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Physiological activity of G protein gated inward rectifier K+ (GIRK, Kir3) channel, dynamically regulated by three key ligands, phosphoinositol-4,5-bisphosphate (PIP2), Gβγ, and Na+, underlies cellular electrical response to multiple hormones and neurotransmitters in myocytes and neurons. In a reducing environment, matching that inside cells, purified GIRK2 (Kir3.2) channels demonstrate low basal activity, and expected sensitivity to the above ligands. However, under oxidizing conditions, anomalous behavior emerges, including rapid loss of PIP2 and Na+-dependent activation and a high basal activity in the absence of any agonists, that is now paradoxically inhibited by PIP2. Mutagenesis identifies two cysteine residues (C65 and C190) as being responsible for the loss of PIP2 and Na+-dependent activity and the elevated basal activity, respectively. The results explain anomalous findings from earlier studies and illustrate the potential pathophysiologic consequences of oxidation on GIRK channel function, as well as providing insight to reversed ligand-dependence of Kir and KirBac channels. Graphical Abstract [ABSTRACT FROM AUTHOR]

Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M1AChR in complex with MT7, a subtype-selective anti-M1AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M1AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M1AChR toward M2AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein–coupled receptor modulators. [ABSTRACT FROM AUTHOR]

The article discusses research on the role of muscarinic acetylcholine receptors as G protein-coupled receptors (GPCRs). Topics include the role of iperoxo binding pockets in the receptors M1R and M2R, the role of chimeric receptors and site-directed mutagenesis in research on muscarinic receptors, and the structure of a nucleotide-free G-protein subtype.

The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and the native Gt purified from bovine retinas. We demonstrate that the light-activated rhodopsin in this complex contains a covalently bound unprotonated retinal and therefore corresponds to the active metarhodopin II state; that the isolated complex is active and dissociates upon addition of GTPγS; and that the stoichiometry corresponds to a 1∶1 molar ratio of rhodopsin to the heterotrimeric G-protein. And finally, we show that the rhodopsin also forms stable complex with Gi. This complex has significantly higher thermostability than RhoM257Y/Gt complex and is resistant to a variety of detergents. Overall, our data suggest that the RhoM257Y/Gi complex is an ideal target for future structural and mechanistic studies of signaling in the visual system. [ABSTRACT FROM AUTHOR]

Highlights: [•] Recent methodologies have rendered structure determination of GPCR more feasible. [•] Interdisciplinary collaboration has promoted the structural biology of GPCR. [•] Growing number of GPCR structures in different states has become available. [•] The structure of an active GPCR and G-protein complex has been determined. [Copyright &y& Elsevier]

Gap junctions consist of arrays of intercellular channels composed of integral membrane proteins called connexin in vertebrates. Gap junction channels regulate the passage of ions and biological molecules between adjacent cells and, therefore, are critically important in many biological activities, including development, differentiation, neural activity, and immune response. Mutations in connexin genes are associated with several human diseases, such as neurodegenerative disease, skin disease, deafness, and developmental abnormalities. The activity of gap junction channels is regulated by the membrane voltage, intracellular microenvironment, interaction with other proteins, and phosphorylation. Each connexin channel has its own property for conductance and molecular permeability. A number of studies have tried to reveal the molecular architecture of the channel pore that should confer the connexin-specific permeability/selectivity properties and molecular basis for the gating and regulation. In this review, we give an overview of structural studies and describe the structural and functional relationship of gap junction channels. [ABSTRACT FROM AUTHOR]

X-ray analysis of the human connexin26 gap junction channel has provided structural details of its open state. The gap junction channel is formed by paired hemichannels on two adjacent cells; each hemichannel consists of six protomers, and exhibits a six-fold symmetry. The protomer folds in a typical four-helix bundle. The amino-terminal region folds in a short helix and is inserted into the lumen to form a funnel structure. The structure of the amino-terminal region could explain the channel''s gating mechanism. Extensive interactions between two hemichannels allow the gap junction channel to tightly connect two adjacent cells. The gap junction, which consists of hundreds of gap junction channels, could both serve as an intracellular channel and contribute to cellular adhesion. [Copyright &y& Elsevier]

Intercellular signalling is an essential characteristic of multicellular organisms. Gap junctions, which consist of arrays of intercellular channels, permit the exchange of ions and small molecules between adjacent cells. Here, the structural determination of a gap junction channel composed of connexin 26 (Cx26) at 3.5 Å resolution is described. During each step of the purification process, the protein was examined using electron microscopy and/or dynamic light scattering. Dehydration of the crystals improved the resolution limits. Phase refinement using multi-crystal averaging in conjunction with noncrystallographic symmetry averaging based on strictly determined noncrystallographic symmetry operators resulted in an electron-density map for model building. The amino-acid sequence of a protomer structure consisting of the aminoterminal helix, four transmembrane helices and two extracellular loops was assigned to the electron-density map. The amino-acid assignment was confirmed using six selenomethionine (SeMet) sites in the difference Fourier map of the SeMet derivative and three intramolecular disulfide bonds in the anomalous difference Fourier map of the native crystal. [ABSTRACT FROM AUTHOR]

