Petra Weissgerber

Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4

Mast cells are key effector cells in allergic reactions. Aggregation of the receptor FcεRI in mast cells triggers the influx of calcium (Ca2+) and the release of inflammatory mediators. Here we show that transient receptor potential TRPM4 proteins acted as calcium-activated nonselective cation channels and critically determined the driving force for Ca2+ influx in mast cells. Trpm4−/− bone marrow–derived mast cells had more Ca2+ entry than did TRPM4+/+ cells after FcεRI stimulation. Consequently, Trpm4−/− bone marrow–derived mast cells had augmented degranulation and released more histamine, leukotrienes and tumor necrosis factor. Trpm4−/− mice had a more severe IgE-mediated acute passive cutaneous anaphylactic response, whereas late-phase passive cutaneous anaphylaxis was not affected. Our results establish the physiological function of TRPM4 channels as critical regulators of Ca2+ entry in mast cells.

Reduced Cardiac L-Type Ca2+ Current in Cavβ2−/− Embryos Impairs Cardiac Development and Contraction With Secondary Defects in Vascular Maturation

Cardiac myocyte contraction depends on transmembrane L-type Ca2+ currents and the ensuing release of Ca2+ from the sarcoplasmic reticulum. Here we show that these L-type Ca2+ currents are essential for cardiac pump function in the mouse at developmental stages where the functional significance of the heart becomes imperative to blood flow and to the continuing growth and survival of the embryo. Disruption of the Cavβ2 gene, which encodes for the predominant ancillary β subunit of cardiac Ca2+ channels, resulted in diminished L-type Ca2+ currents in cardiomyocytes of embryonic day 9.5 (E9.5). This led to a functionally compromised heart, causing defective remodeling of intra- and extraembryonic blood vessels and embryonic death following E10.5. The defects in vascular remodeling were also observed when the Cavβ2 gene was selectively targeted in cardiomyocytes, demonstrating that they are secondary to cardiac failure rather than a result of the lack of Cavβ2 proteins in the vasculature. Partial rescue of the Ca2+ channel currents by a Ca2+ channel agonist significantly postponed embryonic death in Cavβ2−/− mice. Taken together, these data strongly support the essential role of L-type Ca2+ channel activity in cardiomyocytes for normal heart development and function and that this is a prerequisite for proper maturation of the vasculature.

Male fertility depends on Ca2+ absorption by TRPV6 in epididymal epithelia

Production of functional spermatozoa requires regulation of the Ca2+ concentration in epididymal fluid by TRPV6. A Good Environment Is Crucial Male infertility can result from decreased production of or functional deficits in sperm. Ca2+ signaling plays a crucial role in sperm function, and, here, Weissgerber et al. uncovered a role for the Ca2+-selective TRPV6 channel in regulating Ca2+ concentration in the lumen of the epididymis as well as sperm motility and survival. Sperm maturation—including the acquisition of motility—occurs in the epididymis, after their exit from the testis. Male transgenic mice bearing an inactive form of TRPV6 showed decreased fertility, and the motility, viability, and in vitro capacity to fertilize eggs of sperm isolated from their caudal epididymides were impaired. TRPV6 was present in epididymal epithelial cells but not in the sperm themselves, and the Ca2+ concentration in the lumens of the epididymides of transgenic mice was 10 times higher than in wild-type mice. Moreover, sperm exposed to comparable extracellular Ca2+ concentrations showed an increase in intracellular Ca2+ concentration. The authors thus conclude that TRPV6 channels function to decrease the Ca2+ concentration of the intraluminal fluid in the epididymis and propose that the impaired function and survival of sperm in the transgenic mice results from the disturbed microenvironment in the epididymal fluid. TRPV6 [transient receptor potential vanilloid 6] is a calcium ion (Ca2+)–selective channel originally identified in the duodenal epithelium and in placenta; replacement of a negatively charged aspartate in the pore-forming region with an uncharged alanine (D541A) renders heterologously expressed TRPV6 channels nonfunctional. We found that male, but not female, mice homozygous for this mutation (Trpv6D541A/D541A) showed severely impaired fertility. The motility and fertilization capacity of sperm were markedly reduced, despite intact spermatogenesis. Trpv6 was expressed in epididymal epithelium where the protein was detected in the apical membrane, whereas it was not expressed in spermatozoa or the germinal epithelium. The Ca2+ concentration of the fluid in the cauda epididymis of Trpv6D541A/D541A males was 10 times higher than that of wild-type mice, which was accompanied by a seven- to eightfold decrease in Ca2+ absorption through the epididymal epithelium and was associated with reduced sperm viability. Thus, appropriate Ca2+ absorption and a consequent TRPV6-mediated decrease in the extracellular Ca2+ concentration toward the distal segments of the epididymal duct are essential for the acquisition of basic functions and the survival of spermatozoa.

