Interaction of a peptide nanotube with a water-membrane interface

Inserting peptide nanotubes into lipid bilayers modulates the permeability properties of the cell wall, thus conferring potential bacteriocidal capability. Interaction of a peptide nanotube formed by eight cyclo[RRKWLWLW] subunits with the surface of a hydrated dimyristoylphosphatidylcholine bilayer is investigated using molecular dynamics simulations. The present sequence of alternated D-L-alpha-amino acids has been shown to yield remarkable antibacterial in vitro activity, and the chosen topoisomer corresponds to the optimum amphipathy of the tubular structure, whereby non-polar and charged side chains are segregated by the aqueous interface. The cohesion of the nanotube is ensured by a scaffold of intermolecular hydrogen bonds between adjacent cyclic peptides, supplemented by favorable like-charged contacts of arginine side chains. It is further reinforced by interactions of charged residues with the lipid head groups and of non-polar residues with the lipid acyl chains. The simulation reveals a partial breaking of the synthetic channel accompanying its early insertion into the lipid bilayer. The latter opens new questions about how peptide nanotubes permeate the membrane, in particular whether or not (i) self-assembly precedes partitioning and (ii) translocation occurs with the complete tubular structure.     

Influence of hydrophobic mismatch on structures and dynamics of gramicidin a and lipid bilayers

Gramicidin A (gA) is a 15-amino-acid antibiotic peptide with an alternating L-D sequence, which forms (dimeric) bilayer-spanning, monovalent cation channels in biological membranes and synthetic bilayers. We performed molecular dynamics simulations of gA dimers and monomers in all-atom, explicit dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers. The variation in acyl chain length among these different phospholipids provides a way to alter gA-bilayer interactions by varying the bilayer hydrophobic thickness, and to determine the influence of hydrophobic mismatch on the structure and dynamics of both gA channels (and monomeric subunits) and the host bilayers. The simulations show that the channel structure varied little with changes in hydrophobic mismatch, and that the lipid bilayer adapts to the bilayer-spanning channel to minimize the exposure of hydrophobic residues. The bilayer thickness, however, did not vary monotonically as a function of radial distance from the channel. In all simulations, there was an initial decrease in thickness within 4–5 Å from the channel, which was followed by an increase in DOPC and POPC or a further decrease in DLPC and DMPC bilayers. The bilayer thickness varied little in the monomer simulations—except one of three independent simulations for DMPC and all three DLPC simulations, where the bilayer thinned to allow a single subunit to form a bilayer-spanning water-permeable pore. The radial dependence of local lipid area and bilayer compressibility is also nonmonotonic in the first shell around gA dimers due to gA-phospholipid interactions and the hydrophobic mismatch. Order parameters, acyl chain dynamics, and diffusion constants also differ between the lipids in the first shell and the bulk. The lipid behaviors in the first shell around gA dimers are more complex than predicted from a simple mismatch model, which has implications for understanding the energetics of membrane protein-lipid interactions.     

Synthetic Chloride-Selective Carbon Nanotubes Examined by Using Molecular and Stochastic Dynamics

Synthetic channels, such as nanotubes, offer the possibility of ion-selective nanoscale pores which can broadly mimic the functions of various biological ion channels, and may one day be used as antimicrobial agents, or for treatment of cystic fibrosis. We have designed a carbon nanotube that is selectively permeable to anions. The virtual nanotubes are constructed from a hexagonal array of carbon atoms (graphene) rolled up to form a tubular structure, with an effective radius of 4.53 Å and length of 34 Å. The pore ends are terminated with polar carbonyl groups. The nanotube thus formed is embedded in a lipid bilayer and a reservoir containing ionic solutions is added at each end of the pore. The conductance properties of these synthetic channels are then examined with molecular and stochastic dynamics simulations. Profiles of the potential of mean force at 0 mM reveal that a cation moving across the pore encounters an insurmountable free energy barrier of ∼25 kT in height. In contrast, for anions, there are two energy wells of ∼12 kT near each end of the tube, separated by a central free energy barrier of 4 kT. The conductance of the pore, with symmetrical 500 mM solutions in the reservoirs, is 72 pS at 100 mV. The current saturates with an increasing ionic concentration, obeying a Michaelis-Menten relationship. The pore is normally occupied by two ions, and the rate-limiting step in conduction is the time taken for the resident ion near the exit gate to move out of the energy well.     

