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Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.
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Canales de Calcio , Calcio , Ratones , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Calcio/metabolismo , Páncreas/metabolismo , Exocitosis/fisiología , Vesículas Secretoras/genéticaRESUMEN
Transient receptor potential (TRP) ion channels play important roles in fundamental biological processes throughout the body of humans and mice. TRP channel dysfunction manifests in different disease states, therefore, these channels may represent promising therapeutic targets in treating these conditions. Many TRP channels are expressed in several organs suggesting multiple functions and making it challenging to untangle the systemic pathophysiology of TRP dysfunction. Detailed characterization of the expression pattern of the individual TRP channels throughout the organism is thus essential to interpret data such as those derived from systemic phenotyping of global TRP knockout mice. Murine TRP channel reporter strains enable reliable labeling of TRP expression with a fluorescent marker. Here we present an optimized method to visualize primary TRP-expressing cells with single cell resolution throughout the entire organism. In parallel, we methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest. The TRP protein expression data are then correlated with the GFP reporter expression data. The combined methodological approach presented here can be adopted to generate expression data for other genes of interest and reporter mice.â¢We present an optimized method to systemically characterize gene expression in fluorescent reporter mouse strains with a single cell resolution.â¢We methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest in mice.
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To identify TRPV6 expression in the whole mouse with a cellular resolution we took advantage of TRPV6-IRES-Cre knock-in mice crossed with the enhanced ROSA26-τGFP reporter line. In the resulting TRPV6-IC/eR26-τGFP animals, TRPV6-expressing cells are labeled with τGFP. Data were collected from organs prepared from fixed experimental adult and juvenile TRPV6-IC/eR26τGFP and Cre-negative eR26-τGFP control animals of both sexes. Organ cryosections from each age and sex were stained for GFP and imaged with a slide scanner. Here, we describe reporter gene expression in a large number of tissues. We also document the absence of τGFP signal in the corresponding Cre-negative control tissues, including controls for the TRPV6 expression data described in [1]. The data reported here and in [1] constitute the TRPV6 expression atlas for the mouse. Our data offer a wealth of information to enable investigation of the functional role of TRPV6 channels in different tissues.
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Independent of its function as a subunit of voltage-gated Ca2+ channels, the Cavß3 subunit desensitizes fibroblasts and pancreatic ß-cells to low concentrations of inositol-1,4,5-trisphosphate (IP3). This alters agonist-induced Ca2+ signaling and cellular functions, for example, insulin secretion and wound healing. A total of four Cavß subunits exist, Cavß1, Cavß2, Cavß3, and Cavß4. To investigate whether the other Cavß subunits, like Cavß3, can desensitize cells to IP3 and thereby modulate Ca2+ signaling, we expressed the cDNAs of Cavß1, Cavß2, Cavß3, and Cavß4 in COS-7 cells lacking endogenous Cavß proteins. ATP stimulation of these cells results in the release of Ca2+ from intracellular stores. This receptor-mediated Ca2+ release is significantly decreased by Cavß3 but not by Cavß1, Cavß2, and Cavß4. Electrophysiological recordings of voltage-dependent Ca2+ currents from fibroblasts show a small Ca2+ current, the amplitude of which is slightly but not significantly smaller in fibroblasts from Cavß2 gene-deficient animals than in fibroblasts from wild-type animals. Compared with fibroblasts from wild-type animals, Ca2+ release is not significantly increased in Cavß2-deficient fibroblasts, in contrast to Ca2+ release in Cavß3-deficient fibroblasts. In summary, our results show that desensitization of cells to low concentrations of IP3 is a specific property of Cavß3 that is not shared by other Cavß subunits.
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Canales de Calcio , Células Secretoras de Insulina , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Fenómenos Electrofisiológicos , Fibroblastos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismoRESUMEN
Recently, we reported a case of an infant with neonatal severe under-mineralizing skeletal dysplasia caused by mutations within both alleles of the TRPV6 gene. One mutation results in an in frame stop codon (R510stop) that leads to a truncated, nonfunctional TRPV6 channel, and the second in a point mutation (G660R) that, surprisingly, does not affect the Ca2+ permeability of TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child by coexpressing the TRPV6 G660R and R510stop mutants and combinations with wild type TRPV6. We show that both the G660R and R510stop mutant subunits are expressed and result in decreased calcium uptake, which is the result of the reduced abundancy of functional TRPV6 channels within the plasma membrane. We compared the proteomic profiles of a healthy placenta with that of the diseased infant and detected, exclusively in the latter two proteases, HTRA1 and cathepsin G. Our results implicate that the combination of the two mutant TRPV6 subunits, which are expressed in the placenta of the diseased child, is responsible for the decreased calcium uptake, which could explain the skeletal dysplasia. In addition, placental calcium deficiency also appears to be associated with an increase in the expression of proteases.
