TRPV6 channels function as epithelial Ca²⁺ entry pathways in the epididymis, prostate, and placenta. However, the identity of the endogenous TRPV6 protein relies on predicted gene coding regions and is only known to a certain level of approximation. We show that in vivo the TRPV6 protein has an extended N terminus. Translation initiates at a non-AUG codon, at ACG, which is decoded by methionine and which is upstream of the annotated AUG, which is not used for initiation. The in vitro properties of channels formed by the extended full-length TRPV6 proteins and the so-far annotated and smaller TRPV6 are similar, but the extended N terminus increases trafficking to the plasma membrane and represents an additional scaffold for channel assembly. The increased translation of the smaller TRPV6 cDNA version may overestimate the in vivo situation where translation efficiency may represent an additional mechanism to tightly control the TRPV6-mediated Ca²⁺ entry to prevent deleterious Ca²⁺ overload.

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.

Background: The syncytiotrophoblast (STB) layer of the placenta is formed by cell fusion of cytotrophoblasts, acts as a feto-maternal barrier, is required for the production of pregnancy hormones such as chorionic gonadotropin, estradiol and progesterone and is also responsible for feto-maternal mineral exchange such as calcium. Adequate mineral supply and placental hormone production are essential for the maintenance of pregnancy, and disturbances in trophoblast integrity are associated with pregnancy complications. Since knowledge about the identity and expression levels of proteins in trophoblast and syncytiotrophoblast cells is limited so far, we analyzed the proteomes of trophoblast-like and syncytiotrophoblast-like BeWo cells under different calcium conditions. The investigation of protein expression profiles in combination with hormone assays can provide a better understanding of calcium-dependent cellular processes in trophoblasts and syncytiotrophoblasts. Methods: Here, we combine human trophoblast model cell cultures, hormone assays, antibody-based detection methods and high-resolution mass spectrometry analyzes to assess changes in cellular processes during syncytialization. Results: We monitored the changes in protein expression profiles during forskolin induced syncytialization of trophoblast-like cells in an unbiased manner and show that the expression of numerous proteins is strongly altered. Among them are enzymes of the glucocorticoid and sex hormones synthesis pathways such as cytochrome P450 (CYP) 19A1, CYP11A1, adrenodoxin (FDX1), hydroxysteroid dehydrogenase (HSD) 11β2 and HSD17β1, whose expression is strongly induced by syncytialization. The production of beta human chorionic gonadotropin (ß-hCG), progesterone and estradiol increase during syncytialization, while the secretion and synthesis of ß-hCG and the expression of several protein syncytiotrophoblast markers show a clear calcium dependence. Conclusion: The broad applicability of semi-quantitative proteome profiling of cytotrophoblast- and syncytiotrophoblast-like cells provides new insights into signaling processes that occur in cytotrophoblasts /syncytiotrophoblasts during pregnancy.

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.