Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K⁺ channel by Gα_i^GDP and Gβγ

Abstract

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of G i/o proteins via G protein-coupled receptors (GPCRs). Although Gα i 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 Gα i3 decreases the basal activity (I basal ) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the 'inactive' GDP-bound Gα i3 GDP and involves the formation of Gα i3 βγ 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 Gα i3 , Gα i3 G203A, which strongly binds Gβγ, but not by the 'constitutively active' mutant, Gα i3 Q204L, or by Gβγ-scavenging proteins. Furthermore, regulation by Gα i3 G203A 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 Gα i3 GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gβγ effector, showing low I basal and strong Gβγ-dependent activation. Expression of Gα i3 G203A did not affect either I basal or Gβγ-induced activation. In contrast, homomeric GIRK1 * (a pore mutant able to form functional homomeric channels) exhibited large I basal and was poorly activated by Gβγ. Expression of Gα i3 GDP reduced I basal 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 Gα i3 GDP and Gβγ, while GIRK2 is a Gβγ-effector insensitive to Gα i3 GDP .

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

“…3, hK) (24 mM K ϩ , isotonically replacing NaCl in ND96) as described (12) (see supplemental methods). Differences in current amplitude, resulting from variations in GIRK expression, were corrected to channel expression, determined by the quantitation of YFP fluorescence as described (12). All currents were normalized to the control group.…”

“…G1NC was constructed as described (12), with an 8-amino acid linker GSTASGST replacing the transmembrane segment (amino acids 85-184). Doubly labeled (DL)-GIRK1 and DL-GIRK2 were created by fusing CFP to the NT and YFP to the CT of the GIRK subunit, via linkers (see supplemental methods).…”

“…1B). Moreover, G1NC bound purified G␤ 1 ␥ 2 (12 G1NC. Here, we further characterized the interaction of GIRK1 with G␣ i3 GDP and also with G␣ i3 GTP .…”

“…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).…”