Use of RGS-insensitive Galpha subunits to study endogenous RGS protein action on G-protein modulation of N-type calcium channels in sympathetic neurons.
Regulators of G-protein signaling (RGS) proteins are a large family of signaling proteins that control both the magnitude and temporal characteristics of heterotrimeric G-protein-mediated signaling. A current challenge is to define how endogenous RGS protein function impacts G-protein modulation of ionic channels in mammalian neurons. The experimental strategy described here utilizes distinct mutations in Galpha subunits that confer Bordetella pertussis toxin (PTX) and RGS protein insensitivity. The native signaling pathway in rat sympathetic neurons that mediates voltage-dependent modulation of N-type Ca2+ channels is ablated by PTX treatment and the signaling is reconstituted by expressing a PTX/RGS-insensitive Galpha mutant along with Gbeta and Ggamma subunits. As neurons are resistant to conventional transfection modalities, heterologous expression is accomplished by the direct microinjection of plasmids into the nucleus of the neuron. An advantage of this approach is that knowledge of the specific RGS subtypes participating in the pathway is not required. From the resulting alterations in the kinetics and pharmacology of G-protein-coupled receptor modulation of N-type Ca2+ channels, we can infer the role endogenous RGS proteins play in the signaling pathway.
Ikeda
Stephen R
SR
Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Insitutes of Health, Bethesda, Maryland 20892, USA.
Jeong
Seong-Woo
SW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020864:Calcium Channels, N-Type / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D017124:Conserved Sequence; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D037342:Pertussis Toxin / Q000235:genetics / Q000494:pharmacology; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
D020864:Calcium Channels, N-Type; D020710:RGS Proteins; D012694:Serine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go