Regulation of channel function due to physical energetic coupling with a lipid bilayer

Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions. This study provides a new insight helping better understand of the underlying mechanisms of membrane protein functions in cell membrane leading to important biological implications.     

SGTx1, a Kv Channel Gating-Modifier Toxin, Binds to the Interfacial Region of Lipid Bilayers

SGTx1 is a gating-modifier toxin that has been shown to inhibit the voltage-gated potassium channel Kv2.1. SGTx1 is thought to bind to the S3b-S4a region of the voltage-sensor, and is believed to alter the energetics of gating. Gating-modifier toxins such as SGTx1 are of interest as they can be used to probe the structure and dynamics of their target channels. Although there are experimental data for SGTx1, its interaction with lipid bilayer membranes remains to be characterized. We performed atomistic and coarse-grained molecular dynamics simulations to study the interaction of SGTx1 with a POPC and a 3:1 POPE/POPG lipid bilayer membrane. We reveal the preferential partitioning of SGTx1 into the water/membrane interface of the bilayer. We also show that electrostatic interactions between the charged residues of SGTx1 and the lipid headgroups play an important role in stabilizing SGTx1 in a bilayer environment.     

SGTx1, a Kv channel gating-modifier toxin, binds to the interfacial region of lipid bilayers

SGTx1 is a gating-modifier toxin that has been shown to inhibit the voltage-gated potassium channel Kv2.1. SGTx1 is thought to bind to the S3b-S4a region of the voltage-sensor, and is believed to alter the energetics of gating. Gating-modifier toxins such as SGTx1 are of interest as they can be used to probe the structure and dynamics of their target channels. Although there are experimental data for SGTx1, its interaction with lipid bilayer membranes remains to be characterized. We performed atomistic and coarse-grained molecular dynamics simulations to study the interaction of SGTx1 with a POPC and a 3:1 POPE/POPG lipid bilayer membrane. We reveal the preferential partitioning of SGTx1 into the water/membrane interface of the bilayer. We also show that electrostatic interactions between the charged residues of SGTx1 and the lipid headgroups play an important role in stabilizing SGTx1 in a bilayer environment.     

New insights of membrane environment effects on MscL channel mechanics from theoretical approaches

The prokaryotic mechanosensitive channel of large conductance (MscL) is a remarkable integral membrane protein. During hypo-osmotic shock, it responses to membrane tension through large conformational changes, that lead to an open state of the pore. The structure of the channel from Mycobacterium tuberculosis has been resolved in the closed state. Numerous experiments have attempted to trap the channel in its open state but they did not succeed in obtaining a structure. A gating mechanism has been proposed based on different experimental data but there is no experimental technique available to follow this process in atomic details. In addition, it has been shown that a decrease of the lipid bilayer thickness lowered MscL activation energy and stabilized a structurally distinct closed channel intermediate. Here, we use atomistic molecular dynamics simulations to investigate the effect of the lipid bilayer thinning on our model of the structure of the Escherichia coli. We thoroughly analyze simulations of the channel embedded in two pre-equilibrated membranes differing by their hydrophobic tail length (DMPE and POPE). The MscL structure remains stable in POPE, whereas a distinct structural state is obtained in DMPE in response to hydrophobic mismatch. This latter is obtained by tilts and kinks of the transmembrane helices, leading to a widening and a diminution of the channel height. Part of these motions is guided by a competition between solvent and lipids for the interaction with the periplasmic loops. We finally conduct a principal component analysis of the simulation and compare anharmonic motions with harmonic ones, previously obtained from a coarse-grained normal mode analysis performed on the same structural model. Significant similarities exist between low-frequency harmonic motions and those observed with essential dynamics in DMPE. In summary, change in membrane thickness permits to accelerate the conformational changes involved in the mechanics of the E. coli channel, providing a closed structural intermediate en route to the open state. These results give clues for better understanding why the channel activation energy is lowered in a thinner membrane.     