Gap junctions consist of arrays of intercellular channels between adjacent cells that permit the exchange of ions and small molecules. Here we report the crystal structure of the gap junction channel formed by human connexin 26 (Cx26, also known as GJB2) at 3.5 Å resolution, and discuss structural determinants of solute transport through the channel. The density map showed the two membrane-spanning hemichannels and the arrangement of the four transmembrane helices of the six protomers forming each hemichannel. The hemichannels feature a positively charged cytoplasmic entrance, a funnel, a negatively charged transmembrane pathway, and an extracellular cavity. The pore is narrowed at the funnel, which is formed by the six amino-terminal helices lining the wall of the channel, which thus determines the molecular size restriction at the channel entrance. The structure of the Cx26 gap junction channel also has implications for the gating of the channel by the transjunctional voltage. [ABSTRACT FROM AUTHOR]

This article focuses on a study related to purification, complete amino acid sequence and structural characterization of the heat-stable sweet protein, mabinlin II. All experiments, except for the defatted proeedure, were carried out at 4°C. 400 mg decorticated seeds, free from pulps of the fruit, were ground into powder. The supernatant was colorless. Ammonium sulfate fractionation was carried out by addition of solid ammonium sulfate to the pooled solution to bring about 70% saturation. The eluted protein was monitored by absorption at 280 nm.

A new sweet protein, named mabinlin II, was extracted with 0.5 M NaCI solution from the seeds of Capparis masaikai Lévl. and purified by ammonium sulfate fractionation, carboxymethylcellulose- Sepharose ion-exchange chromatography and gel filtration. The sweetness of mabinlin II was unchanged by at least 48 h incubation at nearly boiling temperature. Purified mabinlin II thus obtained gave a single band having a molecular mass of 14 kDa on SDS/PAGE. In the presence of dithiothreitol, mabinlin II gave two bands having molecular masses of 4.6 kDa and 5.2 kDa on SDS/PAGE. Two peptides (A chain and B chain) were separated from reduced and S-carboxamidomethylated mabinlin II by HPLC. The amino acid sequences of the A chain and B chain were determined by the automatic Edman-degradation method. The A chain and B chain consist of 33-amino-acid and 72-amino-acid residues, respectively. The A chain is mostly composed of hydrophilic amino acid residues and the B chain also contains many hydrophilic residues. High similarity was found between the amino acid sequences of mabinlin 11 and 2S seed storage proteins, especially 2S albumin AT2S3 in Arabidopsis thaliana (mouse-ear cress). [ABSTRACT FROM AUTHOR]

We present comprehensive maps at single-amino acid resolution of the residues stabilizing the human Gαi1 subunit in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of alanine mutations on the stability of Gαi1 and the rhodopsin-Gαi1 complex. We identified stabilization clusters in the GTPase and helical domains responsible for structural integrity and the conformational changes associated with activation. In activation cluster I, helices α1 and α5 pack against strands β1-β3 to stabilize the nucleotide-bound states. In the receptor-bound state, these interactions are replaced by interactions between α5 and strands β4-β6. Key residues in this cluster are Y320, which is crucial for the stabilization of the receptor-bound state, and F336, which stabilizes nucleotide-bound states. Destabilization of helix α1, caused by rearrangement of this activation cluster, leads to the weakening of the interdomain interface and release of GDP. [ABSTRACT FROM AUTHOR]

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with β-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in β-arrestin recruitment and GPCR-β-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for β-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of β-arrestin and stabilize GPCR-β-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-β-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for β-arrestin recruitment, this provides a mechanism for β-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling. [Display omitted] • β-Arrestins coincidentally detect membrane PIPs for recruitment to GPCRs • GPCR phosphorylation affects dependence on membrane PIPs for β-arrestin recruitment • Membrane PIPs are allosteric modulators of GPCR-β-arrestin complexes • Plasma membrane PIPs alone promote β-arrestin activation By acting as allosteric regulators of arrestin conformation, membrane phosphoinositides potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes. Membrane PIPs can therefore drive GPCR conformational diversity beyond what is seen from GPCR phosphorylation alone and provide spatial control for GPCR-arrestin complex formation. [ABSTRACT FROM AUTHOR]

Investigates the role of a family of membrane proteins in the assembly of human connexin 26. Malfunctioning of connexin channels causing various diseases; Structural and functional properties of connexin; Solubilization in dodecyl maltoside.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH 3 -ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH 3 -ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations. • Analysis of conformational dynamics throughout the M2R using 13CH 3 -ε-methionine NMR • Ligands with various efficacies for G protein and arrestin signaling were examined • Each ligand stabilized a distinct conformation of the M2R • There is no clear linear correlation between ligand efficacy and chemical shifts Xu et al. reveal that the M2 muscarinic receptor is highly dynamic, with a complex energy landscape in which ligands with different signaling profiles stabilize distinct receptor conformations. [ABSTRACT FROM AUTHOR]

Summary Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein G i. Activation of CB1-G i signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of G i activation by CB1. Here, we present the structure of the CB1-G i signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in G i. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor. Graphical Abstract Highlights • 3 Å cryo-EM structure of the CB1-G i complex bound to potent agonist MDMB-Fubinaca • MDMB-Fubinaca locks "toggle switch" residues F2003.36/W3566.48 in active conformation • Quantum mechanics calculations reveal the mechanism for the high affinity of Fubinaca • Molecular dynamic simulations reveal a path for ligand entry between TM1 and TM7 Looking at how a toxic, synthetic ligand locks cannabinoid receptor 1 into a signaling conformation points to ways to understand and modulate receptor activity. [ABSTRACT FROM AUTHOR]