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Papers In Press, published online ahead of print October 23, 2002
Department of Biology, Syracuse University, Syracuse, NY 13244
Corresponding Author: gebreitw{at}syr.edu
Activation of the calcium sensing receptor (CaR) by small increments in extracellular calcium (Ca2+e) induces intracellular calcium (Ca2+i) oscillations that are dependent on thapsigargin-sensitive intracellular calcium stores. Phenylalkylamines such as NPS R-568 are allosteric modulators (calcimimetics) which 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 Ca2+i oscillations in HEK-293 cells expressing human CaR. NPS R-568 was more potent than Ca2+e at eliciting Ca2+i oscillations, particularly at low [Ca2+]e (as low as 0.1 mM). The oscillation frequencies elicited by NPS R-568 varied over a two-fold range from peak to peak intervals of 60-70 s to 30-45 s, depending upon the concentrations of both Ca2+e and NPS R-568. Finally, NPS R-568 induced sustained (>15 min after drug removal) Ca2+i oscillations, suggesting slow release of the drug from its binding site. We exploited the potency of NPS R-568 for eliciting Ca2+i oscillations for structural studies. Truncation of the CaR carboxyl terminus from 1077 to 886 amino acids had no effect on the ability of Ca2+ or NPS R-568 to induce Ca2+i oscillations, but further truncation (to 868 amino acids) eliminated both highly cooperative Ca2+-dependent activation and regular Ca2+i oscillations. Alanine scanning within the amino acid sequence from R873 to H879 reveals a linkage between the cooperativity for Ca2+ dependent activation and establishment and maintenance of intracellular Ca2+ 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.
J. Biol. Chem, 10.1074/jbc.M205578200
Submitted on June 5, 2002
Revised on October 23, 2002
Accepted on October 23, 2002
Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations
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