Exploring near-infrared autofluorescence properties in parathyroid tissue: an analysis of fresh and paraffin-embedded thyroidectomy specimens.

Significance: Near-infrared autofluorescence (NIRAF) utilizes the natural autofluorescence of parathyroid glands (PGs) to improve their identification during thyroid surgeries, reducing the risk of inadvertent removal and subsequent complications such as hypoparathyroidism. This study evaluates NIRAF's effectiveness in real-world surgical settings, highlighting its potential to enhance surgical outcomes and patient safety. Aim: We evaluate the effectiveness of NIRAF in detecting PGs during thyroidectomy and central neck dissection and investigate autofluorescence characteristics in both fresh and paraffin-embedded tissues. Approach: We included 101 patients diagnosed with papillary thyroid cancer who underwent surgeries in 2022 and 2023. We assessed NIRAF's ability to locate PGs, confirmed via parathyroid hormone assays, and involved both junior and senior surgeons. We measured the accuracy, speed, and agreement levels of each method and analyzed autofluorescence persistence and variation over 10 years, alongside the expression of calcium-sensing receptor (CaSR) and vitamin D. Results: NIRAF demonstrated a sensitivity of 89.5% and a negative predictive value of 89.1%. However, its specificity and positive predictive value (PPV) were 61.2% and 62.3%, respectively, which are considered lower. The kappa statistic indicated moderate to substantial agreement (kappa = 0.478; P < 0.001 ). Senior surgeons achieved high specificity (86.2%) and PPV (85.3%), with substantial agreement (kappa = 0.847; P < 0.001 ). In contrast, junior surgeons displayed the lowest kappa statistic among the groups, indicating minimal agreement (kappa = 0.381; P < 0.001 ). Common errors in NIRAF included interference from brown fat and eschar. In addition, paraffin-embedded samples retained stable autofluorescence over 10 years, showing no significant correlation with CaSR and vitamin D levels. Conclusions: NIRAF is useful for PG identification in thyroid and neck surgeries, enhancing efficiency and reducing inadvertent PG removals. The stability of autofluorescence in paraffin samples suggests its long-term viability, with false positives providing insights for further improvements in NIRAF technology.

The calcium-sensing-receptor (CaSR) in adipocytes contributes to sex-differences in the susceptibility to high fat diet induced obesity and atherosclerosis.

BACKGROUND: Female mice are more resistant to obesogenic effects of a high-fat diet (HFD), compared to male mice. Although the underlying mechanisms are poorly understood, sex hormones seem to play an important role. Interestingly, the activity of the oestrogen receptor-α (ERα) is affected by the calcium-sensing-receptor (CaSR). Therefore, we investigated sex-differences upon diet-induced obesity and the role of adipocyte-specific CaSR herein. METHODS: Adipocyte-specific Casr deficient mice (AdipoqCre+Casrflox) and control mice (Casrflox) were injected with AAV8-PCSK9 to make them prone to develop atherosclerosis and fed an obesity-inducing diet for 12 weeks. FINDINGS: Female mice have lower visceral white adipose tissue (vWAT) mass compared to male mice, while this sex-difference is abolished upon adipocyte-specific Casr deficiency. Furthermore, while females showed elevated levels of inflammatory cytokines and CD3+CD8+ T cell accumulation in vWAT, compared to males, adipocyte-specific Casr deficiency abrogated this sex-phenotype and demonstrated an inhibition of inflammatory signalling pathways. The expression of Erα, as well as associated genes involved in adipocyte differentiation, was increased in female mice in a mostly adipocyte-specific Casr dependent manner. Interestingly, circulating lipid levels were reduced in female compared to male mice, which correlated with decreased atherosclerotic plaque formation. These systemic effects were abrogated upon adipocyte-specific Casr deficiency. INTERPRETATION: Our findings indicate that female mice show a more pronounced vWAT dysfunction compared to males upon obesity. This sex effect is abolished upon adipocyte-specific Casr deficiency. In contrast, females show diminished atherosclerotic plaque formation compared to males, an effect that was abrogated by adipocyte-specific Casr deficiency. FUNDING: This work was supported by a grant from the Interdisciplinary Center for Clinical Research within the faculty of Medicine at the RWTH Aachen University, by the Corona Foundation, by the Deutsche Forschungsgemeinschaft (DFG), the BMBF and Free State of Bavaria and the DZHK.

