Bone and Mineral Research Unit, Servicio de Metabolismo Oseo y Mineral, Hospital Universitario Central de Asturias, Instituto Reina Sofía de Investigación, RedinRen del ISCIII, Universidad de Oviedo, C/ Julián Clavería s/n, 33006 Oviedo, Spain.
Calcif Tissue Int. 2010 Mar;86(3):227-33. doi: 10.1007/s00223-009-9298-7. Epub 2010 Feb 4.
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 EC(50) 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 muM 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 (EC(50) = 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.
在钙敏感受体 (CaR) 的七跨膜域的细胞外环中，存在一个与钙敏活性相关的区域 (I819-E837)。由于天然存在的变异体 Ala826Thr 位于这个重要区域内，因此可以假设这种变化可能会影响 CaR 对钙和 R-568 的反应。使用瞬时转染三种不同人 CaR（野生型 [A826]、变异等位基因 [T826] 和人工突变体 [W826]）的人胚肾 (HEK-293) 细胞来测试钙单独或与钙敏激动剂 R-568 联合调节 CaR 活性的能力。通过使用荧光探针的流式细胞术检测 CaR 激活。单独测量细胞外钙或不同 R-568 浓度变化时细胞内钙的变化。将 826 位的丙氨酸（A826）替换为苏氨酸（T826）改变了钙敏感性，使 EC50 值从 2.34 +/- 0.48 mM（A826，野生型）增加到 2.96 +/- 0.75 mM（T826）（P < 0.05）。T826 受体与野生型受体相比，用 1 μM R-568 达到相似的反应。相反，在相同位置引入色氨酸（W826）不会影响钙敏感性（EC50 = 2.64 +/- 0.81 mM），但降低了受体对 R-568 的反应能力。结果表明 826 位残基在 CaR 对钙和钙敏激动剂的反应中非常重要。由于 A826T 改变被描述为天然变异，因此观察到的等位基因之间对钙和钙敏剂的反应差异可能具有潜在的临床影响。
