Calcium-sensing Receptor Research Articles

Multiscale effects of the calcimimetic drug, etelcalcetide on bone health of rats with secondary hyperparathyroidism induced by chronic kidney disease

Chronic kidney disease-induced secondary hyperparathyroidism (CKD-SHPT) heightens fracture risk through impaired mineral homeostasis and elevated levels of uremic toxins (UTs), which in turn enhance bone remodeling. Etelcalcetide (Etel), a calcium-sensing receptor (CaSR) agonist, suppresses parathyroid hormone (PTH) in hyperparathyroidism to reduce excessive bone resorption, leading to increased bone mass. However, Etel's effect on bone quality, chemical composition, and strength is not well understood. To address these gaps, we established a CKD-SHPT rat model and administered Etel at a human-equivalent dose concurrently with disease induction. The effects on bone and mineral homeostasis were compared with a CKD-SHPT (vehicle-treated group) and a control group (rats without SHPT). Compared with vehicle-treated CKD-SHPT rats, Etel treatment improved renal function, reduced circulating UT levels, improved mineral homeostasis parameters, decreased PTH levels, and prevented mineralization defects. The upregulation of mineralization-promoting genes by Etel in CKD-SHPT rats might explain its ability to prevent mineralization defects. Etel preserved both trabecular and cortical bones with attendant suppression of osteoclast function, besides increasing mineralization. Etel maintained the number of viable osteocytes to the control level, which could also contribute to its beneficial effects on bone. CKD-SHPT rats displayed increased carbonate substitution of matrix and mineral, decreased crystallinity, mineral-to-matrix ratio, and collagen maturity, and these changes were mitigated by Etel. Further, Etel treatment prevented CKD-SHPT-induced deterioration in bone strength and mechanical behavior. Based on these findings, we conclude that in CKD-SHPT rats, Etel has multiscale beneficial effects on bone that involve remodeling suppression, mineralization gene upregulation, and preservation of osteocytes.

Renal calcium and magnesium handling during pregnancy: modeling and analysis.

Pregnancy is associated with elevated demand of most nutrients, with many trace elements and minerals critical for the development of fetus. In particular, calcium (Ca2+) and magnesium (Mg2+) are essential for cellular function, and their deficiency can lead to impaired fetal growth. A key contributor to the homeostasis of these ions is the kidney, which in a pregnant rat undergoes major changes in morphology, hemodynamics, and molecular structure. The goal of this study is to unravel the functional implications of these pregnancy-induced changes in renal handling of Ca2+ and Mg2+, two cations that are essential in a healthy pregnancy. To achieve that goal, we developed computational models of electrolyte and water transport along the nephrons of a rat in mid and late pregnancy. Model simulations reveal a substantial increase in the reabsorption of Mg2+ along the proximal tubules and thick ascending limbs. In contrast, the reabsorption of Ca2+ is increased in the proximal tubules but decreased in the thick ascending limbs, due to the lower transepithelial concentration gradient of Ca2+ along the latter. Despite the enhanced transport capacity, the marked increase in glomerular filtration rate results in elevated urinary excretions of Ca2+ and Mg2+ in pregnancy. Furthermore, we conducted simulations of hypocalcemia and hypomagnesemia. We found that hypocalcemia lowers Ca2+ excretion substantially more than Mg2+ excretion, with this effect being more pronounced in virgin rats than in pregnant ones. Conversely, hypomagnesemia reduces the excretion of Mg2+ and Ca2+ to more similar degrees. These differences can be explained by the greater sensitivity of the calcium-sensing receptor (CaSR) to Ca2+ compared with Mg2+.NEW & NOTEWORTHY A growing fetus' demands of minerals, notably calcium and magnesium, necessitate adaptations in pregnancy. In particular, the kidney undergoes major changes in morphology, hemodynamics, and molecular structure. This computational modeling study provides insights into how these pregnancy-induced renal adaptation impact calcium and magnesium transport along different nephron segments. Model simulations indicate that, despite the enhanced transport capacity, the marked increase in glomerular filtration rate results in elevated urinary excretions of calcium and magnesium in pregnancy.

