A Hydrophobic Gate in an Ion Channel: The Closed State of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (nAChR) is the prototypic member of the `Cys-loop' superfamily of ligand-gated ion channels which mediate synaptic neurotransmission, and whose other members include receptors for glycine, gamma-aminobutyric acid, and serotonin. Cryo-electron microscopy has yielded a three dimensional structure of the nAChR in its closed state. However, the exact nature and location of the channel gate remains uncertain. Although the transmembrane pore is constricted close to its center, it is not completely occluded. Rather, the pore has a central hydrophobic zone of radius about 3 A. Model calculations suggest that such a constriction may form a hydrophobic gate, preventing movement of ions through a channel. We present a detailed and quantitative simulation study of the hydrophobic gating model of the nicotinic receptor, in order to fully evaluate this hypothesis. We demonstrate that the hydrophobic constriction of the nAChR pore indeed forms a closed gate. Potential of mean force (PMF) calculations reveal that the constriction presents a barrier of height ca. 10 kT to the permeation of sodium ions, placing an upper bound on the closed channel conductance of 0.3 pS. Thus, a 3 A radius hydrophobic pore can form a functional barrier to the permeation of a 1 A radius Na+ ion. Using a united atom force field for the protein instead of an all atom one retains the qualitative features but results in differing conductances, showing that the PMF is sensitive to the detailed molecular interactions.Comment: Accepted by Physical Biology; includes a supplement and a supplementary mpeg movie can be found at http://sbcb.bioch.ox.ac.uk/oliver/download/Movies/watergate.mp

Formality and informality in the summative assessment of motor vehicle apprentices: a case study

This article explores the interaction of formal and informal attributes of competence‐based assessment. Specifically, it presents evidence from a small qualitative case study of summative assessment practices for competence‐based qualifications within apprenticeships in the motor industry in England. The data are analysed through applying an adaptation of a framework for exploring the interplay of formality and informality in learning. This analysis reveals informal mentoring as a significant element which influences not only the process of assessment, but also its outcomes. We offer different possible interpretations of the data and their analysis, and conclude that, whichever interpretation is adopted, there appears to be a need for greater capacity‐building for assessors at a local level. This could acknowledge a more holistic role for assessors; recognise the importance of assessors’ informal practices in the formal retention and achievement of apprentices; and enhance awareness of inequalities that may be reinforced by both informal and formal attributes of assessment practices

Superluminal Expansion of 3C 273

Figure 1 shows hybrid maps of the core of 3C 273B at five epochs, made with arrays of 4 or 5 VLBI antennas. The maps span a period of 3.5 years. They all show a bright eastern peak and a lower-brightness extension to the west. There is a local maximum in the western extension between 6 and 8 milliarcsec from the main peak. This “blob” moves steadily further away from the main peak along a roughly straight line in PA −116° ± 2°. Compare this with the position angle of the 25-arcsec optical jet, −137°. The maps show a slight curvature to the south with increasing separation from the main peak. Lower-resolution VLBI maps at lower frequencies show that this curvature continues at greater separations, suggesting a smooth connection between the milli-arcsecond position angle and the position angle of the optical jet. In our latest map (1981.09) the blob is no longer detectable with the limited dynamic range of the VLBI network (about 20:1)

Moebius strip enterprises and expertise in the creative industries: new challenges for lifelong learning?

The paper argues that the emergence of a new mode of production – co-configuration is generating new modes of expertise that EU policies for lifelong learning are not designed to support professionals to develop. It maintains that this change can be seen most clearly when we analyse Small and Medium Size (SMEs) enterprises in the creative industries. Drawing on concepts from Political Economy - ‘Moebius strip enterprise/expertise’ and Cultural Historical Activity Theory - project-object’ and the ‘space of reasons’, the paper highlights conceptually and through a case study of an SME in the creative industries what is distinctive about the new modes of expertise, before moving on to reconceptualise expertise and learning and to consider the implications of this reconceptualisation for EU policies for lifelong learning. The paper concludes that the new challenge for LLL is to support the development of new forms expertise that are difficult to credentialise, yet, are central to the wider European goal of realising a knowledge economy

Temperature and population density influence SARS-CoV-2 transmission in the absence of nonpharmaceutical interventions

As COVID-19 continues to spread across the world, it is increasingly important to understand the factors that influence its transmission. Seasonal variation driven by responses to changing environment has been shown to affect the transmission intensity of several coronaviruses. However, the impact of the environment on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains largely unknown, and thus seasonal variation remains a source of uncertainty in forecasts of SARS-CoV-2 transmission. Here we address this issue by assessing the association of temperature, humidity, ultraviolet radiation, and population density with estimates of transmission rate (R). Using data from the United States, we explore correlates of transmission across US states using comparative regression and integrative epidemiological modeling. We find that policy intervention ("lockdown") and reductions in individuals' mobility are the major predictors of SARS-CoV-2 transmission rates, but, in their absence, lower temperatures and higher population densities are correlated with increased SARS-CoV-2 transmission. Our results show that summer weather cannot be considered a substitute for mitigation policies, but that lower autumn and winter temperatures may lead to an increase in transmission intensity in the absence of policy interventions or behavioral changes. We outline how this information may improve the forecasting of COVID-19, reveal its future seasonal dynamics, and inform intervention policies

