SUN-211 L-Amino Acids Bind in the Calcium-Sensing Receptor's FlyTrap site but Signaling Responsiveness Depends upon Autocrine Competition

Abstract Disclosure: A.D. Conigrave: None. M.A. Goolam: None. Q. Fan: None. D.T. Ward: None. S.J. Simpson: None. The calcium-sensing receptor (CaSR) is critical for calcium homeostasis by mediating calcium-stimulated inhibition of PTH secretion and promotion of renal calcium excretion. In a second major endocrine action, the CaSR is required for L-amino acid stimulated release of gastrointestinal hormones including GLP-1, GIP and PYY, with impacts on insulin release and postprandial blood glucose levels, and also energy intake. Amino acids (AAs) and calcium (Ca2+) interact mutually via linked CaSR binding sites to promote potency (AAs on Ca2+) and efficacy (Ca2+ on AAs), supporting interactions between calcium and amino acids upon the digestion of complex foods. Previous work has demonstrated that AAs robustly raise intracellular Ca2+ levels(1) and either stimulate (GLP-1, GIP, PYY (2)) or inhibit (PTH (3)) hormone secretion. Surprisingly, AAs have been reported to have no effect on accumulation of the inositol phosphate IP1 which, like Ca2+ mobilization, measures receptor-dependent activation of Gq/11 and PI-PLC. In the present study we hypothesized that autocrine production and release of local activators of the CaSR AA binding site might compete with exogenous AAs in static cell culture-based assays but not in assays in which cells are continuously perifused. In static cultures we found that washing cultured HEK-293 cells that were stably transfected with the CaSR with fresh AA-free medium markedly reduced Ca2+ sensitivity and exposed AA stimulation of IP1 accumulation. Since glutathione has been reported to stimulate the CaSR via the AA binding site(4), we also tested whether inhibition of glutathione synthesis with buthionine sulfoximine might expose AA stimulated IP1 accumulation. The effects of BSO treatment exceeded those of concerted washing.We conclude that the effects of AAs in CaSR expressing cells are dependent on the following factors: (i) Ca2+ concentration; (ii) AA concentration; (iii) local release of competing autocrine activators; (iv) the rate of exchange with systemic plasma. 1. Proc Natl Acad Sci USA. 2000;97:4814-92. J Physiol. 2012;590:2917-363. J Biol Chem. 2004;279:38151-94. J Biol Chem. 2011;286:8786-97 Presentation: Sunday, July 13, 2025