Summary G-protein-gated K+ channels (Kir3.1–Kir3.4) control electrical excitability in many different cells. Among their functions relevant to human physiology and disease, they regulate the heart rate and govern a wide range of neuronal activities. Here we present the first crystal structures of a G-protein-gated K+ channel. By comparing the wild-type structure to that of a constitutively active mutant, we identify a global conformational change through which G-proteins could open a G-loop gate in the cytoplasmic domain. The structures of both channels in the absence and presence of PIP2 show that G-proteins open only the G-loop gate in the absence of PIP2, but in the presence of PIP2 the G-loop gate and a second inner helix gate become coupled, so that both gates open. We also identify a strategically located Na+ ion-binding site, which would allow intracellular Na+ to modulate GIRK channel activity. These data provide a mechanistic description of multi-ligand regulation of GIRK channel gating.

G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo-and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the ␤2-adrenergic receptor (␤2AR), can be incorporated into a reconstituted highdensity lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single ␤2AR is incorporated per rHDL particle. The monomeric ␤2AR efficiently activates Gs and displays GTP-sensitive allosteric ligandbinding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.␤2-adrenergic receptor ͉ single-molecule spectroscopy ͉ oligomerization

G protein-gated inward rectifier K+ (GIRK) channels allow neurotransmitters, via G protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity. We present the 3.5 Å resolution crystal structure of the mammalian GIRK2 channel in complex with βγ G protein subunits, the central signaling complex that links G protein-coupled receptor stimulation to K+ channel activity. Short-range atomic and long-range electrostatic interactions stabilize four βγ G protein subunits at the interfaces between four K+ channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation that is intermediate between the closed and constitutively active mutant, open conformations. The resultant structural picture is compatible with “membrane delimited” activation of GIRK channels by G proteins and the characteristic burst kinetics of channel gating. The structures also permit a conceptual understanding of how the signaling lipid PIP2 and intracellular Na+ ions participate in multi-ligand regulation of GIRK channels.

G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the ␤2 adrenergic receptor (␤2AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The ␤2AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist-and Gs-induced conformational changes. In the absence of an agonist, the ␤2AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the ␤2AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the ␤ 2 AR and the structural basis for ligand efficacy.adrenergic ͉ constitutive activity ͉ receptor ͉ conformation ͉ inverse agonist