Reassessment of the Ca2+ Sensing Property of a Type I Metabotropic Glutamate Receptor by Simultaneous Measurement of Inositol 1,4,5-Trisphosphate and Ca2+ in Single Cells

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1α with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cδ1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca2+ within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1α receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca2+, increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca2+ concentration. Under nominally Ca2+-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca2+ concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca2+ concentration absent. Depletion of intracellular Ca2+ stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca2+ and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1α receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca2+ concentration. However, prolonged activation of phospholipase C requires extracellular Ca2+, while the initial burst of activity is highly dependent on Ca2+ mobilization from intracellular stores.