Structural Model of K+ Channel Activation by the Beta-Gamma Subunits of G-Proteins

Abstract

The first known effectors of the βγ subunits of heterotrimeric G-proteins (Gβγ) were the G-protein-gated inwardly rectifying K+ (GIRK) channels which underlie acetylcholine-induced decrease in heart rate (IKACh). How Gβγ subunits specifically regulate the conformations of their effector proteins to alter activity is not understood at a molecular level. Although several GIRK crystal structures have been published, attempts to co-crystallize Gβγ have failed precluding knowledge of the reciprocal interactions between the two proteins.We have employed a computational approach that combines several known methods in protein-protein docking to produce experimentally testable models of the protein complex. The best scoring model of the GIRK1-Gβγ complex predicted a ∼1800 A2 interaction surface that includes key interactions of the channel's LM and DE loops with Gβ residues that are known to interact with the helical N-terminus of Gα-GDP in the structure of the inactive heterotrimeric G-protein. The channel-Gβγ interactions predicted by the model could be disrupted by mutation of one protein and rescued by additional mutation of reciprocal residues in the other protein. Channel activity was found to be stimulated by Gβγ interactions that enlarged the cleft between the LM and DE loops of the channel and stabilized the LM loop in a “raised” position seen in the “open intracellular gate” conformation of the GIRK1 crystal structure.GIRK4 displayed differences from the GIRK1 with respect to the pattern of responses to Gβγ mutants but the physiologically relevant heteromeric GIRK1/4 channel behaved similarly to GIRK1. The proposed site of action of Gβγ in the channel's DE-LM cleft is also shared with alcohols and is consistent with a previously described cascade of PIP2-driven changes in intramolecular interactions of the channel leading to stabilization of the open conformation of its intracellular G-loop gate.