OmpA: a pore or not a pore? Simulation and modeling studies

The bacterial outer membrane protein OmpA is composed of an N-terminal 171-residue beta-barrel domain (OmpA(171)) that spans the bilayer and a periplasmic, C-terminal domain of unknown structure. OmpA has been suggested to primarily serve a structural role, as no continuous pore through the center of the barrel can be discerned in the crystal structure of OmpA(171). However, several groups have recorded ionic conductances for bilayer-reconstituted OmpA(171). To resolve this apparent paradox we have used molecular dynamics (MD) simulations on OmpA(171) to explore the conformational dynamics of the protein, in particular the possibility of transient formation of a central pore. A total of 19 ns of MD simulations of OmpA(171) have been run, and the results were analyzed in terms of 1) comparative behavior of OmpA(171) in different bilayer and bilayer-mimetic environments, 2) solvation states of OmpA(171), and 3) pore characteristics in different MD simulations. Significant mobility was observed for residues and water molecules within the beta-barrel. A simulation in which putative gate region side chains of the barrel interior were held in a non-native conformation led to an open pore, with a predicted conductance similar to experimental measurements. The OmpA(171) pore has been shown to be somewhat more dynamic than suggested by the crystal structure. A gating mechanism is proposed to explain its documented channel properties, involving a flickering isomerization of Arg138, forming alternate salt bridges with Glu52 (closed state) and Glu128 (open state).     

Membrane electroporation: a molecular dynamics simulation

We present results of molecular dynamics simulations of lipid bilayers under a high transverse electrical field aimed at investigating their electroporation. Several systems are studied, namely 1), a bare bilayer, 2), a bilayer containing a peptide nanotube channel, and 3), a system with a peripheral DNA double strand. In all systems, the applied transmembrane electric fields (0.5 V.nm(-1) and 1.0 V.nm(-1)) induce an electroporation of the lipid bilayer manifested by the formation of water wires and water channels across the membrane. The internal structures of the peptide nanotube assembly and that of the DNA strand are hardly modified under field. For system 2, no perturbation of the membrane is witnessed at the vicinity of the channel, which indicates that the interactions of the peptide with the nearby lipids stabilize the bilayer. For system 3, the DNA strand migrates to the interior of the membrane only after electroporation. Interestingly enough, switching of the external transmembrane potential in cases 1 and 2 for few nanoseconds is enough to allow for complete resealing and reconstitution of the bilayer. We provide evidence that the electric field induces a significant lateral stress on the bilayer, manifested by surface tensions of magnitudes in the order of 1 mN.m(-1). This study is believed to capture the essence of several dynamical phenomena observed experimentally and provides a framework for further developments and for new applications.     

Simulations of electrophoretic RNA transport through transmembrane carbon nanotubes

The study of interactions between carbon nanotubes and cellular components, such as membranes and biomolecules, is fundamental for the rational design of nanodevices interfacing with biological systems. In this work, we use molecular dynamics simulations to study the electrophoretic transport of RNA through carbon nanotubes embedded in membranes. Decorated and naked carbon nanotubes are inserted into a dodecane membrane and a dimyristoylphosphatidylcholine lipid bilayer, and the system is subjected to electrostatic potential differences. The transport properties of this artificial pore are determined by the structural modifications of the membrane in the vicinity of the nanotube openings and they are quantified by the nonuniform electrostatic potential maps at the entrance and inside the nanotube. The pore is used to transport electrophoretically a short RNA segment and we find that the speed of translocation exhibits an exponential dependence on the applied potential differences. The RNA is transported while undergoing a repeated stacking and unstacking process, affected by steric interactions with the membrane headgroups and by hydrophobic interaction with the walls of the nanotube. The RNA is structurally reorganized inside the nanotube, with its backbone solvated by water molecules near the axis of the tube and its bases aligned with the nanotube walls. Upon exiting the pore, the RNA interacts with the membrane headgroups and remains attached to the dodecane membrane while it is expelled into the solvent in the case of the lipid bilayer. The results of the simulations detail processes of molecular transport into cellular compartments through manufactured nanopores and they are discussed in the context of applications in biotechnology and nanomedicine.     

