Functional Sites of Interaction Between G-Protein βγ Subunits and GIRK Channels

Abstract

G-proteins transduce extracellular signals received by cell surface receptors into cellular responses. Each of the α and the βγ subunits of G-proteins propagate signaling by activating or inhibiting various effector molecules in order to modulate cellular function. The first reported Gβγ effectors were the G-protein coupled inwardly rectifying K+ (GIRK or Kir3) channels. Previous studies have shown that Gβγ plays a necessary role in stimulation of both basal as well as agonist-induced components of GIRK channel currents. Work from our lab has shown that mutation of specific residues on the channel or Gβγ may preferentially affect one or the other of these components of GIRK current, leading to our working hypothesis: distinct interactions of Gβγ and GIRK channels contribute to basal versus agonist-induced currents.Through a novel combination of existing protein-protein docking methods, we are predicting sites of interaction between Gβγ and the GIRK1 channel. Initial rigid body docking results from ZDock and ZRank were filtered using steric restraints posed by the membrane and the Gβγ prenyl moiety. Clustering analysis (ClusPro server) was employed for selection of candidate structures for further flexible side-chain refinement with RosettaDock. Residue contacts predicted to be important for protein-protein interaction are tested and functionally characterized by site directed mutagenesis and two-electrode voltage clamp recordings in oocytes. Interaction sites are assessed for their contribution to basal activity, agonist-induced activity, or both. Predicted sites of contact include several residues on the channel or Gβγ previously shown to individually affect channel function, although the residues on both proteins comprising a complete interaction site remain unknown to date. These preliminary results give us confidence that the inclusion of further experimental restraints will further elucidate the distinct residue interactions involved in the mediation of agonist-induced versus basal currents.