Tight steric closure at the intracellular activation gate of a voltage-gated K(+) channel.

In voltage-gated K(+) channels (Kv), an intracellular gate regulates access from the cytoplasm to the pore by organic channel blockers and by chemical modifiers. But is ion flow itself controlled instead by constriction of the narrow selectivity filter near the extracellular surface? We find that the intracellular gate of Kv channels is capable of regulating access even by the small cations Cd(2+) and Ag(+). It can also exclude small neutral or negatively charged molecules, indicating that the gate operates by steric exclusion rather than electrostatically. Just intracellular to the gated region, channel closure does not restrict access even to very large reagents. Either these Kv channels have a broader inner entrance than seen in the KcsA crystal, even in the closed state, or the region is highly flexible (but nevertheless remains very securely closed nearby).     

Electrostatic control and chloride regulation of the fast gating of ClC-0 chloride channels

The opening and closing of chloride (Cl-) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as "fast gating" is regulated by both external and internal Cl- ions. Although the external Cl- effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl- regulation remains to be characterized. In this study, we examine the internal Cl- effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising [Cl-]i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of >1 M, a value very different from the one observed in the external Cl- regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties-the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of [Cl-]i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl- on the fast gating may also be due to the competition of Cl- with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl-. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of [Cl-]i and charges in the inner pore region. The very different nature of external and internal Cl- regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.     

Effects of intracellular K+ and Rb+ on gating of embryonic rat telencephalon Ca(2+)-activated K+ channels.

We have investigated the effects of intracellular K+ and Rb+ on single-channel currents recorded from the large-conductance Ca(2+)-activated K+ (BK) channel of the embryonic rat telencephalon using the inside-out patch-clamp technique. Our novel observation concerns the effects of these ions on rapid flickering of channel openings. Specifically, flicker gating was voltage dependent, i.e., it was reduced by depolarization in the -60 to -10 mV range with equimolar concentrations of K+ ions (150 Ko+/150 Ki+). Removal of Ki+ resulted in significant flickering at all potentials in this voltage range. In other words, the voltage dependence of flicker gating was effectively eliminated by the removal of Ki+. This suggests that a K+ ion entering the channel from the intracellular medium binds, in a voltage-dependent manner, at a site that locks the flicker gate in its open position. No effects of changes in Ki+ were observed on the primary, voltage-dependent gate of the channel. The change in flickering did not cause a change in the mean burst duration, which indicates that the primary gate is stochastically independent of the flicker gate. Intracellular Rb+ can substitute for--and is even more effective than--Ki+ with regard to suppression of flickering. Substitution of Rbi+ for Ki+ also increased the mean burst duration for V > or = -30 mV. Both effects of Rbi+ were removed by membrane hyperpolarization.     

Observations on the origin of the non-linear van't Hoff behaviour of polypeptides in hydrophobic environments.

In this paper we describe a general procedure to determine the thermodynamic parameters associated with the interaction of polypeptides or proteins with immobilised lipophilic compounds such as non-polar n-octyl groups. To this end, the binding behaviour of an all L-alpha-polypeptide, 1, and its retro-inverso-isomer, 2, has been investigated with an n-octylsilica and water-organic solvent mixture containing different percentages of acetonitrile or methanol over the temperature range of 278-338 K. The results confirm that non-linear van'ts Hoff plots occur with this pair of polypeptide isomers, depending on the solvent composition. These findings are consistent with the changes in the thermodynamic parameters, enthalpy of association, delta Hoassoc,i, entropy of association, delta Soassoc,i, and heat capacity, delta Cop,i, all having significant temperature dependencies. Theoretical relationship linking the changes in the delta Hoassoc,i, delta Soassoc,i and delta Cop,i values of these polypeptide-non-polar ligate systems, as a function of temperature, T, have been validated. Significant differences were observed in the magnitudes of these thermodynamic quantities when acetonitrile or methanol was employed as the organic solvent. The origin of these solvent-dependent effects can be attributed to the hydrogen-bonding propensity of the respective solvent. Involvement of enthalpy-entropy compensation effects associated with the interaction of these polypeptides with the hydrophobic ligates has also been documented. Analysis of empirical extra-thermodynamic relationships associated with molecular structural properties of these polypeptides, such as the slope term, S, derived from the plots of the logarithmic capacity factor, log k'i, of these polypeptides vs. the volume fraction of the organic solvent, [symbol: see text] as a function of temperature, T, has also revealed similar correlations in terms of the interactive behaviour of polypeptides 1 and 2 under these experimental conditions. These findings provide an extended thermodynamic and extra-thermodynamic framework to examine the solvational, conformational and other equilibrium processes that polypeptides (or proteins) can undergo in the presence of n-alkylsilicas or other classes of immobilised hydrophobic surfaces. The experimental approach utilised in this study with these topologically similar polypeptides thus represents a generic procedure to explore the behaviour of polypeptides or proteins in non-polar environments in terms of their molecular properties and the associated linear free energy relationships that determine their interactive behaviour.     

Effects of acute or chronic exposure to dietary organic anions on secretion of methotrexate and salicylate by Malpighian tubules of Drosophila melanogaster larvae

The effects of dietary exposure to organic anions on the physiology of isolated Malpighian tubules and on tubule gene expression were examined using larvae of Drosophila melanogaster. Acute (24 h) or chronic (7 d) exposure to type I organic anions (fluorescein or salicylate) was associated with increased fluid secretion rates and increased fluxes of both salicylate and the type II organic anion methotrexate. By contrast, chronic exposure to dietary methotrexate was associated with increased fluid secretion rate and increased flux of methotrexate, but not salicylate. Exposure to methotrexate in the diet resulted in increases in the expression of a multidrug efflux transporter gene (MET; CG30344) in the Malpighian tubules. There were also increases in expression of genes for either a Drosophila multidrug resistance–associated protein (dMRP; CG6214) or an organic anion transporting polypeptide (OATP; CG3380), depending on the concentration of methotrexate in the diet. Exposure to salicylate in the diet was associated with an increase in expression of dMRP and with decreases of MET and OATP. Exposure to dietary salicylate or methotrexate was also associated with different patterns of expression of heat shock protein genes. The results suggest that exposure to specific type I or type II organic anions has multiple effects and results not only in increased organic anion transport but also in increased rates of inorganic ion transport, which drives osmotically‐obliged fluid secretion. Increased fluid secretion may enhance secretion of organic anions by eliminating diffusive backflux from the tubule lumen to the hemolymph. © 2010 Wiley Periodicals, Inc.     

Bio-inspired photoresponse of porphyrin-attached gold nanoparticles on a field-effect transistor

A bio-inspired photoresponse was engineered in porphyrin-attached Au nanoparticles (AuNPs) on a field-effect transistor (FET). The system mimics photosynthetic electron transfer, using porphyrin derivatives as photosensitizers and AuNPs as photoelectron counting devices. Porphyrin-protected AuNPs were immobilized onto the gate of an FET via the formation of self-assembled monolayers. Photoinduced electron transfer from the porphyrin led to single electron transfer at the Au nanoparticles, which was monitored via a changing gate voltage on the FET in the presence of organic electrolyte. The further attachment of other functional molecules to this system should enable various other potential functionalities. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Coupling of activation and inactivation gate in a K+‐channel: potassium and ligand sensitivity

Potassium (K⁺)‐channel gating is choreographed by a complex interplay between external stimuli, K⁺ concentration and lipidic environment. We combined solid‐state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K⁺, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH‐induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K⁺ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K⁺ ion on the inactivation gate modulate activation‐gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K⁺‐channel pore.     

Fabrication and characteristics of MOSFET protein chip for detection of ribosomal protein

A metal oxide silicon field effect transistor (MOSFET) protein chip for the easy detection of protein was fabricated and its characteristics were investigated. Generally, the drain current of the MOSFET is varied by the gate potential. It is expected that the formation of an antibody-antigen complex on the gate of MOSFET would lead to a detectable change in the charge distribution and thus, directly modulate the drain current of MOSFET. As such, the drain current of the MOSFET protein chip can be varied by ribosomal proteins absorbed by the self-assembled monolayer (SAM) immobilized on the gate (Au) surface, as ribosomal protein has positive charge, and these current variations then used as the response of the protein chip. The gate of MOSFET protein chip is not directly biased by an external voltage source, so called open gate or floating gate MOSFET, but rather chemically modified by immobilized molecular receptors called self-assembled monolayer (SAM). In our experiments, the current variation in the proposed protein chip was about 8% with a protein concentration of 0.7 mM. As the protein concentration increased, the drain current also gradually increased. In addition, there were some drift of the drain current in the device. It is considered that these drift might be caused by the drift from the MOSFET itself or protein absorption procedures that are relied on the facile attachment of thiol (-S) ligands to the gate (Au) surface. We verified the formation of SAM on the gold surface and the absorption of protein through the surface plasmon resonance (SPR) measurement.     

Inactivation in HCN channels results from reclosure of the activation gate: desensitization to voltage

Hyperpolarization-activated HCN channels are modulated by direct binding of cyclic nucleotides. For HCN2 channels, cAMP shifts the voltage dependence for activation, with relatively little change in the maximal conductance. By contrast, in spHCN channels, cAMP relieves a rapid inactivation process and produces a large increase in maximum conductance. Our results suggest that these two effects of cAMP represent the same underlying process. We also find that spHCN inactivation occurs not by closure of a specialized inactivation gate, as for other voltage-dependent channels, but by reclosure of the same intracellular gate opened upon activation. Effectively, the activation gate exhibits a "desensitization to voltage," perhaps by slippage of the coupling between the voltage sensors and the gate. Differences in the initial coupling efficiency could allow cAMP to produce either the inactivation or the shift phenotype by strengthening effective coupling: a shift would naturally occur if coupling is already strong in the absence of cAMP.     

Analysing temperature response of decomposition of organic matter

In order to analyse temperature effects on decomposition of organic matter, we tested the following hypothesis: Can an Arrhenius type of equation with constant parameter values for the temperature response of decomposer growth rate adequately describe decomposition of organic matter or must some additional properties be made functions of temperature? Possible temperature effects were analysed by aggregating data in different ways from an experiment with ¹⁴C‐labelled wheat material incubated in the laboratory at different temperatures and with soil materials collected from seven coniferous forest stands in Europe. Our analysis shows that it is possible to let all the temperature dependence reside in the decomposer growth rate. The analysis also supports the use of an Arrhenius type of equation for the temperature response of decomposer growth rate but with the parameters specific for each soil, or at least a distinction between organic and mineral horizons seems necessary.     

Gates and oscillators II: zeitgebers and the network model of the brain clock

Circadian rhythms in physiology and behavior are regulated by the SCN. When assessed by expression of clock genes, at least 2 distinct functional cell types are discernible within the SCN: nonrhythmic, light-inducible, retinorecipient cells and rhythmic autonomous oscillator cells that are not directly retinorecipient. To predict the responses of the circadian system, the authors have proposed a model based on these biological properties. In this model, output of rhythmic oscillator cells regulates the activity of the gate cells. The gate cells provide a daily organizing signal that maintains phase coherence among the oscillator cells. In the absence of external stimuli, this arrangement yields a multicomponent system capable of producing a self-sustained consensus rhythm. This follow-up study considers how the system responds when the gate cells are activated by an external stimulus, simulating a response to an entraining (or phase-setting) signal. In this model, the authors find that the system can be entrained to periods within the circadian range, that the free-running system can be phase shifted by timed activation of the gate, and that the phase response curve for activation is similar to that observed when animals are exposed to a light pulse. Finally, exogenous triggering of the gate over a number of days can organize an arrhythmic system, simulating the light-dependent reappearance of rhythmicity in a population of disorganized, independent oscillators. The model demonstrates that a single mechanism (i.e., the output of gate cells) can account for not only free-running and entrained rhythmicity but also other circadian phenomena, including limits of entrainment, a PRC with both delay and advance zones, and the light-dependent reappearance of rhythmicity in an arrhythmic animal.     

Organic Field‐Effect Transistor for Energy‐Related Applications: Low‐Power‐Consumption Devices,Near‐Infrared Phototransistors,and Organic Thermoelectric Devices

The organic field‐effect transistor (OFET) is the basic building block of integrated circuits. The charge carrier mobility and operating frequency of OFETs have continued to increase; therefore, the power dissipation of OFETs can no longer be ignored. Many research efforts have been made to develop low‐power‐consumption OFETs and complementary circuits. Despite the switching function, OFETs can also be utilized in emerging energy‐related applications, such as near‐infrared (NIR) photodetectors and organic thermoelectric devices. Organic phototransistors show considerably higher photo responsivity than other photodetector architectures due to field‐effect charge modulation. The photoinduced gate modulating largely suppresses the dark current while simultaneously providing gain. These characteristics may favor NIR light detection and suggest that the organic phototransistor is a promising candidate for optoelectronic applications in the NIR regime. For organic thermoelectric applications, OFETs can work as a powerful tool for examining the charge and energy transport in the organic semiconductor, thus giving insight into organic thermoelectric studies. In this review, the authors highlight recent advances in OFET‐ related energy topics, including low‐power‐consumption OFETs, NIR photodetectors, and organic thermoelectric devices. The remaining challenges in the field will also be discussed.     

Organic Farming: Biodiversity Impacts Can Depend on Dispersal Characteristics and Landscape Context

Organic farming, a low intensity system, may offer benefits for a range of taxa, but what affects the extent of those benefits is imperfectly understood. We explored the effects of organic farming and landscape on the activity density and species density of spiders and carabid beetles, using a large sample of paired organic and conventional farms in the UK. Spider activity density and species density were influenced by both farming system and surrounding landscape. Hunting spiders, which tend to have lower dispersal capabilities, had higher activity density, and more species were captured, on organic compared to conventional farms. There was also evidence for an interaction, as the farming system effect was particularly marked in the cropped area before harvest and was more pronounced in complex landscapes (those with little arable land). There was no evidence for any effect of farming system or landscape on web-building spiders (which include the linyphiids, many of which have high dispersal capabilities). For carabid beetles, the farming system effects were inconsistent. Before harvest, higher activity densities were observed in the crops on organic farms compared with conventional farms. After harvest, no difference was detected in the cropped area, but more carabids were captured on conventional compared to organic boundaries. Carabids were more species-dense in complex landscapes, and farming system did not affect this. There was little evidence that non-cropped habitat differences explained the farming system effects for either spiders or carabid beetles. For spiders, the farming system effects in the cropped area were probably largely attributable to differences in crop management; reduced inputs of pesticides (herbicides and insecticides) and fertilisers are possible influences, and there was some evidence for an effect of non-crop plant species richness on hunting spider activity density. The benefits of organic farming may be greatest for taxa with lower dispersal abilities generally. The evidence for interactions among landscape and farming system in their effects on spiders highlights the importance of developing strategies for managing farmland at the landscape-scale for most effective conservation of biodiversity.     

Ion channel gating: insights via molecular simulations

Ion channels are gated, i.e. they can switch conformation between a closed and an open state. Molecular dynamics simulations may be used to study the conformational dynamics of ion channels and of simple channel models. Simulations on model nanopores reveal that a narrow (<4 A) hydrophobic region can form a functionally closed gate in the channel and can be opened by either a small (approximately 1 A) increase in pore radius or an increase in polarity. Modelling and simulation studies confirm the importance of hydrophobic gating in K channels, and support a model in which hinge-bending of the pore-lining M2 (or S6 in Kv channels) helices underlies channel gating. Simulations of a simple outer membrane protein, OmpA, indicate that a gate may also be formed by interactions of charged side chains within a pore, as is also the case in ClC channels.     

Decreased rate of sodium conductance inactivation in the node of Ranvier induced by a polypeptide toxin from sea anemone.

The effects of two toxins extracted from the tentacles of Anemonia sulcata on ionic currents have been tested on the nodal membrane of myelinated nerve fibres from Rana esculenta. While external application of Toxin I at 100 muM leaves both specific ionic currents unmodified, Toxin II at 10 muM reacts with a receptor site associated with the sodium conductance inactivation gating. Since internal application by diffusion of Toxin II at a concentration of 700 muM leaves the ionic currents unchanged, the receptor site is most likely located on the external side of the nodal membrane. An equilibrium dissociation constant for the effects of Toxin II was estimated as 20 muM. The on-reaction is fast (rate constant for the on-reaction roughly equal to 3.103 M-1) suggesting a readily accesible receptor site for the toxin. The kinetics characteristics of the sodium currents recorded in the presence of Toxin II suggest that there are at least two steps in the reaction leading to Na+ -channels with the inactivation gate completely immobilized. The relatively fast reversibility of the intermediate stage of the reaction and the rather slow but, in the end, complete reversal of the toxin effects suggest that the toxin acts by modifying the energy profile for the transition "inactivation gate in the open configuration to inactivation gate in the closed configuration." Toxin II at higher concentrations (greater than 100 muM) also inhibits the potassium currents but these effects were not studied in any detail.     

Structural determinants of lateral gate opening in the protein translocon

The heterotrimeric SecY/Sec61 complex is a protein-conducting channel that provides a passage for proteins across the membrane as well as a means to integrate nascent proteins into the membrane. While the first function is common among membrane protein channels and transporters, the latter is unique. Insertion of nascent membrane proteins, one transmembrane segment at a time, by SecY likely occurs through a lateral gate in the channel. Molecular dynamics simulations have been used to investigate the mechanism of gate opening. Opening and closing the gate under different conditions allowed us to identify structural elements that resist opening as well as those that aid closure. SecE, considered to act as a clamp keeping the lateral gate closed, was found to play no such role. Loosening of the plug by lateral gate opening, a potential step in channel gating, was also observed. The simulations revealed that lipids on time scales of up to 1 micros do not flood channels with an open lateral gate.     

溶解性有机质对土壤中有机污染物环境行为的影响

土壤中溶解性有机质(DOM)是生物活性和物理化学反应活性都很活跃的有机组分,主要通过疏水吸附、分配、氢键、电荷转移、共价键、范德华力等多种作用与有机污染物结合,提高溶液中有机污染物的溶解度,改变土壤中有机污染物的吸附-解吸、迁移-转化等环境行为．DOM对有机污染物的吸附-解吸、迁移-转化过程的影响有双重性：一方面,DOM与有机污染物在土壤表面的共吸附可增加土壤对有机污染物的吸附容量,促进有机污染物在土壤中的吸持;另一方面,DOM对有机污染物的增溶作用,有利于土壤中有机污染物的解吸,提高移动性．作为光敏剂,DOM能提高土壤中有机物的光解反应速率．在一定条件下,DOM也可影响土壤中有机污染物的水解过程．DOM对土壤中有机污染物环境行为的影响与DOM和有机污染物的性质及其相互作用的介质条件密切相关．     

Enzyme field effect transistor (ENFET) for estimation of triglycerides using magnetic nanoparticles

Ion-selective field effect transistor (ISFET) is a robust platform to develop biosensors. A variety of methods are used including covalent attachment or polymer entrapment, to associate enzymes or antibodies to the gate surface of a FET. We have employed a novel method of retaining the enzyme molecules at the gate surface by immobilizing the enzyme on magnetic nickelferrite nanoparticles and applying a permanent magnet below the gate of the FET. We were able to estimate the triglyceride concentrations in the range of 0.1–1.5% by immobilizing a thermostable lipase on nanoparticles. Tributyrin, trioctanoate and triolein have given similar results. The reaction volume could be scaled down to 0.2 ml without a loss in slope or sensitivity. Ionic strength (>150 mM NaCl) has a strong influence on the sensitivity of the measurement. The advantages of this configuration of enzyme biosensor are reduction of mass transfer problems, increasing the amount of enzyme at the gate surface besides providing an opportunity to use a single FET device for multiple analyte detection.     

植物、土壤及土壤管理对土壤微生物群落结构的影响

土壤微生物是土壤生态系统的重要组成部分,对土壤微生物群落结构多样性的研究是近年来土壤生态学研究的热点。本文综述了有关植物、土壤类型以及土壤管理措施对土壤微生物群落结构影响的最新研究结果,指出植物的作用因植物群落结构多样性、植物种类、同种植物不同的基因型,甚至同一植物不同根的区域而异;而土壤的作用与土壤质地和有机质含量等因素有关;植物和土壤类型在对土壤微生物群落结构影响上的作用存在互作关系。不同的土壤管理措施对土壤微生物群落结构影响较大,长期连作、大量的外援化学物质的应用降低了土壤微生物的多样性;而施用有机肥、免耕可以增加土壤微生物群落结构多样性,有利于维持土壤生态系统的功能。     

Immobilizing the moving parts of voltage-gated ion channels

Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzophenone-4-carboxamidocysteine methanethiosulfonate, that can be tethered to cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium channel. The benzophenone-labeled inactivation gate of the sodium channel can be trapped selectively either in an open or closed state by ultraviolet irradiation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depolarization moves the benzophenone adduct out of a restricted environment. Immobilization of the S4 segment of the second domain of sodium channels prevents channels from opening. By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation. Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.