Gi- and Gs-coupled GPCRs show different modes of G-protein binding.

More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR-Gs structures, providing insight into specificity determinants

Chronic Nicotine Selectively Enhances α4β2* Nicotinic Acetylcholine Receptors in the Nigrostriatal Dopamine Pathway

These electrophysiological experiments, in slices and intact animals, study the effects of in vivo chronic exposure to nicotine on functional α4β2* nAChRs in the nigrostriatal dopaminergic (DA) pathway. Recordings were made in wild-type and α4 nicotinic acetylcholine receptor (nAChR) subunit knock-out mice. Chronic nicotine enhanced methyllycaconitine citrate hydrate-resistant, dihydro-β-erythroidine hydrobromide-sensitive nicotinic currents elicited by 3–1000 µM ACh in GABAergic neurons of the substantia nigra pars reticulata (SNr), but not in DA neurons of the substantia nigra pars compacta (SNc). This enhancement leads to higher firing rates of SNr GABAergic neurons and consequently to increased GABAergic inhibition of the SNc DA neurons. In the dorsal striatum, functional α4* nAChRs were not found on the neuronal somata; however, nicotine acts via α4β2* nAChRs in the DA terminals to modulate glutamate release onto the medium spiny neurons. Chronic nicotine also increased the number and/or function of these α4β2* nAChRs. These data suggest that in nigrostriatal DA pathway, chronic nicotine enhancement of α4β2* nAChRs displays selectivity in cell type and in nAChR subtype as well as in cellular compartment. These selective events augment inhibition of SNc DA neurons by SNr GABAergic neurons and also temper the release of glutamate in the dorsal striatum. The effects may reduce the risk of excitotoxicity in SNc DA neurons and may also counteract the increased effectiveness of corticostriatal glutamatergic inputs during degeneration of the DA system. These processes may contribute to the inverse correlation between tobacco use and Parkinson's disease

Bitopic binding mode of an M1 muscarinic acetylcholine receptor agonist associated with adverse clinical trial outcomes

The realisation of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (M1 mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702, described previously as a potent M1 receptor allosteric agonist, which showed pro-cognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side-effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702 together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. We conclude that these properties, whilst imparting beneficial effects on learning and memory, are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data supports the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses

The distribution of charged amino acid residues and the Ca(2+) permeability of nicotinic acetylcholine receptors: a predictive model

Nicotinic acetylcholine receptors (nAChRs) are cation-selective ligand-gated ion channels exhibiting variable Ca(2+) permeability depending on their subunit composition. The Ca(2+) permeability is a crucial functional parameter to understand the physiological role of nAChRs, in particular considering their ability to modulate Ca(2+)-dependent processes such as neurotransmitter release. The rings of extracellular and intracellular charged amino acid residues adjacent to the pore-lining TM2 transmembrane segment have been shown to play a key role in the cation selectivity of these receptor channels, but to date a quantitative relationship between these structural determinants and the Ca(2+) permeability of nAChRs is lacking. In the last years the Ca(2+) permeability of several nAChR subtypes has been experimentally evaluated, in terms of fractional Ca(2+) current (Pf, i.e., the percentage of the total current carried by Ca(2+) ions). In the present study, the available Pf-values of nAChRs are used to build a simplified modular model describing the contribution of the charged residues in defined regions flanking TM2 to the selectivity filter controlling Ca(2+) influx. This model allows to predict the currently unknown Pf-values of existing nAChRs, as well as the hypothetical Ca(2+) permeability of subunit combinations not able to assemble into functional receptors. In particular, basing on the amino acid sequences, a Pf > 50% would be associated with homomeric nAChRs composed by different α subunits, excluding α7, α9, and α10. Furthermore, according to the model, human α7β2 receptors should have Pf-values ranging from 3.6% (4:1 ratio) to 0.1% (1:4 ratio), much lower than the 11.4% of homomeric α7 nAChR. These results help to understand the evolution and the function of the large diversity of the nicotinic receptor family

Mapping the druggable allosteric space of G-protein coupled receptors: a fragment-based molecular dynamics approach.

To address the problem of specificity in G-protein coupled receptor (GPCR) drug discovery, there has been tremendous recent interest in allosteric drugs that bind at sites topographically distinct from the orthosteric site. Unfortunately, structure-based drug design of allosteric GPCR ligands has been frustrated by the paucity of structural data for allosteric binding sites, making a strong case for predictive computational methods. In this work, we map the surfaces of the beta1 (beta1AR) and beta2 (beta2AR) adrenergic receptor structures to detect a series of five potentially druggable allosteric sites. We employ the FTMAP algorithm to identify 'hot spots' with affinity for a variety of organic probe molecules corresponding to drug fragments. Our work is distinguished by an ensemble-based approach, whereby we map diverse receptor conformations taken from molecular dynamics (MD) simulations totaling approximately 0.5 micros. Our results reveal distinct pockets formed at both solvent-exposed and lipid-exposed cavities, which we interpret in light of experimental data and which may constitute novel targets for GPCR drug discovery. This mapping data can now serve to drive a combination of fragment-based and virtual screening approaches for the discovery of small molecules that bind at these sites and which may offer highly selective therapies

The relationship between apamin binding and channel block in KCa2 potassium channels.

Small conductance calcium-activated potassium channels (KCa2.1,2.2,2.3) are widely distributed throughout the body and are involved in diverse physiological processes including the regulation of neuronal firing and smooth muscle contraction. They are also potential targets in the treatment of cardiac arrhythmia. The KCa2.2 and 2.3 members of the family are blocked by the peptide toxin apamin at low concentrations, however, the mechanism of block by apamin is unclear. In electrophysiological experiments apamin has been reported to block Kca2.2 and 2.3 with IC50 ~100 pM and ~1nM respectively. In contrast, in ligand binding experiments using [125I]-mono-iodoapamin it has been found that apamin does not discriminate between Kca2.2 and 2.3 and that it binds with significantly higher affinity ( ~5pM). This discrepancy has led to the suggestion that, rather than acting as a classical pore blocker, apamin exerts its action by an allosteric mechanism. It is notable that the ligand binding studies reported so far have been conducted with isolated cell membranes in non-physiological solution with low ionic strength. We have investigated this discrepancy between results from ligand binding and electrophysiological studies by comparing binding of [125I]-mono-iodoapamin and inhibition of KCa2 current in intact HEK 293 cells using identical physiological solutions. In these conditions we found that apamin bound to KCa2.1 and KCa 2.3 with KL 60 and 606 pM, close to values of IC50 from electrophysiological experiments. We also compared the ability of some known SK channel blockers, UCL 1848, UCL 1684, gallamine and dequalinium, to displace labelled apamin and inhibit KCa2 current. With these compounds we found a good correlation between K¬i and IC50. These findings suggest that the discrepancy between binding and block might arise from differences in the experimental protocols used. To examine this we examined apamin block of KCa2 current in low ionic strength solutions in which NaCl was iso-osmotically replaced by sucrose. In these conditions 100 pM apamin caused 92 ± 0.1 % block as against 51 ± 5 % block in physiological ionic strength. We conclude that binding data obtained from membrane preparations must be interpreted with care when making comparisons with data from functional experiments and that this has implications for current views on the mechanism of action of apamin as an SK channel blocke

A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins avß3, avß5, avß6, avß8, a5ß1, aIIbß3, using homogenous ELISA-like solid phase binding assay.Postprint (published version