Internal Ca2 Research Articles

STIM Proteins: The Gas and Brake of Calcium Entry in Neurons.

Stromal interaction molecules (STIM)s are Ca2+ sensors in internal Ca2+ stores of the endoplasmic reticulum. They activate the store-operated Ca2+ channels, which are the main source of Ca2+ entry in non-excitable cells. Moreover, STIM proteins interact with other Ca2+ channel subunits and active transporters, making STIMs an important intermediate molecule in orchestrating a wide variety of Ca2+ influxes into excitable cells. Nevertheless, little is known about the role of STIM proteins in brain functioning. Being involved in many signaling pathways, STIMs replenish internal Ca2+ stores in neurons and mediate synaptic transmission and neuronal excitability. Ca2+ dyshomeostasis is a signature of many pathological conditions of the brain, including neurodegenerative diseases, injuries, stroke, and epilepsy. STIMs play a role in these disturbances not only by supporting abnormal store-operated Ca2+ entry but also by regulating Ca2+ influx through other channels. Here, we review the present knowledge of STIMs in neurons and their involvement in brain pathology.

Connexin 25 maintains self-renewal and functions of airway basal cells for airway regeneration

BackgroundThe formation of stem cell clones enables close contact of stem cells inside. The gap junctions in such clone spheres establish a microenvironment that allows frequent intercellular communication to maintain self-renewal and functions of stem cells. Nevertheless, the essential gap junction protein for molecular signaling in clones is poorly known.MethodsPrimary human airway basal cells (hBCs) were isolated from brushing samples through bronchoscopy and then cultured. A tightly focused femtosecond laser was used to excite the local Ca2+ in an individual cell to initiate an internal Ca2+ wave in a clone to screen gap junction proteins. Immunoflourescence staining and clonogenicity assay were used to evaluate self-renewal and functions. RNA and protein levels were assessed by PCR and Western blot. Air–liquid interface assay was conducted to evaluate the differentiation potential. A Naphthalene injury mouse model was used to assess the regeneration potential.ResultsHerein, we identify Connexin 25 (Cx25) dominates intercellular Ca2+ communications in clones of hBCs in vitro to maintain the self-renewal and pluripotency of them. The self-renewal and in vitro differentiation functions and in vivo regeneration potential of hBCs in an airway damage model are both regulated by Cx25. The abnormal expression of Cx25 is validated in several diseases including IPF, Covid-19 and bronchiectasis.ConclusionCx25 is essential for hBC clones in maintaining self-renewal and functions of hBCs via gap junctions.

A PACAP-activated network for secretion requires coordination of Ca2+ influx and Ca2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.

Mitochondria control IP3-evoked Ca2+ release triggered by voltage-dependent Ca2+ entry in intact resistance arteries

