Assays of RGS protein modulation by phosphatidylinositides and calmodulin.
Regulator of G-protein signaling (RGS) proteins are a family of proteins that accelerate intrinsic GTP hydrolysis on alpha subunits of trimeric G proteins. They play crucial roles in the physiological regulation of G-protein-mediated cell signaling. If RGS proteins were active unrestrictedly, they would completely suppress G-protein-mediated signaling. Therefore, it is important to understand how the actions of RGS proteins are regulated under different physiological conditions. We have discovered a physiological mode of regulation of a RGS protein in cardiac myocytes. The voltage-dependent formation of Ca2+/calmodulin (CaM) facilitated the GTPase activity of RGS proteins by removing intrinsic inhibition mediated by the phospholipid phosphatidylinositol-3,4,5-trisphosphate. This modulation of RGS protein action underlies the characteristic "relaxation" behavior of G-protein-gated K+ channels in native cardiac myocytes. This article describes briefly the discovery of this novel mode of RGS protein modulation in native cardiac myocytes and then gives details of the biochemical and electrophysiological assays used for the functional investigation of this modulation. These assays would be useful for dissecting the physiological modes of action of RGS proteins in controlling G-protein-mediated signaling machinery.
Ishii
Masaru
M
Department of Pharmacology, Osaka University Graduate School of Medicine, Osaka, Japan
Kurachi
Yoshihisa
Y
D000818:Animals; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000502:physiology; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D032383:Myocytes, Cardiac / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010716:Phosphatidylinositols / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated / Q000502:physiology; D020710:RGS Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction
D002147:Calmodulin; D010716:Phosphatidylinositols; D024642:Potassium Channels, Voltage-Gated; D020710:RGS Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium