The Location of a Closed Channel Gate in the GABAA Receptor Channel

Considerable controversy surrounds the location of the closed channel gate in members of the Cys-loop receptor family of neurotransmitter-gated ion channels that includes the GABAA, glycine, acetylcholine, and 5-HT3 receptors. Cysteine-accessibility studies concluded that the gate is near the cytoplasmic end of the channel in acetylcholine and GABAA receptors but in the middle of the 5-HT3A receptor channel. Zn2+ accessibility studies in a chimeric 5-HT3-ACh receptor suggested the gate is near the channel's cytoplasmic end. In the 4-Å resolution structure of the acetylcholine receptor closed state determined by cryoelectron microscopy, the narrowest region, inferred to be the gate, is in the channel's midsection from 9' to 14' but the M1–M2 loop residues at the channel's cytoplasmic end were not resolved in that structure. We used blocker trapping experiments with picrotoxin, a GABAA receptor open channel blocker, to determine whether a gate exists at a position more extracellular than the picrotoxin binding site, which is in the vicinity of α1Val257 (2') near the channel's cytoplasmic end. We show that picrotoxin can be trapped in the channel after removal of GABA. By using the state-dependent accessibility of engineered cysteines as reporters for the channel's structural state we infer that after GABA washout, with picrotoxin trapped in the channel, the channel appears to be in the closed state. We infer that a gate exists between the picrotoxin binding site and the channel's extracellular end, consistent with a closed channel gate in the middle of the channel. Given the homology with acetylcholine and 5-HT3 receptors there is probably a similar gate in those channels as well. This does not preclude the existence of an additional gate at a more cytoplasmic location

Modular Design of Cys-loop Ligand-gated Ion Channels: Functional 5-HT3 and GABA ρ1 Receptors Lacking the Large Cytoplasmic M3M4 Loop

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the α-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT3A) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT3A-glvM3M4 receptors were comparable to 5-HT3A wild type. The channels remained cation-selective but the 5-HT3A-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT3 and acetylcholine receptors, significantly attenuated 5-HT–induced currents with wild-type 5-HT3A but not 5-HT3A-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-ρ1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains

Defining the propofol binding site location on the GABAA receptor

ABSTRACT The GABA A receptor is a target of many general anesthetics. The low affinity of general anesthetics has complicated the search for the location of anesthetic binding sites. Attention has focused on two pairs of residues near the extracellular ends of the M2 and M3 membrane-spanning segments, ␣ 1 Ser270/␤ 2 Asn265 (15Ј M2) and ␣ 1 Ala291/␤ 2 Met286 (M3). In the 4-Å resolution acetylcholine receptor structure, the aligned positions are separated by ϳ10 Å. To determine whether these residues are part of a binding site for propofol, an intravenous anesthetic, we probed propofol's ability to protect cysteines substituted for these residues from modification by the sulfhydryl-specific reagent p-chloromercuribenzenesulfonate (pCMBS Ϫ ). pCMBS Ϫ reacted with cysteines substituted at the four positions in the absence and presence of GABA. Because propofol binding induces conformational change in the GABA A receptor, we needed to establish a reference state of the receptor to compare reaction rates in the absence and presence of propofol. We compared reaction rates in the presence of GABA with those in the presence of propofol ϩ GABA. The GABA concentration was reduced to give a similar fraction of the maximal GABA current in both conditions. Propofol protected, in a concentration-dependent manner, the cysteine substituted for ␤ 2 Met286 from reaction with pCMBS Ϫ . Propofol did not protect the cysteine substituted for the aligned ␣ 1 subunit position or the 15Ј M2 segment Cys mutants in either subunit. We infer that propofol may bind near the extracellular end of the ␤ subunit M3 segment

Effectiveness of school-based nutrition interventions promoted in the League of Arab States: a systematic review.

Arab countries are doubly burdened with undernutrition as well as overweight and obesity. To provide guidance to those looking to address concerns of obesity and overweight among children in this region, the current review bridges an existing knowledge gap and systematically characterizes and evaluates the available scientific evidence pertaining to school-based nutrition interventions completed to date across the Arab world. Using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and relevant keywords, terms and phrases, a search of the literature across 14 databases for school-based nutrition interventions implemented in this region was conducted. Out of 1568 articles from 14 databases, 38 full-text articles meeting the inclusion criteria were retrieved. Further assessment for eligibility excluded 23 articles and included one article from hand-searching references, leaving 16 articles in the final analysis. Most articles (n = 7) with the implementation of 5 months or longer found strong positive and significant impacts on limiting sweets consumption, increased dietary knowledge, self-efficacy, breakfast and fruit and vegetable consumption, among others. We also found evidence (n = 5) that teachers trained by experts such as dietitians and pediatricians can effectively implement nutrition interventions that achieve the intended outcomes. Training teachers to implement theory-based nutrition and health promotion curricula serves as a potential solution to improving nutrition knowledge, health behaviors and dietary practices among children living in Arab countries. Appropriately designed and implemented nutrition interventions can positively impact nutrition knowledge, health and diet-related behaviors in children and adolescents in this region

