Polycystin-2 Mediated Calcium Signalling in the Dictyostelium Model for Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, PKD1) and Polycystin-2 (PC2, PKD2) contain mutations. PC1 is a large membrane receptor that can interact and form a complex with the calcium-permeable cation channel PC2. This complex localizes to the plasma membrane, primary cilia and ER. Dysregulated calcium signalling and consequential alterations in downstream signalling pathways in ADPKD are linked to cyst formation and expansion; however, it is not completely understood how PC1 and PC2 regulate calcium signalling. We have studied Polycystin-2 mediated calcium signalling in the model organism Dictyostelium discoideum by overexpressing and knocking down the expression of the endogenous Polycystin-2 homologue, Polycystin-2. Chemoattractant-stimulated cytosolic calcium response magnitudes increased and decreased in overexpression and knockdown strains, respectively, and analysis of the response kinetics indicates that Polycystin-2 is a significant contributor to the control of Ca2+ responses. Furthermore, basal cytosolic calcium levels were reduced in Polycystin-2 knockdown transformants. These alterations in Ca2+ signalling also impacted other downstream Ca2+-sensitive processes including growth rates, endocytosis, stalk cell differentiation and spore viability, indicating that Dictyostelium is a useful model to study Polycystin-2 mediated calcium signalling.

Unraveling the significance of PPP1R1A gene in pancreatic ß-cell function: A study in INS-1 cells and human pancreatic islets.

BACKGROUND AND AIMS: The protein phosphatase 1 regulatory inhibitor subunit 1A (PPP1R1A) has been linked with insulin secretion and diabetes mellitus. Yet, its full significance in pancreatic ß-cell function remains unclear. This study aims to elucidate the role of the PPP1R1A gene in ß-cell biology using human pancreatic islets and rat INS-1 (832/13) cells. RESULTS: Disruption of Ppp1r1a in INS-1 cells was associated with reduced insulin secretion and impaired glucose uptake; however, cell viability, ROS, apoptosis or proliferation were intact. A significant downregulation of crucial ß-cell function genes such as Ins1, Ins2, Pcsk1, Cpe, Pdx1, Mafa, Isl1, Glut2, Snap25, Vamp2, Syt5, Cacna1a, Cacna1d and Cacnb3, was observed upon Ppp1r1a disruption. Furthermore, silencing Pdx1 in INS-1 cells altered PPP1R1A expression, indicating that PPP1R1A is a target gene for PDX1. Treatment with rosiglitazone increased Ppp1r1a expression, while metformin and insulin showed no effect. RNA-seq analysis of human islets revealed high PPP1R1A expression, with α-cells showing the highest levels compared to other endocrine cells. Muscle tissues exhibited greater PPP1R1A expression than pancreatic islets, liver, or adipose tissues. Co-expression analysis revealed significant correlations between PPP1R1A and genes associated with insulin biosynthesis, exocytosis machinery, and intracellular calcium transport. Overexpression of PPP1R1A in human islets augmented insulin secretion and upregulated protein expression of Insulin, MAFA, PDX1, and GLUT1, while silencing of PPP1R1A reduced Insulin, MAFA, and GLUT1 protein levels. CONCLUSION: This study provides valuable insights into the role of PPP1R1A in regulating ß-cell function and glucose homeostasis. PPP1R1A presents a promising opportunity for future therapeutic interventions.

Non-ionotropic voltage-gated calcium channel signaling.

