Voltage-regulated water flux through aquaporin channels in silico

Aquaporins (AQPs) facilitate the passive flux of water across biological membranes in response to an osmotic pressure. A number of AQPs, for instance in plants and yeast, have been proposed to be regulated by phosphorylation, cation concentration, pH change, or membrane-mediated mechanical stress. Here we report an extensive set of molecular dynamics simulations of AQP1 and AQP4 subject to large membrane potentials in the range of ±1.5 V, suggesting that AQPs may in addition be regulated by an electrostatic potential. As the regulatory mechanism we identified the relative population of two different states of the conserved arginine in the aromatic/arginine constriction region. A positive membrane potential was found to stabilize the arginine in an up-state, which allows rapid water flux, whereas a negative potential favors a down-state, which reduces the single-channel water permeability.     

Correlated ion flux through parallel pores: Application to channel subconductance states

Summary Many ion channels that normally gate fully open or shut have recently been observed occasionally to display well-defined subconductance states with conductances much less than those of the fully open channel. One model of this behavior is a channel consisting of several parallel pores with a strong correlation between the flux in each pore such that, normally, they all conduct together but, under special circumstances, the pores may transfer to a state in which only some of them conduct. This paper introduces a general technique for modeling correlated pores, and explores in detail by computer simulation a particular model based upon electric interaction between the pores. Correlation is obtained when the transient electric field of ions passing through the pores acts upon a common set of ionizable residues of the channel protein, causing transient changes in their effective pK and hence in their charged state. The computed properties of such a correlated parallel pore channel with single occupation of each pore are derived and compared to those predicted for a single pore that can contain more than one ion at a time and also to those predicted for a model pore with fluctuating barriers. Experiments that could distinguish between the present and previous models are listed.R.M.B. is grateful to the S.E.R.C. for the award of a graduate studentship.     

Cycle flux algebra for ion and water flux through the KcsA channel single-file pore links microscopic trajectories and macroscopic observables

In narrow pore ion channels, ions and water molecules diffuse in a single-file manner and cannot pass each other. Under such constraints, ion and water fluxes are coupled, leading to experimentally observable phenomena such as the streaming potential. Analysis of this coupled flux would provide unprecedented insights into the mechanism of permeation. In this study, ion and water permeation through the KcsA potassium channel was the focus, for which an eight-state discrete-state Markov model has been proposed based on the crystal structure, exhibiting four ion-binding sites. Random transitions on the model lead to the generation of the net flux. Here we introduced the concept of cycle flux to derive exact solutions of experimental observables from the permeation model. There are multiple cyclic paths on the model, and random transitions complete the cycles. The rate of cycle completion is called the cycle flux. The net flux is generated by a combination of cyclic paths with their own cycle flux. T.L. Hill developed a graphical method of exact solutions for the cycle flux. This method was extended to calculate one-way cycle fluxes of the KcsA channel. By assigning the stoichiometric numbers for ion and water transfer to each cycle, we established a method to calculate the water-ion coupling ratio (CR(w-i)) through cycle flux algebra. These calculations predicted that CR(w-i) would increase at low potassium concentrations. One envisions an intuitive picture of permeation as random transitions among cyclic paths, and the relative contributions of the cycle fluxes afford experimental observables.     

Transmembrane domains of viral ion channel proteins: a molecular dynamics simulation study

Nanosecond molecular dynamics simulations in a fully solvated phospholipid bilayer have been performed on single transmembrane alpha-helices from three putative ion channel proteins encoded by viruses: NB (from influenza B), CM2 (from influenza C), and Vpu (from HIV-1). alpha-Helix stability is maintained within a core region of ca. 28 residues for each protein. Helix perturbations are due either to unfavorable interactions of hydrophobic residues with the lipid headgroups or to the need of the termini of short helices to extend into the surrounding interfacial environment in order to form H-bonds. The requirement of both ends of a helix to form favorable interactions with lipid headgroups and/or water may also lead to tilting and/or kinking of a transmembrane alpha-helix. Residues that are generally viewed as poor helix formers in aqueous solution (e.g., Gly, Ile, Val) do not destabilize helices, if located within a helix that spans a lipid bilayer. However, helix/bilayer mismatch such that a helix ends abruptly within the bilayer core destabilizes the end of the helix, especially in the presence of Gly and Ala residues. Hydrogen bonding of polar side-chains with the peptide backbone and with one another occurs when such residues are present within the bilayer core, thus minimizing the energetic cost of burying such side-chains.     

