Conformational changes involved in MscL channel gating measured using FRET spectroscopy

We demonstrate that fluorescence resonance energy transfer spectroscopy is a powerful tool for in situ structural analysis of multimeric membrane proteins by measuring the conformational changes involved in gating the mechanosensitive ion channel of large conductance. Ensemble analysis is used to analyze the intensity of light emitted by AlexaFluor-labeled cysteine mutants reconstituted into artificial liposomes before and after acceptor photobleaching. The diameter of the protein is found to increase by 16 angstrom upon channel activation.

Scaling of decoherence effects in quantum computers

The scaling of decoherence rates with qubit number N is studied for a simple model of a quantum computer in the situation where N is large. The two state qubits are localized around well-separated positions via trapping potentials and vibrational centre of mass motion of the qubits occurs. Coherent one and two qubit gating processes are controlled by external classical fields and facilitated by a cavity mode ancilla. Decoherence due to qubit coupling to a bath of spontaneous modes, cavity decay modes and to the vibrational modes is treated. A non-Markovian treatment of the short time behaviour of the fidelity is presented, and expressions for the characteristic decoherence time scales obtained for the case where the qubit/cavity mode ancilla is in a pure state and the baths are in thermal states. Specific results are given for the case where the cavity mode is in the vacuum state and gating processes are absent and the qubits are in (a) the Hadamard state (b) the GHZ state.

Endogenous Presentation of CD8+ T Cell Epitopes from Epstein-Barr Virus–encoded Nuclear Antigen 1

Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type-dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8(+) T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8(+) T cell epitopes front EBNA1.

Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

Neuroprotectant effects of iso-osmolar D-mannitol to prevent Pacific ciguatoxin-1 induced alterations in neuronal excitability: A comparison with other osmotic agents and free radical scavengers

The basis for the neuroprotectant effect of D-mannitol in reducing the sensory neurological disturbances seen in ciguatera poisoning, is unclear. Pacific ciguatoxin-1 (P-CTX-1), at a concentration 10 nM, caused a statistically significant swelling of rat sensory dorsal root ganglia (DRG) neurons that was reversed by hyperosmolar 50 MM D-mannitol. However, using electron paramagnetic resonance (EPR) spectroscopy, it was found that P-CTX-1 failed to generate hydroxyl free radicals at concentrations of toxin that caused profound effects on neuronal excitability. Whole-cell patch-clamp recordings from DRG neurons revealed that both hyper- and iso-osmolar 50 MM D-mannitol prevented the membrane depolarisation and repetitive firing of action potentials induced by P-CTX-1. In addition, both hyper- and iso-osmolar 50 MM D-mannitol prevented the hyperpolarising shift in steady-state inactivation and the rise in leakage current through tetrodotoxin (TTX)-sensitive Na-v channels, as well as the increased rate of recovery from inactivation of TTX-resistant Nav channels induced by P-CTX-1. D-Mannitol also reduced, but did not prevent, the inhibition of peak TTX-sensitive and TTX-resistant I-Na amplitude by P-CTX-1. Additional experiments using hyper- and isoosmolar D-sorbitol, hyperosmolar sucrose and the free radical scavenging agents Trolox (R) and L-ascorbic acid showed that these agents, unlike D-mannitol, failed to prevent the effects of P-CTX-1 on spike electrogenesis and Na-v channel gating. These selective actions of D-mannitol indicate that it does not act purely as an osmotic agent to reduce swelling of nerves, but involves a more complex action dependent on the Nav channel subtype, possibly to alter or reduce toxin association. (c) 2005 Elsevier Ltd. All rights reserved.

The effects of parabens on the mechanosensitive channels of E. coli

Parabens are alkyl esters of p-hydroxybenzoic acid used as preservatives in a wide range of food, pharmaceutical, and cosmetic products (Soni et al. Food Chem. Toxicol. 39:513-532, 2001). Despite their common use for over 50 years, their mechanism of action is still unclear. In this study we examined the effects of ethyl and propyl paraben, on gating of the E. coli mechanosensitive channel of large conductance (MscL) reconstituted into azolectin liposomes. We found that propyl and ethyl paraben spontaneously activate MscL. Moreover, the addition of propyl paraben caused an increase in MscL activity and the lowering Of P-1/2, the pressure at which the MscL was opened 50% of the time, the AGO, the free energy required to open the MscL, and the parameter a, which describes the channel sensitivity to pressure. In addition, in silico studies showed that propyl paraben binds to the channel gate of the MscL. The mechanosensitive channel of small conductance was also found to be spontaneously activated by parabens. In summary, our study indicates that one of the previously unidentified mechanisms of action of parabens as antimicrobial agents is via an interaction with the mechanosensitive channels to upset the osmotic gradients in bacteria.

A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop.

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg(271) Lys(276)) toward the N-terminal end of the homomeric alpha 1 GlyR M2 - M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound ( MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr(6') residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2 - M3 loop are mediated allosterically. This suggests that the M2 - M3 loop responds differently to the occupation of different binding sites.

Block of voltage-gated potassium channels by Pacific ciguatoxin-1 contributes to increased neuronal excitability in rat sensory neurons

The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-clamp recording techniques. Under current-clamp conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Na-v,.) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Na-v channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons, P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Na-v currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-clamp conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that Occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP. and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Na-v. channel gating, observed clinically in response to ciguatera poisoning. (c) 2004 Elsevier Inc. All rights reserved.

Ligand-gated channels

Ligand-gated ion channels (LGICs) are fast-responding channels in which the receptor, which binds the activating molecule (the ligand), and the ion channel are part of the same nanomolecular protein complex. This paper will describe the properties and functions of the nicotinic acetylcholine LGIC superfamily, which plays a critical role in the fast chemical transmission of electrical signals between nerve cells and between nerve and muscle cells. The superfamily will mainly be exemplified by the excitatory nicotinic acetylcholine receptor (nAChR) and the inhibitory glycine receptor (GlyR) channels.

Structural plasticity in MS channels

Gating of the mechanosensitive channel MscS involves cooperative action of glycine and alanine residues along the pore-lining transmembrane helix. Opening of the channel is facilitated by an iris-like rotation and tilt of the pore-lining helices. Site-directed mutagenesis indicates that substantial structural plasticity can be tolerated by MscS without impairing its function.

Behavioural characterization of Vitamin D receptor knockout mice

Vitamin D (calcitriol) is a nuclear transcription regulator acting via a nuclear hormone receptor (VDR). In addition to its role in the regulation of calcium and phosphate horneostasis and in bone formation, Vitamin D is also thought to be involved in brain function. The aim of this study was to behaviourally phenotype VDR knockout mice. We characterized the behaviour of VDR null mutant mice and wildtype littermate controls by subjecting them to a range of tests including a primary behavioural screen (using the SHIRPA protocol), rotarod, gait analysis, Y-maze, marble burying test, bedding test, holeboard test, elevated plus maze, open field test and prepulse inhibition of the acoustic startle response. There were no effects of genotype on most of the scores from the SHIRPA protocol except that VDR -/- mice had alopecia, were shorter and weighed less than VDR +/+ mice. VDR -/- mice had a shorter gait as well as impairments on the rotarod, in the bedding test and impaired habituation in both the open field and on the acoustic startle response. The VDR -/- mice had normal acoustic startle responses but had impaired PPI at long (256 ms) but not short (64 ms) prepulse to pulse intervals. The VDR -/- mice were less active in the open field and buried fewer marbles in the marble burying test. However, there were no differences in the time spent on the open arms of the elevated plus maze or in working memory as assessed by repeat arm entries on the Y-maze. Therefore, it appears that VDR -/- mice have muscular and motor impairments that significantly affects locomotor behaviour but seemingly no impairments in cognition as indicated by exploration, working memory or anxiety. (C) 2004 Elsevier B.V. All rights reserved.