短杆菌肽A-DMPC通道内离子输运的分子动力学模拟

用最近提出的构建膜体系初始构象的有效方法 ,构建了在DMPC脂膜环境下短杆菌肽A通道模型 (GA -DMPC)。通过对Na 、Ca2 、Cl-三种不同离子在GA -DMPC通道内不同位置的分子动力学模拟 ,研究离子在通道内输运过程中与通道及通道内水分子的相互作用 ,从分子动力学的角度阐明离子在通道内的输运机制。主要计算结果表明 :(1)离子在通道内的输运使GA的构象发生变化 ,GA的柔性是离子在通道内通透的重要因素 ;(2)Cl- 离子可扩大通道半径 ,Na 离子和Ca2 离子则减小通道半径。Cl-离子不能在GA通道内通透 ;(3)离子的出现使通道内水分子的偶极方向发生变化。上述结果均与实验相符。     

Conductance channels in neutral lipid bilayers

Summary A model channel for the conduction of ions (positive or negative but not both) through a lipid bilayer is presented. The transition-state theory is used to relate the current with voltage and ionic concentrations. Sites within the channel are considered to act cooperatively so that the ion is subjected to a ligand field in which it has complete freedom along the channel axis. The ions in the channel are treated as an ionic gas. Effects due to space-charges within the channel arising from the conducting ions are considered whereas surface-charge effects are neglected.The ionic specificity of the channel is indicated and the theory compared to that in which equilibrium free energy changes are the dominant influence.     

Mechanism of blocking K+-channels with tetraethylammonium

Using the patch-voltage-clamp method action of tetraethylammonium on the fast (30 pS) and slow K+ channels was investigated. The slow K+ channels were presented by two types: with whole (30 pS) and decreased (20 pS) conductance. In all cases tetraethylammonium decreased the current magnitude and modified the channel kinetic parameters. Apparent blocking constants determined from the current decreasing are as 8-50 and 4-12 mM for the slow K+ channels with whole and decreased conductance, respectively, and 0.05-0.08 mM--for the fast K+ channel. The potential dependency of the blocking constants correlates with that of the channel conductance. Probability of the channel open state for the slow K+ channels decreases, and that for the fast K+ channel increases under application of tetraethylammonium. It is concluded that there are two sites of tetraethylammonium binding: the first site is into the channel pore, and the second one--into the regulatory centre responsible for the channel kinetic behaviour. Blocking of general conductance of the slow channels is accompanied by proportional decrease of the channel substate conductances without change of their number and cooperatively. Block of the fast K+ channel occurs without change of the channel elementary conductance but with decrease of the number of the channel substates and reversible violation of the channel transition cooperativity. The data are discussed from the point of the hypothesis on the channel clustery organization.     

Single potential-dependent K+ channels and their oligomers in molluscan glial cells

Single K+ channels were studied using the patch-clamp method. A potential-dependent K+ channel of large conductance (about 100 pS at 100 mM of KCl on both membrane sides) was detected. Some properties of the channel (current-voltage relations, kinetic parameters, etc.) are presented. The channel was found to have about 16 resolvable quantized conductance substates. The data are confirmed by spontaneous channel degradation, i.e., spontaneous splitting of the channel conductance into independent conductance oligomers. Some properties of the conductance oligomers of different order are described. The degree of potential dependency of the conductance oligomer parameters is a function of potential dependency. The data obtained are in agreement with a hypothesis that the channels studied are clusters (aggregates) of elementary channel subunits.     

Four cases of direct ion channel gating by cyclic nucleotides

Four different nucleotide-gated ion channels are discussed in terms of their biophysical properties and their importance in cell physiology. Channels activated directly by cGMP are present in vertebrate and invertebrate photoreceptors. In both cases cGMP increases the fraction of time the channel remains in the open state. At least three cGMP molecules are involved in channel opening in vertebrate photoreceptors and the concentration of the cyclic nucleotide to obtain the half maximal effect is about 15 µM. The light-dependent channel of both vertebrates and invertebrates is poorly cation selective. The vertebrate channel allows divalent cations to pass through 10–15-fold more easily than monovalent ions. In agreement with their preference for divalent cations, this channel is blocked byl-cis Dialtazem, a molecule that blocks certain types of calcium channels. In olfactory neurons a channel activated by both cAMP and cGMP is found and, as in the light-dependent channel, several molecules of the nucleotide are needed to open the channel with a half maximal effect obtained in the range of 1–40 µM. The channel is poorly cationic selective. A K⁺ channel directly and specifically activated by cAMP is found inDrosophila larval muscle. At least three cAMP molecules are involved in the opening reaction. Half-maximal effect is obtained at about 50 µM. This channel is blocked by micromolar amount of tetraethylammonium applied internally. Interestingly, this channel has a probability of opening 10–20-fold larger in the mutantdunce, a mutant that possesses abnormally elevated intracellular cAMP level, than in the wild type.     

The plug domain of the SecY protein stabilizes the closed state of the translocation channel and maintains a membrane seal

Proteins are translocated across membranes through a channel that is formed by the prokaryotic SecY or eukaryotic Sec61 complex. The crystal structure of the SecY channel from M. jannaschii revealed a plug domain that appears to seal the channel in its closed state. However, the role of the plug remains unclear, particularly because plug deletion mutants in S. cerevisiae are functional. Here, we demonstrate that plug deletion mutants in E. coli SecY are also functional and even efficiently translocate proteins with defective or missing signal sequences. The crystal structures of equivalent plug deletions in SecY of M. jannaschii show that, although the overall structures are maintained, new plugs are formed. These lack many interactions that normally stabilize the closed channel, explaining why the channels can open for proteins with signal-sequence mutations. Our data show that the plug domain is required to maintain a closed state of the channel and suggest a mechanism for channel gating.     

The action of a carbonsuboxide dimerized gramicidin A on lipid bilayer membranes

Gramicidin A was dimerized with carbonsuboxide as bifunctional reagent. The effect of the resulting malonyl-bis-desformylgramicidin on lipid bilayer membranes was investigated and compared with the effect of the monomer gramicidin. It was found that the single channel conductance and the ion selectivity are very similar to the behaviour of the monomer molecule, whereas the channel forming kinetics and the life time of the single channel of the malonyl-bis-desformylgramicidin differ strongly from the behaviour of the monomer gramicidin.The electrical relaxations are very small and possibly associated with some structural changes of the membrane after a voltage jump. The single channel lifetime of the malonyl-bis-desformylgramicidin is measured in minutes, whereas for the same lipid system the single channel lifetime in the case of the monomer gramicidin is restricted to 1–2 s. It is concluded that the malonyl-bis-desformylgramicidin-molecule itself (as a single molecule) forms an ionic channel without further association.     

The envelope anion channel involved in chloroplast protein import is associated with Tic110.

An anion channel of the chloroplast envelope was previously shown to be involved in protein import. Some gating characteristics of the channel are presented. The pore size of the channel is estimated to be around 6.5 A. Antibodies raised to Tic110 completely inactivate the protein import-related channel. These observations suggest that the channel is associated with the Tic machinery and can function as the protein conducting channel of the inner envelope membrane.     

Alamethicin channel kinetics: Studies using fluctuation analysis and multifractal fluctuation analysis

The kinetics of the alamethicine channel incorporated into BLM was studied using detrended fluctuation analysis and multifractal detrended fluctuation analysis. Detrended fluctuation analysis has shown that both the event series formed with dwell times on fixed channel conductivity levels and the event series formed with opened states on all channel conductivity levels are random processes. However, multifractal detrended fluctuation analysis indicated that only dwell times series on fixed channel conductivity levels are random, but the event series formed with opened states on all channel conductivity levels are the correlated multifractal processes.     

Permeation of ions across the potassium channel: brownian dynamics studies

The physical mechanisms underlying the transport of ions across a model potassium channel are described. The shape of the model channel corresponds closely to that deduced from crystallography. From electrostatic calculations, we show that an ion permeating the channel, in the absence of any residual charges, encounters an insurmountable energy barrier arising from induced surface charges. Carbonyl groups along the selectivity filter, helix dipoles near the oval chamber, and mouth dipoles near the channel entrances together transform the energy barrier into a deep energy well. Two ions are attracted to this well, and their presence in the channel permits ions to diffuse across it under the influence of an electric field. Using Brownian dynamics simulations, we determine the magnitude of currents flowing across the channel under various conditions. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength causes a steady decrease in the channel conductance. The current also decreases systematically when the effective dielectric constant of the channel is lowered. The conductance with the optimal choice of dipoles reproduces the experimental value when the dielectric constant of the channel is assumed to be 60. The current-voltage relationship obtained with symmetrical solutions is linear when the applied potential is less than approximately 100 mV but deviates from Ohm's law at a higher applied potential. The reversal potentials obtained with asymmetrical solutions are in agreement with those predicted by the Nernst equation. The conductance exhibits the saturation property observed experimentally. We discuss the implications of these findings for the transport of ions across the potassium channels and membrane channels in general.     

Disulfonic stilbene permeation and block of the anion channel from the sarcoplasmic reticulum of rabbit skeletal muscle

Block of a sarcoplasmic reticulum anion channel (SCl channel) by disulfonic stilbene derivatives [DIDS, dibenzamidostilbene-2,2'-disulfonic acid (DBDS), and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)] was investigated in planar bilayers using SO4(2-) as the conducting ion. All molecules caused reversible voltage-dependent channel block when applied to either side of the membrane. DIDS also produced nonreversible channel block from both sides within 1-3 min. Reversible inhibition was associated with a decrease in channel open probability and mean open duration but not with any change in channel conductance. The half inhibitory concentration for cis- and trans-inhibition had voltage dependencies with minima of 190 nM and 33 microM for DBDS and 3.4 and 55 microM for DNDS. Our data supports a permeant blocker mechanism, in which stilbenes block SCl channels by lodging in the permeation pathway, where they may dissociate to either side of the membrane and thus permeate the channel. The stilbenes acted as open channel blockers where the binding of a single molecule occludes the channel. DBDS and DNDS, from opposite sides of the membrane, competed for common sites on the channel. Dissociation rates exhibited biphasic voltage dependence, indicative of two dissociation processes associated with ion movement in opposite directions within the trans-membrane electric field. The kinetics of DNDS and DBDS inhibition predict that there are two stilbene sites in the channel that are separated by 14-24 A and that the pore constriction is approximately 10 A in diameter.     

Low-conductance chloride channel from crayfish skeletal muscle incorporated into planar lipid bilayers.

Low-conductance chloride channel from skeletal muscle SR vesicles of the crayfish Astacus fluviatilis was incorporated into planar lipid bilayers and its basic characteristics were investigated. The channel has a relatively low unitary conductance of 26 pS in symmetrical 160 mmol/l choline-chloride. The dependence of the channel conductance on Cl- concentration shows saturating behavior with a maximum conductance of 37 pS and an ionic activity for half-maximum conductance Km = 75 mmol/l. The channel exhibits a complex kinetics with several modes of activity. Open state probability slightly decreases with the increasing absolute value of voltage. The channel activity does not appear to be dependent on the presence of Ca2+ ions. The channel is effectively inhibited by DIDS, a stilbene derivative. The permeability properties of the channel are similar to the specific behavior of the "double-barrelled" channel from Torpedo electroplax described by Miller and White (1984).     

The action of a carbonsuboxide dimerized gramicidin A on lipid bilayer membranes.

Gramicidin A was dimerized with carbonsuboxide as bifunctional reagent. The effect of the resulting malonyl-bis-desformylgramicidin on lipid bilayer membranes was investigated and compared with the effect of the monomer gramicidin. It was found that the single channel conductance and the ion selectivity are very similar to the behaviour of the monomer molecule, whereas the channel forming kinetics and the life time of the single channel of the malonyl-bis-desformylgramicidin differ strongly from the behaviour of the monomer gramicidin. The electrical relaxations are very small and possibly associated with some structural changes of the membrane after a voltage jump. The single channel lifetime of the malonyl-bis-desformylgramicidin is measured in minutes, whereas for the same lipid system the single channel lifetime in the case of the monomer gramicidin is restricted to 1-2 s. It is concluded that the malonyl-bis-desformylgramicidin-molecule itself (as a single molecule) forms an ionic channel without further association.     

Biophysical characterization of a large conductance anion channel in hypodermal membranes of the gastrointestinal nematode,Ascaris suum

Patch-clamp recordings from muscle- and cuticle-facing hypodermal membranes of the gastrointestinal nematode Ascaris suum reveal a high-conductance, voltage- sensitive Ca(2+) -dependent Cl(-) channel. The hypodermal channel has a conductance of 195 pS in symmetrical 160 mM NaCl. The open probability of the channel is highly voltage-sensitive, and channel activity is not observed when Ca(2+) is reduced to <100 microM. The channel is permeable to organic anions that are major end-products of carbohydrate metabolism in A. suum, including acetate, butyrate and 2-methylvalerate. The conductances and relative permeabilities of these organic anions are inversely related to size, with 2-methylvalerate being only approximately 3% as permeable as Cl(-). The diameter of the channel pore was 12.3+/-0.2 A, calculated from the relative permeability coefficients of Cl(-) and the organic anions. Results of this study are consistent with the hypothesis that the large conductance anion channel in A. suum hypodermal membranes provides a low energy pathway for organic anion excretion from the hypodermal compartment, followed by diffusion across the aqueous channels of the cuticle matrix.     

Probes of the conduction process of a voltage-gated Cl- channel from Torpedo electroplax

The open-channel conductance properties of a voltage-gated Cl- channel derived from Torpedo californica electroplax and incorporated into planar bilayers were studied by several approaches. In neutral bilayers the channel conductance saturates with Cl- activity according to a rectangular hyperbolic relation with a half-saturation activity of 75 mM and a maximum conductance of 32 pmho. The observation of identical behavior in charged membranes implies that ions permeating the channel do not sense the surface potential of the bulk membrane. The Cl-:Br- permeability ratio, measured under biionic conditions, is independent of salt concentration. SCN- ion reversibly blocks the channel. The voltage dependence of the block implies the existence of two separate blocking sites within the channel: one accessible from the cis side only (the side to which vesicles are added) and the other accessible from the trans side only. The block at each site is competitive with Cl-. The results are consistent with a single-ion Eyring model of the conduction process in which the ion must traverse three kinetic barriers as it permeates the channel and in which the channel can accommodate at most one ion at a time.     

Hierarchical approach to predicting permeation in ion channels

A hierarchical computational strategy combining molecular modeling, electrostatics calculations, molecular dynamics, and Brownian dynamics simulations is developed and implemented to compute electrophysiologically measurable properties of the KcsA potassium channel. Models for a series of channels with different pore sizes are developed from the known x-ray structure, using insights into the gating conformational changes as suggested by a variety of published experiments. Information on the pH dependence of the channel gating is incorporated into the calculation of potential profiles for K(+) ions inside the channel, which are then combined with K(+) ion mobilities inside the channel, as computed by molecular dynamics simulations, to provide inputs into Brownian dynamics simulations for computing ion fluxes. The open model structure has a conductance of approximately 110 pS under symmetric 250 mM K(+) conditions, in reasonable agreement with experiments for the largest conducting substate. The dimensions of this channel are consistent with electrophysiologically determined size dependence of quaternary ammonium ion blocking from the intracellular end of this channel as well as with direct structural evidence that tetrabutylammonium ions can enter into the interior cavity of the channel. Realistic values of Ussing flux ratio exponents, distribution of ions within the channel, and shapes of the current-voltage and current-concentration curves are obtained. The Brownian dynamics calculations suggest passage of ions through the selectivity filter proceeds by a "knock-off" mechanism involving three ions, as has been previously inferred from functional and structural studies of barium ion blocking. These results suggest that the present calculations capture the essential nature of K(+) ion permeation in the KcsA channel and provide a proof-of-concept for the integrated microscopic/mesoscopic multitiered approach for predicting ion channel function from structure, which can be applied to other channel structures.     

The binding site of sodium in the gramicidin A channel: comparison of molecular dynamics with solid-state NMR data.

The location of the main binding site for sodium in the gramicidin A (GA) channel was investigated with molecular dynamics simulations, using an atomic model of the channel embedded in a fully hydrated dimyristoyl phosphatidycholine (DMPC) bilayer. Twenty-four separate simulations in which a sodium was restrained at different locations along the channel axis were generated. The results are compared with carbonyl 13C chemical shift anisotropy solid-state NMR experimental data previously obtained with oriented GA:DMPC samples. Predictions are made for other solid-state NMR properties that could be observed experimentally. The combined information from experiment and simulation strongly suggests that the main binding sites for sodium are near the channel's mouth, approximately 9.2 A from the center of the dimer channel. The 13C chemical shift anisotropy of Leu10 is the most affected by the presence of a sodium ion in the binding site. In the binding site, the sodium ion is lying off-axis, making contact with two carbonyl oxygens and two single-file water molecules. The main channel ligand is provided by the carbonyl group of the Leu10-Trp11 peptide linkage, which exhibits the largest deviation from the ion-free channel structure. Transient contacts with the carbonyl group of Val8 and Trp15 are also present. The influence of the tryptophan side chains on the channel conductance is examined based on the current information about the binding site.     

Binding of the cGMP-gated channel to the Na/Ca-K exchanger in rod photoreceptors

The intracellular Ca(2+) concentration in rod outer segments of vertebrate photoreceptors is controlled by Ca(2+) influx through cGMP-gated channels and by Ca(2+) efflux driven by Na/Ca-K exchangers. Previously, we suggested that channel and exchanger are associated (Bauer, P. J., and Drechsler, M. (1992) J. Physiol. (Lond. ) 451, 109-131). This suggestion has been thoroughly examined using a variety of biochemical approaches. First, we took advantage of the fact that cGMP-gated channels bind calmodulin (CaM). Using CaM affinity chromatographic purification of the channel in 10 mm CHAPS, a significant fraction of exchanger was co-eluted with the channel indicating a binding affinity between channel and exchanger. Binding of channel and exchanger was examined more directly by cross-linking of proteins in the rod outer segment membranes. Activation of the channel with cyclic 8-bromo-GMP lead to exposure of a cysteine, which allowed cross-linking of the channel to the exchanger with the thiol-specific reagent dl-1,4-bismaleimido-2,3-butanediol. Cleavage of the cross-links and electrophoretic analysis indicated that a cross-link between the alpha-subunit of the channel and the exchanger formed. Furthermore, a cross-link between two adjacent alpha-subunits of the channel was found, suggesting that the alpha-subunits of the native channel are dimerized. Further support for an interaction between alpha-subunit and exchanger was obtained by in vitro experiments. Specific binding of the exchanger to the alpha-subunit but not to the beta-subunit of the channel was observed in Western blots of purified channel incubated with purified exchanger. This study suggests that two exchanger molecules bind to one cGMP-gated channel and, more specifically, that binding of exchanger molecules occurs at the alpha-subunits, which in the native channel are dimerized. The implications of these findings regarding the possibility of local Ca(2+) signaling in vertebrate photoreceptors will be discussed.     

The influence of tidal channels on the distribution of salt marsh plant species in Petaluma Marsh,CA, USA

Tidal channels influence the distribution and composition of salt marsh vegetation in a San Francisco Bay salt marsh. Two channel networks in the Petaluma Marsh, Sonoma County, CA, were mapped and characterized using global positioning and geographic information systems. Plant species abundance was sampled on transects placed perpendicular to and extending away from the channel banks. The vegetation showed significant increases in species richness along channel banks and larger areas of effect which increased approximately linearly with channel size. Composition of species assemblages varies with distance from the channel bank and channel size. These results demonstrate that salt marsh plant assemblages, composed of both major and minor species, are distributed with respect to the channel network in Petaluma Marsh.