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1.
Cell Mol Life Sci ; 79(5): 225, 2022 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-35389104

RESUMO

Zn2+, Mg2+ and Ca2+ 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 Mg2+ regulatory site located in the lower channel gate of TRPM7. Our results suggest that intracellular Mg2+ binds to this site and stabilizes the TRPM7 channel in the closed state, whereas the removal of Mg2+ favours the opening of TRPM7. Hence, our study identifies the structural underpinnings through which the TRPM7 channel is controlled by cytosolic Mg2+, representing a new structure-function relationship not yet explored among TRPM channels.


Assuntos
Canais de Cátion TRPM , Animais , Cátions Bivalentes/metabolismo , Magnésio/metabolismo , Camundongos , Fosfotransferases/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo
2.
Handb Exp Pharmacol ; 278: 71-92, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36639434

RESUMO

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 Ca2+ homeostasis and thus Ca2+ 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.


Assuntos
Canais de Cálcio , Lisossomos , Humanos , Canais de Cálcio/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Sistema Imunitário/metabolismo , Endossomos/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio
3.
Proc Natl Acad Sci U S A ; 117(30): 18068-18078, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661165

RESUMO

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.


Assuntos
Anafilaxia/etiologia , Anafilaxia/metabolismo , Canais de Cálcio/deficiência , Suscetibilidade a Doenças , Mastócitos/imunologia , Mastócitos/metabolismo , Biomarcadores , Sinalização do Cálcio , Degranulação Celular , Citocinas/metabolismo , Predisposição Genética para Doença , Histamina/metabolismo , Imunoglobulina E/imunologia , Mediadores da Inflamação/metabolismo
4.
Arch Toxicol ; 96(10): 2767-2783, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35864199

RESUMO

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-ß1 (TGF-ß1)-induced activation of SMAD transcription factors promotes ECM deposition by enhancing collagen, fibronectin and PAI-1 levels in pulmonary fibroblasts. Hence, counteracting TGF-ß1-induced signaling is a promising approach for the therapy of pulmonary fibrosis. Transient receptor potential cation channel subfamily M Member 7 (TRPM7) supports TGF-ß1-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-ß1 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-ß1 stimulation. In line with these data, inhibition of TRPM7 activity diminished TGF-ß1-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-ß1 signaling in pHPF and propose TRPM7 blockers as new candidates to control excessive ECM levels under pathophysiological conditions conducive to pulmonary fibrosis.


Assuntos
Fibrose Pulmonar , Canais de Cátion TRPM , Colágeno/antagonistas & inibidores , Colágeno/metabolismo , Fibrinolisina/metabolismo , Fibroblastos , Fibronectinas/efeitos adversos , Fibronectinas/antagonistas & inibidores , Fibronectinas/metabolismo , Fibrose , Humanos , Pulmão/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas Serina-Treonina Quinases , Fibrose Pulmonar/induzido quimicamente , Canais de Cátion TRPM/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo
5.
Arterioscler Thromb Vasc Biol ; 38(2): 344-352, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29146750

RESUMO

OBJECTIVE: TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg2+ and Ca2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. APPROACH AND RESULTS: Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice (Trpm7R/R ) causes a marked signaling defect in platelets. Trpm7R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and ß3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. CONCLUSIONS: Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca2+ entry in platelets. The protection of Trpm7R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.


Assuntos
Arteriopatias Oclusivas/sangue , Plaquetas/metabolismo , Sinalização do Cálcio , Cálcio/sangue , Infarto da Artéria Cerebral Média/sangue , Canais de Cátion TRPM/sangue , Trombose/sangue , Animais , Arteriopatias Oclusivas/genética , Arteriopatias Oclusivas/patologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Lectinas Tipo C/sangue , Camundongos Mutantes , Fosfatidilinositol 4,5-Difosfato/sangue , Fosfolipase C beta/sangue , Fosfolipase C gama/sangue , Fosforilação , Glicoproteínas da Membrana de Plaquetas/metabolismo , Mutação Puntual , Receptores Ativados por Proteinase/sangue , Molécula 1 de Interação Estromal/sangue , Sinaptofisina/sangue , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Trombose/genética , Trombose/patologia
6.
J Physiol ; 594(11): 2957-70, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-26660477

RESUMO

KEY POINTS: The Mg(2+) and Ca(2+) conducting transient receptor potential melastatin 7 (TRPM7) channel-enzyme (chanzyme) has been implicated in immune cell function. Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not. As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release. Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function. Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca(2+) and extracellular Mg(2+) sensitivity of mast cell degranulation. ABSTRACT: Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C-terminally located α-kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7(+/∆K) ) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7(KR) ), are not hyperallergic and are resistant to systemic magnesium (Mg(2+) ) deprivation. Since allergic reactions are triggered by mast cell-mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild-type (TRPM7(+/+) ), TRPM7(+/∆K) and TRPM7(KR) mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg(2+) assured unperturbed IgE-DNP-dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE-DNP-dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca(2+) ) in G protein-induced exocytosis. In addition, G protein-coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg(2+) caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca(2+) and affecting granular mobility and/or histamine contents.


Assuntos
Degranulação Celular/fisiologia , Mastócitos/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Células Cultivadas , Ativação Enzimática/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Canais de Cátion TRPM/genética
7.
J Cell Physiol ; 231(5): 1114-29, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26439876

RESUMO

Besides its capacity to inhibit the 1,4,5-trisphosphate (IP3) receptor, the regulatory protein IRBIT (IP3 receptor binding protein released with IP3) is also able to control the activity of numerous ion channels and electrolyte transporters and thereby creates an optimal electrolyte composition of various biological fluids. Since a reliable execution of spermatogenesis and sperm maturation critically depends on the establishment of an adequate microenvironment, the expression of IRBIT in male reproductive tissue was examined using immunohistochemical approaches combined with biochemical fractionation methods. The present study documents that IRBIT is expressed in Leydig and Sertoli cells. In addition, pronounced IRBIT expression was detected in sperm precursors during early stages of spermatogenesis as well as in spermatozoa. Analyzing tissue sections of rodent epididymides, IRBIT was found to co-localize with the proton pumping V-ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of narrow and clear cells. A similar co-localization of IRBIT with CFTR was also observed for Sertoli cells and developing germ cells. Remarkably, assaying caudal sperm in immunogold electron microscopy, IRBIT was found to localize to the acrosomal cap and the flagellum as well as to the sperm nucleus; moreover, a prominent oligomerization was observed for spermatozoa. The pronounced occurrence of IRBIT in the male reproductive system and mature spermatozoa indicates a potential role for IRBIT in establishing the essential luminal environment for a faithful execution of spermatogenesis and epididymal sperm maturation, and suggest a participation of IRBIT during maturation steps after ejaculation and/or the final fertilization process.


Assuntos
Adenosil-Homocisteinase/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Reprodução , Espermatozoides/metabolismo , Animais , Western Blotting , Epididimo/citologia , Epididimo/metabolismo , Células Epiteliais/metabolismo , Immunoblotting , Imuno-Histoquímica , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos Sprague-Dawley , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Espermatozoides/citologia , Testículo/citologia , Testículo/ultraestrutura , ATPases Vacuolares Próton-Translocadoras/metabolismo
8.
Pflugers Arch ; 468(4): 623-34, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26669310

RESUMO

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.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Mibefradil/farmacologia , Canais de Cátion TRPM/agonistas , 1-Naftilamina/análogos & derivados , 1-Naftilamina/farmacologia , Animais , Cálcio/metabolismo , Células HEK293 , Humanos , Magnésio/metabolismo , Camundongos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo
9.
J Biol Chem ; 287(49): 40956-71, 2012 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-23074220