The contractility of vascular smooth muscle cells (VSMCs) is a major contributor of vascular tone and blood pressure. In VSMCs, intracellular Ca2+ level determines the contractile activity that generates the force to contract arteries. A major source of Ca2+ is via influx through voltage dependent Ca2+ channels (VDCCs). While mitochondria are now recognised as key regulators of intracellular Ca2+ homeostasis in several cell types, their role in modulating Ca2+ signalling mediated by VDCC in VSMCs is unresolved. Given the potential physiological significance, and unclarified interaction between mitochondria and VDCCs in VSMCs, we hypothesize that mitochondria directly regulate Ca2+ signalling mediated by VDCCs. The interplay between mitochondria and VDCCs was investigated by imaging and analysing intracellular Ca2+ signals in smooth muscle cells in intact arteries from rat mesentery. Depolarization of the plasma membrane potential, by high potassium (20 mM) physiological saline solution, triggered Ca2+ influx through VDCCs and a sustained increase in intracellular Ca2+ on which repetitive Ca2+ oscillations occurred. All Ca2+ signals were abolished by removal of external Ca2+ and by dihydropyridine inhibitors of VDCCs. Significantly, the repetitive Ca2+ oscillations, but not the sustained Ca2+ signals, were blocked by the IP3 receptor inhibitor 2-APB and SERCA inhibitor cyclopiazonic acid. Neither the repetitive Ca2+ oscillations or the sustained Ca2+ signals were altered by the ryanodine receptor inhibitors ryanodine and dantrolene. These results suggest that Ca2+ influx via VDCC triggers Ca2+-induced Ca2+ release at IP3 receptors in intact mesenteric arteries. Depolarization of the mitochondrial membrane potential (ψm), with the uncoupler CCCP or complex I inhibitor rotenone, but not ATP deprivation with the ATP synthesis blocker oligomycin, inhibited VDCC evoked IP3 mediated Ca2+ oscillations but not the sustained Ca2+ signals. Furthermore, in intact arteries, depolarization of ψm directly suppressed inositol triphosphate receptors (IP3Rs) mediated Ca2+ release from the internal Ca2+ store evoked by photolysis of caged IP3. These results suggest that mitochondria regulate Ca2+ release via IP3R triggered by Ca2+ entry via VDCC. In return, Ca2+ entry via VDCCs did not alter ψm, but upregulated ROS production. Together, these results suggest that mitochondria regulate Ca2+-induced Ca2+ release at IP3 receptors triggered by Ca2+ influx via VDCCs but do not directly regulate VDCC activity. British Heart Foundation (RG/F/20/110007; PG/20/9/34859). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ca2+ signals in human umbilical endothelial cells derived from pregnancy with fetal growth restriction associated with hypertensive disorder.

Fetal growth restriction associated with hypertensive disorders of pregnancy (FGR-HDP) is a prevalent pathology with a higher risk of perinatal morbimortality. In this condition, placental insufficiency and endothelial dysfunction serve key roles. The present prospective cohort study monitored 11 patients with an FGR-HDP and 15 with full-term normotensive pregnancies and studied post-natal intracellular calcium concentration ([Ca2+]i) signals in human umbilical vein endothelial cells (HUVECs). Small fetuses with placental insufficiency were identified using fetal biometry with Doppler velocimetry. Mean gestational age and birth weight were 31.8±4.1 weeks and 1,260±646 g for FGR-HDP and 39.2±0.8 weeks and 3,320±336 g for normal births, respectively. Abnormal umbilical artery Doppler waveforms were found in 64% of neonates with FGR-HDP. A significant percentage (86%) of FGR newborns were admitted to the neonatal intensive care unit at Gustavo Fricke hospital, Viña del Mar, Chile, with one case of death after birth. [Ca2+]i signals were measured by microfluorimetry in Fluo-3-loaded HUVECs from primary cultures. Altered [Ca2+]i signals were observed in HUVECs from FGR-HDP, where the sustained phase of ATP-induced [Ca2+]i responses was significantly reduced compared with the normotensive group. Also, the [Ca2+]i signals induced with 10 mM Ca2+ after depletion of internal Ca2+ stores were significantly higher. The present study provides a better comprehension of the role of altered cytosolic Ca2+ dynamics in endothelial dysfunction and an in vitro model to assess novel therapeutic approaches for decreasing or preventing complications in FGR-HDP.

Store-operated calcium entry in the satellite glial cells of rat sympathetic ganglia.

Satellite glial cells (SGCs), a major type of glial cell in the autonomic ganglia, closely envelop the cell body and even the synaptic regions of a single neuron with a very narrow gap. This structurally unique organization suggests that autonomic neurons and SGCs may communicate reciprocally. Glial Ca2+ signaling is critical for controlling neural activity. Here, for the first time we identified the machinery of store-operated Ca2+ entry (SOCE) which is critical for cellular Ca2+ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative realtime PCR and immunostaining analyses showed that Orai1 and stromal interaction molecules 1 (STIM1) proteins are the primary components of SOCE machinery in the sympathetic ganglia. When the internal Ca2+ stores were depleted in the absence of extracellular Ca2+, the number of plasmalemmal Orai1 puncta was increased in neurons and SGCs, suggesting activation of the Ca2+ entry channels. Intracellular Ca2+ imaging revealed that SOCE was present in SGCs and neurons; however, the magnitude of SOCE was much larger in the SGCs than in the neurons. The SOCE was significantly suppressed by GSK7975A, a selective Orai1 blocker, and Pyr6, a SOCE blocker. Lipopolysaccharide (LPS) upregulated the glial fibrillary acidic protein and Toll-like receptor 4 in the sympathetic ganglia. Importantly, LPS attenuated SOCE via downregulating Orai1 and STIM1 expression. In conclusion, sympathetic SGCs functionally express the SOCE machinery, which is indispensable for intracellular Ca2+ signaling. The SOCE is highly susceptible to inflammation, which may affect sympathetic neuronal activity and thereby autonomic output.

