title: Two distinct aspects of coupling between Gα(i) protein and G protein-activated K+ channel (GIRK) revealed by fluorescently labeled Gα(i3) protein subunits.
abstract: G protein-activated K(+) channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (I(basal)) and kinetics of the response elicited by activation by G protein-coupled receptors (I(evoked)). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gα(i/o) interaction. We investigated the mechanisms of regulation of GIRK by Gα(i/o) using wild-type Gα(i3) (Gα(i3)WT) and Gα(i3) labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gα(i3)xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gα(i3)xFP (Gα(i3)NT) closely mimicked all aspects of Gα(i3)WT regulation except for a weaker regulation of I(basal). Gα(i3) labeled with YFP within the Gα helical domain preserved regulation of I(basal) but failed to restore fast I(evoked). Titrated expression of Gα(i3)NT and Gα(i3)WT confirmed that regulation of I(basal) and of the kinetics of I(evoked) of GIRK1/2 are independent functions of Gα(i). FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gα(i3). Thus, Gα(i/o)βγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gα(i/o) actively regulates GIRK. Although regulation of I(basal) is a function of Gα(i)(GDP), our new findings indicate that regulation of kinetics of I(evoked) is mediated by Gα(i)(GTP).
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Shai
authors.initials: S
authors.lastname: Berlin
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Tsemakhovich
authors.forename: Ruth
authors.initials: R
authors.lastname: Castel
authors.forename: Tatiana
authors.initials: T
authors.lastname: Ivanina
authors.forename: Carmen W
authors.initials: CW
authors.lastname: Dessauer
authors.forename: Tal
authors.initials: T
authors.lastname: Keren-Raifman
authors.forename: Nathan
authors.initials: N
authors.lastname: Dascal
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism*; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism*; 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; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D037342:Pertussis Toxin / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism*; D051381:Rats; D013194:Staining and Labeling; D017186:Titrimetry; D014981:Xenopus
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008164:Luminescent Proteins; D021122:Protein Subunits; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D037342:Pertussis Toxin; C498819:GNAI3 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go