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The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain-containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. We show that TREK-1 binds the AC9:POPDC1 complex and copurifies in a POPDC1-dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP-independent, TREK-1 association with AC9 and POPDC1 is reduced upon stimulation of the β-adrenergic receptor (βAR). AC9 activity is required for βAR reduction of TREK-1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK-1 currents. Finally, deletion of the gene-encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress-induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK-1 to regulate heart rate control.

Key points The G-protein coupled inwardly rectifying potassium (GIRK) channel is an important mediator of neurotransmission via Gβγ subunit of the heterotrimeric Gi/o protein released by G-protein coupled receptor (GPCR) activation. Channels containing the GIRK1 subunit exhibit high basal currents, whereas channels that are formed by the GIRK2 subunit have very low basal currents. GIRK1-containing channels, but not channels consisting of GIRK2 only, recruit Gβγ to the plasma membrane. The Gα subunit of the G protein is not recruited by either GIRK1/2 or GIRK2. The unique distal C terminus of GIRK1 (G1-dCT) endows the channel with strong interaction with Gβγ, and deletion of G1-dCT abolishes the Gβγ recruitment and reduces the basal currents. These findings suggest that the basal activity of GIRK channels depends on channel-induced recruitment of Gβγ. The unique C terminus of GIRK1 subunit plays an important role in Gβγ recruitment. Abstract The G-protein coupled inwardly rectifying potassium (GIRK, or Kir3) channels are important mediators of inhibitory neurotransmission via activation of G-protein coupled receptors (GPCRs). GIRK channels are tetramers comprising combinations of subunits (GIRK1–4), activated by direct binding of the Gβγ subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, Gβγ-dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed Gβγ. Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by Gβγ. The high Ibasal of GIRK1 seems to be associated with its unique distal C terminus (G1-dCT), which is not present in the other subunits. We investigated the role of G1-dCT using electrophysiological and fluorescence assays in Xenopus laevis oocytes and protein interaction assays. We show that expression of GIRK1/2 increases the plasma membrane level of coexpressed Gβγ (a phenomenon we term ‘Gβγ recruitment’) but not of coexpressed Gαi3. All GIRK1-containing channels, but not GIRK2 homomers, recruited Gβγ to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and Gβγ, but not Gαi3. Nevertheless, the truncation of G1-dCT does not impair activation by Gβγ. In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes Gβγ recruitment and decreases Ibasal. Thus, we conclude that G1-dCT carries an essential role in Gβγ recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a Gβγ anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by Gβγ.

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of Gi/o proteins via G protein-coupled receptors (GPCRs). Although Gαi subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gαi3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the ‘inactive’ GDP-bound Gαi3GDP and involves the formation of Gαi3βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ‘away’ from the channel. The regulation of basal and Gβγ-evoked current was produced by the ‘constitutively inactive’ mutant of Gαi3, Gαi3G203A, which strongly binds Gβγ, but not by the ‘constitutively active’ mutant, Gαi3Q204L, or by Gβγ-scavenging proteins. Furthermore, regulation by Gαi3G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gαi3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ‘classical’ Gβγ effector, showing low Ibasal and strong Gβγ-dependent activation. Expression of Gαi3G203A did not affect either Ibasal or Gβγ-induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gβγ. Expression of Gαi3GDP reduced Ibasal and restored the ability of Gβγ to activate GIRK1*, like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1*. These results demonstrate that GIRK1 containing channels are regulated by both Gαi3GDP and Gβγ, while GIRK2 is a Gβγ-effector insensitive to Gαi3GDP.

Relaxin has been shown previously to stimulate cyclic AMP production and the activation of MAPK. We reported that phosphoinositide-3 kinase (PI3K) activity is required for biphasic stimulation of cAMP by relaxin and that relaxin treatment increased PI3K activity in THP-1 cells. A downstream target of PI3K is protein kinase C zeta (PKCzeta). Relaxin stimulated translocation of PKCzeta to the plasma membrane in THP-1, MCF-7, pregnant human myometrial (PHM1-31), and mouse mesangial (MMC) cells. PKCzeta translocation is PI3K dependent and independent of cAMP production. Pharmacological and antisense approaches, utilized to inhibit or knock down PKCzeta, resulted in a 40% inhibition of relaxin-stimulated cAMP production. The stimulation of PKCzeta by relaxin therefore is downstream of PI3K leading to increased cAMP production. To determine the role of PI3K/PKCzeta stimulation by relaxin on downstream-mediated events, we examined the increase in vascular endothelial growth factor (VEGF) gene expression by relaxin. Treatment of THP-1 or MMC cells with the PI3K inhibitor, LY294002, abolished the relaxin-mediated stimulation of VEGF transcript levels. In summary, relaxin has pleiotropic signaling effects in THP-1 cells activating ERK1/2, cAMP, PI3K, and PKCzeta. We have described a novel bifurcated pathway by which relaxin stimulates Gs alpha and PI3K/PKCzeta leading to increased cAMP production and increased VEGF gene expression. Some, but not all, of these pathways are detected in other cell lines which may cause the unique diversity of downstream responses from this interesting hormone.

1. In order to find out the functional roles of cytosolic regions of a G protein-activated, inwardly rectifying potassium channel subunit we studied block of GIRK channels, expressed in Xenopus laevis oocytes, by synthetic peptides in isolated inside-out membrane patches. 2. A peptide (DS6) derived from the very end of the C-terminus of GIRK1 reversibly blocked GIRK activity with IC50 values of 7.9 +/- 2.0 or 3.5 +/- 0.5 micrograms ml-1 (corresponding to 3.7 +/- 0.9 or 1.7 +/- 0.2 mumol l-1) for GIRK1/GIRK5 or GIRK1/GIRK4 channels, respectively. 3. Dose dependency studies of GIRK activation by purified beta gamma subunits of the G protein (G beta gamma) showed that DS6 block of GIRK channels is not the result of competition of the peptide with functional GIRK channels for the available G beta gamma. 4. Burst duration of GIRK channels was reduced, whereas long closed times between bursts were markedly increased, accounting for the channel block observed. 5. Block by the DS6 peptide was slightly voltage dependent, being stronger at more negative potentials. 6. These data support the hypothesis that the distal part of the carboxy-terminus of GIRK1 is a part of the intrinsic gate that keeps GIRK channels closed in the absence of G beta gamma.