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 × 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

How Merkel cells transduce mechanical stimuli: A biophysical model of Merkel cells.

Merkel cells combine with Aβ afferents, producing slowly adapting type 1(SA1) responses to mechanical stimuli. However, how Merkel cells transduce mechanical stimuli into neural signals to Aβ afferents is still unclear. Here we develop a biophysical model of Merkel cells for mechanical transduction by incorporating main ingredients such as Ca2+ and K+ voltage-gated channels, Piezo2 channels, internal Ca2+ stores, neurotransmitters release, and cell deformation. We first validate our model with several experiments. Then we reveal that Ca2+ and K+ channels on the plasma membrane shape the depolarization of membrane potentials, further regulating the Ca2+ transients in the cells. We also show that Ca2+ channels on the plasma membrane mainly inspire the Ca2+ transients, while internal Ca2+ stores mainly maintain the Ca2+ transients. Moreover, we show that though Piezo2 channels are rapidly adapting mechanical-sensitive channels, they are sufficient to inspire sustained Ca2+ transients in Merkel cells, which further induce the release of neurotransmitters for tens of seconds. Thus our work provides a model that captures the membrane potentials and Ca2+ transients features of Merkel cells and partly explains how Merkel cells transduce the mechanical stimuli by Piezo2 channels.

Transient receptor potential channel TRPM4 favors oxidized low-density lipoprotein-induced coronary endothelial cell dysfunction via a mechanism involving ferroptosis

Accelerating the repair of damaged endothelium can effectively inhibit the progression of atherosclerosis (AS). Transient receptor potential channel TRPM4 is a non-selective cation channel activated by internal Ca2+, which is expressed in endothelial cells. This study aimed to reveal the potential role of TRPM4 in AS along with the mechanism. Human coronary artery endothelial cells (HCAECs) induced by ox-LDL was regarded as an in vitro model. The impacts of TRPM4 knockdown on cellular inflammation response, oxidative stress, normal endothelial function and lipid peroxidation were evaluated. Given that ferroptosis promotes AS progression, the effects of TRPM4 on intracellular iron ions and ferroptosis-related proteins was determined. Afterwards, HCAECs were treated with ferroptosis inducer erastin, and the influence of ferroptosis in the cellular model was revealed. TRPM4 was elevated in response to ox-LDL treatment in HCAECs. TRPM4 knockdown reduced the inflammation response, oxidative stress and lipid peroxidation caused by ox-LDL, and maintained the normal function of HCAECs. Erastin treatment destroyed the impacts of TRPM4 knockdown that are beneficial for cells to resist ox-LDL, showing the enhancement of the above adverse factors. Together, this study found that TRPM4 knockdown reduced ox-LDL-induced inflammation, oxidative stress, and dysfunction in HCAECs, possibly via a mechanism involving Fe2+ and ferroptosis-related proteins.