Rare genetic disorders that impair parathyroid hormone synthesis, secretion, or bioactivity provide insights into the diagnostic utility of different parathyroid hormone assays.

Parathyroid hormone (PTH) is the major peptide hormone regulator of blood calcium homeostasis. Abnormal PTH levels can be observed in patients with various congenital and acquired disorders, including chronic kidney disease (CKD). This review will focus on rare human diseases caused by PTH mutations that have provided insights into the regulation of PTH synthesis and secretion as well as the diagnostic utility of different PTH assays. Over the past years, numerous diseases affecting calcium and phosphate homeostasis have been defined at the molecular level that are responsible for reduced or increased serum PTH levels. The underlying genetic mutations impair parathyroid gland development, involve the PTH gene itself, or alter function of the calcium-sensing receptor (CaSR) or its downstream signaling partners that contribute to regulation of PTH synthesis or secretion. Mutations in the pre sequence of the mature PTH peptide can, for instance, impair hormone synthesis or intracellular processing, while amino acid substitutions affecting the secreted PTH(1-84) impair PTH receptor (PTH1R) activation, or cause defective cleavage of the pro-sequence and thus secretion of a pro- PTH with much reduced biological activity. Mutations affecting the secreted hormone can alter detection by different PTH assays, thus requiring detailed knowledge of the utilized diagnostic test. Rare diseases affecting PTH synthesis and secretion have offered helpful insights into parathyroid biology and the diagnostic utility of commonly used PTH assays, which may have implications for the interpretation of PTH measurements in more common disorders such as CKD.

Effect of CaSR Deletion in the Esophagus on Gene Expression and Immune Function

Background: The calcium sensing receptor (CaSR) regulates Ca2+ concentration in plasma by regulating parathyroid hormone secretion. In other tissues, CaSR plays roles in cellular differentiation and migration, and in secretion and absorption. We reported previously that CaSR can be conditionally deleted in the mouse esophagus. This conditional knock-out ( EsoCaSR−/−) model showed significant reduction in levels of adherens and tight junction proteins and had significant build-up of bacteria on the luminal esophageal surface. Objectives: We aimed to further understand the role of CaSR and to examine the modification in gene expression induced by CaSR deletion in the esophagus. Methods: We performed mRNA sequencing and bioinformatic analyses of mice esophageal tissues. Gene expression was characterized using an unbiased pathway approach in EsoCaSR−/− and control mouse esophageal mucosae. Next generation sequencing was used to obtain a profile of the differences in RNA expression between tissues. RNA expression data was used to analyze differences in pathways and functional networks between EsoCaSR−/− and control esophageal tissues. Results: Our data indicated significant upregulation of gene sets involved in DNA replication and cell cycle in EsoCaSR−/−. This is accompanied by marked downregulation of gene sets involved in the innate immune response and in protein homeostasis including peptide elongation and protein traffcking. Ingenuity Pathway Analysis (IPA) demonstrated that these genes are mapped to important biological networks including Calcium and RhoA signaling pathways. Conclusions: The major findings of this study are that CaSR impacts important pathways of cell proliferation, differentiation, cell cycle and innate immune response in esophageal epithelium. The disruption of these pathways also causes significant modifications of the microbiome. Funding: U54 GM104940 grant from NIH, VA Merit grant, Paul Teschan Research grant, Carol Lavin Bernick grant, Tulane institutional grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hyaluronan Fragments Induce Lung Alveolar Edema by Activating Calcium-Sensing Receptor