Diversity and developmental expression of L-type calcium channel beta2 proteins and their influence on calcium current in murine heart.

By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVβ2 proteins are the major CaVβ components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVβ3 proteins are also detectable. At least two CaVβ2 variants of ∼68 and ∼72 kDa are expressed. To identify the underlying CaVβ2 variants, cDNA libraries were constructed from poly(A)+ RNA isolated from hearts of 7-day-old and adult mice. Screening identified 60 independent CaVβ2 cDNA clones coding for four types of CaVβ2 proteins only differing in their 5′ sequences. CaVβ2-N1, -N4, and -N5 but not -N3 were identified in isolated cardiomyocytes by RT-PCR and were sufficient to reconstitute the CaVβ2 protein pattern in vitro. Significant L-type Ca2+ currents (ICa) were recorded in HEK293 cells after co-expression of CaV1.2 and CaVβ2. Current kinetics were determined by the type of CaVβ2 protein, with the ∼72-kDa CaVβ2a-N1 shifting the activation of ICa significantly to depolarizing potentials compared with the other CaVβ2 variants. Inactivation of ICa was accelerated by CaVβ2a-N1 and -N4, which also lead to slower activation compared with CaVβ2a-N3 and -N5. In summary, this study reveals the molecular LTCC composition in mouse heart and indicates that expression of various CaVβ2 proteins may be used to adapt the properties of LTCCs to changing myocardial requirements during development and that CaVβ2a-N1-induced changes of ICa kinetics might be essential in embryonic heart.

The transient receptor potential channel TRPV6 is dynamically expressed in bone cells but is not crucial for bone mineralization in mice

Bone is the major store for Ca2+ in the body and plays an important role in Ca2+ homeostasis. During bone formation and resorption Ca2+ must be transported to and from bone by osteoblasts and osteoclasts, respectively. However, little is known about the Ca2+ transport machinery in these bone cells. In this study, we examined the epithelial Ca2+ channel TRPV6 in bone. TRPV6 mRNA is expressed in human and mouse osteoblast‐like cells as well as in peripheral blood mononuclear cell‐derived human osteoclasts and murine tibial bone marrow‐derived osteoclasts. Also other transcellular Ca2+ transport genes, calbindin‐D9k and/or ‐D28K, Na+/Ca2+ exchanger 1, and plasma membrane Ca2+ ATPase (PMCA1b) were expressed in these bone cell types. Immunofluorescence and confocal microscopy on human osteoblasts and osteoclasts and mouse osteoclasts revealed TRPV6 protein at the apical domain and PMCA1b at the osteoidal domain of osteoblasts, whereas in osteoclasts TRPV6 was predominantly found at the bone‐facing site. TRPV6 was dynamically expressed in human osteoblasts, showing maximal expression during mineralization of the extracellular matrix. 1,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) did not change TRPV6 expression in both mineralizing and non‐mineralizing SV‐HFO cultures. Lentiviral transduction‐mediated overexpression of TRPV6 in these cells did not alter mineralization. Bone microarchitecture and mineralization were unaffected in Trpv6D541A/D541A mice in which aspartate 541 in the pore region was replaced with alanine to render TRPV6 channels non‐functional. In summary, TRPV6 and other proteins involved in transcellular Ca2+ transport are dynamically expressed in bone cells, while TRPV6 appears not crucial for bone metabolism and matrix mineralization in mice. J. Cell. Physiol. 227: 1951–1959, 2012.

Moderate Calcium Channel Dysfunction in Adult Mice with Inducible Cardiomyocyte-specific Excision of the cacnb2 Gene

The major L-type voltage-gated calcium channels in heart consist of an α1C (CaV1.2) subunit usually associated with an auxiliary β subunit (CaVβ2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of CaVβ2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the CaVβ2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, CaVβ2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include CaVβ2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other CaVβ proteins or changes of CaV1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, CaVβ2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, CaVβ2 down-regulation in cardiomyocytes is well tolerated by the adult mice.

Excision of Trpv6 gene leads to severe defects in epididymal Ca2+ absorption and male fertility much like single D541A pore mutation.