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Canales de Calcio/genética , Catepsina G/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Mutación , Osteocondrodisplasias/patología , Placenta/patología , Proteoma/metabolismo , Canales Catiónicos TRPV/genética , Secuencia de Aminoácidos , Animales , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Estudios de Casos y Controles , Catepsina G/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Humanos , Lactante , Ratones Noqueados , Osteocondrodisplasias/etiología , Osteocondrodisplasias/metabolismo , Placenta/metabolismo , Embarazo , Proteoma/análisis , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/fisiologíaRESUMEN
The transient receptor potential vanilloid 6 (TRPV6) channel is highly Ca2+-selective and has been implicated in mediating transcellular Ca2+ transport and thus maintaining the Ca2+ balance in the body. To characterize its physiological function(s), a detailed expression profile of the TRPV6 channel throughout the body is essential. Capitalizing on a recently established murine Trpv6-reporter strain, we identified primary TRPV6 channel-expressing cells in an organism-wide manner. In a complementary experimental approach, we characterized TRPV6 expression in different tissues of wild-type mice by TRPV6 immunoprecipitation (IP) followed by mass spectrometry analysis and correlated these data with the reporter gene expression. Taken together, we present a TRPV6 expression atlas throughout the entire body of juvenile and adult mice, providing a novel resource to investigate the role of TRPV6 channels in vivo.
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Canales de Calcio , Canales Catiónicos TRPV , Animales , Calcio/metabolismo , Canales de Calcio/genética , Expresión Génica , Ratones , Canales Catiónicos TRPV/genéticaRESUMEN
Early embryogenesis depends on proper control of intracellular homeostasis of ions including Ca2+ and Mg2+. Deletion of the Ca2+ and Mg2+ conducting the TRPM7 channel is embryonically lethal in mice but leaves compaction, blastomere polarization, blastocoel formation, and correct specification of the lineages of the trophectoderm and inner cell mass unaltered despite that free cytoplasmic Ca2+ and Mg2+ is reduced at the two-cell stage. Although Trpm7-/- embryos are able to hatch from the zona pellucida, no expansion of Trpm7-/- trophoblast cells can be observed, and Trpm7-/- embryos are not identifiable in utero at E6.5 or later. Given the proliferation and adhesion defect of Trpm7-/- trophoblast stem cells and the ability of Trpm7-/- ESCs to develop to embryos in tetraploid embryo complementation assays, we postulate a critical role of TRPM7 in trophectoderm cells and their failure during implantation as the most likely explanation of the developmental arrest of Trpm7-deficient mouse embryos.
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Calcio/metabolismo , Adhesión Celular , Proliferación Celular , Magnesio/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Canales Catiónicos TRPM/deficiencia , Trofoblastos/metabolismo , Animales , Muerte Celular , Linaje de la Célula , Células Cultivadas , Implantación del Embrión , Desarrollo Embrionario , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/patología , Transducción de Señal , Canales Catiónicos TRPM/genética , Trofoblastos/patologíaRESUMEN
Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in fetal labyrinth trophoblasts as part of the feto-maternal barrier, necessary for sufficient calcium supply, embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact labyrinth morphology of Trpv6-deficient placentae, and reduced Ca2+ uptake of primary trophoblasts upon functional deletion of TRPV6. Trpv6-/- trophoblasts show a distinct calcium-dependent phenotype. Deep proteomic profiling of wt and Trpv6-/- primary trophoblasts using label-free quantitative mass spectrometry leads to the identification of 2778 proteins. Among those, a group of proteases, including high-temperature requirement A serine peptidase 1 (HTRA1) and different granzymes are more abundantly expressed in Trpv6-/- trophoblast lysates, whereas the extracellular matrix protein fibronectin and the fibronectin-domain-containing protein 3A (FND3A) were markedly reduced. Trpv6-/-placenta lysates contain a higher intrinsic proteolytic activity increasing fibronectin degradation. Our results show that the extracellular matrix formation of the placental labyrinth depends on TRPV6; its deletion in trophoblasts correlates with the increased expression of proteases controlling the extracellular matrix in the labyrinth during pregnancy.