One of the hallmarks of lung injury is the breakdown of the cell matrix hygroscopic high molecular weight hyaluronan (HMW-HA >106 kD) into smaller fragments of low molecular weight (LMW-HA < 500 kD) which mediate the onset and progression of acute lung injury. We have found that instillation of very low dose (1μg) of LMW-HA to wt. C57/bl6 mice was suffcient to inhibit lung epithelial ions and water transport and cause lung edema assessed by measuring lungs wet/dry ratio. LMW-HA IC50 for the induction of lung edema, the inhibition of alveolar fluid clearance (AFC) and epithelial sodium channels (ENaC) in alveolar epithelial cells, AEC1 and AEC2, in-situ, in freshly cut lung slices, was 120 nM. We found the expression of calcium-sensing receptor (CaSR), a C class G-protein coupled receptor, was activated by LMW-HA. CD44 is known to be the main membrane receptor of hyaluronan expressed by most cells including lung alveolar epithelial cells, however, because it lacks the intrinsic kinase activity necessary for the transduction of extracellular signal carried by LMW-HA, we hypothesized that it cooperated with CaSR to transduce LMW-HA injurious signal and activated intracellular signaling cascades that induced lung edema by inhibiting ENaC and AFC at 24 h. These effects were prevented by NPS2143 (1 μM), a CaSR inhibitor or calcilytic, instilled 6 h post LMW-HA, and were absent in CD44 knockout mouse. The lack of an effect on W/D ratio, AFC, or ENaC in CD44−/− mouse is evidence that LMW-HA does not directly interact with CaSR, but rather activates CD44 which cooperate with CaSR to trigger intracellular signaling cascades that cause the observed effects. Four hours post mouse airway epithelial cells confluent monolayers were incubated with 1 μg LMW-HA, CaSR expression was increased 4-fold, while amiloride sensitive short-circuit current and αβγ-ENaC subunits expression, at the apical membranes, were significantly reduced. These effects were prevented when CaSR, PLC, or PKC were inhibited with 1 μM NPS2143, 10 μM U73122, or 100 μM Rottlerin, applied 4 h prior to cells incubation with 1 μg LMW-HA, respectively. Our data show that the deleterious effect of LMW-HA on ENaC are mediated by intricate interactions between CD44-LMW-HA complex with CaSR in lung alveolar epithelial cells. The use of calcilytics to decouple the molecular complex formed by CD44-LMW-HA and CaSR may prove to be effective in restoring alveolar sodium ion reabsorption required for lung edema clearance. Department of Anesthesiology and Perioperative Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

New insights into renal calcium-sensing receptor activation.

Activation of the calcium-sensing receptor (CASR) in the parathyroid gland suppresses the release of parathyroid hormone (PTH). Furthermore, activation of the renal CASR directly increases the urinary excretion of calcium, by inhibiting transepithelial calcium transport in the nephron. Gain-of-function mutations in the CASR gene lead to autosomal dominant hypocalcemia 1 (ADH1), with inappropriately low PTH levels and hypocalcemia, indicative of excessive activation of the parathyroid CASR. However, hypercalciuria is not always observed. The reason why the manifestation of hypercalciuria is not uniform among ADH1 patients is not well understood. Direct activation of the CASR in the kidney has been cumbersome to study, and an indirect measure to effectively estimate the degree of CASR activation following chronic hypercalcemia or genetic gain-of-function CASR activation has been lacking. Studies have shown that expression of the pore-blocking claudin-14 is strongly stimulated by the CASR in a dose-dependent manner. This stimulatory effect is abolished after renal Casr ablation in hypercalcemic mice, suggesting that claudin-14 abundance may gauge renal CASR activation. Using this marker has led to unexpected discoveries regarding renal CASR activation. These new studies have informed on renal CASR activation thresholds and the downstream CASR-regulated calcium transport mechanisms.