Targeting protein receptors and enzymes for precision management of urolithiasis: A comprehensive review.

Urolithiasis, characterized by the formation of solid crystalline structures within the urinary tract, presents a significant global health burden with high recurrence rates and limited treatment efficacy. Recent research has identified various protein receptors and enzymes implicated in the pathogenesis of urolithiasis, offering potential targets for therapeutic intervention. Protein receptors such as the calcium-sensing receptor and vasopressin V2 receptor play crucial roles in regulating urinary calcium excretion and water reabsorption, respectively, influencing stone formation. Additionally, modulation of receptors like the angiotensin II receptor and aldosterone receptor can impact renal function and electrolyte balance, contributing to stone prevention. Furthermore, enzymes such as urease inhibitors and xanthine oxidase inhibitors offer targeted approaches to prevent the formation of specific stone types. This review discusses the potential of targeting these receptors and enzymes for the treatment of urolithiasis, exploring associated drugs and their mechanisms of action. Despite promising avenues for personalized and precision medicine approaches, challenges such as the need for robust clinical evidence and ensuring cost-effectiveness must be addressed for the translation of these interventions into clinical practice. By overcoming these challenges, receptor-targeted therapies and enzyme inhibitors hold promise for revolutionizing the management of urolithiasis and reducing its global burden.

The Interaction of Calcium-Sensing Receptor with KIF11 Enhances Cisplatin Resistance in Lung Adenocarcinoma via BRCA1/cyclin B1 pathway.

Cisplatin (DDP) is commonly used in the treatment of non-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD), and the primary cause for its clinical inefficacy is chemoresistance. Here, we aimed to investigate a novel mechanism of chemoresistance in LUAD cells, focusing on the calcium-sensing receptor (CaSR). In this study, high CaSR expression was detected in DDP-resistant LUAD cells, and elevated CaSR expression is strongly correlated with poor prognosis in LUAD patients receiving chemotherapy. LUAD cells with high CaSR expression exhibited decreased sensitivity to cisplatin, and the growth of DDP-resistant LUAD cells was inhibited by cisplatin treatment in combination with CaSR suppression, accompanied by changes in BRCA1 and cyclin B1 protein expression both in vitro and in vivo. Additionally, an interaction between CaSR and KIF11 was identified. Importantly, suppressing KIF11 resulted in decreased protein levels of BRCA1 and cyclin B1, enhancing the sensitivity of DDP-resistant LUAD cells to cisplatin with no obvious decrease in CaSR. Here, our findings established the critical role of CaSR in promoting cisplatin resistance in LUAD cells by modulating cyclin B1 and BRCA1 and identified KIF11 as a mediator, highlighting the potential therapeutic value of targeting CaSR to overcome chemoresistance in LUAD.

Novel osteogenic peptide from bovine bone collagen hydrolysate: Targeted screening, molecular mechanism, and stability analysis.

This study aimed to screen for a novel osteogenic peptide based on the calcium-sensing receptor (CaSR) and explore its molecular mechanism and gastrointestinal stability. In this study, a novel osteogenic peptide (Phe-Ser-Gly-Leu, FSGL) derived from bovine bone collagen hydrolysate was successfully screened by molecular docking and synthesised by solid phase peptide synthesis for further analysis. Cell experiments showed that FSGL significantly enhanced the osteogenic activity of MC3T3-E1 cells by acting on CaSR, including proliferation (152.53%), differentiation, and mineralization. Molecular docking and molecular dynamics further demonstrated that FSGL was a potential allosteric activator of CaSR, that turned on the activation switch of CaSR by closing the Venus flytrap (VFT) domain and driving the two protein chains in the VFT domain to easily form dimers. In addition, 96.03% of the novel osteogenic peptide FSGL was stable during gastrointestinal digestion. Therefore, FSGL showed substantial potential for enhancing the osteogenic activity of osteoblasts. This study provided new insights for the application of CaSR in the targeted screening of osteogenic peptides to improve bone health.