RESUMO

Sensory neuron-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are primate-specific proteins that are exclusively expressed in primary sensory neurons and provoke pain in humans. Hence, MRGPR-X1 represent promising targets for future pain therapy, but signaling pathways activated by MRGPR-X1 are poorly understood. The transient receptor potential cation channel V1 (TRPV1) is also expressed in primary sensory neurons and detects painful stimuli such as protons and heat. G(q)-promoted signaling has been shown to sensitize TRPV1 via protein kinase C (PKC)-dependent phosphorylation. In addition, recent studies suggested TRPV1 activation via a G(q)-mediated mechanism involving diacylglycerol (DAG) or phosphatidylinositol-4,5-bisphosphate (PIP(2)). However, it is not clear if DAG-promoted TRPV1 activation occurs independently from classic TRPV1 activation modes induced by heat and protons. Herein, we analyzed putative functional interactions between MRGPR-X1 and TRPV1 in a previously reported F11 cell line stably over-expressing MRGPR-X1. First, we found that MRGPR-X1 sensitized TRPV1 to heat and protons in a PKC-dependent manner. Second, we observed direct MRGPR-X1-mediated TRPV1 activation independent of MRGPR-X1-induced Ca(2+)-release and PKC activity or other TRPV1 affecting enzymes such as lipoxygenase, extracellular signal-regulated kinases-1/2, sarcoma, or phosphoinositide 3-kinase. Investigating several TRPV1 mutants, we observed that removal of the TRPV1 binding site for DAG and of the putative PIP(2) sensor decreased MRGPR-X1-induced TRPV1 activation by 71 and 43%, respectively. Therefore, we demonstrate dual functional interactions between MRGPR-X1 and TRPV1, resulting in PKC-dependent TRPV1 sensitization and DAG/PIP(2)-mediated activation. The molecular discrimination between TRPV1 sensitization and activation may help improve the specificity of current pain therapies.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Analgésicos/farmacologia , Animais , Linhagem Celular , Dor Crônica/tratamento farmacológico , Diglicerídeos/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Humanos , Manganês/farmacologia , Camundongos , Manejo da Dor , Fosfatidilinositóis/metabolismo , Proteína Quinase C/metabolismo , Ratos , Transdução de Sinais
10.
Mucosal Immunol ; 16(2): 180-193, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36634819

RESUMO

The blockade or deletion of the pro-inflammatory P2X7 receptor channel has been shown to reduce tissue damage and symptoms in models of inflammatory bowel disease, and P2X7 receptors on enteric neurons were suggested to mediate neuronal death and associated motility changes. Here, we used P2X7-specific antibodies and nanobodies, as well as a bacterial artificial chromosome transgenic P2X7-EGFP reporter mouse model and P2rx7-/- controls to perform a detailed analysis of cell type-specific P2X7 expression and possible overexpression effects in the enteric nervous system of the distal colon. In contrast to previous studies, we did not detect P2X7 in neurons but found dominant expression in glia and macrophages, which closely interact with the neurons. The overexpression of P2X7 per se did not induce significant pathological effects. Our data indicate that macrophages and/or glia account for P2X7-mediated neuronal damage in inflammatory bowel disease and provide a refined basis for the exploration of P2X7-based therapeutic strategies.


Assuntos
Colite , Doenças Inflamatórias Intestinais , Camundongos , Animais , Colite/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios , Doenças Inflamatórias Intestinais/metabolismo , Camundongos Transgênicos , Macrófagos/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo
11.
Function (Oxf) ; 4(6): zqad053, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37786778

RESUMO

Cyclooxygenase-2 (COX-2) is a key regulator of inflammation. High constitutive COX-2 expression enhances survival and proliferation of cancer cells, and adversely impacts antitumor immunity. The expression of COX-2 is modulated by various signaling pathways. Recently, we identified the melastatin-like transient-receptor-potential-7 (TRPM7) channel-kinase as modulator of immune homeostasis. TRPM7 protein is essential for leukocyte proliferation and differentiation, and upregulated in several cancers. It comprises of a cation channel and an atypical α-kinase, linked to inflammatory cell signals and associated with hallmarks of tumor progression. A role in leukemia has not been established, and signaling pathways are yet to be deciphered. We show that inhibiting TRPM7 channel-kinase in chronic myeloid leukemia (CML) cells results in reduced constitutive COX-2 expression. By utilizing a CML-derived cell line, HAP1, harboring CRISPR/Cas9-mediated TRPM7 knockout, or a point mutation inactivating TRPM7 kinase, we could link this to reduced activation of AKT serine/threonine kinase and mothers against decapentaplegic homolog 2 (SMAD2). We identified AKT as a direct in vitro substrate of TRPM7 kinase. Pharmacologic blockade of TRPM7 in wildtype HAP1 cells confirmed the effect on COX-2 via altered AKT signaling. Addition of an AKT activator on TRPM7 kinase-dead cells reconstituted the wildtype phenotype. Inhibition of TRPM7 resulted in reduced phosphorylation of AKT and diminished COX-2 expression in peripheral blood mononuclear cells derived from CML patients, and reduced proliferation in patient-derived CD34+ cells. These results highlight a role of TRPM7 kinase in AKT-driven COX-2 expression and suggest a beneficial potential of TRPM7 blockade in COX-2-related inflammation and malignancy.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide , Canais de Cátion TRPM , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Ciclo-Oxigenase 2/genética , Canais de Cátion TRPM/genética , Leucócitos Mononucleares/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Inflamação , Proteínas Serina-Treonina Quinases/genética
12.
JCI Insight ; 8(3)2023 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-36574297

