Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission

Cholesterol is a structural component of cellular membranes particularly enriched in synapses but its role in synaptic transmission remains poorly understood. We used rat hippocampal cultures and their acute cholesterol depletion by methyl-β-cyclodextrin as a tool to describe the physiological role of cholesterol in glutamatergic synaptic transmission. Cholesterol proved to be a key molecule for the function of synapses as its depletion resulted in a significant reduction of both NMDA receptor (NMDAR) and AMPA/kainate receptor-mediated evoked excitatory postsynaptic currents (eEPSCs), by 94% and 72%, respectively. We identified two presynaptic and two postsynaptic steps of synaptic transmission which are modulated by cholesterol and explain together the above-mentioned reduction of eEPSCs. In the postsynapse, we show that physiological levels of cholesterol are important for maintaining the normal probability of opening of NMDARs and for keeping NMDARs localized in synapses. In the presynapse, our results favour the hypothesis of a role of cholesterol in the propagation of axonal action potentials. Finally, cholesterol is a negative modulator of spontaneous presynaptic glutamate release. Our study identifies cholesterol as an important endogenous regulator of synaptic transmission and provides insight into molecular mechanisms underlying the neurological manifestation of diseases associated with impaired cholesterol synthesis or decomposition

Ivermectin, an unconventional agonist of the glycine receptor chloride channel

The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha (1) homomeric and alpha (1)beta heteromeric glycine receptors (GlyRs), At low (0.03 muM) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (greater than or equal to0.03 muM) concentrations it irreversibly activated both alpha (1) homomeric and alpha (1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin, Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism

Rimsky-Korsakov's Antar Symphony : a biographical and analytical study

Rimsky-Korsakov composed his Symphony No. 2, Antar, in 1868, during his apprenticeship to Balakirev and involvement with The Five. Based on an oriental tale by Osip Senkovsky, it was the first multi-movement, programmatic orchestral work in Russian music. Rimsky-Korsakov revised Antar twice, in 1875 – by which time he was a professor at the Saint Petersburg Conservatory – and 1897. This thesis will examine the original 1868 version of Antar by placing it in the context of his career and the influences that shaped his style. The first chapter presents a biographical sketch of Rimsky-Korsakov, with emphasis on his education and training, including his studies with Balakirev in the 1860s and his rigorous course of self-education in harmony, counterpoint, form, and orchestration in the 1870s. The second chapter begins with a summary of the tale by Senkovsky upon which the music is based, followed by an analysis of the themes, harmony, and orchestration of each movement of Antar. I will show how the form and character of the work are indebted to ideas espoused by Balakirev and The Five, including their fondness for program music, orientalism, unconventional scales and modes, and harmonic progressions based on common tones. I will also draw attention to aspects of the tonal organization and scoring that are elaborated in the composer’s textbook, the Practical Manual of Harmony. My objective is to show that Antar is both a summary of the fruits of Rimsky-Korsakov’s apprenticeship within Balakirev’s circle, as well as a foretaste of his mature compositional and theoretical interests.Arts, Faculty ofMusic, School ofGraduat

The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the central nervous system, underlie the induction of synaptic plasticity, and their malfunction is associated with human diseases. Native NMDARs are tetramers composed of two obligatory GluN1 subunits and various combinations of GluN2A-D or, more rarely, GluN3A-B subunits. Each subunit consists of an amino-terminal, ligand-binding, transmembrane and carboxyl-terminal domain. The ligand-binding and transmembrane domains are interconnected via polypeptide chains (linkers). Upon glutamate and glycine binding, these receptors undergo a series of conformational changes leading to the opening of the Ca2+-permeable ion channel. Here we report that different deletions and mutations of amino acids in the M3-S2 linkers of the GluN1 and GluN2B subunits lead to constitutively open channels. Irrespective of whether alterations were introduced in the GluN1 or the GluN2B subunit, application of glutamate or glycine promoted receptor channel activity; however, responses induced by the GluN1 agonist glycine were larger, on average, than those induced by glutamate. We observed the most prominent effect when residues GluN1(L657) and GluN2B(I655) were deleted or altered to glycine. In parallel, molecular modeling revealed that two interacting pairs of residues, the LILI motif (GluN1(L657) and GluN2B(I655)), form a functional unit with the TTTT ring (GluN1(T648) and GluN2B(T647)), described earlier to control NMDAR channel gating. These results provide new insight into the structural organization and functional interplay of the LILI and the TTTT ring during the course of NMDAR channel opening and closing

Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner

Synthesis of novel azanorbornylpurine derivatives

Azanorbornylpurine derivatives were prepared by Mitsunobu reaction of appropriate hydroxyazanorbornane derivative with 6-chloropurine or construction of purine base at azanorbornylamines. The prepared target compounds were evaluated for antiviral activity and effect on neuronal and muscle nicotinic acetylcholine receptors. (C) 2011 Published by Elsevier Ltd.status: publishe

Adenylate cyclase toxin translocates across target cell membrane without forming a pore

The adenylate cyclase toxin-haemolysin of Bordetella (CyaA) targets CD11b(+) myeloid phagocytes and translocates across their cytoplasmic membrane an adenylate cyclase (AC) enzyme that catalyses conversion of cytosolic ATP into cAMP. In parallel, CyaA acts as a cytolysin forming cation-selective pores, which permeabilize cell membrane and eventually provoke cell lysis. Using cytolytic activity, potassium efflux and patch-clamp assays, we show that a combination of substitutions within the pore-forming (E570Q) and acylation-bearing domain (K860R) ablates selectively the cell-permeabilizing activity of CyaA. At the same time, however, the capacity of such mutant CyaA to translocate the AC domain across cytoplasmic membrane into cytosol of macrophage cells and to elevate cellular cAMP concentrations remained intact. Moreover, the combination of E570Q+K860R substitutions suppressed the residual cytolytic activity of the enzymatically inactive CyaA/OVA/AC(-) toxoid on CD11b-expressing monocytes, while leaving unaffected the capacity of the mutant toxoid to deliver in vitro a reporter CD8(+) T cell epitope from ovalbumin (OVA) to the cytosolic pathway of dendritic cells for MHC class I-restricted presentation and induce in vivo an OVA-specific cytotoxic T cell response. CyaA, hence, employs a mechanism of AC enzyme domain translocation across cellular membrane that avoids passage across the cytolytic pore formed by toxin oligomers

Data_Sheet_1.docx

<p>N-methyl-D-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the central nervous system, underlie the induction of synaptic plasticity, and their malfunction is associated with human diseases. Native NMDARs are tetramers composed of two obligatory GluN1 subunits and various combinations of GluN2A-D or, more rarely, GluN3A-B subunits. Each subunit consists of an amino-terminal, ligand-binding, transmembrane and carboxyl-terminal domain. The ligand-binding and transmembrane domains are interconnected via polypeptide chains (linkers). Upon glutamate and glycine binding, these receptors undergo a series of conformational changes leading to the opening of the Ca²⁺-permeable ion channel. Here we report that different deletions and mutations of amino acids in the M3-S2 linkers of the GluN1 and GluN2B subunits lead to constitutively open channels. Irrespective of whether alterations were introduced in the GluN1 or the GluN2B subunit, application of glutamate or glycine promoted receptor channel activity; however, responses induced by the GluN1 agonist glycine were larger, on average, than those induced by glutamate. We observed the most prominent effect when residues GluN1(L657) and GluN2B(I655) were deleted or altered to glycine. In parallel, molecular modeling revealed that two interacting pairs of residues, the LILI motif (GluN1(L657) and GluN2B(I655)), form a functional unit with the TTTT ring (GluN1(T648) and GluN2B(T647)), described earlier to control NMDAR channel gating. These results provide new insight into the structural organization and functional interplay of the LILI and the TTTT ring during the course of NMDAR channel opening and closing.</p

Helix/Coil Nucleation: A Local Response to Global Demands

A complete description of protein structure and function must include a proper treatment of mechanisms that lead to cooperativity. The helix/coil transition serves as a simple example of a cooperative folding process, commonly described by a nucleation-propagation mechanism. The prevalent view is that coil structure must first form a short segment of helix in a localized region despite paying a free energy cost (nucleation). Afterward, helical structure propagates outward from the nucleation site. Both processes entail enthalpy-entropy compensation that derives from the loss in conformational entropy on helix formation with concomitant gain in favorable interactions. Nucleation-propagation models inherently assume that cooperativity arises from a sequential series of local events. An alternative distance constraint model asserts there is a direct link between available degrees of freedom and cooperativity through the nonadditivity in conformational entropy. That is, helix nucleation is a concerted manifestation of rigidity propagating through atomic structure. The link between network rigidity and nonadditivity of conformational entropy is shown in this study by solving the distance constraint model using a simple global constraint counting approximation. Cooperativity arises from competition between excess and deficiency in available degrees of freedom in the coil and helix states respectively