Voltage-gated calcium channels (VGCCs) are the major conduits for calcium ions (Ca2+) within excitable cells. Recent studies have highlighted the non-ionotropic functionality of VGCCs, revealing their capacity to activate intracellular pathways independently of ion flow. This non-ionotropic signaling mode plays a pivotal role in excitation-coupling processes, including gene transcription through excitation-transcription (ET), synaptic transmission via excitation-secretion (ES), and cardiac contraction through excitation-contraction (EC). However, it is noteworthy that these excitation-coupling processes require extracellular calcium (Ca2+) and Ca2+ occupancy of the channel ion pore. Analogous to the "non-canonical" characterization of the non-ionotropic signaling exhibited by the N-methyl-D-aspartate receptor (NMDA), which requires extracellular Ca2+ without the influx of ions, VGCC activation requires depolarization-triggered conformational change(s) concomitant with Ca2+ binding to the open channel. Here, we discuss the contributions of VGCCs to ES, ET, and EC coupling as Ca2+ binding macromolecules that transduces external stimuli to intracellular input prior to elevating intracellular Ca2+. We emphasize the recognition of calcium ion occupancy within the open ion-pore and its contribution to the excitation coupling processes that precede the influx of calcium. The non-ionotropic activation of VGCCs, triggered by the upstroke of an action potential, provides a conceptual framework to elucidate the mechanistic aspects underlying the microseconds nature of synaptic transmission, cardiac contractility, and the rapid induction of first-wave genes.

OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

Mitochondrial Calcium Uniporter (MCU) is Involved in an Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mice.

The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-KATP) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca2+ present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca2+ concentration, mitochondrial membrane potential, and N-methyl-D-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca2+ concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca2+ concentrations against I/R injury.

Advances in TRPV6 inhibitors for tumors by targeted therapies: Macromolecular proteins, synthetic small molecule compounds, and natural compounds.

TRPV6, a Ca2+-selective member of the transient receptor potential vanilloid (TRPV) family, plays a key role in extracellular calcium transport, calcium ion reuptake, and maintenance of a local low calcium environment. An increasing number of studies have shown that TRPV6 is involved in the regulation of various diseases. Notably, overexpression of TRPV6 is closely related to the occurrence of various cancers. Research confirmed that knocking down TRPV6 could effectively reduce the proliferation and invasiveness of tumors by mainly mediating the calcium signaling pathway. Hence, TRPV6 has become a promising new drug target for numerous tumor treatments. However, the development of TRPV6 inhibitors is still in the early stage, and the existing TRPV6 inhibitors have poor selectivity and off-target effects. In this review, we focus on summarizing and describing the structure characters, and mechanisms of existing TRPV6 inhibitors to provide new ideas and directions for the development of novel TRPV6 inhibitors.

Coordinating activation of endo-lysosomal two-pore channels and TRP mucolipins.

Two-pore channels and TRP mucolipins are ubiquitous endo-lysosomal cation channels of pathophysiological relevance. Both are Ca2+-permeable and regulated by phosphoinositides, principally PI(3,5)P2. Accumulating evidence has uncovered synergistic channel activation by PI(3,5)P2 and endogenous metabolites such as the Ca2+ mobilizing messenger NAADP, synthetic agonists including approved drugs and physical cues such as voltage and osmotic pressure. Here, we provide an overview of this coordination.

Synthesis and biological evaluation of lycoctonine derivatives with cardiotonic and calcium channels inhibitory activities.

In our previous study, a screening of a variety of lycotonine-type diterpenoid alkaloids were screened for cardiotonic activity revealed that lycoctonine had moderate cardiac effect. In this study, a series of structurally diverse of lycoctonine were synthesized by modifying on B-ring, D-ring, E-ring, F-ring, N-atom or salt formation on lycoctonine skeleton. We evaluated the cardiotonic activity of the derivatives by isolated frog heart, aiming to identify some compounds with significantly enhanced cardiac effects, among which compound 27 with a N-isobutyl group emerged as the most promising cardiotonic candidate. Furthermore, the cardiotonic mechanism of compound 27 was preliminarily investigated. The result suggested that the cardiotonic effect of compound 27 is related to calcium channels. Patch clamp technique confirmed that the compound 27 had inhibitory effects on CaV1.2 and CaV3.2, with inhibition rates of 78.52 % ± 2.26 % and 79.05 % ± 1.59 % at the concentration of 50 µM, respectively. Subsequently, the protective effect of 27 on H9c2 cells injury induced by cobalt chloride was tested. In addition, compound 27 can alleviate CoCl2-induced myocardial injury by alleviating calcium overload. These findings suggest that compound 27 was a new structural derived from lycoctonine, which may serve as a new lead compound for the treatment of heart failure.