Subunit-specific inhibition of BK channels by piperine

Piperine is the principal alkaloid present in black pepper and is well-known for its diverse pharmacological effects, including inhibition of different ion channels. Large conductance Ca2+-activated K+ channels (BK) are widely expressed across several tissues and play a vital role in many physiological functions. In this study, we investigated the pharmacological effects of piperine on various BK channel subunit compositions (BKα, BKαβ1,4, BKαγ1,3) expressed in HEK293T cells. Piperine in zero Ca2+ reversibly inhibited currents from the pore-forming BKα channels in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 4.8 μM. Elevating the internal Ca2+ concentration from 0 to 100 μM significantly attenuated the inhibitory effects of piperine on BKα channels. The mutation G311S in the pore domain failed to alter the modulatory effects of piperine, whereas deletion of the entire cytoplasmic domain from BKα channels ablated its inhibitory effects. Addition of either BKβ1 or β4 regulatory subunits did not alter the efficacy of piperine on BKα channels. Interestingly, co-expression of either BKγ1 or BKγ3 subunits greatly diminished the ability of piperine to inhibit BKα channels. Our findings demonstrate that piperine is a potent natural modulator of BKα/BKαβ1,4 subunits but not BKαγ1,3 subunits. The mechanism of piperine modulation appeared to be allosteric and differs from that of other BK pore blockers (paxilline, peptide toxins, and quaternary ammonium compounds). Together, our results unravel the potential of piperine to inhibit BK channels, providing a new tool to explore mechanisms underlying the effects of regulatory subunits.

CDN1163, a SERCA activator, causes intracellular Ca2+ leak, mitochondrial hyperpolarization and cell cycle arrest in mouse neuronal N2A cells

ObjectiveActivity or expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) is diminished in some disease states such as cardiac failure and diabetes mellitus. A newly developed activator of SERCA, CDN1163, reportedly rescued or alleviated pathological conditions attributed to dysfunctional SERCA. We examined whether CDN1163 could relieve mouse neuronal N2A cell growth inhibition caused by cyclopiazonic acid (CPA, SERCA inhibitor). We also examined how CDN1163 affected cytosolic Ca2+, mitochondrial Ca2+ and mitochondrial membrane potential. MethodsCell viability was measured by MTT assay and trypan blue exclusion test. Cytosolic Ca2+, mitochondrial Ca2+ and mitochondrial membrane potential were measured using fura 2, Rhod-2 and JC-1, respectively, as fluorescent probes. ResultsCDN1163 (10 μM) itself suppressed cell proliferation, and did not alleviate CPA’s inhibitory effect (and vice versa). Cell cycle was arrested at the G1 phase after CDN1163 treatment. CDN1163 treatment caused a slow yet persistent cytosolic [Ca2+] elevation partly due to Ca2+ release from an internal store other than the CPA-sensitive endoplasmic reticulum (ER). Treatment with CDN1163 for 3 h raised mitochondrial Ca2+ level and such increase was suppressed by MCU-i4 (an inhibitor of mitochondria Ca2+ uniporter, MCU), suggesting Ca2+ entered the mitochondrial matrix through MCU. Treatment of cells with CDN1163 up to 2 days resulted in mitochondrial hyperpolarization. ConclusionCDN1163 caused internal Ca2+ leak, cytosolic Ca2+ overload, mitochondrial Ca2+ elevation and hyperpolarization, cell cycle arrest and cell growth inhibition.

Carotid artery stenting after balloon aortic valvuloplasty in cervical internal carotid artery stenosis associated with severe aortic stenosis: A case report

AbstractCarotid artery stenting (CAS) is contraindicated in patients with severe aortic stenosis (AS). However, it is required for treating those with symptomatic internal CA (ICA) stenosis and severe AS. This is the first report of successful CAS after balloon aortic valvuloplasty (BAV) in a patient having ICA stenosis accompanied with severe AS. An 82‐year‐old man with a history of severe AS presented with amaurosis fugax due to severe stenosis at the left ICA. We performed CAS just after BAV. No perioperative complications were reported. It might be a useful treatment strategy for symptomatic ICA stenosis with severe AS.

Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop.

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+ in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE Clostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.

