Palonosetron-5-HT3Receptor Interactions As Shown by a Binding Protein Cocrystal Structure.

Palonosetron is a potent 5-HT3receptor antagonist and an effective therapeutic agent against emesis. Here we identify the molecular determinants of compound recognition in the receptor binding site by obtaining a high resolution structure of palonosetron bound to an engineered acetylcholine binding protein that mimics the 5-HT3receptor binding site, termed 5-HTBP, and by examining the potency of palonosetron in a range of 5-HT3receptors with mutated binding site residues. The structural data indicate that palonosetron forms a tight and effective wedge in the binding pocket, made possible by its rigid tricyclic ring structure and its interactions with binding site residues; it adopts a binding pose that is distinct from the related antiemetics granisetron and tropisetron. The functional data show many residues previously shown to interact with agonists and antagonists in the binding site are important for palonosetron binding, and indicate those of particular importance are W183 (a cation-π interaction and a hydrogen bond) and Y153 (a hydrogen bond). This information, and the availability of the structure of palonosetron bound to 5-HTBP, should aid the development of novel and more efficacious drugs that act via 5-HT3receptors.Supported by a grant from the MRC (MR/L02/676) to S.C.R.L

Risk of gastrointestinal bleeding with direct oral anticoagulants: a systematic review and network meta-analysis

Background: Direct oral anticoagulants are increasingly used for a wide range of indications. However, data are conflicting about the risk of major gastrointestinal bleeding with these drugs. We compared the risk of gastrointestinal bleeding with direct oral anticoagulants, warfarin, and low-molecular-weight heparin. Methods: For this systematic review and meta-analysis, we searched MEDLINE and Embase from database inception to April 1, 2016, for prospective and retrospective studies that reported the risk of gastrointestinal bleeding with use of a direct oral anticoagulant compared with warfarin or low-molecular-weight heparin for all indications. We also searched the Cochrane Library for systematic reviews and assessment evaluations, the National Health Service (UK) Economic Evaluation Database, and ISI Web of Science for conference abstracts and proceedings (up to April 1, 2016). The primary outcome was the incidence of major gastrointestinal bleeding, with all gastrointestinal bleeding as a secondary outcome. We did a Bayesian network meta-analysis to produce incidence rate ratios (IRRs) with 95% credible intervals (CrIs). Findings: We identified 38 eligible articles, of which 31 were included in the primary analysis, including 287 692 patients exposed to 230 090 years of anticoagulant drugs. The risk of major gastrointestinal bleeding with direct oral anticoagulants did not differ from that with warfarin or low-molecular-weight heparin (factor Xa vs warfarin IRR 0·78 [95% CrI 0·47−1·08]; warfarin vs dabigatran 0·88 [0·59−1·36]; factor Xa vs low-molecular-weight heparin 1·02 [0·42−2·70]; and low-molecular-weight heparin vs dabigatran 0·67 [0·20−1·82]). In the secondary analysis, factor Xa inhibitors were associated with a reduced risk of all severities of gastrointestinal bleeding compared with warfarin (0·25 [0.07–0.76]) or dabigatran (0.24 [0.07–0.77]). Interpretation: Our findings show no increase in risk of major gastrointestinal bleeding with direct oral anticoagulants compared with warfarin or low-molecular-weight heparin. These findings support the continued use of direct oral anticoagulants. Funding: Leeds Teaching Hospitals Charitable Foundation

Varenicline Interactions at the 5-HT3 Receptor Ligand Binding Site are Revealed by 5-HTBP.

Cys-loop receptors are the site of action of many therapeutic drugs. One of these is the smoking cessation agent varenicline, which has its major therapeutic effects at nicotinic acetylcholine (nACh) receptors but also acts at 5-HT3 receptors. Here, we report the X-ray crystal structure of the 5-HT binding protein (5-HTBP) in complex with varenicline, and test the predicted interactions by probing the potency of varenicline in a range of mutant 5-HT3 receptors expressed in HEK293 cells and Xenopus oocytes. The structure reveals a range of interactions between varenicline and 5-HTBP. We identified residues within 5 Å of varenicline and substituted the equivalent residues in the 5-HT3 receptor with Ala or a residue with similar chemical properties. Functional characterization of these mutant 5-HT3 receptors, using a fluorescent membrane potential dye in HEK cells and voltage clamp in oocytes, supports interactions between varenicline and the receptor that are similar to those in 5-HTBP. The structure also revealed C-loop closure that was less than in the 5-HT-bound 5-HTBP, and hydrogen bonding between varenicline and the complementary face of the binding pocket via a water molecule, which are characteristics consistent with partial agonist behavior of varenicline in the 5-HT3 receptor. Together, these data reveal detailed insights into the molecular interaction of varenicline in the 5-HT3 receptor.Supported by grants from the Wellcome Trust (81925) and the MRC to S.C.R.L.This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/cn500369

