B, two small molecule inhibitors of mitochondrial fission protein dunamin-related protein 1 (Drp-1), in neuronal injury induced by oxygen-glucose deprivation (OGD) in PC12 cells. We found that mdivi A and mdivi B inhibited OGD-induced neuronal injury through attenuating apoptotic cell death. These two inhibitors also preserved mitochondrial function, as evidenced by reduced reactive oxygen species (ROS) generation and cytochrome c release, as well as prevented loss of mitochondrial membrane potential (MMP). Moreover, mdivi A and mdivi B significantly suppressed mitochondrial Ca²⁺ uptake, but had no effect on cytoplasmic Ca²⁺ after OGD injury. The results of calcium imaging and immunofluorescence staining showed that Drp-1 inhibitors attenuated endoplasmic reticulum (ER) Ca²⁺ release and prevented ER morphological changes induced by OGD. These results demonstrate that Drp-1 inhibitors protect against ischemic neuronal injury through inhibiting mitochondrial Ca²⁺ uptake from the ER store and attenuating mitochondrial dysfunction.     

Mitochondrial Ca2+ Dynamics in MCU Knockout C. elegans Worms

Mitochondrial [Ca²⁺] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca²⁺ overload. This regulation depends on Ca²⁺ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca²⁺ uniporter (MCU). Here, we have studied the mitochondrial Ca²⁺ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca²⁺ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca²⁺] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca²⁺ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca²⁺ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca²⁺ pathway.     

A Calcium Guard in the Outer Membrane: Is VDAC a Regulated Gatekeeper of Mitochondrial Calcium Uptake?

Already in the early 1960s, researchers noted the potential of mitochondria to take up large amounts of Ca²⁺. However, the physiological role and the molecular identity of the mitochondrial Ca²⁺ uptake mechanisms remained elusive for a long time. The identification of the individual components of the mitochondrial calcium uniporter complex (MCUC) in the inner mitochondrial membrane in 2011 started a new era of research on mitochondrial Ca²⁺ uptake. Today, many studies investigate mitochondrial Ca²⁺ uptake with a strong focus on function, regulation, and localization of the MCUC. However, on its way into mitochondria Ca²⁺ has to pass two membranes, and the first barrier before even reaching the MCUC is the outer mitochondrial membrane (OMM). The common opinion is that the OMM is freely permeable to Ca²⁺. This idea is supported by the presence of a high density of voltage-dependent anion channels (VDACs) in the OMM, forming large Ca²⁺ permeable pores. However, several reports challenge this idea and describe VDAC as a regulated Ca²⁺ channel. In line with this idea is the notion that its Ca²⁺ selectivity depends on the open state of the channel, and its gating behavior can be modified by interaction with partner proteins, metabolites, or small synthetic molecules. Furthermore, mitochondrial Ca²⁺ uptake is controlled by the localization of VDAC through scaffolding proteins, which anchor VDAC to ER/SR calcium release channels. This review will discuss the possibility that VDAC serves as a physiological regulator of mitochondrial Ca²⁺ uptake in the OMM.     

Electrophysiological Properties of Endogenous Single Ca2+ Activated Cl− Channels Induced by Local Ca2+ Entry in HEK293

Microdomains formed by proteins of endoplasmic reticulum and plasma membrane play a key role in store-operated Ca²⁺ entry (SOCE). Ca²⁺ release through inositol 1,4,5-trisphosphate receptor (IP₃R) and subsequent Ca²⁺ store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca²⁺, which, in turn, open the Orai channels in plasma membrane. Downstream to this process could be activated TRPC (transient receptor potential-canonical) calcium permeable channels. Using single channel patch-clamp technique we found that a local Ca²⁺ entry through TRPC1 channels activated endogenous Ca²⁺-activated chloride channels (CaCCs) with properties similar to Anoctamin6 (TMEM16F). Our data suggest that their outward rectification is based on the dependence from membrane potential of both the channel conductance and the channel activity: (1) The conductance of active CaCCs highly depends on the transmembrane potential (from 3 pS at negative potentials till 60 pS at positive potentials); (2) their activity (NPo) is enhanced with increasing Ca²⁺ concentration and/or transmembrane potential, conversely lowering of intracellular Ca²⁺ concentration reduced the open state dwell time; (3) CaCC amplitude is only slightly increased by intracellular Ca²⁺ concentration. Experiments with Ca²⁺ buffering by EGTA or BAPTA suggest close local arrangement of functional CaCCs and TRPC1 channels. It is supposed that Ca²⁺-activated chloride channels are involved in Ca²⁺ entry microdomains.     

The Orai Pore Opening Mechanism

Cell survival and normal cell function require a highly coordinated and precise regulation of basal cytosolic Ca²⁺ concentrations. The primary source of Ca²⁺ entry into the cell is mediated by the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Its action is stimulated in response to internal Ca²⁺ store depletion. The fundamental constituents of CRAC channels are the Ca²⁺ sensor, stromal interaction molecule 1 (STIM1) anchored in the endoplasmic reticulum, and a highly Ca²⁺-selective pore-forming subunit Orai1 in the plasma membrane. The precise nature of the Orai1 pore opening is currently a topic of intensive research. This review describes how Orai1 gating checkpoints in the middle and cytosolic extended transmembrane regions act together in a concerted manner to ensure an opening-permissive Orai1 channel conformation. In this context, we highlight the effects of the currently known multitude of Orai1 mutations, which led to the identification of a series of gating checkpoints and the determination of their role in diverse steps of the Orai1 activation cascade. The synergistic action of these gating checkpoints maintains an intact pore geometry, settles STIM1 coupling, and governs pore opening. We describe the current knowledge on Orai1 channel gating mechanisms and summarize still open questions of the STIM1–Orai1 machinery.     

Computational Analyses of the AtTPC1 (Arabidopsis Two-Pore Channel 1) Permeation Pathway

Two Pore Channels (TPCs) are cation-selective voltage- and ligand-gated ion channels in membranes of intracellular organelles of eukaryotic cells. In plants, the TPC1 subtype forms the slowly activating vacuolar (SV) channel, the most dominant ion channel in the vacuolar membrane. Controversial reports about the permeability properties of plant SV channels fueled speculations about the physiological roles of this channel type. TPC1 is thought to have high Ca²⁺ permeability, a conclusion derived from relative permeability analyses using the Goldman–Hodgkin–Katz (GHK) equation. Here, we investigated in computational analyses the properties of the permeation pathway of TPC1 from Arabidopsis thaliana. Using the crystal structure of AtTPC1, protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we identified a free energy minimum for Ca²⁺, but not for K⁺, at the luminal side next to the selectivity filter. Residues D269 and E637 coordinate in particular Ca²⁺ as demonstrated in in silico mutagenesis experiments. Such a Ca²⁺-specific coordination site in the pore explains contradicting data for the relative Ca²⁺/K⁺ permeability and strongly suggests that the Ca²⁺ permeability of SV channels is largely overestimated from relative permeability analyses. This conclusion was further supported by in silico electrophysiological studies showing a remarkable permeation of K⁺ but not Ca²⁺ through the open channel.     

TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts

TRPM7 plays an important role in cellular Ca²⁺, Zn²⁺ and Mg²⁺ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca²⁺ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg²⁺ ([Mg²⁺]_i), were reduced by elevated [Mg²⁺]_i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg²⁺]_i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca²⁺ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca²⁺ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca²⁺ uptake and as an independent Ca²⁺ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca²⁺ transport in amelogenesis.     

Intracellular Ca2+ Signaling in Protozoan Parasites: An Overview with a Focus on Mitochondria

Ca²⁺ signaling has been involved in controling critical cellular functions such as activation of proteases, cell death, and cell cycle control. The endoplasmatic reticulum plays a significant role in Ca²⁺ storage inside the cell, but mitochondria have long been recognized as a fundamental Ca²⁺ pool. Protozoan parasites such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi display a Ca²⁺ signaling toolkit with similarities to higher eukaryotes, including the participation of mitochondria in Ca²⁺-dependent signaling events. This review summarizes the most recent knowledge in mitochondrial Ca²⁺ signaling in protozoan parasites, focusing on the mechanism involved in mitochondrial Ca²⁺ uptake by pathogenic protists.     

Phenolic Compounds Cannabidiol,Curcumin and Quercetin Cause Mitochondrial Dysfunction and Suppress Acute Lymphoblastic Leukemia Cells

Anticancer activity of different phenols is documented, but underlying mechanisms remain elusive. Recently, we have shown that cannabidiol kills the cells of acute lymphoblastic leukemia (ALL) by a direct interaction with mitochondria, with their consequent dysfunction. In the present study, cytotoxic effects of several phenolic compounds against human the T-ALL cell line Jurkat were tested by means of resazurin-based metabolic assay. To unravel underlying mechanisms, mitochondrial membrane potential (∆Ψ_m) and [Ca²⁺]_m measurements were undertaken, and reactive oxygen species generation and cell death were evaluated by flow cytometry. Three out of eight tested phenolics, cannabidiol, curcumin and quercetin, which displayed a significant cytotoxic effect, also dissipated the ∆Ψ_m and induced a significant [Ca²⁺]_m increase, whereas inefficient phenols did not. Dissipation of the ∆Ψ_m by cannabidiol was prevented by cyclosporine A and reverted by Ru360, inhibitors of the permeation transition pore and mitochondrial Ca²⁺ uniporter, respectively. Ru360 prevented the phenol-induced [Ca²⁺]_m rise, but neither cyclosporine A nor Ru360 affected the curcumin- and quercetin-induced ∆Ψ_m depolarization. Ru360 impeded the curcumin- and cannabidiol-induced cell death. Thus, all three phenols exert their antileukemic activity via mitochondrial Ca²⁺ overload, whereas curcumin and quercetin suppress the metabolism of leukemic cells by direct mitochondrial uncoupling.     