The influence of gravimetric moisture content on studded shoe–surface interactions in soccer

It is desirable for the studs of a soccer shoe to penetrate the sport surface and provide the player with sufficient traction when accelerating. Mechanical tests are often used to measure the traction of shoe–surface combinations. Mechanical testing offers a repeatable measure of shoe–surface traction, eliminating the inherent uncertainties that exist when human participant testing is employed, and are hence used to directly compare the performance of shoe–surface combinations. However, the influence specific surface characteristics has on traction is often overlooked. Examining the influence of surface characteristics on mechanical test results improves the understanding of the traction mechanisms at the shoe–surface interface. This allows footwear developers to make informed decisions on the design of studded outsoles. The aim of this paper is to understand the effect gravimetric moisture content has on the tribological mechanisms at play during stud–surface interaction. This study investigates the relationships between: the gravimetric moisture content of a natural sand-based soccer surface; surface stiffness measured via a bespoke impact test device; and surface traction measured via a bespoke mechanical test device. Regression analysis revealed that surface stiffness decreases linearly with increased gravimetric moisture content (p = 0.04). Traction was found to initially increase and then decrease with gravimetric moisture content. It was observed that: a surface of low moisture content provides low stud penetration and therefore reduced traction; a surface of high moisture content provides high stud penetration but also reduced traction due to a lubricating effect; and surfaces with moisture content in between the two extremes provide increased traction. In this study a standard commercially available stud was used and other studs may provide slightly different results. The results provide insight into the traction mechanisms at the stud–surface interface which are described in the paper. The variation between traction measurements shows the influence gravimetric moisture content will have on player performance. This highlights the requirement to understand surface conditions prior to making comparative shoe–surface traction studies and the importance of using a studded outsole that is appropriate to the surface condition during play

Rare Variant Enrichment analysis Supports

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by aplasia of the female reproductive tract; the syndrome can include renal anomalies, absence or dysgenesis, and skeletal anomalies. While functional models have elucidated several candidate genes, onl

An Efficient Human Activity Recognition Technique Based on Deep Learning

In this paper, we present a new deep learning-based human activity recognition technique. First, we track and extract human body from each frame of the video stream. Next, we abstract human silhouettes and use them to create binary space-time maps (BSTMs) which summarize human activity within a defined time interval. Finally, we use convolutional neural network (CNN) to extract features from BSTMs and classify the activities. To evaluate our approach, we carried out several tests using three public datasets: Weizmann, Keck Gesture and KTH Database. Experimental results show that our technique outperforms conventional state-of-the-art methods in term of recognition accuracy and provides comparable performance against recent deep learning techniques. It’s simple to implement, requires less computing power, and can be used for multi-subject activity recognition

Cross-tolerance to abiotic stresses in halophytes: Application for phytoremediation of organic pollutants

International audienceHalopytes are plants able to tolerate high salt concentrations but no clear definition was retained for them. In literature, there are more studies that showed salt-enhanced tolerance to other abiotic stresses compared to investigations that found enhanced salt tolerance by other abiotic stresses in halophytes. The phenomenon by which a plant resistance to a stress induces resistance to another is referred to as cross-tolerance. In this work, we reviewed cross-tolerance in halophytes at the physiological, biochemical, and molecular levels. A special attention was accorded to the cross-tolerance between salinity and organic pollutants that could allow halophytes a higher potential of xenobiotic phytoremediation in comparison with glycophytes

Differential Effects of Bartonella henselae on Human and Feline Macro- and Micro-Vascular Endothelial Cells

Bartonella henselae, a zoonotic agent, induces tumors of endothelial cells (ECs), namely bacillary angiomatosis and peliosis in immunosuppressed humans but not in cats. In vitro studies on ECs represent to date the only way to explore the interactions between Bartonella henselae and vascular endothelium. However, no comparative study of the interactions between Bartonella henselae and human (incidental host) ECs vs feline (reservoir host) ECs has been carried out because of the absence of any available feline endothelial cell lines