Background: The TRPV6D541A pore mutation abrogates epididymal Ca2+ absorption causing hypofertility in mice, raising the possibility of residual TRPV6D541A channel activity. Results: Trpv6 deletion reduces fertility parameters to the same extent as the D541A pore mutation. Conclusion: The D541A pore mutation leads to complete inactivation of TRPV6 channels in epididymal epithelium. Significance: Targeted mutations in mice help to understand the function of TRPV6 proteins in native systems. Replacement of aspartate residue 541 by alanine (D541A) in the pore of TRPV6 channels in mice disrupts Ca2+ absorption by the epididymal epithelium, resulting in abnormally high Ca2+ concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilization capacity, raising the possibility of residual activity of channels formed by TRPV6D541A proteins (Weissgerber, P., Kriebs, U., Tsvilovskyy, V., Olausson, J., Kretz, O., Stoerger, C., Vennekens, R., Wissenbach, U., Middendorff, R., Flockerzi, V., and Freichel, M. (2011) Sci. Signal. 4, ra27). It is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared with the deletion of the corresponding protein. Therefore, we generated TRPV6-deficient mice (Trpv6−/−) by deleting exons encoding transmembrane domains with the pore-forming region and the complete cytosolic C terminus harboring binding sites for TRPV6-associated proteins that regulate its activity and plasma membrane anchoring. Using this strategy, we aimed to determine whether the TRPV6D541A pore mutant still contributes to residual channel activity and/or channel-independent functions in vivo. Trpv6−/− males reveal severe defects in fertility and motility and viability of sperm and a significant increase in epididymal luminal Ca2+ concentration that is mirrored by a lack of Ca2+ uptake by the epididymal epithelium. Therewith, Trpv6 excision affects epididymal Ca2+ handling and male fertility to the same extent as the introduction of the D541A pore mutation, arguing against residual functions of the TRPV6D541A pore mutant in epididymal epithelial cells.

Functional Characterization of Transient Receptor Potential (TRP) Channel C5 in Female Murine Gonadotropes

Gonadotrope cells in the anterior pituitary gland secrete gonadotropins regulating gonadal function in mammals. Recent results have implicated transient receptor potential (TRP) cation channels in pituitary physiology; however, if and how TRP channels contribute to gonadotrope function is not known. Here, we report that 14 out of 28 TRP channels encoded in the mouse genome are expressed in murine gonadotropes with highest expression levels found for canonical TRP (TRPC) channel 5 in juvenile females. We show that TRP channel expression in these cells exhibits considerable plasticity and that it depends on the sex and the developmental and hormonal status of the animal. We then combine different genetic strategies including genetic confocal Ca2+ imaging in whole-mount pituitary gland preparations to characterize TRPC5 channel function in gonadotropes from juvenile females. We show that the TRPC5 agonist Englerin A activates a cytosolic Ca2+ signal and a whole-cell current in these cells, which is absent in TRPC5-deficient mice, and demonstrate that TRPC5 forms functional heteromultimers with TRPC1 in gonadotropes. We further show that the Englerin A-activated TRPC5-dependent Ca2+ signal is mediated by Ca2+ influx both via TRPC5 and via l-type voltage-gated Ca2+ channels, activated by the depolarization through TRPC5-mediated cation influx. Finally, we demonstrate that the gonadotropin-releasing hormone (GnRH)-mediated net depolarization is significantly reduced in gonadotropes isolated from TRPC5-deficient mice. In conclusion, our data suggest that TRPC5 contributes to depolarization of the plasma membrane in gonadotropes upon GnRH stimulation and increases the intracellular Ca2+ concentration via its own Ca2+ permeability and via the activation of voltage-gated Ca2+ channels.

TRPV6: From identification to function

• TRPV6 is one of the most Ca 2+ selective Trp ion channels. It is mainly expressed in Ca 2+ -transporting epithelia e.g. epididymis and intestine, where it is assumed to play an important role in Ca 2+ (re)absorption.

Influence of the β2-Subunit of L-Type Voltage-Gated Cav Channels on the Structural and Functional Development of Photoreceptor Ribbon Synapses.

PURPOSE The cacnb2 gene encodes the β2 subunit (Cavβ2) of voltage-gated Ca2+ channels in photoreceptors, and its targeted deletion in mice has previously been shown to cause altered retinal morphology and synaptic transmission. The purpose of this study was to provide a detailed morphologic study combined with experiments on the altered functions of photoreceptor ribbon synapses lacking Cavβ2. METHODS A cacnb2-deficient mouse strain was generated and deletion of the Cavβ2 in the retina documented by biochemical and immunhistochemical approaches. Ultrastructural changes of photoreceptor ribbon synapses were examined by electronmicroscopy and functional implications of the lack of Cavβ2 studied by depolarization-induced Ca2+ influx into isolated photoreceptor cells and electroretinography. RESULTS Voltage-gated Ca2+ influx into rod photoreceptors lacking Cavβ2 was abolished and the typical rod ribbon-type active zones were absent in Cavβ2-deficient retinas. The active zone and the architecture of the presynaptic terminals were severely altered in rod synapses. Cone photoreceptor and the bipolar cell ribbon synapses were largely spared from ultrastructural changes although peanut agglutinin (PNA) labelling and photopic ERG analyses demonstrated that also cone pathways were disturbed in Cavβ2-deficient retinas. CONCLUSIONS The presence of the Cavβ2 is essential for the structural integrity and function of the rod photoreceptor synapse. The Cavβ2 is less essential for the morphology of cone and bipolar cell ribbon synapses, although the impaired photopic electroretinogram suggests a functional alteration also of the cone-mediated signaling in Cavβ2-deficient retinas.