Co-staining of KCa3.1 Channels in NSCLC Cells with a Small-Molecule Fluorescent Probe and Antibody-Based Indirect Immunofluorescence

The Ca²⁺ activated potassium channel 3.1 (K_Ca3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of K_Ca3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole-based BODIPY dye with a derivative of the K_Ca3.1 channel inhibitor senicapoc via linkers of different length. Patch-clamp experiments revealed the inhibition of K_Ca3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain K_Ca3.1 channels in non-small-cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre-incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2 . Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody-based indirect immunofluorescence yielded identical or very similar densities of stained K_Ca3.1 channels. However, co-staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel.     

Ginsenoside Rd Attenuates Mitochondrial Permeability Transition and Cytochrome c Release in Isolated Spinal Cord Mitochondria: Involvement of Kinase-Mediated Pathways

Ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. However, the effects of Rd on spinal cord mitochondrial dysfunction and underlying mechanisms are still obscure. In this study, we sought to investigate the in vitro effects of Rd on mitochondrial integrity and redox balance in isolated spinal cord mitochondria. We verified that Ca²⁺ dissipated the membrane potential, provoked mitochondrial swelling and decreased NAD(P)H matrix content, which were all attenuated by Rd pretreatment in a dose-dependent manner. In contrast, Rd was not able to inhibit Ca²⁺ induced mitochondrial hydrogen peroxide generation. The results of Western blot showed that Rd significantly increased the expression of p-Akt and p-ERK, but had no effects on phosphorylation of PKC and p38. In addition, Rd treatment significantly attenuated Ca²⁺ induced cytochrome c release, which was partly reversed by antagonists of Akt and ERK, but not p-38 inhibitor. The effects of bisindolylmaleimide, a PKC inhibitor, on Rd-induced inhibition of cytochrome c release seem to be at the level of its own detrimental activity on mitochondrial function. Furthermore, we also found that pretreatment with Rd in vivo (10 and 50 mg/kg) protected spinal cord mitochondria against Ca²⁺ induced mitochondrial membrane potential dissipation and cytochrome c release. It is concluded that Rd regulate mitochondrial permeability transition pore formation and cytochrome c release through protein kinases dependent mechanism involving activation of intramitochondrial Akt and ERK pathways.     

Augmented KCa2.3 Channel Feedback Regulation of Oxytocin Stimulated Uterine Strips from Nonpregnant Mice

Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant risk to the infant. Current tocolytic therapies aimed at suppressing premature uterine contractions are largely ineffective and cause serious side effects. Calcium (Ca²⁺) dependent contractions of uterine smooth muscle are physiologically limited by the opening of membrane potassium (K⁺) channels. Exploiting such inherent negative feedback mechanisms may offer new strategies to delay labor and reduce risk. Positive modulation of small conductance Ca²⁺-activated K⁺ (K_Ca2.3) channels with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), effectively decreases uterine contractions. This study investigates whether the receptor agonist oxytocin might solicit K_Ca2.3 channel feedback that facilitates CyPPA suppression of uterine contractions. Using isometric force myography, we found that spontaneous phasic contractions of myometrial tissue from nonpregnant mice were suppressed by CyPPA and, in the presence of CyPPA, oxytocin failed to augment contractions. In tissues exposed to oxytocin, depletion of internal Ca²⁺ stores with cyclopiazonic acid (CPA) impaired CyPPA relaxation, whereas blockade of nonselective cation channels (NSCC) using gadolinium (Gd³⁺) had no significant effect. Immunofluorescence revealed close proximity of K_Ca2.3 channels and ER inositol trisphosphate receptors (IP₃Rs) within myometrial smooth muscle cells. The findings suggest internal Ca²⁺ stores play a role in K_Ca2.3-dependent feedback control of uterine contraction and offer new insights for tocolytic therapies.     

Tamoxifen Sensitizes Acute Lymphoblastic Leukemia Cells to Cannabidiol by Targeting Cyclophilin-D and Altering Mitochondrial Ca2+ Homeostasis

Cytotoxic effects of cannabidiol (CBD) and tamoxifen (TAM) have been observed in several cancer types. We have recently shown that CBD primarily targets mitochondria, inducing a stable mitochondrial permeability transition pore (mPTP) and, consequently, the death of acute lymphoblastic leukemia (T-ALL) cells. Mitochondria have also been documented among cellular targets for the TAM action. In the present study we have demonstrated a synergistic cytotoxic effect of TAM and CBD against T-ALL cells. By measuring the mitochondrial membrane potential (ΔΨm), mitochondrial calcium ([Ca²⁺]_m) and protein-ligand docking analysis we determined that TAM targets cyclophilin D (CypD) to inhibit mPTP formation. This results in a sustained [Ca²⁺]_m overload upon the consequent CBD administration. Thus, TAM acting on CypD sensitizes T-ALL to mitocans such as CBD by altering the mitochondrial Ca²⁺ homeostasis.