Modeling negative ion defect migration through the gramicidin A channel

The results of potential of mean force (PMF) calculations for the distinct stages of proton conduction through the gramicidin A channel, including proton migration, reorientation of the water file and negative ion defect migration, are presented. The negative ion defect migration mechanism was hypothesized in experimental studies but was not considered previously in molecular dynamics simulations. The model system consisted of the peptide chains constructed on the base of the structure PDBID:1JNO, the inner file of nine water molecules and external clusters of water molecules placed at both ends of the channel. Potential energy functions were computed with the CHARMM/PM6/TIP3P parameters. The results obtained for proton migration and water file reorientation are basically consistent with those reported previously by Pómès and Roux (Biophys J 82:2304, 2002) within the similar approach. For the newly considered mechanism of negative ion defect migration from the channel center to the end of the water file we obtain the energy 3.8 kcal mol⁻¹ which is not considerably different from the activation energy of water reorientation, 5.4 kcal mol⁻¹. Therefore this mechanism may principally compete for the rate-limiting step in proton conduction in gramicidin.     

Analysis of the ion transfer through the channel of 9,11,13,15-phenylalanylgramicidin A

The behavior of an analogue of gramicidin A in which all four tryptophanyl residues are substituted by phenylalanyl and which shows a strong voltage effect on the single channel conductance is analyzed on the basis of a 'three-barrier--two-site' model. It is shown that in the gramicidin family the side chains of some amino acids, in spite of their location, which point outside the channel can play a major role in the binding of ions in the channel and thus can significantly modify the energy profile of the channel.     

The passage of an ion through a synthetic ion channel

The simulated system consisted of a fatty acid bilayer membrane dividing two electrolyte layers each containing ions, and a channel composed of linked 15-crown-5 ether rings. The Na⁺ and F^?  ions in the aqueous electrolyte layers were too large to enter the channel, but the Li⁺ ions entered and were transported. Conditions that optimised the passive, electric-field-induced transport of Li⁺ ions through the channel were investigated. It was calculated and rationalised that the higher the numerical value of the electrostatic charge on the oxygen atoms of the crown ether rings, the more easily does the channel convey the Li⁺ ions.     

A simple model for surface charge on ion channel proteins.

We present a simple two-parameter model for surface charge directly associated with ion channels. A spherically symmetric "charged shell" models a distribution of surface charge arrayed about the channel entrance, with a corresponding set of image charges behind the plane of the membrane. The transition between a regime of buffered conductance and a regime of rapidly falling conductance at very low ionic strength is found to depend on the magnitude of the surface charge as well as the separation between the charge and the channel entrance. This resolves an apparent discrepancy between the experimental findings of Naranjo and Latorre (1993. Biophys. J. 64:1038-1050) and previous theoretical computations. The charged-shell model is used in a comparative study of the toad skeletal muscle conductance data of Naranjo and Latorre, the rat skeletal muscle conductances of Ravindran et al. (1992. Biophys. J. 61:494-508), and a second set of rat muscle conductances presented in this paper.     

Demonstrating the intrinsic ion channel activity of virally encoded proteins

This review summarizes the types of evidence that can be invoked in order to demonstrate that a virally encoded protein possesses ion channel activity that is intrinsic to the life cycle of the virus. Ion channel activity has been proposed to be a key step in the life cycle of influenza virus, and the protein responsible for this activity has been proposed to be the M2 protein encoded by the virus. This review contrasts the evidence supporting the conclusion that the A/M2 protein of influenza A virus has intrinsic ion channel activity with the evidence that the 3AB protein encoded by the human rhinovirus possesses intrinsic ion channel activity.     

