Ann Pediatr Endocrinol Metab.  2021 Mar;26(1):24-30. 10.6065/apem.2040022.011.

A cell function study on calcium regulation of a novel calcium-sensing receptor mutation (p.Tyr825Phe)

• Moon JE
• Yang HY
• Wee G
• ParK SH
• Ko CW

• KMID: 2514149
• DOI: http://doi.org/10.6065/apem.2040022.011

Abstract

Purpose
Autosomal dominant hypocalcemia with hypercalciuria is a genetic disease characterized by hypoparathyroidism with hypercalciuria. We discovered a novel variant (p.Tyr825Phe[Y825F]) of the CASR gene in a neonate with congenital hypoparathyroidism and hypercalciuria and conducted a cell function study to determine whether the CASR-Y825F variant was pathogenic.
Methods
To perform a functional study on CaSR-Y825F, we constructed expression vectors expressing wild-type (WT) CASR and CASR-Y825F. After transfection of each expression vector into HEK293 cells, we examined alterations in intracellular signaling. Mitogen-activated protein kinase (MAPK) signaling activity of HEK293 cells expressing CASR-WT or CASR-Y825F was determined. Changes in intracellular calcium ions ([Ca²⁺]i) by extracellular calcium ion ([Ca²⁺]e) stimulation were quantitatively compared and analyzed.
Results
Cells expressing CASR-Y825F showed elevated of MAPK signaling (phospho-ERK [pERK], phospho-JNK [pJNK], phospho-p38 [pp38]) and increased [Ca²⁺]i levels at low [Ca²⁺]e stimulation compared with cells expressing CASR-WT. Additionally, [Ca²⁺]i levels in HEK293 cells expression CASR-WT and CASR-Y825F were determined at 340 nm/380 nm wavelength ratios using Fura-2 AM. At [Ca²⁺]e concentrations of 2.5 mM and 3 mM, the ratios of CASR-Y825F cells were higher (2.6 and 3.5, respectively) than those of CASR-WT cells (1.04 and 1.40, respectively).
Conclusion
This cell function study proved that the CASR-Y825F expressed in HEK293 cells elevated MAPK signaling (pERK, pJNK, pp38) and increased [Ca²⁺]i to induce hypocalcemia.

Keyword

Hypercalciuric Hypocalcemia; Hypoparathyroidism; Ca-sensing receptor; Point mutation

Figure

• 

  Fig. 1. Western blot analysis of wild-type CASR (CASR-WT) and Y825F-mutant CASR (CASR-Y825F). HEK293 human pancreatic cells were transfected with CASR-WT or CASR-Y825F expression plasmids and examined by western blot. There was no difference in the intracellular protein expression structure of CASR-WT and CASR-Y825F. β-actin was used as loading control.

• 

  Fig. 2. Bioanalysis results of mitogen-activated protein kinase (MAPK) signaling depending on extracellular calcium ions ([Ca2+]e) concentrations of wild-type (WT) CASR and Y825F-mutant CASR (CASR-Y825F) in HEK293 cells. (A) MAPK activity was determined using antibodies against phosphorylated ERK (Thr202/Tyr204), JNK (Thr183/Tyr185), and p38 (Thr180/Tyr182) antibodies. After 10 minutes of calcium treatment, cells were lysed and western blot analysis was conducted. (B) Quantification of phospho-ERK/JNK/p38 based on the expression of total-ERK/JNK/p38 using densitometry analysis (with Image J software). The values were normalized to the expression of wild-type protein in cells not treated with calcium. The level of phospho-ERK/JNK activation was increased in HEK293 cells expressing CASR-Y825F at [Ca2+]e concentrations of 0, 2, 4, and 10 mM. The level of phospho-p38 was increased at a [Ca2+]e concentration of 2 mM. *P<0.05.

• 

  Fig. 3. Measurement of [Ca2+]i concentration in cells expressing wild-type CASR (CASR-WT) or Y825F-mutant CASR (CASR-Y825F). After transfection of CASR-WT and CASR-Y825F, HEK293 cells were treated with [Ca2+]e at different concentrations, and [Ca2+]i concentrations were measured using Fura-2 AM fluorescent dye. The [Ca2+]e concentrations included the range that leads to mitogen-activated protein kinase signaling. The average of [Ca2+]i concentrations were calculated from 3 determinations of 340 nm/380 nm wavelength ratios. The [Ca2+]i level of HEK293 cells transfected with CASR-Y825F was higher at concentrations between 2 mM and 4 mM of [Ca2+ ]e compared with cells expressing CASR-WT. *P<0.05.

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