The A986S calcium sensing receptor variant supports the adaptation of breast cancer cells to high calcium — a novel paradigm in breast cancer progression

Breast cancer and other solid tumors that metastasize to bone are frequently accompanied by a progressive increase in circulating calcium as the disease becomes more malignant. The increase in circulating calcium not only disrupts systemic calcium homeostasis but also alters the ability of cells to sense slight changes in extracellular Ca2+ via the ubiquitous calcium sensing receptor (CaSR). Of the CASR polymorphisms at rs1801725 (p.A986S), rs1801726 (p.Q1011E), and rs1042636 (p.R990G) in exon 7 of the receptor, previous studies have shown that only the A986S CaSR variant is associated with calcium in breast, prostate and skin cancers. In this study, we assessed if exon 7 inactivating A986S (AS-CaSR) and Q1011E (QE-CaSR) variants of the receptor influence the survival of breast tumor cells at higher Ca2+. We generated isogenic HEK293T and BT-549 cells expressing these CaSR variants and carried out bulk RNA sequencing of high Ca2+ adapted control and breast cancer cells. CaSR expression was detected by western blotting using anti-flag M2 (Sigma) antibody. By using phospho-ERK1/2 as the readout, the activation of AS-CaSR required higher concentrations of Ca2+ (EC50 ~5.28 mM) compared to the QE-CaSR (EC50 of ~3.81 mM) and the wild type (WT-CaSR; EC50 ~3.69 mM) receptor. When the CaSR variants transfected cells initially cultured at 7.5 mM for 6 days, and then sub-cultured in medium containing 7.5 to 20 mM Ca2+, the viability of empty vector control and WT-CaSR transfected cells strongly decreased at Ca2+ levels beyond 7.5 mM and these cells were mostly necrotic at ≥15 mM Ca2+. The QE-CaSR variant transfected cells on the other hand exhibited limited cell death beyond 10 mM Ca2+, while the surviving colonies of the AS-CaSR variant were viable at up to 15 mM Ca2+ with invasive tendencies. Analysis of differentially expressed genes in high Ca2+ adapted mesenchymal-like BT-549 and epithelial MDA-MB-468 breast cancer cells compared to MCF10A control cells revealed a small set of differentially expressed genes that were significantly associated with relapse free survival of basal-like breast cancer patients. Among these genes are ligands and negative regulators of the WNT/FZD pathway. Our data suggest that the A986S-CaSR variant with reduced sensitivity to Ca2+ supported adaptation of TNBC cells at high Ca2+, and that high Ca2+ adaptation of breast cancer cells may be mediated via a panel of high Ca2+ inducible genes with implications in bone metastasis. This study is supported by NIH SC1GM139814, T32AI007281, T32GM144927, T32HL007737; and the American Cancer Society. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Calcium-sensing receptor signaling is amplified in patients with a FAM111A gene mutation

Introduction: A six-year-old female presented with seizures, low blood calcium (Ca2+), low parathyroid hormone (PTH) and increased urinary Ca2+ excretion. She was diagnosed with Autosomal Dominant Hypocalcemia (ADH), a childhood disorder characterized by low blood Ca2+ and inappropriately low PTH. She had no mutations in genes known to cause ADH (Ca2+-sensing receptor ( CASR) and G-protein subunit alpha 11 ( GNA11)). Consequently, we performed whole exome sequencing and a trio analysis that identified a novel FAM111A gene mutation (c.1454G>A, p.C485Y). Although FAM111A is a protease, the specific protein targets are unknown. In addition, FAM111A regulates DNA replication and thus gene expression. Interestingly, FAM111A gene mutations cause Kenny Caffey syndrome (KCS) and Osteocraniostenosis (OCS), conditions characterized by low blood Ca2+, low PTH, and bony abnormalities. The molecular mechanism mediating these phenotypes are unknown. We hypothesize that mutations in FAM111A cause increased CASR signaling, resulting in low blood Ca2+ and high urine Ca2+ levels. The CASR is a G-protein coupled receptor, which upon activation by extracellular Ca2+, initiates signaling cascades that increase intracellular Ca2+ levels and decrease cyclic adenosine monophosphate (cAMP). CASR activation also increases the expression of the tight-junction protein claudin-14 (CLDN14). In the kidney, CLDN14 blocks Ca2+ reabsorption leading to increased urine Ca2+ excretion and lower blood Ca2+. Our objective was to determine if FAM111A wild-type (WT) suppresses CASR signaling and if the mutants fail to do so. Methods: Human embryonic cells (HEK293) were transfected with empty vector (EV) as a control, CASR and EV, FAM111A WT with EV or CASR and FAM111A WT or mutants (C485Y, ADH; Y511H, KCS; R569H, KCS; T338A, OCS; P527R, OCS; D528G, OCS; S541A, inactivated protease). We performed Fura2AM imaging of intracellular Ca2+ in transfected cells in the presence of increasing extracellular Ca2+ (0.5-11.3 mM) levels. Also, as an indication of CASR activity, we measured CLDN14 expression and cAMP levels, in transfected cells incubated in 0.5 mM or 5 mM extracellular Ca2+, via a dual luciferase assay and cAMP assay kit, respectively. Results: The change in peak intracellular Ca2+ concentration was two times higher (p<0.05) in cells with CASR plus EV or FAM111A mutants, compared to EV or FAM111A WT plus CASR. Similarly, luciferase activity as an indication of CLDN14 levels, was significantly higher (p<0.05) in CASR plus EV compared to EV and FAM111A WT plus CASR. Some mutants (C485Y, T338A, P527T, D528G, S542A) had similar CLDN14 levels to CASR plus EV, while others (Y511H, R569H) showed similar levels to FAM111A plus CASR. The cAMP levels were not different in cells with CASR and FAM111A WT compared to CASR with FAM111A mutants. Conclusions: FAM111A WT attenuates CASR activity. All FAM111A mutations assessed, enhanced CASR activity when measured by an increase in intracellular Ca2+. Some mutants increase CLDN14 expression, while others may affect a different CASR signaling pathway. Elucidating the mechanism of how FAM111A affects CASR activity and Ca2+ homeostasis will provide a better understanding of our patient’s condition as well as those with KCS or OCS. Canadian Institute of Health Research; Stollery Children’s Hospital Foundation through the Women and Children's Health Research Institute (WCHRI); Alberta Innovates Graduate Student Scholarship; WCHRI Graduate Studentship Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The screened compounds from Ligustri Lucidi Fructus using the immobilized calcium sensing receptor column exhibit osteogenic activity in vitro

Calcium sensing receptor (CaSR) has become the novel target of treating osteoporosis with herbal medicine Ligustri Lucidi Fructus (LLF), however, the bioactive compounds responsible for anti-osteoporosis are hard to clarify due to the complexity and diversity of chemical constituents in it. Herein, the immobilized CaSR column was packed with stationary phase materials, which were derived from integrating CLIP-tagged CaSR directly out of crude cell lysates onto the surface of silica gels (5.83 mg/g) in a site-specific covalent manner. The column had a great specificity of recognizing agonists and kept a good stability for at least 3 weeks. The two compounds from LLF extract were screened and identified as olenuezhenoside and ligustroflavone using the immobilized CaSR column in conjunction with mass spectrometry. Molecular docking predicted that both compounds were bound in venus flytrap (VFT) domain of CaSR by the formation of hydrogen bonds. Cellular results showed that both compounds exhibited the distinct osteogenic activity by enhancing the proliferation, differentiation and mineralization of osteoblastic cells. Our study demonstrated that, the immobilized protein column enables to screen the bioactive compounds rapidly from herbal extract, and the newly discovered natural product ligands towards CaSR, including olenuezhenoside and ligustroflavone, will be the candidates for the treatment of osteoporosis.

Molecular regulation of calcium-sensing receptor (CaSR)-mediated signaling.