Mercury intoxication disrupts tonic signaling in B cells, and may promote autoimmunity due to abnormal phosphorylation of STIM-1 and other autoimmunity risk associated phosphoproteins involved in BCR signaling

Epidemiological studies link exposure to mercury with autoimmune disease. Unfortunately, in spite of considerable effort, no generally accepted mechanistic understanding of how mercury functions with respect to the etiology of autoimmune disease is currently available. Nevertheless, autoimmune disease often arises because of defective B cell signaling. Because B cell signaling is dependent on phosphorylation cascades, in this report, we have focused on how mercury intoxication alters phosphorylation of B cell proteins in antigen-non stimulated (tonic) mouse (BALB/c) splenic B cells.Specifically, we utilized mass spectrometric techniques to conduct a comprehensive unbiased global analysis of the effect of inorganic mercury (Hg2+) on the entire B cell phosphoproteome. We found that the effects were pleotropic in the sense that large numbers of pathways were impacted. However, confirming our earlier work, we found that the B cell signaling pathway stood out from the rest, in that phosphoproteins which had sites which were affected by Hg2+, exhibited a much higher degree of connectivity, than components of other pathways. Further analysis showed that many of these BCR pathway proteins had been previously linked to autoimmune disease. Finally, dose response analysis of these BCR pathway proteins showed STIM1_S575, and NFAT2_S259 are the two most Hg2+ sensitive of these sites. Because STIM1_S575 controls the ability of STIM1 to regulate internal Ca2+, we speculate that STIM1 may be the initial point of disruption, where Hg2+ interferes with B cell signaling leading to systemic autoimmunity, with the molecular effects pleiotropically propagated throughout the cell by virtue of Ca2+ dysregulation.

Disruption of ER ion homeostasis maintained by an ER anion channel CLCC1 contributes to ALS-like pathologies

Although anion channel activities have been demonstrated in sarcoplasmic reticulum/endoplasmic reticulum (SR/ER), their molecular identities and functions remain unclear. Here, we link rare variants of Chloride Channel CLIC Like 1 (CLCC1) to amyotrophic lateral sclerosis (ALS)-like pathologies. We demonstrate that CLCC1 is a pore-forming component of an ER anion channel and that ALS-associated mutations impair channel conductance. CLCC1 forms homomultimers and its channel activity is inhibited by luminal Ca2+ but facilitated by phosphatidylinositol 4,5-bisphosphate (PIP2). We identified conserved residues D25 and D181 in CLCC1 N-terminus responsible for Ca2+ binding and luminal Ca2+-mediated inhibition on channel open probability and K298 in CLCC1 intraluminal loop as the critical PIP2-sensing residue. CLCC1 maintains steady-state [Cl–]ER and [K+]ER and ER morphology and regulates ER Ca2+ homeostasis, including internal Ca2+ release and steady-state [Ca2+]ER. ALS-associated mutant forms of CLCC1 increase steady-state [Cl–]ER and impair ER Ca2+ homeostasis, and animals with the ALS-associated mutations are sensitized to stress challenge-induced protein misfolding. Phenotypic comparisons of multiple Clcc1 loss-of-function alleles, including ALS-associated mutations, reveal a CLCC1 dosage dependence in the severity of disease phenotypes in vivo. Similar to CLCC1 rare variations dominant in ALS, 10% of K298A heterozygous mice developed ALS-like symptoms, pointing to a mechanism of channelopathy dominant-negatively induced by a loss-of-function mutation. Conditional knockout of Clcc1 cell-autonomously causes motor neuron loss and ER stress, misfolded protein accumulation, and characteristic ALS pathologies in the spinal cord. Thus, our findings support that disruption of ER ion homeostasis maintained by CLCC1 contributes to ALS-like pathologies.

TRPM4: New actor in osteogenic differentiation of human valvular interstitial cells