Effects of clitorienolactones from Clitoria ternatea root on calcium channel mediating hippocampal long-term potentiation in rats induced chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH) is a common mechanism of acute brain injury due to impairment of blood flow to the brain. Moreover, a prolonged lack of oxygen supply may result in cerebral infarction or global ischemia, which subsequently causes long-term memory impairment. Research on using Clitoria ternatea root extract for treating long-term memory has been studied extensively. However, the bioactive compound contributing to its neuroprotective effects remains uncertain. In the present study, we investigate the effects of clitorienolactone A (CLA) and B (CLB) from the roots of Clitoria ternatea extract on hippocampal neuroplasticity in rats induced by CCH. CLA and CLB were obtained using column chromatography. The rat model of CCH was induced using two-vessel occlusion surgery (2VO). The 2VO rats were given 10 mg/kg of CLA and CLB orally, followed by hippocampal neuroplasticity recording using in vivo electrophysiological. Rats received CLA and CLB (10 mg/kg) significantly reversed the impairment of long-term potentiation following 2VO surgery. Furthermore, we investigate the effect of CLA and CLB on the calcium channel using the calcium imaging technique. During hypoxia, CLA and CLB sustain the increase in intracellular calcium levels. We next predict the binding interactions of CLA and CLB against NMDA receptors containing GluN2A and GluN2B subunits using in silico molecular docking. Our result found that both CLA and CLB exhibited lower binding affinity against GluN2A and GluN2B subunits. Our findings demonstrated that bioactive compounds from Clitoria ternatea improved long-term memory deficits in the chronic cerebral hypoperfusion rat model via calcium uptake. Hence, CLA and CLB could be potential therapeutic tools for treating cognitive dysfunction.

Platelet-activating factor contracts guinea pig esophageal muscularis mucosae by stimulating extracellular Ca2+ influx through diltiazem-insensitive Ca2+ channels.

Platelet-activating factor (PAF) is expected to increase esophageal motility. However, to the best of our knowledge, this has not been examined. Thus, we investigated the contractile effects of PAF on guinea pig (GP) esophageal muscularis mucosae (EMM) and the extracellular Ca2+ influx pathways responsible. PAF (10-9-10-6 M) contracted EMM in a concentration-dependent manner. PAF (10-6 M)-induced contractions were almost completely suppressed by apafant (a PAF receptor antagonist, 3 × 10-5 M). In EMM strips, PAF receptor and PAF-synthesizing/degrading enzyme mRNAs were detected. PAF (10-6 M)-induced contractions were abolished by extracellular Ca2+ removal but were not affected by diltiazem [a voltage-dependent Ca2+ channel (VDCC) inhibitor, 10-5 M]. PAF (10-6 M)-induced contractions in the presence of diltiazem were significantly suppressed by LOE-908 [a receptor-operated Ca2+ channel (ROCC) inhibitor, 3 × 10-5 M], SKF-96365 [an ROCC and store-operated Ca2+ channel (SOCC) inhibitor, 3 × 10-5 M], and LOE-908 plus SKF-96365. Among the tested ROCC/SOCC-related mRNAs, Trpc3, Trpc6, and Trpv4/Orai1, Orai3, and Stim2 were abundantly expressed in EMM strips. These results indicate that PAF potently induces GP EMM contractions that are dependent on extracellular Ca2+ influx through ROCCs/SOCCs, and VDCCs are unlikely to be involved.

A comprehensive update on the ADMET considerations for α2δ calcium channel ligand medications for treating restless legs syndrome.

INTRODUCTION: Restless legs syndrome/Willis-Ekbom disease (RLS/WED) is a sleep-related sensory-motor disorder associated with poor sleep quality and impaired daily functioning. In patients affected by chronic RLS/WED, a pharmacological therapy is recommended. International guidelines suggest to start the treatment with a α2δ calcium channel ligand in most cases, unless contraindicated. AREAS COVERED: The present review is based on an extensive Internet and PubMed search from 1986 to 2024. Our purpose is to describe the absorption, distribution, metabolism, and toxicology (ADMET) of the α2δ ligands, with common consideration for the therapeutic class, specificities of different compounds, efficacy, and safety in relation to other treatment options. EXPERT OPINION: α2δ ligands are quite similar in their ADMET profiles, sharing most of the pharmacokinetics and potential adverse effects. However, we highlight the linear kinetic of gabapentin enacarbil and pregabalin, differently from gabapentin. α2δ ligands are safe and effective for the treatment of RLS/WED. Additional benefits can be obtained in comorbid insomnia, chronic pain syndromes, history of impulse control disorder, and comorbid anxiety. The use of α2δ ligands is associated with poor risk of augmentation. We still need new long-term safe and effective treatments, which could be developed along with our knowledge of RLS/WED pathophysiology.

