Gα_i3 primes the G protein‐activated K⁺ channels for activation by coexpressed Gβγ in intact Xenopus oocytes

Abstract

G protein-activated K + channels (GIRK) mediate postsynaptic inhibitory effects of neurotransmitters in the atrium and in the brain by coupling to G protein-coupled receptors (GPCRs). In neurotransmitter-dependent GIRK signalling, Gβγ is released from the heterotrimeric Gαβγ complex upon GPCR activation, activating the channel and attenuating its rectification. Now it becomes clear that Gα is more than a mere Gβγ donor. We have proposed that Gα i3 -GDP regulates GIRK gating, keeping its basal activity low but priming (predisposing) the channel for activation by agonist in intact cells, and by Gβγ in excised patches. Here we have further investigated GIRK priming by Gα i3 using a model in which the channel was activated by coexpression of Gβγ, and the currents were measured in intact Xenopus oocytes using the two-electrode voltage clamp technique. This method enables the bypass of GPCR activation during examination of the regulation of the channel in intact cells. Using this method, we further characterize the priming phenomenon. We tested and excluded the possibility that our estimates of priming are affected by artifacts caused by series resistance or large K + fluxes. We demonstrate that both Gα i3 and membrane-attached Gβγ scavenger protein, m-phosducin, reduce the basal channel activity. However, Gα i3 allows robust channel activation by coexpressed Gβγ, in sharp contrast to m-phosducin, which causes a substantial reduction in the total Gβγ-induced current. Furthermore, Gα i3 also does not impair the Gβγ-dependent attenuation of the channel rectification, in contrast to m-phosducin, which prevents this Gβγ-induced modulation. The Gα i3 -induced enhancement of direct activation of GIRK by Gβγ, demonstrated here for the first time in intact cells, strongly supports the hypothesis that Gα i regulates GIRK gating under physiological conditions.

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

“…In contrast, coexpression of G␤␥ did not reverse the m-phosducin-induced reduction in i-FRET and in I ␤ ␥ (Figs. 3B and 4A), supporting the differentiation of the actions of G␣ i3 GA from those of a simple G␤␥ scavenger (11). Expression of G␣ i3 QL did not significantly alter the basal i-FRET and currents, but further coexpression of G␤␥ led to a strong increase in i-FRET (ϳ30%), significantly greater than under all other conditions ( Fig.…”

“…Preactivated (resting) states of GIRK1/2 probably reflect a mixture of conformations of GIRK1/2 channels, associated with a variable number of G␣ i ␤␥ heterotrimers and/or G␤␥ that lack matching G␣ i GDP (Fig. 6, b and d), as suggested by functional data (11). Correspondingly, DL-G1/G2 and G1/DL-G2 expressed alone at high levels yield high I basal (10) and an intermediate i-FRET signal.…”

“…Recently, we have demonstrated that this regulation is exerted by the GDP-bound form of G␣ i , G␣ i GDP (12); no role for G␣ i GTP in GIRK gating could be assigned so far. We proposed that regulation of GIRK by G␣ i relies upon the formation of the G␣ i GDP -G␤␥ heterotrimer, which forms a persistent, dynamic signaling complex with GIRK to ensure proper gating with low I basal and high signal-to-background ratio upon G␤␥ activation (11,12).We hypothesized that within a multiprotein signaling complex, the partner proteins alter each other's conformation and activity at various stages of the signaling process. Thus, G␣ i and G␤␥ may regulate the conformation of the channel both before and after activation.…”

“…Recently, we have demonstrated that this regulation is exerted by the GDP-bound form of G␣ i , G␣ i GDP (12); no role for G␣ i GTP in GIRK gating could be assigned so far. We proposed that regulation of GIRK by G␣ i relies upon the formation of the G␣ i GDP -G␤␥ heterotrimer, which forms a persistent, dynamic signaling complex with GIRK to ensure proper gating with low I basal and high signal-to-background ratio upon G␤␥ activation (11,12).…”

Structural elements in the Girk1 subunit that potentiate G protein–gated potassium channel activity

“…The impact of the distal C terminus of Girk1 on basal activity might relate to channel interactions with Gα subunits. Gα i3 was found to interact with the distal Girk1 C terminus, leading to a reduction in basal activity and increased Gβγ-dependent activation of Girk1(F137S) homomers (22). In contrast, basal activity of Girk2 homomers was insensitive to Gα i3 -dependent modulation.…”