Although ammonia is a well‐known neuropathogenic factor, the cellular mechanisms of ammonia toxicity are less characterized. Up to now, the main focus of ammonia toxicity has been on astrocytes and ...neurons. Despite the significance of microglia in neurodegenerative diseases, little is known about their responsiveness to ammonia. In the present study, we found that ammonia triggered mitosis at concentrations between 30 μm and 3.0 mm but apoptosis at concentrations ≥ 1.0 mm in the murine microglial cell line BV‐2. Most apoptotic cells showed an accumulation of condensed chromatin at the nuclear envelope, blebbing of the plasma membrane, formation of apoptotic bodies and an increase in caspase 3/7 activity. Blockade of caspase 3/7 activity by Ac‐DEVD‐CHO suppressed ammonia‐induced apoptosis. Surprisingly, some BV‐2 cells exposed to ammonia displayed clear signs of mitotic catastrophe, a type of cell death occurring during mitosis. In a further series of experiments, we found that cyclic adenosine 3′,5′‐monophosphate (cAMP) mediated the apoptogenic effects of ammonia, because (i) ammonia dose‐dependently elevated the intracellular cAMP level, (ii) blockade of the adenylyl cyclase by SQ‐22536 suppressed ammonia‐induced apoptosis, (iii) inhibition of phosphodiesterases (PDEs) by the nonselective PDE inhibitor IBMX, or by the PDE4‐selective inhibitor rolipram, increased the relative number of apoptotic cells, and (iv) the cAMP analogues 8‐bromoadenosine cAMP and Sp‐cAMP mimicked the effect of ammonia and triggered apoptosis. Taken together, our results indicate that distinct concentrations of ammonia trigger opposite signalling pathways in microglial cells.
TPC Functions in the Immune System Steiner, Philip; Arlt, Elisabeth; Boekhoff, Ingrid ...
Handbook of experimental pharmacology,
2023, Letnik:
278
Journal Article
Recenzirano
Two-pore channels (TPCs) are novel intracellular cation channels, which play a key role in numerous (patho-)physiological and immunological processes. In this chapter, we focus on their function in ...immune cells and immune reactions. Therefore, we first give an overview of the cellular immune response and the partaking immune cells. Second, we concentrate on ion channels which in the past have been shown to play an important role in the regulation of immune cells. The main focus is then directed to TPCs, which are primarily located in the membranes of acidic organelles, such as lysosomes or endolysosomes but also certain other vesicles. They regulate Ca
homeostasis and thus Ca
signaling in immune cells. Due to this important functional role, TPCs are enjoying increasing attention within the field of immunology in the last few decades but are also becoming more pertinent as pharmacological targets for the treatment of pro-inflammatory diseases such as allergic hypersensitivity. However, to uncover the precise molecular mechanism of TPCs in immune cell responses, further molecular, genetic, and ultrastructural investigations on TPCs are necessary, which then may pave the way to develop novel therapeutic strategies to treat diseases such as anaphylaxis more specifically.
•Channel-kinases TRPM6 and TRPM7 control the organismal balance of divalent cations.•Impaired functions of TRPM6 and TRPM7 affect normal prenatal development and healthy adulthood.•Pharmacological ...agents are required for selective targeting of TRPM6 and TRPM7 in preclinical disease models.•Iloperidone, ifenprodil, and VER155008 were identified as specific and potent inhibitors of the TRPM6 and TRPM7 channels.