Stimulation of the calcium-sensing receptor induces relaxations through CGRP and NK1 receptor-mediated pathways in male rat mesenteric arteries.

Stimulation of the calcium-sensing receptor (CaSR) regulates vascular contractility, but cellular mechanisms involved remain unclear. This study investigated the role of perivascular sensory nerves in CaSR-induced relaxations of male rat mesenteric arteries. In fluorescence studies, colocalisation between synaptophysin, a synaptic vesicle marker, and the CaSR was present in the adventitial layer of arterial segments. Using wire myography, increasing external Ca2+ concentration ([Ca2+]o) from 1 to 10 mM induced vasorelaxations, previously shown to involve the CaSR, which were inhibited by pretreatment with capsaicin. [Ca2+]o-induced vasorelaxations were partially reduced by the calcitonin gene-related peptide (CGRP) receptor blockers, CGRP 8-37 and BIBN 4096, and the neurokinin 1 (NK1) receptor blocker L733,060. The inhibitory effect of CGRP 8-37 required a functional endothelium whereas the inhibitory action of L733,060 did not. Complete inhibition of [Ca2+]o-induced vasorelaxations occurred when CGRP 8-37 and L733,060 were applied together. [Ca2+]o-induced vasorelaxations in the presence of capsaicin were abolished by the ATP-dependent K+ channel (KATP) blocker PNU 37883, but unaffected by the endothelium nitric oxide synthase (eNOS) inhibitor L-NAME. We suggest that the CaSR on perivascular sensory nerves mediate relaxations in rat mesenteric arteries via endothelium-dependent and -independent mechanisms involving CGRP and NK1 receptor-activated NO production and KATP channels, respectively.

Glutathione-Based Photoaffinity Probe Identifies Caffeine as a Positive Allosteric Modulator of the Calcium-Sensing Receptor.

The calcium-sensing receptor (CaSR), abundantly expressed in the parathyroid gland and kidney, plays a central role in calcium homeostasis. In addition, CaSR exerts multimodal roles, including inflammation, muscle contraction, and bone remodeling, in other organs and tissues. The diverse functions of CaSR are mediated by many endogenous and exogenous ligands, including calcium, amino acids, glutathione, cinacalcet, and etelcalcetide, that have distinct binding sites in CaSR. However, strategies to evaluate ligand interactions with CaSR remain limited. Here, we developed a glutathione-based photoaffinity probe, DAZ-G, that analyzes ligand binding to CaSR. We showed that DAZ-G binds to the amino acid binding site in CaSR and acts as a positive allosteric modulator of CaSR. Oxidized and reduced glutathione and phenylalanine effectively compete with DAZ-G conjugation to CaSR, while calcium, cinacalcet, and etelcalcetide have cooperative effects. An unexpected finding was that caffeine effectively competes with DAZ-G's conjugation to CaSR and acts as a positive allosteric modulator of CaSR. The effective concentration of caffeine for CaSR activation (<10 µM) is easily attainable in plasma by ordinary caffeine consumption. Our report demonstrates the utility of a new chemical probe for CaSR and discovers a new protein target of caffeine, suggesting that caffeine consumption can modulate the diverse functions of CaSR.

Analysis of Expression of the ANG1, CaSR and FAK Proteins in Uterine Fibroids.

Understanding the molecular factors involved in the development of uterine myomas may result in the use of pharmacological drugs instead of aggressive surgical treatment. ANG1, CaSR, and FAK were examined in myoma and peripheral tissue samples taken from women after myoma surgery and in normal uterine muscle tissue samples taken from the control group. Tests were performed using tissue microarray immunohistochemistry. No statistically significant differences in ANG1 expression between the tissue of the myoma, the periphery, and the normal uterine muscle tissue of the control group were recorded. The CaSR value was reduced in the myoma and peripheral tissue and normal in the group of women without myomas. FAK expression was also lower in the myoma and periphery compared to the healthy uterine myometrium. Calcium supplementation could have an effect on stopping the growth of myomas.

Ca2+-sensing receptor-TRP channel-mediated Ca2+ signaling: Functional diversity and pharmacological complexity.

