Molecular and clinical analysis of a neonatal severe hyperparathyroidism case caused by a stop mutation in the calcium-sensing receptor extracellular domain representing in effect a human ‘knockout’

ObjectiveLoss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaRR392X), in effect a full CAR ‘knockout’.Case reportThe infant (daughter of distant cousins) presented with hypercalcaemia (5.5–6 mmol/l corrected calcium (2.15–2.65)) and elevated PTH concentrations (650–950 pmol/l (12–81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels.Design and methodsThe R392X stop codon was inserted into human CAR and the resulting mutant (CaRR392X) expressed transiently in HEK-293 cells.ResultsCaRR392X expressed as a 54 kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaRR392X-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity.ConclusionsIntriguingly, the patient remained normocalcaemic throughout childhood (2.5 mM corrected calcium, 11 pg/ml PTH (10–71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.

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Needle in a Haystack—Parathyroid Gland in a 10-Day Old Infant: A Case Report and Literature Review

ISRN Pediatrics ◽

10.5402/2011/678070 ◽

2011 ◽

Vol 2011 ◽

pp. 1-3 ◽

Cited By ~ 1

Author(s):

Adel Ismail ◽

Tariq O. Abbas ◽

Fawziya Al-Khalaf

Keyword(s):

Parathyroid Gland ◽

Autosomal Recessive ◽

Autosomal Recessive Disorder ◽

Surgical Removal ◽

Total Parathyroidectomy ◽

Calcium Sensor ◽

Calcium Sensing Receptor ◽

Calcium Sensing ◽

Thyroid C Cells ◽

Neonatal Severe Primary Hyperparathyroidism

Neonatal severe primary hyperparathyroidism (NSPHT) is a rare autosomal recessive disorder of calcium homeostasis. It presents shortly after birth and is characterized by striking hyperparathyroidism, marked hypercalcemia, and hyperparathyroid bone disease. It is caused by mutations of the calcium-sensing receptor (CASR), the ionized calcium sensor for the parathyroid cells, the parafollicular thyroid C cells, and the kidney epithelium, as well as cells in bone and intestine. Without early intervention, which frequently requires surgical removal of the hyperplastic parathyroids, the patients often succumb to complications of hypercalcemia and respiratory failure. Finding the parathyroid gland in small neonates is not an easy task. Here we report on a patient with neonatal hyperparathyroidism who was treated by total parathyroidectomy and discuss the various ways of helping to find the parathyroid glands during surgery at this young age.

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Calcium-sensing receptors control CYP27B1-luciferase expression. Transcriptional and post-transcriptional mechanisms

Journal of the Endocrine Society ◽

10.1210/jendso/bvab057 ◽

2021 ◽

Author(s):

Alice Huang ◽

Lenah Binmahfouz ◽

Dale P Hancock ◽

Paul H Anderson ◽

Donald T Ward ◽

...

Keyword(s):

Fold Increase ◽

Firefly Luciferase ◽

Inhibitory Effect ◽

Renilla Luciferase ◽

Luciferase Expression ◽

Mrna Levels ◽

Hek 293 ◽

Hek 293 Cells ◽

Calcium Sensing ◽

293 Cells

Abstract 25-hydroxyvitamin D 1α-hydroxylase (encoded by CYP27B1), which catalyses the synthesis of 1,25-dihydroxyvitamin D3, is subject to negative or positive modulation by extracellular Ca 2+ (Ca 2+o) depending on the tissue. However, the Ca 2+ sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca 2+o-dependent control of 1α-hydroxylase using HEK-293 cells stably expressing the CaSR (HEK-CaSR cells). In HEK-CaSR cells, but not control HEK-293 cells, co-transfected with reporter genes for CYP27B1-Photinus pyralis (firefly) luciferase and control Renilla luciferase, an increase in Ca 2+o from 0.5 to 3.0 mM induced a 2-3 fold increase in firefly-luciferase activity as well as mRNA and protein levels. Surprisingly, firefly-luciferase was specifically suppressed at Ca 2+o ≥ 5.0 mM, demonstrating biphasic Ca 2+o control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca 2+o induced simple monotonic increases in firefly-luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca 2+o was post-transcriptional. Studies with inhibitors and the CaSR C-terminal mutant T888A identified roles for PKC, phosphorylation of T888, and ERK1/2 in high Ca 2+o-dependent suppression of firefly-luciferase. Blockade of both PKC and ERK1/2 abolished Ca 2+o-stimulated firefly-luciferase, demonstrating that either PKC or ERK1/2 is sufficient to stimulate the CYP27B1 promoter. A key CCAAT box (–74 bp to –68 bp), which is regulated downstream of PKC and ERK1/2 was required for both basal transcription and Ca 2+o-mediated transcriptional upregulation. The CaSR mediates Ca 2+o-dependent transcriptional upregulation of 1α-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca 2+o can promote luciferase and possibly 1α-hydroxylase breakdown.

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Inhibition of Na+-K+-2Cl−cotransport by mercury

AJP Cell Physiology ◽

10.1152/ajpcell.1999.277.4.c684 ◽

1999 ◽

Vol 277 (4) ◽

pp. C684-C692 ◽

Cited By ~ 42

Author(s):

Steven C. Jacoby ◽

Edith Gagnon ◽

Luc Caron ◽

John Chang ◽

Paul Isenring

Keyword(s):

Ion Transport ◽

Binding Sites ◽

Essential Role ◽

Species Differences ◽

Transmembrane Domain ◽

Transport Proteins ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH−) groups. The inorganic form (Hg2+) is toxic to epithelial tissues and interacts with various transport proteins including the Na+ pump and Cl− channels. In this study, we determined whether the Na+-K+-Cl−cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg2+ on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg2+inhibits Na+-K+-Cl−cotransport, with inhibitor constant ( K i) values of 25 μM for the shark carrier (sNKCC1) and 43 μM for the human carrier. In further studies, we took advantage of species differences in Hg2+ affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697→Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+binding. We also show that modification of additional SH− groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.

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Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms221810124 ◽

2021 ◽

Vol 22 (18) ◽

pp. 10124

Author(s):

Martin Schepelmann ◽

Nadja Kupper ◽

Marta Sladczyk ◽

Bethan Mansfield ◽

Teresa Manhardt ◽

...

Keyword(s):

Colon Cancer ◽

Cancer Cells ◽

Inflammatory Cytokine ◽

Extracellular Calcium ◽

Colon Cancer Cells ◽

Calcium Sensing Receptor ◽

Hek 293 ◽

Calcium Sensing ◽

293 Cells ◽

Target Effects

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on β-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes.

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Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽

10.4161/auto.25455 ◽

2013 ◽

Vol 9 (9) ◽

pp. 1407-1417 ◽

Cited By ~ 41

Author(s):

Patience Musiwaro ◽

Matthew Smith ◽

Maria Manifava ◽

Simon A. Walker ◽

Nicholas T. Ktistakis

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Anesthesiology ◽

10.1097/00000542-200512000-00009 ◽

2005 ◽

Vol 103 (6) ◽

pp. 1156-1166 ◽

Cited By ~ 7

Author(s):

Kevin J. Gingrich ◽

Son Tran ◽

Igor M. Nikonorov ◽

Thomas J. Blanck

Keyword(s):

Charge Transfer ◽

Calcium Channels ◽

Dependent Manner ◽

Current Time ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Dependent Inactivation ◽

Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

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