Photo-antagonism of the GABAA receptor

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation

Trans−cis Switching Mechanisms in Proline Analogues and Their Relevance for the Gating of the 5-HT3 Receptor

Trans-cis isomerization of a proline peptide bond is a potential mechanism to open the channel of the 5-HT3 receptor. Here, we have used the metadynamics method to theoretically explore such a mechanism. We have determined the free energy surfaces in aqueous solution of a series of dipeptides of proline analogues and evaluated the free energy difference between the cis and trans isomers. These theoretical results were then compared with data from mutagenesis experiments, in which the response of the 5-HT3 receptor was measured when the proline at the apex of the M2-M3 transmembrane domain loop was mutated. The strong correlation between the experimental and the theoretical data supports the existence of a trans-cis proline switch for opening the 5-HT3 receptor ion channel

β Subunit M2–M3 Loop Conformational Changes Are Uncoupled from α1 β Glycine Receptor Channel Gating: Implications for Human Hereditary Hyperekplexia

Hereditary hyperekplexia, or startle disease, is a neuromotor disorder caused mainly by mutations that either prevent the surface expression of, or modify the function of, the human heteromeric α1 β glycine receptor (GlyR) chloride channel. There is as yet no explanation as to why hyperekplexia mutations that modify channel function are almost exclusively located in the α1 to the exclusion of β subunit. The majority of these mutations are identified in the M2–M3 loop of the α1 subunit. Here we demonstrate that α1 β GlyR channel function is less sensitive to hyperekplexia-mimicking mutations introduced into the M2–M3 loop of the β than into the α1 subunit. This suggests that the M2–M3 loop of the α subunit dominates the β subunit in gating the α1 β GlyR channel. A further attempt to determine the possible mechanism underlying this phenomenon by using the voltage-clamp fluorometry technique revealed that agonist-induced conformational changes in the β subunit M2–M3 loop were uncoupled from α1 β GlyR channel gating. This is in contrast to the α subunit, where the M2–M3 loop conformational changes were shown to be directly coupled to α1 β GlyR channel gating. Finally, based on analysis of α1 β chimeric receptors, we demonstrate that the structural components responsible for this are distributed throughout the β subunit, implying that the β subunit has evolved without the functional constraint of a normal gating pathway within it. Our study provides a possible explanation of why hereditary hyperekplexia-causing mutations that modify α1 β GlyR channel function are almost exclusively located in the α1 to the exclusion of the β subunit

Conformational changes in α7 acetylcholine receptors underlying allosteric modulation by divalent cations

Allosteric modulation of membrane receptors is a widespread mechanism by which endogenous and exogenous agents regulate receptor function. For example, several members of the nicotinic receptor family are modulated by physiological concentrations of extracellular calcium ions. In this paper, we examined conformational changes underlying this modulation and compare these with changes evoked by ACh. Two sets of residues in the α7 acetylcholine receptor extracellular domain were mutated to cysteine and analyzed by measuring the rates of modification by the thiol-specific reagent 2-aminoethylmethane thiosulfonate. Using Ba2+ as a surrogate for Ca2+, we found a divalent-dependent decrease the modification rates of cysteine substitutions at M37 and M40, residues at which rates were also slowed by ACh. In contrast, Ba2+ had no significant effect at N52C, a residue where ACh increased the rate of modification. Thus divalent modulators cause some but not all of the conformational effects elicited by agonist. Cysteine substitution of either of two glutamates (E44 or E172), thought to participate in the divalent cation binding site, caused a loss of allosteric modulation, yet Ba2+ still had a significant effect on modification rates of these residues. In addition, the effect of Ba2+ at these residues did not appear to be due to direct occlusion. Our data demonstrate that modulation by divalent cations involves substantial conformational changes in the receptor extracellular domain. Our evidence also suggests the modulation occurs via a binding site distinct from one which includes either (or both) of the conserved glutamates at E44 or E172

Ligand Activation of the Prokaryotic Pentameric Ligand-Gated Ion Channel ELIC

While the pentameric ligand-gated ion channel ELIC has recently provided first insight into the architecture of the family at high resolution, its detailed investigation was so far prevented by the fact that activating ligands were unknown. Here we describe a study on the functional characterization of ELIC by electrophysiology and X-ray crystallography. ELIC is activated by a class of primary amines that include the neurotransmitter GABA at high micro- to millimolar concentrations. The ligands bind to a conserved site and evoke currents that slowly desensitize over time. The protein forms cation selective channels with properties that resemble the nicotinic acetylcholine receptor. The high single channel conductance and the comparably simple functional behavior make ELIC an attractive model system to study general mechanisms of ion conduction and gating in this important family of neurotransmitter receptors

Anaesthetic Impairment of Immune Function Is Mediated via GABAA Receptors

GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem