Vulnerability of Store-Operated Calcium Entry to Inhibitors and Microenvironment in Cells of Different Breast Cancer Subtypes.

The incidence and development of cancer are highly dependent on pathological disturbances in calcium homeostasis of the cell. One of the major pathways for calcium entry is store-operated calcium entry (SOCE), which functions in virtually all cell types. Changes in the expression level of the main proteins organizing SOCE are observed during the development of various cancer types, particularly breast cancer (BC). This leads to unique SOCE with characteristics individual for each type of BC and requires particular therapeutic approaches. In this study, we tested the sensitivity of SOCE in various BC cells to selective ORAI channel inhibitors and the less selective compounds Leflunomide and Teriflunomide, approved by the FDA for clinical use. We also analyzed the vulnerability of SOCE to the influence of factors typical of the tumor microenvironment: hypoxia and acidification. We have observed that the SOCE inhibitors Leflunomide and Teriflunomide suppress SOCE in the triple-negative BC cell line MDA-MB-231, but not in the luminal A BC cell line MCF-7. MDA-MB-231 cells also demonstrate higher pH dependence of SOCE compared to MCF-7 cells. In addition, the oxygen scavenger sodium dithionide also affects SOCE, stimulating it in MDA-MB-231 cells but inhibiting in MCF-7 cells. Overall, our data highlight the importance of considering the different sensitivities of various BC cell types to inhibitors and to microenvironmental factors such as hypoxia and acidification when developing targeted drugs.

Reorganization and Suppression of Store-Operated Calcium Entry in Podocytes of Type 2 Diabetic Rats.

Type 2 diabetes mellitus (DM2) is a widespread metabolic disorder that results in podocyte damage and diabetic nephropathy. Previous studies demonstrated that TRPC6 channels play a pivotal role in podocyte function and their dysregulation is associated with development of different kidney diseases including nephropathy. Here, using single channel patch clamp technique, we demonstrated that non-selective cationic TRPC6 channels are sensitive to the Ca2+ store depletion in human podocyte cell line Ab8/13 and in freshly isolated rat glomerular podocytes. Ca2+ imaging indicated the involvement of ORAI and sodium-calcium exchanger in Ca2+ entry induced upon store depletion. In male rats fed a high-fat diet combined with a low-dose streptozotocin injection, which leads to DM2 development, we observed the reduction of a store-operated Ca2+ entry (SOCE) in rat glomerular podocytes. This was accompanied by a reorganization of store-operated Ca2+ influx such that TRPC6 channels lost their sensitivity to Ca2+ store depletion and ORAI-mediated Ca2+ entry was suppressed in TRPC6-independent manner. Altogether our data provide new insights into the mechanism of SOCE organization in podocytes in the norm and in pathology, which should be taken into account when developing pharmacological treatment of the early stages of diabetic nephropathy.

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.

STIM2 Mediates Excessive Store-Operated Calcium Entry in Patient-Specific iPSC-Derived Neurons Modeling a Juvenile Form of Huntington's Disease.

Huntington's disease (HD) is a severe autosomal-dominant neurodegenerative disorder caused by a mutation within a gene, encoding huntingtin protein. Here we have used the induced pluripotent stem cell technology to produce patient-specific terminally differentiated GABA-ergic medium spiny neurons modeling a juvenile form of HD (HD76). We have shown that calcium signaling is dramatically disturbed in HD76 neurons, specifically demonstrating higher levels of store-operated and voltage-gated calcium uptakes. However, comparing the HD76 neurons with the previously described low-repeat HD models, we have demonstrated that the severity of calcium signaling alterations does not depend on the length of the polyglutamine tract of the mutant huntingtin. Here we have also observed greater expression of huntingtin and an activator of store-operated calcium channels STIM2 in HD76 neurons. Since shRNA-mediated suppression of STIM2 decreased store-operated calcium uptake, we have speculated that high expression of STIM2 underlies the excessive entry through store-operated calcium channels in HD pathology. Moreover, a previously described potential anti-HD drug EVP4593 has been found to attenuate high levels of both huntingtin and STIM2 that may contribute to its neuroprotective effect. Our results are fully supportive in favor of the crucial role of calcium signaling deregulation in the HD pathogenesis and indicate that the cornerstone of excessive calcium uptake in HD-specific neurons is a calcium sensor and store-operated calcium channels activator STIM2, which should become a molecular target for medical treatment and novel neuroprotective drug development.

Impact of the DSP-H1684R Genetic Variant on Ion Channels Activity in iPSC-Derived Cardiomyocytes.

