Gαi Controls the Gating of the G Protein-Activated K+ Channel, GIRK

AbstractGIRK (Kir3) channels are activated by neurotransmitters coupled to G proteins, via a direct binding of Gβγ. The role of Gα subunits in GIRK gating is elusive. Here we demonstrate that Gαi is not only a donor of Gβγ but also regulates GIRK gating. When overexpressed in Xenopus oocytes, GIRK channels show excessive basal activity and poor activation by agonist or Gβγ. Coexpression of Gαi3 or Gαi1 restores the correct gating parameters. Gαi acts neither as a pure Gβγ scavenger nor as an allosteric cofactor for Gβγ. It inhibits only the basal activity without interfering with Gβγ-induced response. Thus, GIRK is regulated, in distinct ways, by both arms of the G protein. Gαi probably acts in its GDP bound form, alone or as a part of Gαβγ heterotrimer

Na+ promotes the dissociation between Galpha GDP and Gbeta gamma, activating G protein-gated K+ channels

G protein-gated K(+) channels (GIRK, or Kir3) are activated by the direct binding of Gbetagamma or of cytosolic Na(+). Na(+) activation is fast, Gbetagamma-independent, and probably via a direct, low affinity (EC(50), 30-40 mm) binding of Na(+) to the channel. Here we demonstrate that an increase in intracellular Na(+) concentration, [Na(+)](in), within the physiological range (5-20 mm), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking the direct Na(+) effect. It is inhibited by a Gbetagamma scavenger, hence it is Gbetagamma-dependent; but it does not require GTP. We hypothesized that Na(+) elevates the cellular concentration of free Gbetagamma by promoting the dissociation of the Galphabetagamma heterotrimer into free Galpha(GDP) and Gbetagamma. Direct biochemical measurements showed that Na(+) causes a moderate decrease (approximately 2-fold) in the affinity of interaction between Galpha(GDP) and Gbetagamma. Furthermore, in accord with the predictions of our model, slow Na(+) activation was enhanced by mild coexpression of Galpha(i3). Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gbetagamma-dependent regulation of GIRK by Na(+). We propose that Na(+) may act as a regulatory factor, or even a second messenger, that regulates effectors via Gbetagamma

Expression of GIRK (Kir3.1/Kir3.4) channels in mouse fibroblast cells with and without β1 integrins

G protein‐activated K⁺ channel (GIRK) subunits possess a conserved extracellular integrin‐binding motif (RGD) and bind directly to β1 integrins. We expressed GIRK1/GIRK4 channels labeled with green fluorescent protein in fibroblast cell lines expressing or lacking β1 integrins. Neither plasma membrane localization nor agonist‐evoked GIRK currents were affected by the absence of β1 integrins or by incubation with externally applied RGD‐containing peptide. Mutation of the aspartate (D) of RGD impaired currents, GIRK glycosylation, and membrane localization, but the interaction with β1 integrins remained intact. Thus, β1 integrins are not essential for functional GIRK expression; and the GIRK‐integrin interactions involve structural elements other than the RGD motif

Expression of GIRK (Kir3.1/Kir3.4) channels in mouse fibroblast cells with and without beta1 integrins

G protein-activated K + channel (GIRK) subunits possess a conserved extracellular integrin-binding motif (RGD) and bind directly to # #1 integrins. We expressed GIRK1/GIRK4 channels labeled with green fluorescent protein in fibroblast cell lines expressing or lacking # #1 integrins. Neither plasma membrane localization nor agonist-evoked GIRK currents were affected by the absence of # #1 integrins or by incubation with externally applied RGD-containing peptide. Mutation of the aspartate (D) of RGD impaired currents, GIRK glycosylation, and membrane localization, but the interaction with # #1 integrins remained intact. Thus, # #1 integrins are not essential for functional GIRK expression ; and the GIRK-integrin interactions involve structural elements other than the RGD motif. z 2000 Federation of European Biochemical Societies. ### ###### Voltage clamp ; Inward rectier K channel ; Fibroblast ; Green uorescent protein 1