MCU-independent Ca2+ uptake mediates mitochondrial Ca2+ overload and necrotic cell death in a mouse model of Duchenne muscular dystrophy.

Mitochondrial Ca2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca2+ influx across the sarcolemma that causes mitochondrial Ca2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca2+ uptake. One strategy for preventing mitochondrial Ca2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial Ca2+ overload. Moreover, myofiber specific Mcub gene deletion did not augment Duchenne MD muscle pathology. Interestingly, we observed MCU-independent Ca2+ uptake in dystrophic mitochondria that was sufficient to drive mitochondrial permeability transition pore (MPTP) activation and skeletal muscle necrosis, and this same type of activity was observed in heart, liver, and brain mitochondria. These results demonstrate that mitochondria possess an uncharacterized MCU-independent Ca2+ uptake mechanism that is sufficient to drive MPTP-dependent necrosis in MD in vivo.

Do calcium channel blockers applied to cardiomyocytes cause increased channel expression resulting in reduced efficacy?

In the initial hours following the application of the calcium channel blocker (CCB) nifedipine to microtissues consisting of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we observe notable variations in the drug's efficacy. Here, we investigate the possibility that these temporal changes in CCB effects are associated with adaptations in the expression of calcium ion channels in cardiomyocyte membranes. To explore this, we employ a recently developed mathematical model that delineates the regulation of calcium ion channel expression by intracellular calcium concentrations. According to the model, a decline in intracellular calcium levels below a certain target level triggers an upregulation of calcium ion channels. Such an upregulation, if instigated by a CCB, would then counteract the drug's inhibitory effect on calcium currents. We assess this hypothesis using time-dependent measurements of hiPSC-CMs dynamics and by refining an existing mathematical model of myocyte action potentials incorporating the dynamic nature of the number of calcium ion channels. The revised model forecasts that the CCB-induced reduction in intracellular calcium concentrations leads to a subsequent increase in calcium ion channel expression, thereby attenuating the drug's overall efficacy. The data and fit models suggest that dynamic changes in cardiac cells in the presence of CCBs may be explainable by induced changes in protein expression, and that this may lead to challenges in understanding calcium based drug effects on the heart unless timings of applications are carefully considered.

Deletion of the auxiliary α2δ1 voltage sensitive calcium channel subunit in osteocytes and late-stage osteoblasts impairs femur strength and load-induced bone formation in male mice.

Osteocytes sense and respond to mechanical force by controlling the activity of other bone cells. However, the mechanisms by which osteocytes sense mechanical input and transmit biological signals remain unclear. Voltage-sensitive calcium channels (VSCCs) regulate calcium (Ca2+) influx in response to external stimuli. Inhibition or deletion of VSCCs impairs osteogenesis and skeletal responses to mechanical loading. VSCC activity is influenced by its auxiliary subunits, which bind the channel's α1 pore-forming subunit to alter intracellular Ca2+ concentrations. The α2δ1 auxiliary subunit associates with the pore-forming subunit via a glycosylphosphatidylinositol anchor and regulates the channel's calcium-gating kinetics. Knockdown of α2δ1 in osteocytes impairs responses to membrane stretch, and global deletion of α2δ1 in mice results in osteopenia and impaired skeletal responses to loading in vivo. Therefore, we hypothesized that the α2δ1 subunit functions as a mechanotransducer, and its deletion in osteocytes would impair skeletal development and load-induced bone formation. Mice (C57BL/6) with LoxP sequences flanking Cacna2d1, the gene encoding α2δ1, were crossed with mice expressing Cre under the control of the Dmp1 promoter (10 kb). Deletion of α2δ1 in osteocytes and late-stage osteoblasts decreased femoral bone quantity (P < .05) by DXA, reduced relative osteoid surface (P < .05), and altered osteoblast and osteocyte regulatory gene expression (P < .01). Cacna2d1f/f, Cre + male mice displayed decreased femoral strength and lower 10-wk cancellous bone in vivo micro-computed tomography measurements at the proximal tibia (P < .01) compared to controls, whereas Cacna2d1f/f, Cre + female mice showed impaired 20-wk cancellous and cortical bone ex vivo micro-computed tomography measurements (P < .05) vs controls. Deletion of α2δ1 in osteocytes and late-stage osteoblasts suppressed load-induced calcium signaling in vivo and decreased anabolic responses to mechanical loading in male mice, demonstrating decreased mechanosensitivity. Collectively, the α2δ1 auxiliary subunit is essential for the regulation of osteoid-formation, femur strength, and load-induced bone formation in male mice.