Molecular dynamics study of MscL interactions with a curved lipid bilayer

Mechanosensitivity is a ubiquitous sensory mechanism found in living organisms. The simplest known mechanotransducing mechanism is found in bacteria in the form of the mechanosensitive membrane channel of large conductance, MscL. This channel has been studied extensively using a variety of methods at a functional and structural level. The channel is gated by membrane tension in the lipid bilayer alone. It serves as a safety valve protecting bacterial cells against hypoosmotic shock. MscL of Escherichia coli embedded in bilayers composed of asymmetric amounts of single-tailed and double-tailed lipids has been shown to gate spontaneously, even in the absence of membrane tension. To gain insight into the effect of the lipid membrane composition and geometry on MscL structure, a fully solvated, all-atom model of MscL in a stress-free curved bilayer composed of double- and single-tailed lipids was studied using a 9.5-ns molecular dynamics simulation. The bilayer was modeled as a domed structure accommodating the asymmetric composition of the monolayers. During the course of the simulation a spontaneous restructuring of the periplasmic loops occurred, leading to interactions between one of the loops and phospholipid headgroups. Previous experimental studies of the role of the loops agree with the observation that opening starts with a restructuring of the periplasmic loop, suggesting an effect of the curved bilayer. Because of limited resources, only one simulation of the large system was performed. However, the results obtained suggest that through the geometry and composition of the bilayer the protein structure can be affected even on short timescales.     

Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants

The three-dimensional structure of recombinants of the isolated membrane attack complex (MAC) of complement with single bilayer dioleoyllecithin (DOL) vesicles and with dimyristoyllecithin (DML) vesicles was determined. A total of four MAC-vesicle complexes were analyzed by imaging negatively stained specimens at various defined tilting angles under minimal dose conditions in the electron microscope and by computer-aided three-dimensional reconstruction. The information on electron micrographs obtained at 6 degrees angular increments from +60 degrees to -60 degrees was digitized by densitometric scanning, Fourier-transformed, corrected for imaging errors, cross-correlated, and synthesized to the three-dimensional image. All four MAC-vesicle recombinants showed stain penetration into the interior of the vesicle, indicating increased permeability of the bilayer to negative stain. The MAC appeared as a hollow structure of 16-nm height, 2.0-nm wall thickness, and a 3.0-nm torus at the free end with an outer and inner diameter of 20.0 nm and 10.0 nm. In MAC-DOL vesicles the hollow core of the MAC terminated at the membrane-binding site, and only small pores of up to 2.0-nm in diameter penetrated the bilayer. In one MAC-DML vesicle lipid discontinuities on the outer circumference of the MAC binding site mediated stain penetration. The second MAC-DML vesicle showed a channel of approximately 4.0 nm connecting the hollow core of the MAC across the bilayer with the vesicle interior. The results suggest the MAC may mediate increased membrane permeability by protein channel formation in addition to lipid reorientation.     

Halloysite clay nanotubes for resveratrol delivery to cancer cells

Halloysite is natural aluminosilicate clay with hollow tubular structure which allows loading with low soluble drugs using their saturated solutions in organic solvents. Resveratrol, a polyphenol known for having antioxidant and antineoplastic properties, is loaded inside these clay nanotubes lumens. Release time of 48?h is demonstrated. Spectroscopic and ζ-potential measurements are used to study the drug loading/release and for monitoring the nanotube layer-by-layer (LbL) coating with polyelectrolytes for further release control. Resveratrol-loaded clay nanotubes are added to breast cell cultures for toxicity tests. Halloysite functionalization with LbL polyelectrolyte multilayers remarkably decrease nanotube self-toxicity. MTT measurements performed with a neoplastic cell lines model system (MCF-7) as function of the resveratrol-loaded nanotubes concentration and incubation time indicate that drug-loaded halloysite strongly increase of cytotoxicity leading to cell apoptosis.     