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Matriz Extracelular/metabolismo , Placenta/metabolismo , Canales Catiónicos TRPV/metabolismo , Transporte Biológico , Biomarcadores , Calcio/metabolismo , Movimiento Celular/genética , Supervivencia Celular/genética , Biología Computacional , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Embarazo , Proteolisis , Proteoma , Proteómica , Canales Catiónicos TRPV/genéticaRESUMEN
BACKGROUND/AIMS: The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca2+ influx into pancreatic beta cells by the opening of voltage-gated Ca2+ channels. Transient Receptor Potential Melastatin 3 proteins build Ca2+ permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca2+ influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release. METHODS: We used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated Ca2+ channels in imaging experiments (FMP, Fura-2) and electrophysiological recordings. In immunoassays, we examined the contribution of TRPM3 channels to pregnenolone sulfate- and glucose-induced insulin release. To confirm our findings, we generated beta cell-specific Trpm3-deficient mice and compared their glucose clearance with the wild type in glucose tolerance tests. RESULTS: TRPM3 channels triggered the activity of voltage-gated Ca2+ channels and both channels together contributed to insulin release after TRPM3 activation. Trpm3-deficient INS-1 cells lacked pregnenolone sulfate-induced Ca2+ signals just like the pregnenolone sulfate-induced insulin release. Both, glucose-induced Ca2+ signals and the glucose-induced insulin release were strongly reduced. Accordingly, Trpm3-deficient mice displayed an impaired decrease of the blood sugar concentration after intraperitoneal or oral administration of glucose. CONCLUSION: The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release.
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Señalización del Calcio , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Canales Catiónicos TRPM/metabolismo , Animales , Línea Celular , Ratones , Ratones Mutantes , Ratas , Canales Catiónicos TRPM/genéticaRESUMEN
In mammalian cells, one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61 channel. While the Sec61 complex facilitates ER import of most precursor polypeptides, the Sec61-associated Sec62/Sec63 complex supports ER import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63 complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified 22 novel Sec62/Sec63 substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides (SP) with comparatively longer but less hydrophobic hydrophobic region of SP and lower carboxy-terminal region of SP (C-region) polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly gating SP and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62/Sec63 requirement. In the case of ERj3, these features were found to be responsible for an additional immunoglobulin heavy-chain binding protein (BiP) requirement and to correlate with sensitivity toward the Sec61-channel inhibitor CAM741. Thus, the human Sec62/Sec63 complex may support Sec61-channel opening for precursor polypeptides with slowly gating SPs by direct interaction with the cytosolic amino-terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked polycystic liver disease. DATABASES: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data (https://data.mendeley.com/datasets/6s5hn73jcv/2).
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Retículo Endoplásmico/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Señales de Clasificación de Proteína , Proteínas de Unión al ARN/metabolismo , Animales , Células HEK293 , Proteínas del Choque Térmico HSP40/metabolismo , Células HeLa , Humanos , Proteínas de Transporte de Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Chaperonas Moleculares/genética , Transporte de Proteínas , Proteoma/metabolismo , Proteómica/métodos , Proteínas de Unión al ARN/genética , Canales de Translocación SEC/genética , Canales de Translocación SEC/metabolismo , Especificidad por SustratoRESUMEN
BACKGROUND & AIMS: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca2+-selective ion channel, in an international cohort of patients and in mice. METHODS: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca2+ imaging assays. CP was induced by repeated injections of cerulein in TRPV6mut/mut mice. RESULTS: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10-8). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10-8). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10-9 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10-5). TRPV6mut/mut mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis. CONCLUSIONS: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca2+ balance in pancreatic cells. TRPV6 regulates Ca2+ homeostasis and pancreatic inflammation.
