Specificity of Gβγ Signaling to Kir3 Channels Depends on the Helical Domain of Pertussis Toxin-sensitive Gα Subunits

Abstract

Acetylcholine signaling through muscarinic type 2 receptors activates atrial G protein-gated inwardly rectifying K ؉ (Kir3) channels via the ␤␥ subunits of G proteins (G␤␥). Different combinations of recombinant G␤␥ subunits have been shown to activate Kir3 channels in a similar manner. In native systems, however, only G␤␥ subunits associated with the pertussis toxinsensitive G␣ i/o subunits signal to K ؉ channels. Additionally, in vitro binding experiments supported the notion that the C terminus of Kir3 channels interacts preferentially with G␣ i over G␣ q . In this study we confirmed in two heterologous expression systems a preference of G␣ i over G␣ q in the activation of K ؉ currents. To identify determinants of G␤␥ signaling specificity, we first exchanged domains of G␣ i and G␣ q subunits responsible for receptor coupling selectivity and swapped their receptor coupling partners. Our results established that the G proteins, regardless of the receptor type to which they coupled, conferred specificity to Kir3 activation. We next tested signaling through chimeras between the G␣ i and G␣ q subunits in which the N terminus, the helical, or the GTPase domains of the G␣ subunits were exchanged. Our results revealed that the helical domain of G␣ i (residues 63-175) in the background of G␣ q could support Kir3 activation, whereas the reverse chimera could not. Moreover, the helical domain of the G␣ i subunit conferred "G␣ i -like" binding of the Kir3 C terminus to the G␣ q subunits that contained it. These results implicate the helical domain of G␣ i proteins as a critical determinant of G␤␥ signaling specificity.

Two Distinct Aspects of Coupling between Gαi Protein and G Protein-activated K+ Channel (GIRK) Revealed by Fluorescently Labeled Gαi3 Protein Subunits

“…Thus, the coupling of G␣ i3 117 to GPCR and to G␤␥ appears intact, and the slowing of GIRK activation probably reflects a problem in the G␣ i3 117-GIRK dialog, in line with the idea that G␣ i actively regulates GIRK gating (12,39). Indeed, interaction with GIRK and specific activation of GIRK by G␣ i/o -coupled GPCRs involve the helical domain of G␣ i/o (57). We propose that xFP insertions in the G␣ helical domain obstruct G␣ i/o -GIRK interaction via the interface between the helical domain of G␣ i/o and the putative G␣-interaction site in GIRK1, interfering with normal regulation of GIRK by G␣ i/o .…”

Heterotrimeric G Proteins Directly Regulate MMP14/Membrane Type-1 Matrix Metalloprotease

“…1T). This decrease in GTP␥S-stimulated MMP14 activity may be attributable to loss of activity of G␤␥ or G␣ i/o subunits (34). However, because G␤␥ is able to directly activate rMMP14 in vitro, the loss in activity in response to pertussis toxin is consistent with a role for G␤␥ subunits.…”

The role of inhibitory heterotrimeric G proteins in the control of in vivo heart rate dynamics

“…The channel is directly activated by G␤␥ (27), and its role has been validated in an experiment with cardiac-restricted overexpression of nonisoprenylated G␥ subunit, which led to reduced membrane G ␤␥ and impaired parasympathetic heart rate control (11). Despite G␤␥ being the direct activator of the channel, recent work has revealed the importance of inhibitory G␣ subunits in determining selectivity of this particular response (25,26,31,38). However, the identity of the inhibitory G␣ isoform(s) involved in vivo remains unclear.…”