title: Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations.
abstract: Activation of the calcium sensing receptor (CaR) by small increments in extracellular calcium (Ca(2+)(e)) induces intracellular calcium (Ca(2+)(i)) oscillations that are dependent on thapsigargin-sensitive intracellular calcium stores. Phenylalkylamines such as NPS R-568 are allosteric modulators (calcimimetics) that activate CaR by increasing the apparent affinity of the receptor for calcium. We determined, by fluorescence imaging with fura-2, whether the calcimimetic NPS R-568 could activate Ca(2+)(i) oscillations in HEK-293 cells expressing human CaR. NPS R-568 was more potent than Ca(2+)(e) at eliciting Ca(2+)(i) oscillations, particularly at low [Ca(2+)](e) (as low as 0.1 mm). The oscillation frequencies elicited by NPS R-568 varied over a 2-fold range from peak to peak intervals of 60-70 to 30-45 s, depending upon the concentrations of both Ca(2+)(e) and NPS R-568. Finally, NPS R-568 induced sustained (>15 min after drug removal) Ca(2+)(i) oscillations, suggesting slow release of the drug from its binding site. We exploited the potency of NPS R-568 for eliciting Ca(2+)(i) oscillations for structural studies. Truncation of the CaR carboxyl terminus from 1077 to 886 amino acids had no effect on the ability of Ca(2+) or NPS R-568 to induce Ca(2+)(i) oscillations, but further truncation (to 868 amino acids) eliminated both highly cooperative Ca(2+)-dependent activation and regular Ca(2+)(i) oscillations. Alanine scanning within the amino acid sequence from Arg(873) to His(879) reveals a linkage between the cooperativity for Ca(2+)-dependent activation and establishment and maintenance of intracellular Ca(2+) oscillations. The amino acid residues critical to both functions of CaR may contribute to interactions with either G proteins or between CaR monomers within the functional dimer.
authors.affiliation: Department of Biology, Syracuse University, 108 College Place, Syracuse, New York 13244, USA.
authors.forename: Susanne
authors.initials: S
authors.lastname: Miedlich
authors.forename: Lucio
authors.initials: L
authors.lastname: Gama
authors.forename: Gerda E
authors.initials: GE
authors.lastname: Breitwieser
mesh_terms: D000495:Allosteric Site; D000814:Aniline Compounds / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000819:agonists / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D008040:Genetic Linkage; D006639:Histidine / Q000737:chemistry; D006801:Humans; D010627:Phenethylamines; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011437:Propylamines; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
chemical_list: D000814:Aniline Compounds; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D006639:Histidine; D019284:Thapsigargin; D001120:Arginine; D002118:Calcium