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Calcif Tissue Int (2010) 86:227233
DOI 10.1007/s00223-009-9298-7
LABORATORY INVESTIGATIONS
Residue 826 in the Calcium-Sensing Receptor Is Implicatedin the Response to Calcium and to R-568 Calcimimetic Compound
Daniellvarez-Hernndez Ignacio Gonzlez-Surez
Jos L. Fernndez-Martn Isabel Rodrguez
igo Santamara Eliecer Coto Jorge B. Cannata-Anda
Received: 30 June 2009 / Accepted: 8 September 2009 / Published online: 4 February 2010 Springer Science+Business Media, LLC 2010
Abstract Within the extracellular loops of the seven-transmembrane domain of the calcium-sensing receptor (CaR) there is a region (I819E837) relevant for calcimimetic activity. As the naturally occurring variant Ala826Thr is within this important region, it may be postulated that this change may inuence the CaR response to calcium and R-568. Human embryonic kidney (HEK-293) cells transiently transfected with three different human
CaRs (wild-type [A826], variant allele [T826], and articial mutant [W826]) were used to test the ability of calcium alone or in combination with the calcimimetic R-568 to modulate CaR activity. CaR activation was detected by ow cytometry using a uorescent probe. Intracellular calcium changes were measured in response to changes in extracellular calcium alone or with different R-568 concentrations. The change of the alanine in the 826 position (A826) for threonine (T826) worsened calcium sensitivity, increasing the EC50 value from 2.34 0.48 mM (A826, wild-type) to 2.96 0.75 mM (T826) (P \ 0.05). The
T826 receptor reached a similar response with 1 lM R-568 compared with the wild-type receptor. On the contrary, the articial introduction of a tryptophan in the same position (W826) did not affect calcium sensitivity (EC50 = 2.64
0.81 mM) but reduced the ability of the receptor to respond to R-568. The results demonstrate the importance of the 826 residue in the CaR response to calcium and calcimimetics. Since the A826T change was described as a natural variant, the differences in the calcium and calcimimetic responses observed between the alleles could have potential clinical impact.
Keywords Parathyroid hormone
Calcium-sensing receptor Calcimimetic HEK-293
Site-directed mutagenesis
The calcium-sensing receptor (CaR) plays an essential role in extracellular calcium ion homeostasis by regulating the rate of parathyroid hormone (PTH) secretion from parathyroid cells and the rate of calcium reabsorption by the kidney in response to extracellular Ca2? [1]. The CaR is a member of G proteincoupled receptor (GPCR) family 3, characterized by a large...