BACKGROUND/AIMS: Mutations of desmosomal genes are known to cause arrhythmogenic cardiomyopathy characterized by arrhythmias and sudden cardiac death. Previously, we described a novel genetic variant H1684R in desmoplakin gene (DSP), associated with a progressive cardiac conduction disease (PCCD). In the present study, we aimed to investigate an effect of the DSP-H1684R genetic variant on the activity of ion channels. METHODS: We used cardiomyocytes derived from induced pluripotent stem cells (iPSC cardiomyocytes) from a patient with DSP-H1684R genetic variant and from two healthy donors. Immunofluorescent staining and western blot analyses were used to characterize patient-specific cardiomyocytes. By the whole-cell voltage-clamp technique we estimated the activity of voltage-gated sodium, calcium, and potassium channels that are responsible for action potential generation and its shape. Action potentials' parameters were measured using whole-cell current-clamp technique. RESULTS: In patient-specific cardiomyocytes we observed both lower amplitudes of currents through sodium Nav1.5 channels and L-type calcium channels, but higher amplitude of current through transient-outward potassium channels in comparison to donor cardiomyocytes. Current-clamp measurements revealed shortening of action-potential in DSP-H1684R-carrying iPSC cardiomyocytes. Therefore, observed alterations in the channels activity might have a great impact on the properties of action potential and development of PCCD. CONCLUSION: Our results show that desmoplakin genetic variants, besides conduction slowing caused by structural heart remodeling, could affect multiple ion channel activity aggravating arrhythmia manifestation in PCCD.

Generation of two iPSC lines (FAMRCi004-A and FAMRCi004-B) from patient with familial progressive cardiac conduction disorder carrying genetic variant DSP p.His1684Arg.

Human iPSC cell lines (FAMRCi004-A and FAMRCi004-B) were generated from patient with progressive cardiac conduction disease and sick sinus syndrome carrying DSP p.His1684Arg genetic variant. Patient-specific adipose tissue-derived mesenchymal multipotent stromal cells were reprogrammed using non-integrative Sendai viruses. Established iPSC lines showed normal karyotype, expressed pluripotent markers and were able to differentiate toward three germ layers in vitro. The reported iPSC lines could be useful tool for in vitro modeling of progressive cardiac conduction disease associated with mutations in desmosomal genes.

Patient-Specific iPSC-Based Models of Huntington's Disease as a Tool to Study Store-Operated Calcium Entry Drug Targeting.

Neurodegenerative pathologies are among the most serious and socially significant problems of modern medicine, along with cardiovascular and oncological diseases. Several attempts have been made to prevent neuronal death using novel drugs targeted to the cell calcium signaling machinery, but the lack of adequate models for screening markedly impairs the development of relevant drugs. A potential breakthrough in this field is offered by the models of hereditary neurodegenerative pathologies based on endogenous expression of mutant proteins in neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs). Here, we study specific features of store-operated calcium entry (SOCE) using an iPSCs-based model of Huntington's disease (HD) and analyze the pharmacological effects of a specific drug targeted to the calcium channels. We show that SOCE in gamma aminobutyric acid-ergic striatal medium spiny neurons (GABA MSNs) was mediated by currents through at least two different channel groups, ICRAC and ISOC. Both of these groups were upregulated in HD neurons compared with the wild-type neurons. Thapsigargin-induced intracellular calcium store depletion in GABA MSNs resulted in predominant activation of either ICRAC or ISOC. The potential anti-HD drug EVP4593, which was previously shown to have neuroprotective activity in different HD models, affected both ICRAC and ISOC.

Axial tubule junctions control rapid calcium signaling in atria.

The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface. Rapid Ca2+ release correlated with colocalization of highly phosphorylated RyR2 clusters at AT-SR junctions and earlier, more rapid shortening of central sarcomeres. In contrast, mice expressing phosphorylation-incompetent RyR2 displayed depressed AM sarcomere shortening and reduced in vivo atrial contractile function. Moreover, left atrial hypertrophy led to AT proliferation, with a marked increase in the highly phosphorylated RyR2-pS2808 cluster fraction, thereby maintaining cytosolic Ca2+ signaling despite decreases in RyR2 cluster density and RyR2 protein expression. AT couplon "super-hubs" thus underlie faster excitation-contraction coupling in health as well as hypertrophic compensatory adaptation and represent a structural and metabolic mechanism that may contribute to contractile dysfunction and arrhythmias.

Hypersensitivity of excitation-contraction coupling in dystrophic cardiomyocytes.

Duchenne muscular dystrophy represents a severe inherited disease of striated muscle. It is caused by a mutation of the dystrophin gene and characterized by a progressive loss of skeletal muscle function. Most patients also develop a dystrophic cardiomyopathy, resulting in dilated hypertrophy and heart failure, but the cellular mechanisms leading to the deterioration of cardiac function remain elusive. In the present study, we tested whether defective excitation-contraction (E-C) coupling contributes to impaired cardiac performance. "E-C coupling gain" was determined in cardiomyocytes from control and dystrophin-deficient mdx mice. To this end, L-type Ca2+ currents (ICaL) were measured with the whole cell patch-clamp technique, whereas Ca2+ transients were simultaneously recorded with confocal imaging of fluo-3. Initial findings indicated subtle changes of E-C coupling in mdx cells despite matched Ca2+ loading of the sarcoplasmic reticulum (SR). However, lowering the extracellular Ca2+ concentration, a maneuver used to unmask latent E-C coupling problems, was surprisingly much better tolerated by mdx myocytes, suggesting a hypersensitive E-C coupling mechanism. Challenging the SR Ca2+ release by slow elevations of the intracellular Ca2+ concentration resulted in Ca2+ oscillations after a much shorter delay in mdx cells. This is consistent with an enhanced Ca2+ sensitivity of the SR Ca2+-release channels [ryanodine receptors (RyRs)]. The hypersensitivity could be normalized by the introduction of reducing agents, indicating that the elevated cellular ROS generation in dystrophy underlies the abnormal RyR sensitivity and hypersensitive E-C coupling. Our data suggest that in dystrophin-deficient cardiomyocytes, E-C coupling is altered due to potentially arrhythmogenic changes in the Ca2+ sensitivity of redox-modified RyRs.