Calcium-sensing receptor (CaSR), a family C G-protein-coupled receptor, plays a crucial role in regulating calcium homeostasis by sensing small concentration changes of extracellular Ca2+, Mg2+, amino acids (e.g., L-Trp and L-Phe), small peptides, anions (e.g., HCO3 - and PO4 3-), and pH. CaSR-mediated intracellular Ca2+ signaling regulates a diverse set of cellular processes including gene transcription, cell proliferation, differentiation, apoptosis, muscle contraction, and neuronal transmission. Dysfunction of CaSR with mutations results in diseases such as autosomal dominant hypocalcemia, familial hypocalciuric hypercalcemia, and neonatal severe hyperparathyroidism. CaSR also influences calciotropic disorders, such as osteoporosis, and noncalciotropic disorders, such as cancer, Alzheimer's disease, and pulmonary arterial hypertension. This study first reviews recent advances in biochemical and structural determination of the framework of CaSR and its interaction sites with natural ligands, as well as exogenous positive allosteric modulators and negative allosteric modulators. The establishment of the first CaSR protein-protein interactome network revealed 94 novel players involved in protein processing in endoplasmic reticulum, trafficking, cell surface expression, endocytosis, degradation, and signaling pathways. The roles of these proteins in Ca2+-dependent cellular physiological processes and in CaSR-dependent cellular signaling provide new insights into the molecular basis of diseases caused by CaSR mutations and dysregulated CaSR activity caused by its protein interactors and facilitate the design of therapeutic agents that target CaSR and other family C G-protein-coupled receptors.

Petrology and Geochemistry of an Unusual Granulite Facies Xenolith of the Late Oligocene Post-Obduction Koum Granodiorite (New Caledonia, Southwest Pacific): Geodynamic Inferences

Pressure–temperature estimates of a xenolith found within a post-obduction granodiorite in southern New Caledonia provide evidence for subcrustal, granulite facies, peak crystallisation conditions (ca. 850 °C—8.5 ± 1.0 kbar), followed by isobaric cooling to 700 °C, and final decompression with partial rehydration at ca. 650 °C—3.5 kbar. The xenolith, dated at 24.7 Ma (U-Pb zircon), i.e., the same age as the granodiorite host rock, has low SiO2 (35.5 wt%) and high Al2O3 (33.2 wt%) contents, suggesting that it is the restite of a previous melting episode, while the elevated Ca (Ba and Sr) contents suggest mantle metasomatism. Although the concentrations of Rb, K, Ca, Ba, and Sr have been strongly modified, some geochemical (REE patterns and some “immobile” trace element ratios) and isotopic (Sr and Nd isotopic ratios, U-Pb zircon age) characteristics of the granulite facies xenolith are similar to those of the xenoliths found in other Late Oligocene intrusions in southern New Caledonia; therefore, this rock is interpreted to be related to an early magmatic episode. The rock protolith was emplaced and equilibrated at the base of the crust where it underwent ductile deformation. Younger ascending magma picked it up and they eventually crystallised together at a shallow crustal level, near the tectonic sole of the ophiolite. The recrystallisation and ductile deformation at ~8.5 kbar suggest that a rheological discontinuity existed at about 25–28 km, probably representing the Moho. It is concluded that a continental crust of normal thickness must have existed beneath New Caledonia at about 24 Ma, i.e., 10 Ma after obduction.

Silicon-Rhodamine Functionalized Evocalcet Probes Potently and Selectively Label Calcium Sensing Receptors In Vitro, In Vivo, and Ex Vivo.

