G Protein-Coupled Receptor Signaling to Kir Channels in Xenopus Oocytes

Abstract

Kir3 (or GIRK) channels have been known for nearly three decades to be activated by direct interactions with the βγ subunits of heterotrimeric G (Gαβγ) proteins in a membrane-delimited manner. Gα also interacts with GIRK channels and since PTX-sensitive Gα subunits show higher affinity of interaction they confer signaling specificity to G Protein-Coupled Receptors (GPCRs) that normally couple to these G protein subunits. In heterologous systems, overexpression of non PTX-sensitive Gα subunits scavenges the available Gβγ and biases GIRK activation through GPCRs that couple to these Gα subunits. Moreover, all Kir channels rely on their direct interactions with the phospholipid PIP2 to maintain their activity. Thus, signals that activate phospholipase C (e.g. through Gq signaling) to hydrolyze PIP2 result in inhibition of Kir channel activity. In this review, we illustrate with experiments performed in Xenopus oocytes that Kir channels can be used efficiently as reporters of GPCR function through Gi, Gs or Gq signaling. The membrane-delimited nature of this expression system makes it highly efficient for constructing dose-response curves yielding highly reproducible apparent affinities of different ligands for each GPCR tested.

Cross-signaling in metabotropic glutamate 2 and serotonin 2A receptor heteromers in mammalian cells

“…To investigate whether mGlu 2 R and 2AR can cross-signal in HEK293 cells, we proceeded to generate stable cell lines expressing the two receptors in the background of the Kir3.1/3.4 (or GIRK1/GIRK4) inwardly rectifying channel, which serves as a reliable reporter for both Gq and Gi signaling [14,23]. HEK293 cells were transfected with the bidirectional expression vector pBI-CMV1, carrying the GIRK1 and GIRK4 subunits of the channel and antibiotic resistant clones were screened by Western blotting for expression of GIRK1 (Fig.…”

“…GIRK channels are known to be directly activated by the Gβγ subunits of Gi/o proteins [24]. GIRK channel activation has been extensively used as a measure of Gi signaling [14,15,34]. In contrast, the activity of GIRK channels is inhibited by Gq/11 signaling via hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP 2 ), a membrane phospholipid critical for the maintenance of channel activity [25].…”

N-Alkylated Analogs of 4-Methylamphetamine (4-MA) Differentially Affect Monoamine Transporters and Abuse Liability

“…Adult male Sprague-Dawley rats (Harlan, Frederick, MD) were used for the synaptosome assays (rats weighing 250-350 g) and for the ICSS procedures (rats weighing 313-360 g at the time of surgery). Rats were group housed with free access to food and water, under a 12 h light/dark cycle with lights on from 0700 to 1900 h. Oocytes for the electrophysiology experiments were harvested and prepared from adult female Xenopus laevis females following standard procedures (Hatcher-Solis et al, 2014;Koren et al, 1990).…”

The Roles of Gβγ and Gα in Gating and Regulation of GIRK Channels

“…However, G s can activate GIRK, via the standard Gβγ-mediated mechanism, under conditions of overexpression of Gα s or the G s -coupled β-adrenergic receptor, both in model cells and cardiomyocytes (Digby et al, 2008;Hatcher-Solis et al, 2014;Lim et al, 1995;Wellner-Kienitz et al, 2001). This activation is not due to a promiscuous β-adrenergic receptor coupling to Gα i/o , because it is not suppressed by PTX (Digby et al, 2008;Hatcher-Solis et al, 2014).…”

“…However, G s can activate GIRK, via the standard Gβγ-mediated mechanism, under conditions of overexpression of Gα s or the G s -coupled β-adrenergic receptor, both in model cells and cardiomyocytes (Digby et al, 2008;Hatcher-Solis et al, 2014;Lim et al, 1995;Wellner-Kienitz et al, 2001). This activation is not due to a promiscuous β-adrenergic receptor coupling to Gα i/o , because it is not suppressed by PTX (Digby et al, 2008;Hatcher-Solis et al, 2014). The proposed reasons for the "reluctant" activation via G s are absence of interaction of Gα s with GIRK (Clancy et al, 2005) (only the CT of GIRK2 was examined), distinct localization of G s but not GIRKs to membrane caveolae/rafts (Schwarzer et al, 2010) (but see Cui, Ho, Kim, & Cho, 2010), or the slow kinetics of dissociation of Gα s from Gβγ compared to Gα i/o (Digby et al, 2008).…”