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 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.
Alvarez-Hernández
Daniel
D
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. danalhe@gmail.com
González-Suárez
Ignacio
I
Fernández-Martín
José L
JL
Rodríguez
Isabel
I
Santamaría
Iñigo
I
Coto
Eliecer
E
Cannata-Andía
Jorge B
JB
D000595:Amino Acid Sequence / Q000187:drug effects / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000814:Aniline Compounds / Q000494:pharmacology*; D002118:Calcium / Q000819:agonists* / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010627:Phenethylamines; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011437:Propylamines; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D044169:Receptors, Calcium-Sensing / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics
D000814:Aniline Compounds; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing; D002118:Calcium