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Edad de Inicio , Canales de Calcio/genética , Pancreatitis Crónica/genética , Canales Catiónicos TRPV/genética , Adolescente , Adulto , Anciano , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Niño , Preescolar , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Mutación INDEL , Lactante , Recién Nacido , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Páncreas/patología , Pancreatitis Crónica/patología , Polimorfismo de Nucleótido Simple , Canales Catiónicos TRPV/metabolismo , Secuenciación del Exoma , Adulto JovenRESUMEN
Transient receptor potential (TRP) proteins form Ca2+-permeable, nonselective cation channels, but their role in neuronal Ca2+ homeostasis is elusive. In the present paper, we show that TRPC channels potently regulate synaptic plasticity by changing the presynaptic Ca2+-homeostasis of hippocampal neurons. Specifically, loss of TRPC1/C4/C5 channels decreases basal-evoked secretion, reduces the pool size of readily releasable vesicles, and accelerates synaptic depression during high-frequency stimulation (HFS). In contrast, primary TRPC5 channel-expressing neurons, identified by a novel TRPC5-τ-green fluorescent protein (τGFP) knockin mouse line, show strong short-term enhancement (STE) of synaptic signaling during HFS, indicating a key role of TRPC5 in short-term plasticity. Lentiviral expression of either TRPC1 or TRPC5 turns classic synaptic depression of wild-type neurons into STE, demonstrating that TRPCs are instrumental in regulating synaptic plasticity. Presynaptic Ca2+ imaging shows that TRPC activity strongly boosts synaptic Ca2+ dynamics, showing that TRPC channels provide an additional presynaptic Ca2+ entry pathway, which efficiently regulates synaptic strength and plasticity.
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Señalización del Calcio , Plasticidad Neuronal , Canales Catiónicos TRPC/fisiología , Animales , Canales de Calcio/metabolismo , Femenino , Glutamina/metabolismo , Hipocampo/metabolismo , Masculino , Ratones Noqueados , Neuronas/metabolismoRESUMEN
BACKGROUND & AIMS: Intestinal Ca2+ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca2+ absorption have been defined in adults after peak bone mass is obtained, but they are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca2+ absorption during this critical period. METHODS: Expression of small intestinal and renal calcium transport genes was assessed by using quantitative polymerase chain reaction. Net calcium flux across small intestinal segments was measured in Ussing chambers, including after pharmacologic inhibition or genetic manipulation of TRPV6 or Cav1.3 calcium channels. Femurs were analyzed by using micro-computed tomography and histology. RESULTS: Net TRPV6-mediated Ca2+ flux across the duodenum was absent in pre-weaned (P14) mice but present after weaning. In contrast, we found significant transcellular Ca2+ absorption in the jejunum at 2 weeks but not 2 months of age. Net jejunal Ca2+ absorption observed at P14 was not present in either Trpv6 mutant (D541A) mice or Cav1.3 knockout mice. We observed significant nifedipine-sensitive transcellular absorption across the ileum at P14 but not 2 months. Cav1.3 knockout pups exhibited delayed bone mineral accrual, compensatory nifedipine-insensitive Ca2+ absorption in the ileum, and increased expression of renal Ca2+ reabsorption mediators at P14. Moreover, weaning pups at 2 weeks reduced jejunal and ileal Cav1.3 expression. CONCLUSIONS: We have detailed novel pathways contributing to transcellular Ca2+ transport across the distal small intestine of mice during development, highlighting the complexity of the multiple mechanisms involved in achieving a positive Ca2+ balance early in life.
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Canales de Calcio Tipo L/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Intestino Delgado/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Calcificación Fisiológica/fisiología , Canales de Calcio/genética , Canales de Calcio Tipo L/genética , Calcio de la Dieta/metabolismo , Femenino , Absorción Intestinal , Mucosa Intestinal/metabolismo , Transporte Iónico , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Canales Catiónicos TRPV/genética , Transcriptoma , DesteteRESUMEN
Very young red blood cells, namely reticulocytes, can be quite easily recognized and labeled by cluster of differentiation antibodies (CD71, transferrin receptor) or by staining remnant RNA with thiazol orange. In contrast, age specific erythrocyte labeling is more difficult in later periods of their life time. While erythrocytes contain band 4.1 protein, a molecular clock, so far it has not been possible to read this clock on individual cells. One concept to track erythrocytes during their life time is to mark them when they are young, either directly in vivo or ex vivo followed by a transfusion. Several methods like biotinylation, use of isotopes or fluorescent labeling have proved to be useful experimental approaches but also have several inherent disadvantages. Genetic engineering of mice provides additional options to express fluorescent proteins in erythrocytes. To allow co-staining with popular green fluorescent dyes like Fluo-4 or other fluorescein-based dyes, we bred a mouse line expressing a tandem red fluorescent protein (tdRFP). Within this Brief Research Report, we provide the initial characterisation of this mouse line and show application examples ranging from transfusion experiments and intravital microscopy to multicolour flow cytometry and confocal imaging. We provide a versatile new tool for erythrocyte research and discuss a range of experimental opportunities to study membrane processes and other aspects of erythrocyte development and aging with help of these animals.
