βγ Subunits of GTP-binding proteins inhibit muscarinic receptor stimulation of phospholipase C

This study examines the mechanism of guanine nucleotide-binding protein (G protein) coupling of receptors to phospholipase C. The Xenopus oocyte has a muscarinic receptor-activated Cl^- current that is mediated by inositol 1,4,5-trisphosphate. Modulation of the muscarinic receptor-evoked Cl^- current was examined under voltage clamp in oocytes injected with resolved G-protein subunits. The presence of an α subunit of G proteins in oocytes was shown by pertussis toxin-labeling of a 41-kDa band in oocyte membranes. The presence of the β subunit of G proteins was demonstrated by immunoblotting experiments with an antiserum (U-49) that is specific for the β subunit. Pertussis toxin treatment of oocytes resulted in the uncoupling of muscarinic receptors from activation of the Cl^- current. Cells microinjected with 1.5 ng of human erythrocyte βγ-subunit complex to 1.0 ng of bovine brain βγ-subunit complex showed approximately a 95% reduction in the evoked Cl^- current. Cells injected with equal volumes of protein storage vehicle showed no change in response. Cells injected with boiled βγ subunits, bovine serum albumin, or resolved α subunits also showed no reduction in response. Cells injected with various concentrations of βγ subunits showed a concentration dependence with half-maximal inhibition of the muscarinic activated Cl^- current at about 10 nM. Cells injected with 1.0 ng of bovine brain βγ subunits could not respond to bath-applied agonist but could generate the Cl^- current on intracellular injection of inositol 1,4,5-trisphosphate. These observations suggest that there is a G protein responsible for muscarinic receptor-mediated signal transduction through phospholipase C and that it is an αβγ heterotrimer. It appears that the mode of action of the G protein in the phospholipase C system may be similar to that of the hormone-activated adenylyl cyclase.