The ability of bone to sense and respond to forces generated during daily physical activities is essential to skeletal health. Although several bone cell types contribute to the maintenance of bone health, osteocytes are thought to be the primary mechanosensitive cells; however, the mechanisms through which these cells perceive mechanical stimuli remains unclear. Previous work has shown that voltage sensitive calcium channels are necessary for bone to sense mechanical force; yet the means by which those channels translate the physical signal into a biochemical signal is unclear. Data within this manuscript demonstrate that the extracellular α2δ1 subunit of voltage sensitive calcium channels is necessary for load-induced bone formation as well as to enable calcium influx within osteocytes. As this subunit enables physical interactions of the channel pore with the extracellular matrix, our data demonstrate the need for the α2δ1 subunit for mechanically induced bone adaptation, thus serving as a physical conduit through which mechanical signals from the bone matrix are transduced into biochemical signals by enabling calcium influx into osteocytes.

The difference in endothelium-dependent relaxation components in proximal and distal thoracic aorta regions of male rats.

Aorta, the largest vessel in the body, is generally considered anatomically homogeneous, yet spatial functional differences exist. In our study, we conducted a comprehensive analysis by reexamining public RNA-SEQ data, comparing expression patterns between thoracic and abdominal aorta. Additionally, we measured acetylcholine-induced relaxations of the different regions of thoracic aorta in Wistar Rats. Our results revealed a distinct percentage difference in acetylcholine-induced relaxation in the proximal and distal segments of the thoracic aorta (p = 1.14e-4). To explain this variation, we performed differential expression analysis of previously published RNA-sequencing data between thoracic and abdominal aorta, which showed 497 differentially expressed genes between these locations. From results of RNA-Seq analysis, we draw a hypothesis that differential expressions of the potassium inward rectifying channels (KIR) and voltage gated calcium channels (VGCC) presumably located on SMC, with higher expression in the distal thoracic segments in comparison with the proximal thoracic segments of aorta, can explain differences in acetylcholine-induced relaxation. Notably, specific blockade of KIR eliminated differences between the proximal and distal regions of thoracic aorta, underscoring their significance in understanding the spatial nuances in aortic behavior, also blockade of VGCC, shows a higher effect on basal tone, in distal region of thoracic aorta in comparison with proximal.

TRPM5 activation depends on a synergistic effect of calcium and PKC phosphorylation.

Transient receptor potential melastatin 5 (TRPM5) is a calcium-activated monovalent-specific ion channel involved in insulin secretion and taste transduction, making it an attractive target for drug development in various pathologies. While TRPM5 activation involves ligand binding to Gq/G-protein coupled receptors (GPCR) and subsequent elevation of intracellular calcium levels, recent reports suggest the need for additional molecular determinants. Hence, the mechanism of TRPM5 activation remains to be elucidated. Here, we show that PKC phosphorylation and the elevation of intracellular Ca2+ levels are required for TRPM5 activation, with PKC phosphorylation being crucial for channel-evoked currents, primarily at physiological membrane potentials. In contrast, physiological relevant calcium levels alone only induce TRPM5 activation at positive voltages. Our findings highlight the necessity of coordinated intracellular calcium release and PKC phosphorylation for TRPM5 activation. Thus, our results suggest that regulation of PKC activity could be a promising therapeutic target for diseases associated with TRPM5 modulation.

STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca2+ entry and mitochondrial Ca2+ homeostasis in cardiomyocytes.

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca2+ sensors that trigger store-operated Ca2+ entry (SOCE) in a variety of cell types. While STIM1 isoform has been the focus of the research in cardiac pathophysiology, the function of the homolog STIM2 remains unknown. Using Ca2+ imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the ISOC current in neonatal rat ventricular cardiomyocytes (NRVMs). Within this cardiomyocyte model, we identified the transcript expression of Stim2.1 and Stim2.2 splice variants, with predominance for Stim2.2. Using conventional and super-resolution confocal microscopy (STED), we found that exogenous STIM2.1 and STIM2.2 formed pre-clusters with a reticular organization at rest. Following SR Ca2+ store depletion, some STIM2.1 and STIM2.2 clusters were translocated to SR-plasma membrane (PM) junctions and co-localized with Orai1. The overexpression strategy revealed that STIM2.1 suppressed Orai1-mediated SOCE and the ISOC current while STIM2.2 enhanced SOCE. STIM2.2-enhanced SOCE was also dependent on TRPC1 and TRPC4. Even if STIM2 KD or splice variants overexpression did not affect cytosolic Ca2+ cycling, we observed, using Rhod-2/AM Ca2+ imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca2+ (mCa2+) uptake, as opposed to STIM2 KD. We also found that STIM2 was present in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by interacting with the inositol trisphosphate receptors (IP3Rs), voltage-dependent anion channel (VDAC), mitochondrial Ca2+ uniporter (MCU), and mitofusin-2 (MNF2). Our results suggested that, in NRVMs, STIM2.1 constitutes the predominant functional variant that negatively regulates Orai1-generated SOCE. It participates in the control of mCa2+ uptake capacity possibly via the STIM2-IP3Rs-VDAC-MCU and MNF2 complex.

Calcium Channel Inhibitory Effect of Marjoram (Origanum majorana L.): Its Medicinal Use in Diarrhea and Gut Hyperactivity.

BACKGROUND: The leaves of Origanum majorana (O. majorana) are traditionally renowned for treating diarrhea and gut spasms. This study was therefore planned to evaluate its methanolic extract. METHODS: Gas chromatography-mass spectrometry (GC-MS) was used to identify the phytochemicals, and Swiss albino mice were used for an in vivo antidiarrheal assay. Isolated rat ileum was used as an ex vivo assay model to study the possible antispasmodic effect and its mechanism(s). RESULTS: The GC-MS analysis of O. majorana detected the presence of 21 compounds, of which alpha-terpineol was a major constituent. In the antidiarrheal experiment, O. majorana showed a substantial inhibitory effect on diarrheal episodes in mice at an oral dosage of 200 mg/kg, resulting in 40% protection. Furthermore, an oral dosage of 400 mg/kg provided even greater protection, with 80% effectiveness. Similarly, loperamide showed 100% protection at oral doses of 10 mg/kg. O. majorana caused complete inhibition of carbachol (CCh, 1 µM) and high K+ (80 mM)-evoked spasms in isolated ileal tissues by expressing significantly higher potency (p < 0.05) against high K+ compared to CCh, similar to verapamil, a Ca++ antagonist. The verapamil-like predominant Ca++ ion inhibitory action of O. majorana was further confirmed in the ileal tissues that were made Ca++-free by incubating the tissues in a physiological salt solution having ethylenediaminetetraacetic acid (EDTA) as a chelating agent. The preincubation of O. majorana at increasing concentrations (0.3 and 1 mg/mL) shifted towards the right of the CaCl2-mediated concentration-response curves (CRCs) with suppression of the maximum contraction. Similarly, verapamil also caused non-specific suppression of Ca++ CRCs towards the right, as expected. CONCLUSIONS: Thus, this study conducted an analysis to determine the chemical constituents of the leaf extract of O. majorana and provided a detailed mechanistic basis for the medicinal use of O. majorana in hyperactive gut motility disorders.