TRPM6

The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (Casr^BCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed Casr^BCH002 mice with PTH deficient mice. Heterozygous Casr^BCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in Casr^BCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, Casr^BCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH.

Pterygium is a hyaline degenerative disease of the conjunctiva characterized by the progression of fibrovascular connective tissue from the bulbar conjunctiva to the cornea. The mechanism of pterygium formation is still not fully understood. Transient receptor potential (TRP) channels are a group of ion channels with distinct characteristics. Recent indications suggest TRP channels may play a significant regulatory role in pterygium development, but previous studies have mainly focused on in silico analysis. Accordingly, in the present study, we aimed to decipher the expression signatures and role of TRP channels in pterygium development.

The study encompassed a cohort of 45 patients matched for age and gender distribution, comprising 30 individuals with primary pterygium (PP) and 15 individuals with recurrent pterygium (RP). The control group consisted of unaffected conjunctival tissue obtained from the same set of patients. High-throughput screening of differentially expressed TRP channels in pterygium tissues was achieved with the help of Fluidigm 96.96 Dynamic Array Expression Chip and reactions were held in BioMark™ HD System Real-Time PCR platform.

Statistically significant increases were found in the expression of 21 genes, mainly TRPA1 (p = 0.021), TRPC2 (p = 0.001), and TRPM8 (p = 0.003), in patients with PP, and in TRPC5 (p = 0.05), TRPM2 (p = 0.029), TRPM4 (p = 0.03), TRPM6 (p = 0.045), TRPM8 (p = 0.038), TRPV1 (p = 0.01) and TRPV4 (p = 0.025) genes in RP tissues.

Collectively, TRP channel proteins appear to play pivotal roles in both the development and progression of pterygium, making them promising candidates for future therapeutic interventions in patients afflicted by this condition.

Disruption of Ca²⁺ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca²⁺ kinetics and gene expression involved in the regulation of Ca²⁺ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1.

CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca²⁺ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca²⁺ regulation ([Ca²⁺]_i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl₃, and GdCl₃. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca²⁺ signaling through RNA sequencing.

EA.hy926 cells, at increasing Ca²⁺ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca²⁺-dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca²⁺ kinetics showed higher amplitudes in Ca²⁺ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca²⁺]_i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca²⁺ ions induced a significantly higher [Ca²⁺]_i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca²⁺-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca²⁺ import and downregulated export genes in EA.hy926.

Our finding show that significant differences in CaEP response and [Ca²⁺]_i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca²⁺]_i changes, which may be linked to overexpression of Ca²⁺-related genes and an inability to mitigate changes in [Ca²⁺]_i. The study offers a bioinformatic basis for selecting EC models based on research objectives.