RESUMO

Most overweight individuals do not develop diabetes due to compensatory islet responses to restore glucose homeostasis. Therefore, regulatory pathways that promote ß cell compensation are potential targets for treatment of diabetes. The transient receptor potential cation channel subfamily M member 7 protein (TRPM7), harboring a cation channel and a serine/threonine kinase, has been implicated in controlling cell growth and proliferation. Here, we report that selective deletion of Trpm7 in ß cells disrupted insulin secretion and led to progressive glucose intolerance. We indicate that the diminished insulinotropic response in ß cell-specific Trpm7-knockout mice was caused by decreased insulin production because of impaired enzymatic activity of this protein. Accordingly, high-fat-fed mice with a genetic loss of TRPM7 kinase activity displayed a marked glucose intolerance accompanied by hyperglycemia. These detrimental glucoregulatory effects were engendered by reduced compensatory ß cell responses because of mitigated protein kinase B (AKT)/ERK signaling. Collectively, our data identify TRPM7 kinase as a potentially novel regulator of insulin synthesis, ß cell dynamics, and glucose homeostasis under obesogenic diet.


Assuntos
Intolerância à Glucose , Canais de Cátion TRPM , Animais , Camundongos , Glucose , Insulina/metabolismo , Camundongos Knockout , Obesidade , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo
13.
J Biol Chem ; 286(45): 39328-35, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-21926172

RESUMO

Transient receptor potential melastatin 7 (TRPM7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. They are expressed abundantly in a variety of human carcinoma cells controlling survival, growth, and migration. These characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. We screened a chemical library of marine organism-derived extracts and identified waixenicin A from the soft coral Sarcothelia edmondsoni as a strong inhibitor of overexpressed and native TRPM7. Waixenicin A activity was cytosolic and potentiated by intracellular free magnesium (Mg(2+)) concentration. Mutating a Mg(2+) binding site on the TRPM7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of Mg(2+). Waixenicin A failed to inhibit the closely homologous TRPM6 channel and did not significantly affect TRPM2, TRPM4, and Ca(2+) release-activated Ca(2+) current channels. Therefore, waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Consistent with TRPM7 inhibition, the compound blocked cell proliferation in human Jurkat T-cells and rat basophilic leukemia cells. Based on the ability of the compound to inhibit cell proliferation through Mg(2+)-dependent block of TRPM7, waixenicin A, or structural analogs may have cancer-specific therapeutic potential, particularly because certain cancers accumulate cytosolic Mg(2+).


Assuntos
Acetatos/farmacologia , Antozoários/química , Proliferação de Células/efeitos dos fármacos , Diterpenos/farmacologia , Magnésio/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Acetatos/química , Animais , Sítios de Ligação , Diterpenos/química , Células HEK293 , Humanos , Células Jurkat , Moduladores de Transporte de Membrana , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína , Ratos , Canais de Cátion TRPM/genética
14.
Cell Calcium ; 106: 102636, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35981457

RESUMO

Localized intracellular calcium fluxes are indispensable for immunologically directed Fc receptor-mediated cellular phagocytosis. A similar dependency on calcium signals has been speculated to occur in efferocytosis, the clearance of non-opsonized apoptotic cell bodies by macrophages. In a recent study published in Nature Communications, Schappe et al. describe the TRPM7 ion channel as mediator of peri­phagosomal calcium currents, ensuring maturation of the acidifying phagosome. The authors identified a fundamental calcium signaling module provided by TRPM7, which is necessary for clearance of apoptotic cells. This finding updates our current molecular understanding of calcium dynamics, tissue maintenance and immunological clean-up.


