International Journal of Molecular Sciences

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23 pages, 3810 KiB

Open AccessArticle

Synthesis and Characterization of Store-Operated Calcium Entry Inhibitors Active in the Submicromolar Range

by Camille Le Guilcher, Tomas Luyten, Jan B. Parys, Mathieu Pucheault and Olivier Dellis

Int. J. Mol. Sci. 2020, 21(24), 9777; https://doi.org/10.3390/ijms21249777 - 21 Dec 2020

Cited by 2 | Viewed by 2750

Abstract

The store-operated calcium entry, better known as SOCE, forms the main Ca²⁺ influx pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation and the immune response. During the past decades, several inhibitors were developed, but they lack [...] Read more.

The store-operated calcium entry, better known as SOCE, forms the main Ca²⁺ influx pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation and the immune response. During the past decades, several inhibitors were developed, but they lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate (2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups. Among them, our compound P11 showed the best inhibitory capacity with a K_i ≈ 75 nM. Furthermore, below 1 µM, P11 was devoid of any inhibitory activity on the two other main cellular targets of 2-APB, the IP₃ receptors, and the SERCA pumps. Interestingly, Jurkat T cells secrete interleukin-2 under phytohemagglutinin stimulation but undergo cell death and stop IL-2 synthesis when stimulated in the presence of increasing P11 concentrations. Thus, P11 could represent the first member of a new and potent family of immunosuppressors. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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25 pages, 4234 KiB

Open AccessArticle

PGRMC1 Inhibits Progesterone-Evoked Proliferation and Ca²⁺ Entry Via STIM2 in MDA-MB-231 Cells

by Carlos Cantonero, Ginés M. Salido, Juan A. Rosado and Pedro C. Redondo

Int. J. Mol. Sci. 2020, 21(20), 7641; https://doi.org/10.3390/ijms21207641 - 15 Oct 2020

Cited by 14 | Viewed by 3072

Abstract

Progesterone receptor membrane component 1 (PGRMC1) has been shown to regulate some cancer hallmarks. Progesterone (P₄) evokes intracellular calcium (Ca²⁺) changes in the triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and BT-20) and in other breast cancer cell lines [...] Read more.

Progesterone receptor membrane component 1 (PGRMC1) has been shown to regulate some cancer hallmarks. Progesterone (P₄) evokes intracellular calcium (Ca²⁺) changes in the triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and BT-20) and in other breast cancer cell lines like the luminal MCF7 cells. PGRMC1 expression is elevated in MDA-MB-231 and MCF7 cells as compared to non-tumoral MCF10A cell line, and PGRMC1 silencing enhances P₄-evoked Ca²⁺ mobilization. Here, we found a new P₄-dependent Ca²⁺ mobilization pathway in MDA-MB-231 cells and other triple-negative breast cancer cells, as well as in MCF7 cells that involved Stromal interaction molecule 2 (STIM2), Calcium release-activated calcium channel protein 1 (Orai1), and Transient Receptor Potential Channel 1 (TRPC1). Stromal interaction molecule 1 (STIM1) was not involved in this novel Ca²⁺ pathway, as evidenced by using siRNA STIM1. PGRMC1 silencing reduced the negative effect of P₄ on cell proliferation and cell death in MDA-MB-231 cells. In line with the latter observation, Nuclear Factor of Activated T-Cells 1 (NFAT1) nuclear accumulation due to P₄ incubation for 48 h was enhanced in cells transfected with the small hairpin siRNA against PGRMC1 (shPGRMC1). These results provide evidence for a novel P₄-evoked Ca²⁺ entry pathway that is downregulated by PGRMC1. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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28 pages, 5651 KiB

Open AccessArticle

Synthesis and Pharmacological Characterization of 2-Aminoethyl Diphenylborinate (2-APB) Derivatives for Inhibition of Store-Operated Calcium Entry (SOCE) in MDA-MB-231 Breast Cancer Cells

by Achille Schild, Rajesh Bhardwaj, Nicolas Wenger, Dominic Tscherrig, Palanivel Kandasamy, Jan Dernič, Roland Baur, Christine Peinelt, Matthias A. Hediger and Martin Lochner

Int. J. Mol. Sci. 2020, 21(16), 5604; https://doi.org/10.3390/ijms21165604 - 5 Aug 2020

Cited by 19 | Viewed by 4424

Abstract

Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca²⁺ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP [...] Read more.

Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca²⁺ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP₃) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca²⁺ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca²⁺ channels that facilitate a measurable Ca²⁺ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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15 pages, 3209 KiB

Open AccessArticle

Luminal STIM1 Mutants that Cause Tubular Aggregate Myopathy Promote Autophagic Processes

by Matthias Sallinger, Adéla Tiffner, Tony Schmidt, Daniel Bonhenry, Linda Waldherr, Irene Frischauf, Victoria Lunz, Isabella Derler, Romana Schober and Rainer Schindl

Int. J. Mol. Sci. 2020, 21(12), 4410; https://doi.org/10.3390/ijms21124410 - 21 Jun 2020

Cited by 21 | Viewed by 2961

Abstract

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca²⁺ sensor protein that induces permeation of Orai Ca²⁺ channels upon endoplasmic reticulum Ca²⁺-store depletion. A drop in luminal Ca²⁺ causes partial unfolding of the N-terminal STIM1 domains and [...] Read more.

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca²⁺ sensor protein that induces permeation of Orai Ca²⁺ channels upon endoplasmic reticulum Ca²⁺-store depletion. A drop in luminal Ca²⁺ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca²⁺ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca²⁺-release-activated Ca²⁺ currents (I_CRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca²⁺ down-stream activation of autophagic processes. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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13 pages, 2325 KiB

Open AccessArticle

The Number and Position of Orai3 Units within Heteromeric Store-Operated Ca²⁺ Channels Alter the Pharmacology of I_CRAC

by Sven Kappel, Tatiana Kilch, Roland Baur, Martin Lochner and Christine Peinelt

Int. J. Mol. Sci. 2020, 21(7), 2458; https://doi.org/10.3390/ijms21072458 - 2 Apr 2020

Cited by 12 | Viewed by 2624

Abstract

Store-operated heteromeric Orai1/Orai3 channels have been discussed in the context of aging, cancer, and immune cell differentiation. In contrast to homomeric Orai1 channels, they exhibit a different pharmacology upon application of reactive oxygen species (ROS) or 2-aminoethoxydiphenyl borate (2-APB) in various cell types. [...] Read more.

Store-operated heteromeric Orai1/Orai3 channels have been discussed in the context of aging, cancer, and immune cell differentiation. In contrast to homomeric Orai1 channels, they exhibit a different pharmacology upon application of reactive oxygen species (ROS) or 2-aminoethoxydiphenyl borate (2-APB) in various cell types. In endogenous cells, subunit composition and arrangement may vary and cannot be defined precisely. In this study, we used patch-clamp electrophysiology to investigate the 2-APB profile of store-operated and store-independent homomeric Orai1 and heteromeric Orai1/Orai3 concatenated channels with defined subunit compositions. As has been shown previous, one or more Orai3 subunit(s) within the channel result(s) in decreased Ca²⁺ release activated Ca²⁺ current (I_CRAC). Upon application of 50 µM 2-APB, channels with two or more Orai3 subunits exhibit large outward currents and can be activated by 2-APB independent from storedepletion and/or the presence of STIM1. The number and position of Orai3 subunits within the heteromeric store-operated channel change ion conductivity of 2-APB-activated outward current. Compared to homomeric Orai1 channels, one Orai3 subunit within the channel does not alter 2-APB pharmacology. None of the concatenated channel constructs were able to exactly simulate the complex 2-APB pharmacology observed in prostate cancer cells. However, 2-APB profiles of prostate cancer cells are similar to those of concatenated channels with Orai3 subunit(s). Considering the presented and previous results, this indicates that distinct subtypes of heteromeric SOCE channels may be selectively activated or blocked. In the future, targeting distinct heteromeric SOCE channel subtypes may be the key to tailored SOCE-based therapies. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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Review

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34 pages, 5634 KiB

Open AccessReview

The Orai Pore Opening Mechanism

by Adéla Tiffner, Lena Maltan, Sarah Weiß and Isabella Derler

Int. J. Mol. Sci. 2021, 22(2), 533; https://doi.org/10.3390/ijms22020533 - 7 Jan 2021

Cited by 16 | Viewed by 3481

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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29 pages, 1129 KiB

Open AccessReview

More Than Just Simple Interaction between STIM and Orai Proteins: CRAC Channel Function Enabled by a Network of Interactions with Regulatory Proteins

by Sascha Berlansky, Christina Humer, Matthias Sallinger and Irene Frischauf

Int. J. Mol. Sci. 2021, 22(1), 471; https://doi.org/10.3390/ijms22010471 - 5 Jan 2021

Cited by 20 | Viewed by 5483

Abstract

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca²⁺ from the endoplasmic reticulum (ER), is critical for Ca²⁺ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of [...] Read more.

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca²⁺ from the endoplasmic reticulum (ER), is critical for Ca²⁺ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca²⁺ concentration, and in turn, are responsible for relaying the signal of Ca²⁺ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca²⁺ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca²⁺ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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18 pages, 1993 KiB

Open AccessReview

STIM Proteins: An Ever-Expanding Family

by Herwig Grabmayr, Christoph Romanin and Marc Fahrner

Int. J. Mol. Sci. 2021, 22(1), 378; https://doi.org/10.3390/ijms22010378 - 31 Dec 2020

Cited by 25 | Viewed by 3595

Abstract

Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An [...] Read more.

Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel. The SOCE pathway is activated by the ligand-induced depletion of the ER calcium store. STIM proteins, acting as calcium sensors, subsequently sense this depletion and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes. This review article is dedicated to the latest advances in the field of STIM proteins. New results of ongoing investigations based on the recently published functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are reported and complemented with a discussion of the latest developments in the research of STIM protein isoforms and their differential functions in regulating SOCE. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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21 pages, 1319 KiB

Open AccessReview

Altered Ca²⁺ Homeostasis in Immune Cells during Aging: Role of Ion Channels

by Dorina Zöphel, Chantal Hof and Annette Lis

Int. J. Mol. Sci. 2021, 22(1), 110; https://doi.org/10.3390/ijms22010110 - 24 Dec 2020

Cited by 12 | Viewed by 3168

Abstract

Aging is an unstoppable process and begins shortly after birth. Each cell of the organism is affected by the irreversible process, not only with equal density but also at varying ages and with different speed. Therefore, aging can also be understood as an [...] Read more.

Aging is an unstoppable process and begins shortly after birth. Each cell of the organism is affected by the irreversible process, not only with equal density but also at varying ages and with different speed. Therefore, aging can also be understood as an adaptation to a continually changing cellular environment. One of these very prominent changes in age affects Ca²⁺ signaling. Especially immune cells highly rely on Ca²⁺-dependent processes and a strictly regulated Ca²⁺ homeostasis. The intricate patterns of impaired immune cell function may represent a deficit or compensatory mechanisms. Besides, altered immune function through Ca²⁺ signaling can profoundly affect the development of age-related disease. This review attempts to summarize changes in Ca²⁺ signaling due to channels and receptors in T cells and beyond in the context of aging. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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