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

Possible Effects of Noncommutative Geometry on Weak CP Violation and Unitarity Triangles

Possible effects of noncommutative geometry on weak CP violation and unitarity triangles are discussed by taking account of a simple version of the momentum-dependent quark mixing matrix in the noncommutative standard model. In particular, we calculate nine rephasing invariants of CP violation and illustrate the noncommutative CP-violating effect in a couple of charged D-meson decays. We also show how inner angles of the deformed unitarity triangles are related to CP-violating asymmetries in some typical B_d and B_s transitions into CP eigenstates. B-meson factories are expected to help probe or constrain noncommutative geometry at low energies in the near future.Comment: RexTev 16 pages. Modifications made. References added. Accepted for publication in Phys. Rev.

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

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

Gating at the Mouth of the Acetylcholine Receptor Channel: Energetic Consequences of Mutations in the αM2-Cap

Gating of nicotinic acetylcholine receptors from a C(losed) to an O(pen) conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where C↔O gating motions generate a low↔high change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the α subunit (‘αM2-cap’). This region contains a binding site for non-competitive inhibitors and a charged ring that influences the conductance of the open pore. αM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three αM2-cap residues (αI260, αP265 and αS268) appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the αM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance

Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine

ELIC, the pentameric ligand-gated ion channel from Erwinia chrysanthemi, is a prototype for Cys-loop receptors. Here we show that acetylcholine is a competitive antagonist for ELIC. We determine the acetylcholine–ELIC cocrystal structure to a 2.9-Å resolution and find that acetylcholine binding to an aromatic cage at the subunit interface induces a significant contraction of loop C and other structural rearrangements in the extracellular domain. The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed. We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel. This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs

Twenty Thousand-Year-Old Huts at a Hunter-Gatherer Settlement in Eastern Jordan

Ten thousand years before Neolithic farmers settled in permanent villages, hunter-gatherer groups of the Epipalaeolithic period (c. 22–11,600 cal BP) inhabited much of southwest Asia. The latest Epipalaeolithic phase (Natufian) is well-known for the appearance of stone-built houses, complex site organization, a sedentary lifestyle and social complexity—precursors for a Neolithic way of life. In contrast, pre-Natufian sites are much less well known and generally considered as campsites for small groups of seasonally-mobile hunter-gatherers. Work at the Early and Middle Epipalaeolithic aggregation site of Kharaneh IV in eastern Jordan highlights that some of these earlier sites were large aggregation base camps not unlike those of the Natufian and contributes to ongoing debates on their duration of occupation. Here we discuss the excavation of two 20,000-year-old hut structures at Kharaneh IV that pre-date the renowned stone houses of the Natufian. Exceptionally dense and extensive occupational deposits exhibit repeated habitation over prolonged periods, and contain structural remains associated with exotic and potentially symbolic caches of objects (shell, red ochre, and burnt horn cores) that indicate substantial settlement of the site pre-dating the Natufian and outside of the Natufian homeland as currently understood

A Unique Human-Fox Burial from a Pre-Natufian Cemetery in the Levant (Jordan)

New human burials from northern Jordan provide important insights into the appearance of cemeteries and the nature of human-animal relationships within mortuary contexts during the Epipalaeolithic period (c. 23,000–11,600 cal BP) in the Levant, reinforcing a socio-ideological relationship that goes beyond predator-prey. Previous work suggests that archaeological features indicative of social complexity occur suddenly during the latest Epipalaeolithic phase, the Natufian (c. 14,500–11,600 cal BP). These features include sedentism, cemeteries, architecture, food production, including animal domestication, and burials with elaborate mortuary treatments. Our findings from the pre-Natufian (Middle Epipalaeolithic) cemetery of ‘Uyun al-Hammam demonstrate that joint human-animal mortuary practices appear earlier in the Epipalaeolithic. We describe the earliest human-fox burial in the Near East, where the remains of dogs have been found associated with human burials at a number of Natufian sites. This is the first time that a fox has been documented in association with human interments pre-dating the Natufian and with a particular suite of grave goods. Analysis of the human and animal bones and their associated artefacts provides critical data on the nature and timing of these newly-developing relationships between people and animals prior to the appearance of domesticated dogs in the Natufian