Assuntos
Canais de Cátion TRPM , Cálcio/metabolismo , Sinalização do Cálcio , Macrófagos/metabolismo , Fagossomos/metabolismo , Canais de Cátion TRPM/metabolismo
15.
Cells ; 11(9)2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35563771

RESUMO

Two-pore channels (TPCs) are ligand-gated cation-selective ion channels that are preserved in plant and animal cells. In the latter, TPCs are located in membranes of acidic organelles, such as endosomes, lysosomes, and endolysosomes. Here, we focus on the function of these unique ion channels in mast cells, which are leukocytes that mature from myeloid hematopoietic stem cells. The cytoplasm of these innate immune cells contains a large number of granules that comprise messenger substances, such as histamine and heparin. Mast cells, along with basophil granulocytes, play an essential role in anaphylaxis and allergic reactions by releasing inflammatory mediators. Signaling in mast cells is mainly regulated via the release of Ca2+ from the endoplasmic reticulum as well as from acidic compartments, such as endolysosomes. For the crosstalk of these organelles TPCs seem essential. Allergic reactions and anaphylaxis were previously shown to be associated with the endolysosomal two-pore channel TPC1. The release of histamine, controlled by intracellular Ca2+ signals, was increased upon genetic or pharmacologic TPC1 inhibition. Conversely, stimulation of TPC channel activity by one of its endogenous ligands, namely nicotinic adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), were found to trigger the release of Ca2+ from the endolysosomes; thereby improving the effect of TPC1 on regulated mast cell degranulation. In this review we discuss the importance of TPC1 for regulating Ca2+ homeostasis in mast cells and the overall potential of TPC1 as a pharmacological target in anti-inflammatory therapy.


Assuntos
Anafilaxia , Canais de Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Endossomos/metabolismo , Histamina , Homeostase , NADP/metabolismo
16.
Cell Calcium ; 106: 102640, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36030694

RESUMO

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.


Assuntos
Canais de Cátion TRPM , Animais , Humanos , Isoxazóis/farmacologia , Magnésio/metabolismo , Camundongos , Piperidinas/farmacologia , Proteínas Serina-Treonina Quinases , Nucleosídeos de Purina/farmacologia , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Canais de Potencial de Receptor Transitório/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/metabolismo
17.
Cardiovasc Res ; 118(5): 1289-1302, 2022 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33881519

RESUMO

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.


Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Animais , Cálcio/metabolismo , Inflamação , Canal de Potássio Kv1.5 , Potenciais da Membrana/fisiologia , Camundongos , Infiltração de Neutrófilos
18.
J Clin Invest ; 132(13)2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35503420

RESUMO

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.


Assuntos
Infecções Bacterianas , Paladar , Animais , Células Epiteliais , Imunidade Inata , Camundongos , Pseudomonas aeruginosa , Transdução de Sinais , Paladar/fisiologia , Traqueia
19.
Nat Commun ; 13(1): 318, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-35031603

RESUMO

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.


Assuntos
Macrófagos Alveolares/enzimologia , Metaloproteinase 12 da Matriz/metabolismo , Elastase Pancreática/metabolismo , Enfisema Pulmonar/enzimologia , Canais de Potencial de Receptor Transitório/deficiência , Animais , Modelos Animais de Doenças , Endossomos/metabolismo , Feminino , Humanos , Pulmão/enzimologia , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Knockout , Elastase Pancreática/genética , Enfisema Pulmonar/genética , Enfisema Pulmonar/metabolismo , Canais de Potencial de Receptor Transitório/genética
20.
Elife ; 102021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34766907

RESUMO

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+, and Ca2+, and thus shapes cellular excitability, plasticity, and metabolic activity. The molecular appearance and operation of TRPM7 channels in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative mass spectrometry (MS) analysis. We found that native TRPM7 channels are high-molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ADP-ribosylation factor-like protein 15 (ARL15). Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that complex formation effectively and specifically impacts TRPM7 activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.


Assuntos
Encéfalo/metabolismo , Proteômica/métodos , Canais de Cátion TRPM/genética , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Canais de Cátion TRPM/metabolismo
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