Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate andCa2+ in single cells

Transient transfection of Chinese hamster ovary or baby hamster kidney cell s expressing the Group I metabotropic glutamate receptor mGlu1 alpha with g reen fluorescent protein-tagged pleckstrin homology domain of phospholipase C delta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loadi ng with Fura-2 enables simultaneous measurement of intracellular Ca2+ withi n the same cell. Using this technique we have studied the extracellular cal cium sensing property of the mGlu1 alpha receptor. Quisqualate, in extracel lular 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 par alleled by concurrent increases in intracellular Ca2+ concentration. Under nominally Ca2+-free conditions similar initial peaks in inositol 1,1,B-tris phosphate and Ca2+ concentration occurred with little change in either agon ist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate pr oduction was substantially reduced and the plateau in Ca(2+)concentration a bsent. Depletion of intracellular Ca2+ stores using thapsigarin abolished q uisqualate-induced increases in intracellular Ca(2+)and markedly reduced in ositol 1,4,5-trisphosphate production. These data suggest that the mGlu1 al pha 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.