Angiotensin II-mediated adaptive and maladaptive remodeling of cardiomyocyte excitation-contraction coupling.

Cardiac hypertrophy is associated with alterations in cardiomyocyte excitation-contraction coupling (ECC) and Ca(2+) handling. Chronic elevation of plasma angiotensin II (Ang II) is a major determinant in the pathogenesis of cardiac hypertrophy and congestive heart failure. However, the molecular mechanisms by which the direct actions of Ang II on cardiomyocytes contribute to ECC remodeling are not precisely known. This question was addressed using cardiac myocytes isolated from transgenic (TG1306/1R [TG]) mice exhibiting cardiac specific overexpression of angiotensinogen, which develop Ang II-mediated cardiac hypertrophy in the absence of hemodynamic overload. Electrophysiological techniques, photolysis of caged Ca(2+) and confocal Ca(2+) imaging were used to examine ECC remodeling at early ( approximately 20 weeks of age) and late ( approximately 60 weeks of age) time points during the development of cardiac dysfunction. In young TG mice, increased cardiac Ang II levels induced a hypertrophic response in cardiomyocyte, which was accompanied by an adaptive change of Ca(2+) signaling, specifically an upregulation of the Na(+)/Ca(2+) exchanger-mediated Ca(2+) transport. In contrast, maladaptation was evident in older TG mice, as suggested by reduced sarcoplasmic reticulum Ca(2+) content resulting from a shift in the ratio of plasmalemmal Ca(2+) removal and sarcoplasmic reticulum Ca(2+) uptake. This was associated with a conserved ECC gain, consistent with a state of hypersensitivity in Ca(2+)-induced Ca(2+) release. Together, our data suggest that chronic elevation of cardiac Ang II levels significantly alters cardiomyocyte ECC in the long term, and thereby contractility, independently of hemodynamic overload and arterial hypertension.

Ca2+ signaling in mouse cardiomyocytes with ablated S100A1 protein.

S100A1 is a Ca2+-binding protein expressed at high levels in the myocardium. It is thought to modulate the Ca2+ sensitivity of the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors or RyRs) and its expression has been shown to be down regulated in various heart diseases. In this study we used S100A1 knock-out (KO) mice to investigate the consequences of chronic S100A1 deficiency on Ca2+ cycling in ventricular cardiomyocytes. Confocal Ca2+ imaging showed that field-stimulated KO myocytes had near normal Ca2+ signaling under control conditions but a blunted response to beta-adrenergic stimulation with 1 micromol/l isoproterenol (ISO). Voltage-clamp experiments revealed that S100A1-deficient cardiomyocytes have elevated ICa under basal conditions. This larger Ca2+ influx was accompanied by augmented Ca2+ transients and elevated SR Ca2+ content, without changes in macroscopic excitation-contraction coupling gain, which suggests impaired fractional Ca2+ release. Exposure of KO and WT cells to ISO led to similar maximal ICa. Thus, the stimulation of the ICa was less pronounced in KO cardiomyocytes, suggesting that changes in basal ICa could underlie the reduced beta-adrenergic response. Taken together, our findings indicate that chronic absence of S100A1 results in enhanced L-type Ca2+ channel activity combined with a blunted SR Ca2+ release amplification. These findings may have implications in a variety of cardiac pathologies where abnormal RyR Ca2+ sensitivity or reduced S100A1 levels have been described.

Modulation of the local SR Ca2+ release by intracellular Mg2+ in cardiac myocytes.

In cardiac muscle, Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca(2+) transient. The global elevation of the intracellular Ca(2+) concentration arises from the spatial and temporal summation of elementary Ca(2+) release events, Ca(2+) sparks. Ca(2+) sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca(2+), Mg(2+), and ATP). Here, we examined by which mechanism the free intracellular Mg(2+) ([Mg(2+)](free)) affects various Ca(2+) spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg(2+) in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg(2+) from the possible activation by ATP and Mg(2+)-ATP via the adenine binding site of the channel. Changes in [Mg(2+)](free) generally led to biphasic alterations of the Ca(2+) spark frequency. For example, lowering [Mg(2+)](free) resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca(2+) depletion. Fitting the Ca(2+) spark inhibition by [Mg(2+)](free) with a Hill equation revealed a K(i) of 0.1 mM. In conclusion, our results support the notion that local Ca(2+) release and Ca(2+) sparks are modulated by Mg(2+) in the intracellular environment. This seems to occur predominantly by hindering Ca(2+)-dependent activation of the RYRs through competitive Mg(2+) occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg(2+) and ATP can change.

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms.

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.