Residue 826 in the Calcium-Sensing Receptor Is Implicated in the Response to Calcium and to R-568 Calcimimetic Compound

Within the extracellular loops of the seven-transmembrane domain of the calcium-sensing receptor (CaR) there is a region (I819–E837) relevant for calcimimetic activity. As the naturally occurring variant Ala826Thr is within this important region, it may be postulated that this change may influence 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 artificial 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 flow cytometry using a fluorescent 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 μM R-568 compared with the wild-type receptor. On the contrary, the artificial 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.