In silico study of the ion channel formed by tolaasin I produced by Pseudomonas tolaasii

A toxin produced by Pseudomonas tolaasii, tolaasin, causes brown blotch disease in mushrooms. Tolaasin forms pores on the cellular membrane and destroys cell structure. Inhibiting the ability of tolaasin to form ion channels may be an effective method to protect against attack by tolaasin. However, it is first necessary to elucidate the three-dimensional structure of the ion channels formed by tolaasin. In this study, the structure of the tolaasin ion channel was determined in silico based on data obtained from nuclear magnetic resonance experiments.     

Molecular dynamics simulation of a synthetic ion channel.

A molecular dynamics simulation has been performed on a synthetic membrane-spanning ion channel, consisting of four alpha-helical peptides, each of which is composed of the amino acids leucine (L) and serine (S), with the sequence Ac-(LSLLLSL)3-CONH2. This four-helix bundle has been shown experimentally to act as a proton-conducting channel in a membrane environment. In the present simulation, the channel was initially assembled as a parallel bundle in the octane portion of a phase-separated water/octane system, which provided a membrane-mimetic environment. An explicit reversible multiple-time-step integrator was used to generate a dynamical trajectory, a few nanoseconds in duration for this composite system on a parallel computer, under ambient conditions. After more than 1 ns, the four helices were found to adopt an associated dimer state with twofold symmetry, which evolved into a coiled-coil tetrameric structure with a left-handed twist. In the coiled-coil state, the polar serine side chains interact to form a layered structure with the core of the bundle filled with H2O. The dipoles of these H2O molecules tended to align opposite the net dipole of the peptide bundle. The calculated dipole relaxation function of the pore H2O molecules exhibits two reorientation times. One is approximately 3.2 ps, and the other is approximately 100 times longer. The diffusion coefficient of the pore H2O is about one-third of the bulk H2O value. The total dipole moment and the inertia tensor of the peptide bundle have been calculated and reveal slow (300 ps) collective oscillatory motions. Our results, which are based on a simple united atom force-field model, suggest that the function of this synthetic ion channel is likely inextricably coupled to its dynamical behavior.     

Computer simulation of ion channel gating: the M(2) channel of influenza A virus in a lipid bilayer

The transmembrane fragment of the influenza virus M(2) protein forms a homotetrameric channel that transports protons. In this paper, we use molecular dynamics simulations to help elucidate the mechanism of channel gating by four histidines that occlude the channel lumen in the closed state. We test two competing hypotheses. In the "shuttle" mechanism, the delta nitrogen atom on the extracellular side of one histidine is protonated by the incoming proton, and, subsequently, the proton on the epsilon nitrogen atom is released on the opposite side. In the "water-wire" mechanism, the gate opens because of electrostatic repulsion between four simultaneously biprotonated histidines. This allows for proton transport along the water wire that penetrates the gate. For each system, composed of the channel embedded in a hydrated phospholipid bilayer, a 1.3-ns trajectory was obtained. It is found that the states involved in the shuttle mechanism, which contain either single-protonated histidines or a mixture of single-protonated histidines plus one biprotonated residue, are stable during the simulations. Furthermore, the orientations and dynamics of water molecules near the gate are conducive to proton transfer. In contrast, the fully biprotonated state is not stable. Additional simulations show that if only two histidines are biprotonated, the channel deforms but the gate remains closed. These results support the shuttle mechanism but not the gate-opening mechanism of proton gating in M(2).     

Roles of heterotrimeric G proteins in guard cell ion channel regulation

Stomata are formed by pairs of surrounding guard cells and perform important roles in photosynthesis, transpiration and innate immunity of terrestrial plants. Ionic solutes in the cytosol of guard cells are important for cell turgor and volume change. Consequently, trans-membrane flux of ions such as K⁺, Cl⁻, and malate2⁻ through K⁺ channels and anion channels of guard cells are a direct driving force for turgor change, while the opening of calcium permeable channels can serve as a trigger of cytosolic free calcium concentration elevations or oscillations, which play second messenger roles. In plants, heterotrimeric G proteins have fewer members than in animals, but they are well investigated and found to regulate these channels and to play fundamental roles in guard cell function. This mini-review focuses on the recent understanding of G-protein regulation of ion channels on the plasma membrane of guard cells and their participation in stomatal movements.Key words: guard cell, heterotrimeric G protein, ion channel, arabidopsis thaliana, stomata, plasma membrane, patch clampHeterotrimeric G proteins, composed of Gα, Gβ and Gγ subunits, are key elements of cellular signal transduction networks. In plant species, fewer members of G proteins are present than in animals. For example, only one Gα subunit (GPA1), one Gβ subunit (AGB1) and two Gγ subunits (AGG1 and AGG2) are reported in Arabidopsis while 23 Gα, 5 Gβ and 12 Gγ subunits have been identified in human.¹ All three kinds of subunits are expressed in guard cells. Ubiquitous expression of GPA1 throughout plant was ascertained by northern and promoter::GUS analyses and RT-PCR results also indicate guard cell expression.^2–4 AGB1 is ubiquitously expressed throughout the plant and its promoter::GUS transgenic lines show strong expression in guard cells.^5–7 For Gγ subunits, RNA blots show AGG1 and AGG2 expression throughout the plant, however, reporter gene analysis shows guard cell expression of AGG2 but not AGG1.^7–9 The guard cell expression of G protein subunits implies the function of G protein in guard cell signaling and stomatal movement regulation.Stomata are microscopic pores in the epidermis of terrestrial plants, which serve as the mouths of plants for gas change since through them CO₂ enters leaves for photosynthesis and water vapor is lost as transpiration.^10–13 In addition, stomatal movements induced by pathogen and pathogen/microbe-associated molecular patterns (PAMPs or MAMPs) are a component of the plant innate immunity system.^14–16 Biotic and abiotic stresses (e.g. water deficiency, cold, pathogens) and their induced phytohormone changes (e.g. abscisic acid [ABA], ethylene) have been widely investigated in stomatal movement regulation, and stomatal apertures are directly regulated by volume change of the surrounding guard cell pairs. The accumulation/release of ionic solutes through ion channels on the guard-cell plasma membrane together with malate production/metabolism induces water influx/efflux driving increase/decrease of cell turgor and volume which co-operates with the radial reinforcement of the guard cell walls to widen/shrink stomatal aperture.^10,17 Given that mature guard cells lack plasmodesmata with neighboring cells, all ion uptake and efflux must pass through ion channels and ion transporters on the plasma membrane.In Arabidopsis guard cells, the model cell type for cell signaling of the model plant species, all three kinds of ion channels (K⁺ channels, anion channels and Ca²⁺-permeable channels) have been investigated and found to be regulated by heterotrimeric G proteins.^10,17 Their ion channel activities can be measured in intact guard cells, guard cell protoplasts, or cell membrane patches using the patch clamp technique.^15,18,19 Patch clamping can be used to measure ion fluxes in whole cells or even through a single ion channel.^20,21 The patch clamp technique under the whole-cell recording configuration can measure the currents through hyperpolarization-activated inward K⁺ channels which account for K⁺ accumulation during stomatal opening, and the depolarizationactivated outward K⁺ channels which, together with R-type and S-type anion channels, mediate solute removal during stomatal closure. Besides these ionic fluxes which directly elicit changes in turgor, Ca²⁺-permeable channels which participate in Ca²⁺ signaling are also regulated by G proteins. For better visualization of the currents through K⁺, anion and Ca²⁺permeable channels, real current traces and their idealized current/voltage relationships are indicated in Figure 1. The G-protein regulation of inward and outward K⁺ channels, S-type anion channels, and Ca²⁺-permeable channels and their significance for stomatal movements will be discussed below, and the genes encoding them which have been explored up to now also will be discussed.Open in a separate windowFigure 1Current traces and idealized current/voltage relationships of wild type guard cell plasma membrane ion channels involved in G-protein regulation (A–C), ABA inhibition of whole-cell inward K⁺ currents. (A) indicates inward K⁺ currents of wild type guard cell protoplasts in response to hyperpolarizing voltages under control conditions [Scale bar is shown in (B)]; (B) indicates inward K+ currents of wild type guard cell protoplasts with ABA treatment; (C) indicates the idealized current/voltage relationship of inward K⁺ currents for control (gray) and ABA treatments (black). (D–F), ABA activation of slow anion currents. (D) indicates anion currents of wild type under control condition and (E) shows current after ABA treatment; (F) indicates the idealized current/voltage relationship of anion currents for control (gray) and ABA treatments (black). (G–I), ABA activation of currents through Ca²⁺-permeable channels. (G) indicates currents through Ca²⁺-permeable channels of wild type under control condition and (H) shows current after ABA treatments; (I) indicates the idealized current/voltage relationship of currents through Ca²⁺-permeable channels for control (gray) and ABA treatments (black).     

Temperature dependence of single channel currents and the peptide libration mechanism for ion transport through the gramicidin A transmembrane channel

Summary A study of the temperature dependence of gramicidin A conductance of K⁺ in diphytanoyllecithin/n-decane membranes shows the plot of In (single channel conductance) as a function of reciprocal temperature to be nonlinear for the most probable set of conductance, states. These results are considered in terms of a series of barriers, of the dynamics of channel conformation,vis-a-vis the peptide libration mechanism, and of the effect of lipid viscosity on side chain motions again as affecting the energetics of peptide libration.     

Phycodnavirus potassium ion channel proteins question the virus molecular piracy hypothesis

Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K(+) channels. To determine if these viral K(+) channels are the product of molecular piracy from their hosts, we compared the sequences of the K(+) channel pore modules from seven phycodnaviruses to the K(+) channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K(+) channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K(+) channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K(+) channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K(+) channels in algae and perhaps even all cellular organisms.     

Molecular dynamics simulation of water permeation through the alpha-hemolysin channel

The alpha-hemolysin (AHL) nanochannel is a non-selective channel that allows for uncontrolled transport of small molecules across membranes leading to cell death. Although it is a bacterial toxin, it has promising applications, ranging from drug delivery systems to nano-sensing devices. This study focuses on the transport of water molecules through an AHL nanochannel using molecular dynamics (MD) simulations. Our results show that AHL can quickly transport water across membranes. The first-passage time approach was used to estimate the diffusion coefficient and the mean exit time. To study the energetics of transport, the potential of mean force (PMF) of a water molecule along the AHL nanochannel was calculated. The results show that the energy barriers of water permeation across a nanopore are always positive along the channel and the values are close to thermal energy (k_BT). These findings suggest that the observed quick permeation of water is due to small energy barriers and a hydrophobic inner channel surface resulting in smaller friction. We speculate that these physical mechanisms are important in how AHL causes cell death.     

Improved 3D continuum calculations of ion flux through membrane channels

A continuum model, based on the Poisson–Nernst–Planck (PNP) theory, is applied to simulate steady-state ion flux through protein channels. The PNP equations are modified to explicitly account (1) for the desolvation of mobile ions in the membrane pore and (2) for effects related to ion sizes. The proposed algorithm for a three-dimensional self-consistent solution of PNP equations, in which final results are refined by a focusing technique, is shown to be suitable for arbitrary channel geometry and arbitrary protein charge distribution. The role of the pore shape and protein charge distribution in formation of basic electrodiffusion properties, such as channel conductivity and selectivity, as well as concentration distributions of mobile ions in the pore region, are illustrated by simulations on model channels. The influence of the ionic strength in the bulk solution and of the externally applied electric field on channel properties are also discussed.