Single-channel behavior of heteromeric α1β glycine receptors: an attempt to detect a conformational change before the channel opens

The α1β heteromeric receptors are likely to be the predominant synaptic form of glycine receptors in the adult. Their activation mechanism was investigated by fitting putative mechanisms to single-channel recordings obtained at four glycine concentrations (10-1000 µM) from rat {alpha}1{beta} receptors, expressed in human embryonic kidney 293 cells. The adequacy of each mechanism, with its fitted rate constants, was assessed by comparing experimental dwell time distributions, open-shut correlations, and the concentration-open probability (Popen) curve with the predictions of the model. A good description was obtained only if the mechanism had three glycine binding sites, allowed both partially and fully liganded openings, and predicted the presence of open-shut correlations. A strong feature of the data was the appearance of an increase in binding affinity as more glycine molecules bind, before the channel opens. One interpretation of this positive binding cooperativity is that binding sites interact, each site sensing the state of ligation of the others. An alternative, and novel, explanation is that agonist binding stabilizes a higher affinity form of the receptor that is produced by a conformational change ("flip") that is separate from, and precedes, channel opening. Both the "interaction" scheme and the flip scheme describe our data well, but the latter has fewer free parameters and above all it offers a mechanism for the affinity increase. Distinguishing between the two mechanisms will be important for our understanding of the structural dynamics of activation in the nicotinic superfamily and is important for our understanding of mutations in these receptors

Determining the neurotransmitter concentration profile at active synapses

Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission

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

Neuregulin and BDNF Induce a Switch to NMDA Receptor-Dependent Myelination by Oligodendrocytes

<div><p>Myelination is essential for rapid impulse conduction in the CNS, but what determines whether an individual axon becomes myelinated remains unknown. Here we show, using a myelinating coculture system, that there are two distinct modes of myelination, one that is independent of neuronal activity and glutamate release and another that depends on neuronal action potentials releasing glutamate to activate NMDA receptors on oligodendrocyte lineage cells. Neuregulin switches oligodendrocytes from the activity-independent to the activity-dependent mode of myelination by increasing NMDA receptor currents in oligodendrocyte lineage cells 6-fold. With neuregulin present myelination is accelerated and increased, and NMDA receptor block reduces myelination to far below its level without neuregulin. Thus, a neuregulin-controlled switch enhances the myelination of active axons. <i>In vivo</i>, we demonstrate that remyelination after white matter damage is NMDA receptor-dependent. These data resolve controversies over the signalling regulating myelination and suggest novel roles for neuregulin in schizophrenia and in remyelination after white matter damage.</p></div

The distribution of line widths of single probe molecules in a crystalline host at milliKelvin temperatures

The line width distributions for single terrylene molecules in a naphthalene crystal have been measured at temperatures down to 30 mK. The line width distribution becomes narrower with decreasing temperature, and has a full-width at half-maximum of approximately 4.3(13) MHz at 30 mK and an average line width of 42.7(3) MHz. (C) 1999 Elsevier Science B.V. All rights reserved

Zero-phonon lines of single molecules in polyethylene down to millikelvin temperatures

Linewidth distributions for single terrylene molecules in polyethylene have been measured in the temperature range from 30 mK to 1.83 K. The temperature dependence of the average linewidth is best described by a linear relationship over the full temperature range. At 30 mK, the linewidth distribution has a full-width at half-maximum of similar to 18.6 MHz and an average linewidth of 42.8(6) MHz. (C) 2000 Elsevier Science B.V. All rights reserved

Fluorescence and absorption detected magnetic resonance of membranes from the green sulfur bacterium Chlorobium limicola. Full assignment of detected triplet states

Optically detected magnetic resonance of chlorosomes-containing membranes from the green sulfur bacterium Chlorobium limicola has been performed both by fluorescence and absorption detection. Triplet states localized in the chlorosomes and in the FMO complex have been characterized. After chemical reduction with dithionite followed by illumination at 200 K, a recombination triplet state localized in the primary donor P840 becomes populated under illumination at low temperature. A reaction center triplet state characterized by slightly different ZFS parameters, grows; up irreversibly after prolonged illumination at low temperature in the presence of reductant. We were able to obtain the T-S spectra of the FMO complex and of the primary donor P840 in their native environment and to compare them to the spectra obtained in isolated complexes previously published, revealing differences in the spectra. Fluorescence detected magnetic resonance measurements demonstrate that the BChl c antenna pigments are connected via energy transfer to the BChl a molecules at the low temperature of the measurements (1.8 K) and that all the pigments carrying the triplet states are sensitive to the redox treatment. Dithionite reduction, in fact, induces an enhancement of the BChl c and, at a major extent, of the BChl a fluorescence yield accompanied by an increase of the yield of all the triplet states of the pigments. Evidence for the presence of BChl c excited states quenchers in the core chlorosome and for their selective effect at low temperature is provided, and the location of the quenchers close to BChl c molecules absorbing at longer wavelengths discussed

Glutamatergic signaling in the brain's white matter

Glutamatergic signaling has been exceptionally well characterized in the brain's gray matter, where it underlies fast information processing, learning and memory, and also generates the neuronal damage that occurs in pathological conditions such as stroke. The role of glutamatergic signaling in the white matter, an area until recently thought to be devoid of synapses, is less well understood. Here we review what is known, and highlight what is not known, of glutamatergic signaling in the white matter. We focus on how glutamate is released, the location and properties of the receptors it acts on, the interacting molecules that may regulate trafficking or signaling of the receptors, the possible functional roles of glutamate in the white matter, and its pathological effects including the possibility of treating white matter disorders with glutamate receptor blockers. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved

Stoichiometry and subunit arrangement of alpha 1 beta glycine receptors as determined by atomic force microscopy

The glycine receptor is an anion-permeable member of the Cys-loop ion channel receptor family. Synaptic glycine receptors predominantly comprise pentameric alpha 1 beta subunit heteromers. To date, attempts to define the subunit stoichiometry and arrangement of these receptors have not yielded consistent results. Here we introduced FLAG and six-His epitopes into alpha 1 and beta subunits, respectively, and imaged single antibody-bound alpha 1 beta receptors using atomic force microscopy. This permitted us to infer the number and relative locations of the respective subunits in functional pentamers. Our results indicate an invariant 2 alpha 1:3 beta stoichiometry with a beta-alpha-beta-alpha-beta subunit arrangement