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Dopamine neurons of the hypothalamic arcuate nucleus (ARC) tonically inhibit the release of the protein hormone prolactin from lactotropic cells in the anterior pituitary gland and thus play a central role in prolactin homeostasis of the body. Prolactin, in turn, orchestrates numerous important biological functions such as maternal behavior, reproduction, and sexual arousal. Here, we identify the canonical transient receptor potential channel Trpc5 as an essential requirement for normal function of dopamine ARC neurons and prolactin homeostasis. By analyzing female mice carrying targeted mutations in the Trpc5 gene including a conditional Trpc5 deletion, we show that Trpc5 is required for maintaining highly stereotyped infraslow membrane potential oscillations of dopamine ARC neurons. Trpc5 is also required for eliciting prolactin-evoked tonic plateau potentials in these neurons that are part of a regulatory feedback circuit. Trpc5 mutant females show severe prolactin deficiency or hypoprolactinemia that is associated with irregular reproductive cyclicity, gonadotropin imbalance, and impaired reproductive capabilities. These results reveal a previously unknown role for the cation channel Trpc5 in prolactin homeostasis of female mice and provide strategies to explore the genetic basis of reproductive disorders and other malfunctions associated with defective prolactin regulation in humans.
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Núcleo Arqueado del Hipotálamo/metabolismo , Neuronas Dopaminérgicas/metabolismo , Enfermedades Genéticas Congénitas/genética , Trastornos de la Lactancia/genética , Prolactina/deficiencia , Prolactina/genética , Canales Catiónicos TRPC/genética , Animales , Núcleo Arqueado del Hipotálamo/patología , Nivel de Alerta/fisiología , Neuronas Dopaminérgicas/patología , Retroalimentación Fisiológica , Femenino , Regulación de la Expresión Génica , Enfermedades Genéticas Congénitas/metabolismo , Enfermedades Genéticas Congénitas/patología , Gonadotropinas/sangre , Gonadotropinas/genética , Homeostasis/genética , Humanos , Trastornos de la Lactancia/metabolismo , Trastornos de la Lactancia/patología , Potenciales de la Membrana/fisiología , Ratones , Mutación , Prolactina/sangre , Prolactina/metabolismo , Reproducción/fisiología , Transducción de Señal , Canales Catiónicos TRPC/deficienciaRESUMEN
Plasma calcium (Ca2+) is maintained by amending the release of parathyroid hormone and through direct effects of the Ca2+ sensing receptor (CaSR) in the renal tubule. Combined, these mechanisms alter intestinal Ca2+ absorption by modulating 1,25-dihydroxy vitamin D3 production, bone resorption, and renal Ca2+ excretion. The CaSR is a therapeutic target in the treatment of secondary hyperparathyroidism and hypocalcemia a common complication of calcimimetic therapy. The CaSR is also expressed in intestinal epithelium, however, a direct role in regulating local intestinal Ca2+ absorption is unknown. Chronic CaSR activation decreased expression of genes involved in Ca2+ absorption. In Ussing chambers, increasing extracellular Ca2+ or basolateral application of the calcimimetic cinacalcet decreased net Ca2+ absorption across intestinal preparations acutely. Conversely, Ca2+ absorption increased with decreasing extracellular Ca2+ concentration. These responses were absent in mice expressing a non-functional TRPV6, TRPV6D541A. Cinacalcet also attenuated Ca2+ fluxes through TRPV6 in Xenopus oocytes when co-expressed with the CaSR. Moreover, the phospholipase C inhibitor, U73122, prevented cinacalcet-mediated inhibition of Ca2+ flux. These results reveal a regulatory pathway whereby activation of the CaSR in the basolateral membrane of the intestine directly attenuates local Ca2+ absorption via TRPV6 to prevent hypercalcemia and help explain how calcimimetics induce hypocalcemia.
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Calcimiméticos/efectos adversos , Canales de Calcio/metabolismo , Calcio/metabolismo , Mucosa Intestinal/metabolismo , Receptores Sensibles al Calcio/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Calcio/agonistas , Calcio/sangre , Canales de Calcio/genética , Cinacalcet/efectos adversos , Modelos Animales de Enfermedad , Estrenos/farmacología , Femenino , Técnicas de Sustitución del Gen , Humanos , Hipercalcemia/inducido químicamente , Hipercalcemia/prevención & control , Hiperparatiroidismo Secundario/inducido químicamente , Hiperparatiroidismo Secundario/tratamiento farmacológico , Hipocalcemia/inducido químicamente , Hipocalcemia/tratamiento farmacológico , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Transgénicos , Oocitos , Hormona Paratiroidea/metabolismo , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/farmacología , Pirrolidinonas/farmacología , Receptores Sensibles al Calcio/agonistas , Canales Catiónicos TRPV/genética , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , XenopusRESUMEN
Embryonic growth and bone development depend on placental Ca2+ transport across the feto-maternal barrier to supply minerals to the fetus. The individual factors and cellular mechanisms that regulate placental Ca2+ transfer, however, are only beginning to emerge. We find that the Ca2+ -selective transient receptor potential vanilloid 6 (TRPV6) channel is expressed in trophoblasts of the fetal labyrinth, in the yolk sac, and in the maternal part of the placenta. Lack of functional TRPV6 channels in the mother leads to a reduced Ca2+ content in both placenta and embryo. Ca2+ uptake in trophoblasts is impaired in the absence of Trpv6. Trpv6-deficient embryos are smaller, have a lower body weight, and shorter and less calcified femurs. The altered cortical bone microarchitecture persists in adulthood. We show that TRPV6's Ca2+ -conducting property causes this embryonic and bone phenotype. Our results show that TRPV6 is necessary for the Ca2+ uptake in trophoblasts and that TRPV6 deficiency in the placenta leads to reduced embryo growth, minor bone calcification, and impaired bone development. © 2019 American Society for Bone and Mineral Research.
Asunto(s)
Densidad Ósea , Desarrollo Óseo , Canales de Calcio/metabolismo , Calcio/metabolismo , Hueso Cortical/embriología , Canales Catiónicos TRPV/metabolismo , Trofoblastos/metabolismo , Animales , Canales de Calcio/genética , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/metabolismo , Ratones , Ratones Noqueados , Canales Catiónicos TRPV/genéticaRESUMEN
Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high-resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from Trpc1/Trpc4/Trpc5-triple-knockout (Trpc1/4/5-/-) mice, lacking any TRPC1-, TRPC4-, or TRPC5-containing channels, action potential-triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. In vivo, Trpc1/4/5-/- mice displayed impaired cross-frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5-/- animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.
Asunto(s)
Hipocampo/fisiología , Memoria a Corto Plazo , Multimerización de Proteína , Transmisión Sináptica , Canales Catiónicos TRPC/metabolismo , Animales , Técnicas de Inactivación de Genes , Hipocampo/metabolismo , Espectrometría de Masas , Ratones , Ratones Noqueados , Canales Catiónicos TRPC/genéticaRESUMEN
Following brain injury astrocytes change into a reactive state, proliferate and grow into the site of lesion, a process called astrogliosis, initiated and regulated by changes in cytoplasmic Ca2+ . Transient receptor potential canonical (TRPC) channels may contribute to Ca2+ influx but their presence and possible function in astrocytes is not known. By RT-PCR and RNA sequencing we identified transcripts of Trpc1, Trpc2, Trpc3, and Trpc4 in FACS-sorted glutamate aspartate transporter (GLAST)-positive cultured mouse cortical astrocytes and subcloned full-length Trpc1 and Trpc3 cDNAs from these cells. Ca2+ entry in cortical astrocytes depended on TRPC3 and was increased in the absence of Trpc1. After co-expression of Trpc1 and Trpc3 in HEK-293 cells both proteins co-immunoprecipitate and form functional heteromeric channels, with TRPC1 reducing TRPC3 activity. In vitro, lack of Trpc3 reduced astrocyte proliferation and migration whereas the TRPC3 gain-of-function moonwalker mutation and Trpc1 deficiency increased astrocyte migration. In vivo, astrogliosis and cortex edema following stab wound injury were reduced in Trpc3-/- but increased in Trpc1-/- mice. In summary, our results show a decisive contribution of TRPC3 to astrocyte Ca2+ signaling, which is even augmented in the absence of Trpc1, in particular following brain injury. Targeted therapies to reduce TRPC3 channel activity in astrocytes might therefore be beneficial in traumatic brain injury.