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 mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), 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 Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) 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 Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.
Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, P. O. Box 138, University Road, Leicester, LE1 9HN, United Kingdom.
M S
MS
Nash
R
R
Saunders
K W
KW
Young
R A
RA
Challiss
S R
SR
Nahorski
D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D016257:Fura-2 / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010750:Phosphoproteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D001798:Blood Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D010750:Phosphoproteins; D018094:Receptors, Metabotropic Glutamate; D011993:Recombinant Fusion Proteins; C104077:metabotropic glutamate receptor type 1; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2