Heterotrimeric Gi protein is associated with the inositol 1,4,5-trisphosphate receptor complex and modulates calcium flux.
In vascular smooth muscle, pertussis toxin (PT) inhibits thrombin-induced Ca2+ release by a mechanism independent of its effect on IP3 formation. Thus, the possibility of a direct role of G alpha i proteins in regulating IP3-sensitive Ca2+ release was investigated by examining whether G alpha i proteins are associated with the IP3 receptor complex. Purified microsomal membranes were prepared and separated by sucrose density gradient centrifugation. The relative density of [3H]-IP3 binding sites between the microsomal fractions was inversely related to the distribution of the plasma membrane marker. The relative distribution of G alpha i3 determined by immunoblotting was closely correlated with the density of [3H]-IP3 binding. Levels of G alpha i2 were more evenly distributed with highest levels present in plasma membrane-enriched fractions. IP3 receptor immunoprecipitated from triton-solubilized microsomal membranes contained G alpha i3 immunoreactivity. To determine whether G alpha i proteins influence IP3-induced Ca2+ release, the effect of PT on Ca2+ release from digitonin-permeabilized cell suspensions using Fluo-3 was examined. Exposure to PT (0.1 microgram/ml, 5 min) attenuated the initial rate of IP3 (1 microM)-induced Ca2+ release. Together, these findings are consistent with the hypothesis that a heterotrimeric G alpha i protein directly regulates IP3-dependent Ca2+ release.
Baker Medical Research Institute, Prahran, Victoria, Australia. neylon@neuro.duke.edu
C B
CB
Neylon
A
A
Nickashin
V A
VA
Tkachuk
A
A
Bobik
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000502:physiology*; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D017136:Ion Transport / Q000187:drug effects; D046911:Macromolecular Substances; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D051381:Rats; D011921:Rats, Inbred WKY; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D046911:Macromolecular Substances; D018160:Receptors, Cytoplasmic and Nuclear; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D004072:Digitonin; D002118:Calcium