Transient receptor potential melastatin 4 (TRPM4) protein, a non-selective cation channel activated by calcium, is involved in cardiac fibroblasts remodeling. Its expression was reported in human valvular interstitial cells (VIC) with similar properties than fibroblasts, and its protein expression is increased on calcified aortic valves from patients suffering of aortic stenosis. VIC osteogenic differentiation is a key element in valvular calcification, which is, at term, responsible of aortic stenosis. The purpose of this study is to evaluate the implication of TRPM4 in human VIC osteogenic differentiation. VIC are isolated by enzymatic digestion from human aortic valves obtained from patients undergoing surgical aortic valve replacement. They are maintained 14 days in procalcifying media (2.7 mM calcium/2.5 mM phosphate), with or without 9-phenanthrol, a pharmacological TRPM4 inhibitor. Channel expression and functionality were followed respectively by PCR, Western Blot and electrophysiological recordings in the inside-out configuration of the patch clamp technique. Osteogenic differentiation of VIC was evaluated by measuring hydroxyapatite crystals by Alizarin red staining and by monitoring the protein expression of the osteogenic markers, BMP2 and RUNX2. A typical TRPM4 current was detected on VIC: its current-voltage relationship was linear with a conductance of 20.3 pS; the channel exhibited similar permeability for Na+ and K+; it was more activated in positive voltages and with increasing concentrations of internal Ca2+; the current was dose-dependently inhibited by 9-phenanthrol. TRPM4 mRNA and protein expression on VIC was confirmed by PCR and Western Blot. When maintained in procalcifying media, VIC developed hydroxyapatite crystals. Application of 9-phenanthrol (3.10-6 M) reduced mineralization by 30% without affecting cell viability. Moreover, it significantly reduced BMP2 and RUNX2 expression. Thereby, TRPM4 participates in osteogenic differentiation of human VIC in culture. Next studies of our unit are to evaluate whether TRPM4 repression by shRNA confirms those results.

Piezo1 regulates expression of BK and TRPV4 channels in mouse cortical collecting duct (CCD)

Within the CCD, an acute increase in tubular fluid flow rate (TFFR) leads to an immediate high amplitude increase in [Ca 2+ ] i , due to release of IP3-sensitive internal Ca 2+ stores coupled to extracellular Ca 2+ entry at the basolateral membrane in both principal (PC) and intercalated (IC) cells therein (Liu et al., 2003, 2005, 2007). This is followed by a decay in [Ca 2+ ] i to a sustained plateau elevation due, at least in part, to luminal Ca 2+ entry through TRPV4 (Berrout et al., 2012). This flow-induced increase in [Ca 2+ ] i is necessary for BK channel-mediated flow induced K + secretion (FIKS) in the microperfused mammalian CCD. We generated a mouse with IC-specific targeted deletion of the mechanosensitive Ca 2+ permeable channel, Piezo1, which is expressed on the basolateral membranes of PCs and ICs in the mouse CCD (Dalghi et al., 2019). Microperfused CCDs isolated from these IC-PIEZO1-KO mice fed a high K (HK, 5% K + ) diet and subject to an increase in TFFR did not exhibit either a peak or plateau elevation in [Ca 2+ ] i in response to flow, and FIKS was eliminated. The absence of FIKS and the plateau elevation in [Ca 2+ ] during sustained flow in the CCDs of KO mice led us to examine whether BK and TRPV4 channel expression was impacted by deletion of PIEZO1.Indirect immunofluorescence (IF) staining was performed to examine expression and localization of the BK and TRPV4 channels in PCs and ICs of IC-PIEZO1-KO mice. Whole cell and apical + subapical fluorescence intensities in cryosections were analyzed using LAS X software from a total of 3 male and 3 female control mice and 3 male and 3 female IC-PIEZO1-KO mice fed HK diet x 10d.IF analysis showed reduced whole cell BKα expression in ICs of both male (p≤0.05) and female (p≤0.01) IC-PIEZO1-KO mice. We also saw reduced BKα expression in PCs from female but not male KOs. In addition, ICs in female but not male KO mice had reduced whole cell TRPV4 expression vs. controls (p≤0.01). Also, whole cell expression of TRPV4 in PCs from female KO mice was reduced vs. female controls (p≤0.01). The ratio of apical-to-whole cell expression of BKα was similar in PCs and ICs of KO and control mice. Male, but not female, KO mice had modestly reduced apical TRPV4 expression in PCs vs. controls (p≤0.01). Piezo1 plays a role in the regulation of BK and TRPV4 channel expression in CCDs. In addition, sex differences exist in whole cell expression of these channels in response to IC-selective deletion of PIEZO1. R01 DK038470 (TK, LMS); R01 DK129285 (TK, LMS); P30 DK079307 (TK) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ca2+-induced myelin pathology precedes axonal spheroid formation and is mediated in part by store-operated Ca2+ entry after spinal cord injury.