TiO_2纳米管阵列的表面特性对猪肾小管上皮细胞生长状态的影响

TiO2纳米管阵列由于其优异的生物相容性及光催化效应,在生物医学领域引起了广泛关注.但能否将肾小管上皮细胞较好地黏附于TiO2纳米管材料并使其发挥肾小管的功能,目前还未见报道.为研究TiO2纳米管材料的表面特性对猪肾小管上皮细胞株（LLC-PK1）的黏附及增殖影响,采用阳极氧化法制备新型高强度的TiO2纳米管阵列,利用荧光显微镜考察了TiO2纳米管阵列光照特性、晶型结构及几何形貌参数对LLC-PK1细胞黏附的影响,采用MTT方法检测了黏附细胞的活性;同时使用扫描电子显微镜观察了4种不同管径上细胞生长的形态,并与纯钛片上细胞的生长形态进行对照.结果表明,管径为70nm的TiO2纳米管阵列膜最有利于LLC-PK1细胞的黏附及增殖,且细胞的活性最高;未经紫外光照射时锐钛矿型的TiO2比无定型更有利于细胞的黏附,然而锐钛矿型TiO2经紫外光照射后会导致细胞凋亡.扫描电子显微镜观察显示,细胞在纳米管上延伸为长条状,而在钛片上则呈堆积平板状.证实TiO2纳米管阵列膜具有良好的生物相容性,有助于改善细胞与材料的黏附.     

Structure and dynamics of the pore-lining helix of the nicotinic receptor: MD simulations in water, lipid bilayers, and transbilayer bundles

Multiple nanosecond duration molecular dynamics simulations on the pore-lining M2 helix of the nicotinic acetylcholine receptor reveal how its structure and dynamics change as a function of environment. In water, the M2 helix partially unfolds to form a molecular hinge in the vicinity of a central Leu residue that has been implicated in the mechanism of ion channel gating. In a phospholipid bilayer, either as a single transmembrane helix, or as part of a pentameric helix bundle, the M2 helix shows less flexibility, but still exhibits a kink in the vicinity of the central Leu. The single M2 helix tilts relative to the bilayer normal by 12 degrees, in agreement with recent solid state NMR data (Opella et al., Nat Struct Biol 6:374-379, 1999). The pentameric helix bundle, a model for the pore domain of the nicotinic receptor and for channels formed by M2 peptides in a bilayer, is remarkably stable over a 2-ns MD simulation in a bilayer, provided one adjusts the pK(A)s of ionizable residues to their calculated values (when taking their environment into account) before starting the simulation. The resultant transbilayer pore shows fluctuations at either mouth which transiently close the channel. Proteins 2000;39:47-55.     

Molecular dynamics investigation of membrane-bound bundles of the channel-forming transmembrane domain of viral protein U from the human immunodeficiency virus HIV-1

Molecular dynamics (MD) simulations have been carried out on bundles of the channel-forming transmembrane (TM) domain of the viral protein U (VPU(1-27) and VPU(6-27)) from the human immunodeficiency virus (HIV-1). Simulations of hexameric and pentameric bundles of VPU(6-27) in an octane/water membrane mimetic system suggested that the pentamer is the preferred oligomer. Accordingly, an unconstrained pentameric helix bundle of VPU(1-27) was then placed in a hydrated palmitoyl-oleyl-3-n-glycero-phosphatidylethanolamine (POPE) lipid bilayer and its structural properties calculated from a 3-ns MD run. Some water molecules, initially inside the channel lumen, were expelled halfway through the simulation and the bundle adopted a conical structure reminiscent of previous MD results obtained for VPU(6-27) in an octane/water system. The pore constriction generated may correspond to a closed state of the channel and underlies the relocation of the W residue toward the pore lumen. The relative positions of the helices with respect to the bilayer and their interactions with the lipids are discussed. The observed structure is stabilized via specific interactions between the VPU helices and the carbonyl oxygen atoms of the lipid molecules, particularly at the Q and S residues.     