The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor (GPCR) that regulates extracellular calcium signals via the parathyroid glands. CaSR has recently also been implicated in noncalcitropic pathophysiologies like asthma, gut inflammation, and cancer. To date, molecular tools that enable the bioimaging of CaSR in tissues are lacking. Based on in silico analyses of available structure-activity relationship data on CaSR ligands, we designed and prepared silicon-rhodamine (SiR) conjugates of the clinically approved drug evocalcet. The new probes EvoSiR4 and EvoSiR6, with differing linker lengths at the evocalcet carboxyl end, both showed a 6-fold and 3-fold increase in potency toward CaSR (EC50 < 45 nM) compared to evocalcet and the evocalcet-linker conjugate, respectively, in an FLIPR-based cellular functional assay. The specificity of the EvoSiR probes toward CaSR binding and the impact of albumin was evaluated in live cell experiments. Both probes showed strong albumin binding, which facilitated the clearance of nonspecific binding interactions. Accordingly, in zebrafish embryos, EvoSiR4 specifically labeled the high CaSR expressing neuromasts of the lateral line in vivo. EvoSiR4 was also assessed in human parathyroid tissues ex vivo, showing a specific absolute CaSR-associated fluorescence compared to that of parathyroid autofluorescence. In summary, functionalization of evocalcet by SiR led to the preparation of potent and specific fluorescent CaSR probes. EvoSiR4 is a versatile small-molecular probe that can be employed in CaSR-related biomedical analyses where antibodies are not applicable.

Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges

G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors.

Autosomal dominant hypercalciuric hypocalcaemia: the calcium-sensing receptor in renal calcium homeostasis and the impact of renal transplantation.

Calcium-sensing receptors (CaSRs) are G protein-coupled receptors that help maintain Ca2+ concentrations, modulating calciotropic hormone release (parathyroid hormone (PTH), calcitonin and 1,25-dihydroxyvitamin D) by direct actions in the kidneys, gastrointestinal tract and bone. Variability in population calcium levels has been attributed to single nucleotide polymorphisms in CaSR genes, and several conditions affecting calcium and phosphate homeostasis have been attributed to gain- or loss-of-function mutations. An example is autosomal dominant hypercalciuric hypocalcaemia, because of a missense mutation at codon 128 of chromosome 3, as reported in our specific case and her family. As a consequence of treating symptomatic hypocalcaemia as a child, this female subject slowly developed progressive end-stage kidney failure because of nephrocalcinosis and nephrolithiasis. After kidney transplantation, she remains asymptomatic, with decreased vitamin D and elemental calcium requirements, stable fluid and electrolyte homeostasis during intercurrent illnesses and has normalised urinary calcium and phosphate excretion, reducing the likelihood of hypercalciuria-induced graft impairment. We review the actions of the CaSR, its role in regulating renal Ca2+ homeostasis along with the impact of a proven gain-of-function mutation in the CaSR gene resulting in autosomal dominant hypercalciuric hypocalcaemia before and after kidney transplantation.

Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Calcium-sensing receptor regulates Kv7 channels via Gi/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.

Neuropathic pain, a debilitating condition with unmet medical needs, can be characterised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage-gated potassium channels type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus excitability, reside under tight control of G protein-coupled receptors (GPCRs). Calcium-sensing receptor (CaSR) is a GPCR that regulates the activity of numerous ion channels, but whether CaSR can control Kv7 channel function has been unexplored until now. Experiments were conducted in recombinant cell models, mouse dorsal root ganglia (DRG) neurons and human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neurons using patch-clamp electrophysiology and molecular biology techniques. Our results demonstrate that CaSR is expressed in recombinant cell models, hiPSC-derived nociceptive-like neurons and mouse DRG neurons, and its activation induced depolarisation via Kv7.2/7.3 channel inhibition. The CaSR-Kv7.2/7.3 channel crosslink was mediated via the Gi/o protein-adenylate cyclase-cyclicAMP-protein kinase A signalling cascade. Suppression of CaSR function demonstrated a potential to rescue hiPSC-derived nociceptive-like neurons from algogenic cocktail-induced hyperexcitability. This study demonstrates that the CaSR-Kv7.2/7.3 channel crosslink, via a Gi/o protein signalling pathway, effectively regulates neuronal excitability, providing a feasible pharmacological target for neuronal hyperexcitability management in neuropathic pain.

ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

Calcium-sensing receptor-mediated macrophage polarization improves myocardial remodeling in spontaneously hypertensive rats.

