Gβ residues that do not interact with Gα underlie agonist-independent activity of K + channels

Abstract

Gβγ subunits interact directly and activate G protein-gated Inwardly Rectifying K + (GIRK) channels. Little is known about the identity of functionally important interactions between Gβγ and GIRK channels. We tested the effects of all mammalian Gβ subunits on channel activity and showed that whereas Gβ1-4 subunits activate heteromeric GIRK channels independently of receptor activation, Gβ5 does not. Gβ1 and Gβ5 both bind the N and C termini of the GIRK1 and GIRK4 channel subunits. Chimeric analysis between the Gβ1 and Gβ5 proteins revealed a 90-amino acid stretch that spans blades two and three of the seven-propeller structure and is required for channel activation. Within this region, eight non-conserved amino acids were critical for the activity of Gβ1, as mutation of each residue to its counterpart in Gβ5 significantly reduced the ability of Gβ1 to stimulate channel activity. In particular, mutation of residues Ser-67 and Thr-128 to the corresponding Gβ5 residues completely abolished Gβ1 stimulation of GIRK channel activity. Mapping these functionally important residues on the three-dimensional structure of Gβ1 shows that Ser-67, Ser-98, and Thr-128 are the only surface accessible residues. Gα i1 interacts with Ser-98 but not with Ser-67 and Thr-128 in the heterotrimeric Gαβγ structure. Further characterization of the three mutant proteins showed that they fold properly and interact with Gγ2. Of the three identified functionally important residues, the Ser-67 and Thr-128 Gβ mutants significantly inhibited basal currents of a channel point mutant that displays Gβγ-mediated basal but not agonist-induced currents. Our findings indicate that the presence of Gβ residues that do not interact with Gα are involved in Gβγ interactions in the absence of agonist stimulation.