The formation of axonal spheroid is a common feature following spinal cord injury. To further understand the source of Ca2+ that mediates axonal spheroid formation, we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca2+. We performed two-photon excitation imaging of spinal cords isolated from Thy1YFP+ transgenic mice and applied the lipophilic dye, Nile red, to record dynamic changes in dorsal column axons and their myelin sheaths respectively. We selectively released Ca2+ from internal stores using the Ca2+ ionophore ionomycin in the presence or absence of external Ca2+. We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 mM Ca2+ artificial cerebrospinal fluid. In contrast, removal of external Ca2+ significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment. Using mice that express a neuron-specific Ca2+ indicator in spinal cord axons, we confirmed that ionomycin induced significant increases in intra-axonal Ca2+, but not in the absence of external Ca2+. Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation. Pretreatment with YM58483 (500 nM), a well-established blocker of store-operated Ca2+ entry, significantly decreased myelin injury and axonal spheroid formation. Collectively, these data reveal that ionomycin-induced depletion of internal Ca2+ stores and subsequent external Ca2+ entry through store-operated Ca2+ entry contributes to pathological changes in myelin and axonal spheroid formation, providing new targets to protect central myelinated fibers.

Store-operated Ca2+ entry regulatory factor alters murine metabolic state in an age-dependent manner via hypothalamic pathways.

Store-operated calcium entry (SOCE) is a vital process aimed at refilling cellular internal Ca2+ stores and a primary cellular signaling driver for transcription factors' entry to the nucleus. SOCE-associated regulatory factor (SARAF)/TMEM66 is an endoplasmic reticulum (ER)-resident transmembrane protein that promotes SOCE inactivation and prevents Ca2+ overfilling of the cell. Here, we demonstrate that mice deficient in SARAF develop age-dependent sarcopenic obesity with decreased energy expenditure, lean mass, and locomotion without affecting food consumption. Moreover, SARAF ablation reduces hippocampal proliferation, modulates the activity of the hypothalamus-pituitary-adrenal (HPA) axis, and mediates changes in anxiety-related behaviors. Interestingly, selective SARAF ablation in the hypothalamus's paraventricular nucleus (PVN) neurons reduces old age-induced obesity and preserves locomotor activity, lean mass, and energy expenditure, suggesting a possible central control with a site-specific role for SARAF. At the cellular level, SARAF ablation in hepatocytes leads to elevated SOCE, elevated vasopressin-induced Ca2+ oscillations, and an increased mitochondrial spare respiratory capacity (SPC), thus providing insights into the cellular mechanisms that may affect the global phenotypes. These effects may be mediated via the liver X receptor (LXR) and IL-1 signaling metabolic regulators explicitly altered in SARAF ablated cells. In short, our work supports both central and peripheral roles of SARAF in regulating metabolic, behavioral, and cellular responses.

Ca2+-Sensitive Potassium Channels.

The Ca2+ ion is used ubiquitously as an intracellular signaling molecule due to its high external and low internal concentration. Many Ca2+-sensing ion channel proteins have evolved to receive and propagate Ca2+ signals. Among them are the Ca2+-activated potassium channels, a large family of potassium channels activated by rises in cytosolic calcium in response to Ca2+ influx via Ca2+-permeable channels that open during the action potential or Ca2+ release from the endoplasmic reticulum. The Ca2+ sensitivity of these channels allows internal Ca2+ to regulate the electrical activity of the cell membrane. Activating these potassium channels controls many physiological processes, from the firing properties of neurons to the control of transmitter release. This review will discuss what is understood about the Ca2+ sensitivity of the two best-studied groups of Ca2+-sensitive potassium channels: large-conductance Ca2+-activated K+ channels, KCa1.1, and small/intermediate-conductance Ca2+-activated K+ channels, KCa2.x/KCa3.1.