Structural correlations in heterogeneous electron transfer at monolayer and multilayer graphene electrodes

As a new form of carbon, graphene is attracting intense interest as an electrode material with widespread applications. In the present study, the heterogeneous electron transfer (ET) activity of graphene is investigated using scanning electrochemical cell microscopy (SECCM), which allows electrochemical currents to be mapped at high spatial resolution across a surface for correlation with the corresponding structure and properties of the graphene surface. We establish that the rate of heterogeneous ET at graphene increases systematically with the number of graphene layers, and show that the stacking in multilayers also has a subtle influence on ET kinetics. © 2012 American Chemical Society

Fabrication and characterization of dual function nanoscale pH-scanning ion conductance microscopy (SICM) probes for high resolution pH mapping

The easy fabrication and use of nanoscale dual function pH-scanning ion conductance microscopy (SICM) probes is reported. These probes incorporate an iridium oxide coated carbon electrode for pH measurement and an SICM barrel for distance control, enabling simultaneous pH and topography mapping. These pH-SICM probes were fabricated rapidly from laser pulled theta quartz pipets, with the pH electrode prepared by in situ carbon filling of one of the barrels by the pyrolytic decomposition of butane, followed by electrodeposition of a thin layer of hydrous iridium oxide. The other barrel was filled with an electrolyte solution and Ag/AgCl electrode as part of a conductance cell for SICM. The fabricated probes, with pH and SICM sensing elements typically on the 100 nm scale, were characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and various electrochemical measurements. They showed a linear super-Nernstian pH response over a range of pH (pH 2–10). The capability of the pH-SICM probe was demonstrated by detecting both pH and topographical changes during the dissolution of a calcite microcrystal in aqueous solution. This system illustrates the quantitative nature of pH-SICM imaging, because the dissolution process changes the crystal height and interfacial pH (compared to bulk), and each is sensitive to the rate. Both measurements reveal similar dissolution rates, which are in agreement with previously reported literature values measured by classical bulk methods

Signal Transduction Pathways in the Pentameric Ligand-Gated Ion Channels

The mechanisms of allosteric action within pentameric ligand-gated ion channels (pLGICs) remain to be determined. Using crystallography, site-directed mutagenesis, and two-electrode voltage clamp measurements, we identified two functionally relevant sites in the extracellular (EC) domain of the bacterial pLGIC from Gloeobacter violaceus (GLIC). One site is at the C-loop region, where the NQN mutation (D91N, E177Q, and D178N) eliminated inter-subunit salt bridges in the open-channel GLIC structure and thereby shifted the channel activation to a higher agonist concentration. The other site is below the C-loop, where binding of the anesthetic ketamine inhibited GLIC currents in a concentration dependent manner. To understand how a perturbation signal in the EC domain, either resulting from the NQN mutation or ketamine binding, is transduced to the channel gate, we have used the Perturbation-based Markovian Transmission (PMT) model to determine dynamic responses of the GLIC channel and signaling pathways upon initial perturbations in the EC domain of GLIC. Despite the existence of many possible routes for the initial perturbation signal to reach the channel gate, the PMT model in combination with Yen's algorithm revealed that perturbation signals with the highest probability flow travel either via the β1-β2 loop or through pre-TM1. The β1-β2 loop occurs in either intra- or inter-subunit pathways, while pre-TM1 occurs exclusively in inter-subunit pathways. Residues involved in both types of pathways are well supported by previous experimental data on nAChR. The direct coupling between pre-TM1 and TM2 of the adjacent subunit adds new insight into the allosteric signaling mechanism in pLGICs. © 2013 Mowrey et al

Dual-barrel conductance micropipet as a new approach to the study of ionic crystal dissolution kinetics

A new approach to the study of ionic crystal dissolution kinetics is described, based on the use of a dual-barrel theta conductance micropipet. The solution in the pipet is undersaturated with respect to the crystal of interest, and when the meniscus at the end of the micropipet makes contact with a selected region of the crystal surface, dissolution occurs causing the solution composition to change. This is observed, with better than 1 ms time resolution, as a change in the ion conductance current, measured across a potential bias between an electrode in each barrel of the pipet. Key attributes of this new technique are: (i) dissolution can be targeted at a single crystal surface; (ii) multiple measurements can be made quickly and easily by moving the pipet to a new location on the surface; (iii) materials with a wide range of kinetics and solubilities are open to study because the duration of dissolution is controlled by the meniscus contact time; (iv) fast kinetics are readily amenable to study because of the intrinsically high mass transport rates within tapered micropipets; (v) the experimental geometry is well-defined, permitting finite element method modeling to allow quantitative analysis of experimental data. Herein, we study the dissolution of NaCl as an example system, with dissolution induced for just a few milliseconds, and estimate a first-order heterogeneous rate constant of 7.5 (±2.5) × 10–5 cm s–1 (equivalent surface dissolution flux ca. 0.5 μmol cm–2 s–1 into a completely undersaturated solution). Ionic crystals form a huge class of materials whose dissolution properties are of considerable interest, and we thus anticipate that this new localized microscale surface approach will have considerable applicability in the future