The transient receptor potential cation channel, subfamily M, members 6 and 7 (TRPM6 and TRPM7) are homologous membrane proteins encompassing cation channel units fused to cytosolic serine/threonine-protein kinase domains. Clinical studies and experiments with animal disease models suggested that selective inhibition of TRPM6 and TRPM7 currents might be beneficial for subjects with immune and cardiovascular disorders, tumours and other pathologies, but the suitable pharmacological toolkit remains underdeveloped. The present study identified small synthetic molecules acting specifically on the channel moieties of TRPM6 and TRPM7. Using electrophysiological analysis in conjunction with Ca2+ imaging, we show that iloperidone and ifenprodil inhibit the channel activity of recombinant TRPM6 with IC50 values of 0.73 and 3.33 µM, respectively, without an impact on the TRPM7 channel. We also found that VER155008 suppresses the TRPM7 channel with an IC50 value of 0.11 µM but does not affect TRPM6. Finally, the effects of iloperidone and VER155008 were found to be suitable for blocking native endogenous TRPM6 and TRPM7 in a collection of mouse and human cell models. Hence, the identification of iloperidone, ifenprodil, and VER155008 allows for the first time to selectively manipulate TRPM6 and TRPM7 currents.
Display omitted
Mg(2+) is the second-most abundant cation in animal cells and is an essential cofactor in numerous enzymatic reactions. The molecular mechanisms controlling Mg(2+) balance in the organism are not ...well understood. In this study, we report identification of TRPM7, a bifunctional protein containing a protein kinase fused to an ion channel, as a key regulator of whole body Mg(2+) homeostasis in mammals. We generated TRPM7-deficient mice with the deletion of the kinase domain. Homozygous TRPM7(Δkinase) mice demonstrated early embryonic lethality, whereas heterozygous mice were viable, but developed signs of hypomagnesaemia and revealed a defect in intestinal Mg(2+) absorption. Cells derived from heterozygous TRPM7(Δkinase) mice demonstrated reduced TRPM7 currents that had increased sensitivity to the inhibition by Mg(2+). Embryonic stem cells lacking TRPM7 kinase domain displayed a proliferation arrest phenotype that can be rescued by Mg(2+) supplementation. Our results demonstrate that TRPM7 is essential for the control of cellular and whole body Mg(2+) homeostasis.
Mg2+ is the second-most abundant cation in animal cells and is an essential cofactor in numerous enzymatic reactions. The molecular mechanisms controlling Mg2+ balance in the organism are not well ...understood. In this study, we report identification of TRPM7, a bifunctional protein containing a protein kinase fused to an ion channel, as a key regulator of whole body Mg2+ homeostasis in mammals. We generated TRPM7-deficient mice with the deletion of the kinase domain. Homozygous TRPM7Δkinase mice demonstrated early embryonic lethality, whereas heterozygous mice were viable, but developed signs of hypomagnesaemia and revealed a defect in intestinal Mg2+ absorption. Cells derived from heterozygous TRPM7Δkinase mice demonstrated reduced TRPM7 currents that had increased sensitivity to the inhibition by Mg2+ . Embryonic stem cells lacking TRPM7 kinase domain displayed a proliferation arrest phenotype that can be rescued by Mg2+ supplementation. Our results demonstrate that TRPM7 is essential for the control of cellular and whole body Mg2+ homeostasis.
Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a bi-functional protein comprising an ion channel moiety covalently linked to a protein kinase domain. Currently, the ...prevailing view is that a decrease in the cytosolic Mg
2+
concentration leads to activation of divalent cation-selective TRPM7 currents. TRPM7 plays a role in immune responses, hypotension, tissue fibrosis, and tumor progression and, therefore, represents a new promising therapeutic target. Because of the dearth of pharmacological tools, our mechanistic understanding of the role of TRPM7 in physiology and pathophysiology still lags behind. Therefore, we have recently carried out a high throughput screen for small-molecule activators of TRPM7. We have characterized the phenanthrene naltriben as a first stimulatory agonist of the TRPM7 channel. Surprisingly, the effect of naltriben on TRPM7 was found to be unaffected by the physiological levels of cytosolic Mg
2+
. Here, we demonstrate that mibefradil and NNC 50–0396, two benzimidazole relatives of the TRPM7 inhibitor NS8593, are positive modulators of TRPM7. Using Ca
2+
imaging and the patch-clamp technique, we show that mibefradil activates TRPM7-mediated Ca
2+
entry and whole-cell currents. The response to mibefradil was fast, reversible, and reproducible. In contrast to naltriben, mibefradil efficiently activates TRPM7 currents only at physiological intracellular Mg
2+
concentrations, and its stimulatory effect was fully abrogated by high internal Mg
2+
levels. Consequently, a TRPM7 variant harboring a gain-of-function mutation was insensitive to further mibefradil activation. Finally, we observed that the effect of mibefradil was selective for TRPM7 when various TRP channels were tested. Taken together, mibefradil acts as a Mg
2+
-regulated agonist of the TRPM7 channel and, hence, uncovers a new class of TRPM7 agonists.
Zn
2+
, Mg
2+
and Ca
2+
are essential divalent cations implicated in many metabolic processes and signalling pathways. An emerging new paradigm is that the organismal balance of these cations ...predominantly depends on a common gatekeeper, the channel-kinase TRPM7. Despite extensive electrophysiological studies and recent cryo-EM analysis, an open question is how the channel activity of TRPM7 is activated. Here, we performed site-directed mutagenesis of mouse TRPM7 in conjunction with patch-clamp assessment of whole-cell and single-channel activity and molecular dynamics (MD) simulations to show that the side chains of conserved N1097 form an inter-subunit Mg
2+
regulatory site located in the lower channel gate of TRPM7. Our results suggest that intracellular Mg
2+
binds to this site and stabilizes the TRPM7 channel in the closed state, whereas the removal of Mg
2+
favours the opening of TRPM7. Hence, our study identifies the structural underpinnings through which the TRPM7 channel is controlled by cytosolic Mg
2+
, representing a new structure–function relationship not yet explored among TRPM channels.
Sustained exposure of the lung to various environmental or occupational toxins may eventually lead to pulmonary fibrosis, a devastating disease with no cure. Pulmonary fibrosis is characterized by ...excessive deposition of extracellular matrix (ECM) proteins such as fibronectin and collagens. The peptidase plasmin degrades the ECM, but protein levels of the plasmin activator inhibitor-1 (PAI-1) are increased in fibrotic lung tissue, thereby dampening plasmin activity. Transforming growth factor-β1 (TGF-β1)-induced activation of SMAD transcription factors promotes ECM deposition by enhancing collagen, fibronectin and PAI-1 levels in pulmonary fibroblasts. Hence, counteracting TGF-β1-induced signaling is a promising approach for the therapy of pulmonary fibrosis. Transient receptor potential cation channel subfamily M Member 7 (TRPM7) supports TGF-β1-promoted SMAD signaling in T-lymphocytes and the progression of fibrosis in kidney and heart. Thus, we investigated possible effects of TRPM7 on plasmin activity, ECM levels and TGF-β1 signaling in primary human pulmonary fibroblasts (pHPF). We found that two structurally unrelated TRPM7 blockers enhanced plasmin activity and reduced fibronectin or PAI-1 protein levels in pHPF under basal conditions. Further, TRPM7 blockade strongly inhibited fibronectin and collagen deposition induced by sustained TGF-β1 stimulation. In line with these data, inhibition of TRPM7 activity diminished TGF-β1-triggered phosphorylation of SMAD-2, SMAD-3/4-dependent reporter activation and PAI-1 mRNA levels. Overall, we uncover TRPM7 as a novel supporter of TGF-β1 signaling in pHPF and propose TRPM7 blockers as new candidates to control excessive ECM levels under pathophysiological conditions conducive to pulmonary fibrosis.
Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of ...inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca2+) signals. Ca2+ release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca2+ homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca2+ uptake and filling of endoplasmic reticulum Ca2+ stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.
Abstract
Aims
Neutrophil trafficking within the vasculature strongly relies on intracellular calcium signalling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to ...maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signalling.
Methods and results
Using in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store-operated Ca2+ entry through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signalling, thereby preventing cellular spreading, adhesion strengthening, and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of Escherichia coli particles by neutrophils in the absence of KV1.3.
Conclusion
We show that the voltage-gated potassium channel KV1.3 is critical for Ca2+ signalling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signalling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration.
Graphical Abstract
PDF