Chronic inflammation is a key element in the progression of essential hypertension (EH). Calcium plays a key role in inflammation, so its receptor, the calcium-sensing receptor (CaSR), is an essential mediator of the inflammatory process. Compelling evidence suggests that CaSR mediates inflammation in tissues and immune cells, where it mediates their activity and chemotaxis. Macrophages (Mφs) play a major role in the inflammatory response process. This study provided convincing evidence that R568, a positive regulator of CaSR, was effective in lowering blood pressure in spontaneously hypertensive rats (SHRs), improving cardiac function by alleviating cardiac hypertrophy and fibrosis. R568 can increase the content of CaSR and M2 macrophages (M2Mφs, exert an anti-inflammatory effect) in myocardial tissue, reduce M1 macrophages (M1Mφs), which have a pro-inflammatory effect in this process. In contrast, NPS2143, a negative state regulator of CaSR, exerted the opposite effect in all of the above experiments. Following this study, R568 increased CaSR content in SHR myocardial tissue, lowered blood pressure, promoted macrophages to M2Mφs and improved myocardial fibrosis, but interestingly, both M1Mφs and M2Mφs were increased in the peritoneal cavity of SHRs, the number of M2Mφs remained lower than M1Mφs. In vitro, R568 increased CaSR content in RAW264.7 cells (a macrophage cell line), regulating intracellular Ca2+ ([Ca2+]i) inhibited NOD-like receptor family protein 3 (NLRP3) inflammasome activation and ultimately prevented its conversion to M1Mφs. The results showed that a decrease in CaSR in hypertensive rats causes further development of hypertension and cardiac damage. EH myocardial remodeling can be improved by CaSR overexpression by suppressing NLRP3 inflammasome activation and macrophage polarization toward M1Mφs and increasing M2Mφs.

Inhibition of calcium-sensing receptor by its antagonist promotes gastrointestinal motility in a Parkinson’s disease mouse model

BackgroundThe Calcium-sensing receptor (CaSR) participates in the regulation of gastrointestinal (GI) motility under normal conditions and might be involved in the regulation of GI dysmotility in patients with Parkinson's disease (PD). MethodsCaSR antagonist-NPS-2143 was applied in in vivo and ex vivo experiments to study the effect and underlying mechanisms of CaSR inhibition on GI dysmotility in the MPTP-induced PD mouse model. FindingsOral intake of NPS-2143 promoted GI motility in PD mice as shown by the increased gastric emptying rate and shortened whole gut transit time together with improved weight and water content in the feces of PD mice, and the lack of influence on normal mice. Meanwhile, the number of cholinergic neurons, the proportion of serotonergic neurons, as well as the levels of acetylcholine and serotonin increased, but the numbers of nitrergic and tyrosine hydroxylase immunoreactive neurons, and the levels of nitric oxide synthase and dopamine decreased in the myenteric plexus in the gastric antrum and colon of PD mice in response to NPS-2143 treatment. Furthermore, the numbers of c-fos positive neurons in the nucleus tractus solitarius (NTS) and cholinergic neurons in the dorsal motor nucleus of the vagus (DMV) increased in NPS-2143 treated PD mice, suggesting the involvement of both the enteric (ENS) and central (CNS) nervous systems. However, ex vivo results showed that NPS-2143 directly inhibited the contractility of antral and colonic strips in PD mice via a non-ENS mediated mechanism. Further studies revealed that NPS-2143 directly inhibited the voltage gated Ca2+ channels, which might, at least in part, explain its direct inhibitory effects on the GI muscle strips. InterpretationCaSR inhibition by its antagonist ameliorated GI dysmotility in PD mice via coordinated neuronal regulation by both ENS and CNS in vivo, although the direct effects of CaSR inhibition on GI muscle strips were suppressive.

Functional evaluation of a novel nonsense variant of the calcium-sensing receptor gene leading to hypocalcemia.

The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897_2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants. To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs. Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT. This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction.