title: G alpha(i) and G betagamma jointly regulate the conformations of a G betagamma effector, the neuronal G protein-activated K+ channel (GIRK).
abstract: Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical G betagamma effector G protein-activated K(+) channel (GIRK; Kir3) physically interacts with G betagamma but also with G alpha(i/o). Whether and how G alpha(i/o) subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, G alpha(i3) and G betagamma. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that G betagamma and G alpha(i3) distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that G betagamma greatly enhances the binding of GIRK1 subunit to G alpha(i3)(GDP) and, unexpectedly, to G alpha(i3)(GTP). i-FRET showed that both G alpha(i3) and G betagamma induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a "constitutively active" G alpha(i3) mutant and G betagamma together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either G alpha(i3) or G betagamma. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-G alpha-G betagamma signaling complex and mediates allosteric interactions between G alpha(i)(GTP) and G betagamma. Our findings imply that G alpha(i/o) and the G alpha(i) betagamma heterotrimer can regulate a G betagamma effector both before and after activation by neurotransmitters.
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. shaiberlin@gmail.com
authors.forename: Shai
authors.initials: S
authors.lastname: Berlin
authors.forename: Tal
authors.initials: T
authors.lastname: Keren-Raifman
authors.forename: Ruth
authors.initials: R
authors.lastname: Castel
authors.forename: Moran
authors.initials: M
authors.lastname: Rubinstein
authors.forename: Carmen W
authors.initials: CW
authors.lastname: Dessauer
authors.forename: Tatiana
authors.initials: T
authors.lastname: Ivanina
authors.forename: Nathan
authors.initials: N
authors.lastname: Dascal
mesh_terms: D000818:Animals; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000737:chemistry*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D046912:Multiprotein Complexes; D009865:Oocytes; D011485:Protein Binding; D011487:Protein Conformation; D014981:Xenopus
chemical_list: D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D046912:Multiprotein Complexes; D019206:GTP-Binding Protein alpha Subunits, Gi-Go