The Ca2+-sensing receptor (CaSR) is a G-protein-coupled receptor activated by elevated concentrations of extracellular Ca2+, and was initially known for its regulation of parathyroid hormone (PTH) release. Ubiquitous expression of CaSR in different tissues and organs was later noted and CaSR participation in various physiological functions was demonstrated. Accumulating evidence has suggested that CaSR functionally interacts with transient receptor potential (TRP) channels, which are mostly non-selective cation channels involved in sensing temperature, pain and stress. This review describes the interactions of CaSR with TRP channels in diverse cell types to trigger a variety of biological responses. CaSR has been known to interact with different types of G proteins. Possible involvements of G proteins, other signaling and scaffolding protein intermediates in CaSR-TRP interaction are discussed. In addition, an attempt will be made to extend the current understanding of biased agonism of CaSR.

Phosphate as an adjunct to calcium in promoting coronary vascular calcification in chronic inflammatory states.

Bone releases calcium and phosphate in response to pro-inflammatory cytokine-mediated inflammation. The body develops impaired urinary excretion of phosphate with age and chronic inflammation given the reduction of the kidney protein Klotho, which is essential to phosphate excretion. Phosphate may also play a role in the development of the resistance of the parathyroid calcium-sensing receptor (CaSR) to circulating calcium thus contributing to calcium retention in the circulation. Phosphate can contribute to vascular smooth muscle dedifferentiation with manifestation of osteoblastogenesis and ultimately endovascular calcium phosphate precipitation. Thus phosphate, along with calcium, contributes to the calcification and inflammation of atherosclerotic plaques and the origin of these elements is likely the bone, which serves as storage for the majority of the body's supply of extracellular calcium and phosphate. Early cardiac evaluation of patients with chronic inflammation and attempts at up-regulating the parathyroid CaSR with calcimimetics or introducing earlier anti-resorptive treatment with bone active pharmacologic agents may serve to delay onset or reduce the quantity of atherosclerotic plaque calcification in these patients.

Calcium-sensing Receptor, a Potential Biomarker Revealed by Large-scale Public Databases and Experimental Verification in Metastatic Breast Cancer.

INTRODUCTION: Breast cancer (BC) is a common cancer characterized by a high molecular heterogeneity. Therefore, understanding its biological properties and developing effective treatments for patients with different molecular features is imperative. Calcium-sensing receptor (CaSR) has been implicated in several regulatory functions in various types of human cancers. However, its underlying pathological mechanism in BC progression remains elusive. METHODS: We utilized The Cancer Genome Atlas and Gene Expression Omnibus databases to explore the function of CaSR in the metastasis of BC. Gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and Gene Set Enrichment Analysis of biological processes and cell signaling pathways revealed that CaSR could be activated or inhibited. Importantly, quantitative reverse transcriptase-polymerase chain reaction and western blotting were used to verify the gene expression of the CaSR. Wound healing and transwell assays were conducted to assess the effect of CaSR on the migration of BC cells. RESULTS: We demonstrated that CaSR expression in metastatic BC was higher than that in non-metastatic BC. It is the first time that database information has been used to reveal the biological process and molecular mechanism of CaSR in BC. Moreover, the CaSR expression in normal breast epithelial cells was notably less compared to that in BC cells. The activation of CaSR by Cinacalcet (a CaSR agonist) significantly enhanced the migration of BC cells, whereas NPS-2143 (a CaSR antagonist) treatment dramatically inhibited these effects. CONCLUSION AND FUTURE PERSPECTIVE: Bioinformatics techniques and experiments demonstrated the involvement of CaSR in BC metastasis. Our findings shed new light on the receptor therapy and molecular pathogenesis of BC, and emphasize the crucial function of CaSR, facilitating the metastasis of BC.

New insights into renal calcium-sensing receptor activation.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: These new studies have informed on renal CASR activation thresholds and the downstream CASR-regulated calcium transport mechanisms.

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

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: 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.

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.

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.

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.

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

BACKGROUND: The 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). METHODS: CaSR 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. FINDINGS: Oral 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. INTERPRETATION: CaSR 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.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSION AND IMPLICATIONS: 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.

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.