Water transport in human aquaporin-4: molecular dynamics (MD) simulations

Aquaporin-4 (AQP4) is the predominant water channel in the central nervous system, where it has been reported to be involved in many pathophysiological roles including water transport. In this paper, the AQP4 tetramer was modeled from its PDB structure file, embedded in a palmitoyl-oleoyl-phosphatidyl-choline (POPC) lipid bilayer, solvated in water, then minimized and equilibrated by means of molecular dynamics simulations. Analysis of the equilibrated structure showed that the central pore along the fourfold axis of the tetramers is formed with hydrophobic amino acid residues. In particular, Phe-195, Leu-191 and Leu-75, form the narrowest part of the pore. Therefore water molecules are not expected to transport through the central pore, which was confirmed by MD simulations. Each monomer of the AQP4 tetramers forms a channel whose walls consist mostly of hydrophilic residues. There are eight water molecules in single file observed in each of the four channels, transporting through the selectivity filter containing Arg-216, His-201, Phe-77, Ala-210, and the two conserved Asn-Pro-Ala (NPA) motifs containing Asn-213 and Asn-97. By using Brownian dynamics fluctuation–dissipation-theorem (BD-FDT), the overall free-energy profile was obtained for water transporting through AQP4 for the first time, which gives a complete map of the entire channel of water permeation.     

Lipid Bilayer Composition Influences the Activity of the Antimicrobial Peptide Dermcidin Channel

Antimicrobial peptides (AMPs) carry great potential as new antibiotics against “superbugs.” Dermcidin (DCD), a broad-spectrum AMP in human sweat, has been recently crystallized in its oligomeric state and showed channel-like properties. In this work, we performed multiscale molecular dynamics simulations to study how the membrane composition influences the behavior of a transmembrane pore formed by the DCD oligomer in the hope of revealing the origin of the membrane selectivity of this AMP toward bacteria. Our results indicate that bilayers composed of various lipids (DMPC, DPPC, and DSPC) with different thicknesses result in different orientations of the DCD oligomer when embedded in lipid bilayers. The thicker the bilayer, the less tilted the channel. Cholesterol makes the bilayers more rigid and thicker, which also affects the orientation of the channel. Furthermore, we observed that the predicted conductance of the channel from computational electrophysiology simulations is related to its orientation in the lipid bilayer: the larger the tilt, the larger the conductance. Our results indicate that the membrane composition has a significant influence on the activity of the DCD channel, with thicker, cholesterol-rich membranes showing lower conductance than that of thinner membranes.     

Understanding pH-dependent selectivity of alamethicin K18 channels by computer simulation

Alamethicin K18 is a covalently linked alamethicin dimer in which the glutamine residue at position 18 in each helix has been replaced by a lysine residue. As described in previous work, channels formed by this peptide show pH-dependent selectivity. The maximum anion selectivity of the putative octameric conducting state is obtained at pH 7 or lower. Inasmuch as no change in selectivity is seen between pH 7 and pH 3, and because protons are expected to be in equilibrium with the open state of the channel during a selectivity measurement, the channel is believed to be fully charged (i.e., all eight lysines protonated) at pH 7. In an effort to understand how such a highly charged channel structure is stable in membranes and why it is not more selective for anions, we have performed a number of computer simulations of the system. Molecular dynamics simulations of 10 ns each of the octameric bundle in a lipid bilayer environment are presented, with either zero, four, or eight lysines charged in the absence of salt, and with eight lysines charged in the presence of 0.5 M and 1 M KCl. When no salt is present and all lysines are charged, on average 1.9 Cl(-) ions are inside the channel and the channel significantly deforms. With 0.5 M KCl present, 2.9 Cl(-) ions are inside the channel. With 1 M KCl present, four Cl(-) ions are present and the channel maintains a regular structure. Poisson-Boltzmann calculations on models of the octameric channel also predict an average of 2-4 Cl(-) ions near the lysine residues as a function of ionic strength. These counterions lower the apparent charge of the channel, which may underlie the decrease in selectivity observed experimentally with increasing salt concentrations. We suggest that to increase the selectivity of Alm K18 channels, positive charges could be engineered in a narrower part of the channel.