Combining mass spectrometry and genetic labeling in mice to report TRP channel expression

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. • We present an optimized method to systemically characterize gene expression in fluorescent reporter mouse strains with a single cell resolution. • 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

Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels

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

Transient Receptor Potential Vanilloid 6 (TRPV6) Proteins Control the Extracellular Matrix Structure of the Placental Labyrinth

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

Mutations that affect the surface expression of TRPV6 are associated with the upregulation of serine proteases in the placenta of an infant

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 (R(510)stop) that leads to a truncated, nonfunctional TRPV6 channel, and the second in a point mutation (G(660)R) that, surprisingly, does not affect the Ca(2+) permeability of TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child by coexpressing the TRPV6 G(660)R and R(510)stop mutants and combinations with wild type TRPV6. We show that both the G(660)R and R(510)stop 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

Mutations That Affect the Surface Expression of TRPV6 Are Associated with the Upregulation of Serine Proteases in the Placenta of an Infant

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

The Evolution of Erythrocytes Becoming Red in Respect to Fluorescence

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

Identification of signal peptide features for substrate specificity in human Sec62/Sec63-dependent ER protein import

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

IP3-dependent Ca2+ signals are tightly controlled by Cavβ3, but not by Cavβ1, 2 and 4

Independent of its function as a subunit of voltage-gated Ca2+ channels, the Cavβ3 subunit desensitizes fibroblasts and pancreatic β-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 Cavβ subunits exist, Cavβ1, Cavβ2, Cavβ3, and Cavβ4. To investigate whether the other Cavβ subunits, like Cavβ3, can desensitize cells to IP3 and thereby modulate Ca2+ signaling, we expressed the cDNAs of Cavβ1, Cavβ2, Cavβ3, and Cavβ4 in COS-7 cells lacking endogenous Cavβ proteins. ATP stimulation of these cells results in the release of Ca2+ from intracellular stores. This receptor-mediated Ca2+ release is significantly decreased by Cavβ3 but not by Cavβ1, Cavβ2, and Cavβ4. 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 Cavβ2 gene-deficient animals than in fibroblasts from wild-type animals. Compared with fibroblasts from wild-type animals, Ca2+ release is not significantly increased in Cavβ2-deficient fibroblasts, in contrast to Ca2+ release in Cavβ3-deficient fibroblasts. In summary, our results show that desensitization of cells to low concentrations of IP3 is a specific property of Cavβ3 that is not shared by other Cavβ subunits