Mechanism Of Ion Permeation Research Articles

Trans-Membrane Permeation Mechanism of Charged Methyl Guanidine

ABSTRACT: The mechanism of trans-membrane ion permeation is studied using charged methyl guanidine as a model ion. With a widely applied reaction coordinate, our umbrella sampling results reveal a significant finite-size effect in small simulation systems and a serious hysteresis in large systems. Therefore, it is important to re-examine the simulation techniques for studying trans-membrane permeation mechanism of ions suggested in previous works. In this work, two novel collective variables are designed to acquire a continuous trajectory of the permeation process and small statistical errors through umbrella sampling. A water-bridge mechanism is discussed in detail. In this mechanism, a continuous water chain (or a chain of water molecules and lipid head groups) is formed across the membrane to conduct the trans-membrane permeation of charged methyl guanidine. We obtain a continuous transition trajectory by combining the two-dimensional umbrella sampling in the local region of the saddle state and a one-dimensional sampling in the out region. Our free energy analysis shows that, with the presence of the water-bridge, the energy barrier of the trans-membrane permeation of ions is reduced significantly. Our analysis suggests that the water-bridge mechanism is common for permeation of ions across thick membranes, including POPC and DPPC membranes.

Molecular Origin of Ion Selectivity in Phaseolus Coccineus Mitochondrial VDAC

The mitochondrial voltage-dependent anion-selective channel (VDAC) is the major permeation pathway for small ions and metabolites through the mitochondrial outer membrane. The deciphering of the mechanism underpinning anion transport through the VDAC and of its selectivity is important as most of the chemical species entering or leaving the mitochondrion are anions that are directly involved in respiration. Although a wealth of electrophysiological data has been obtained on different VDAC species, the physical mechanisms of their ionic selectivity are still elusive. We addressed this issue using electrophysiological experiments performed on plant VDAC.Here we examined the ionic selectivity of the open state of the plant PcVDAC experimentally and theoretically. We demonstrated that a simple one-compartment macroscopic electrodiffusion model including both ion diffusion and an effective fixed charge in the pore can properly describe the selectivity of the channel. Using Brownian Dynamics (BD) simulations performed on plant and mammalian VDACs we also proposed a comprehensive detailed model of the molecular mechanism of ion permeation.

Gating Mechanisms of Store-Operated CRAC Channels

In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. The ensuing Ca2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. The physiological importance of CRAC channels for human health is underscored by studies indicating that mutations in CRAC channel genes produce a spectrum of devastating diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Moreover, from a basic science perspective, CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca2+-selectivity, store-operated gating, and distinct pore properties and therefore serve as fascinating ion channels for understanding the biophysical mechanisms of ion permeation and gating. Studies in the last decade have revealed the cellular and molecular choreography of the CRAC channel activation process, and it is now established that opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins, and the ER Ca2+ sensors, STIM1 and STIM2. Here, I will discuss recent work in our laboratory that seeks to understand the molecular rearrangements in the CRAC channel protein underlying the opening of the pore.

Coupling of Channel Fluctuations in Ion Permeation and Selectivity in Bacterial Sodium Channel NavAb

Even though crystallographic structures of several cation channels are known at atomistic resolution, the molecular basis for selective ion permeation, and in particular, the role of structural fluctuations of the channel in that process, remains unclear. The determination of structures of voltage-gated sodium channels opens the way to elucidating the mechanism of sodium permeation and selectivity. Recent molecular simulation studies of bacterial sodium channel NavAb (Chakrabarti et al., PNAS 110, 11331-11336, 2013) suggest that Na+ binding and permeation through the selectivity filter are coupled to the conformational isomerization of Glu177 side chains from an outfacing conformation to a lumen-facing conformation, resulting in a high rate of Na+ diffusion through the selectivity filter.To clarify the role of channel dynamics on ion permeation and selectivity, we examine the effect of structural constraints systematically. Specifically, we characterize the mechanism of cation permeation in the absence of conformational “dunking” of Glu177 side chains. In addition, we investigate the effect of structural restraints imposed on the pore helices to prevent channel closure, as well as of applied voltage, on channel fluctuations and transport properties. Results of simulations totaling over 100 microseconds indicate that restricting Glu177 conformations, either directly or through global structural restraints on the helices of the pore domain, modulates cation binding and permeation. Further, applying strong external voltage gradients significantly displaces the conformational equilibrium of the Glu177 side chains, thereby also modulating the mechanism of ion permeation in NavAb.

Transmembrane Permeation Mechanism of Charged Methyl Guanidine.

The mechanism of transmembrane ion permeation is studied using charged methyl guanidine as a model ion. With a widely applied reaction coordinate, our umbrella sampling results reveal a significant finite-size effect in small simulation systems and a serious hysteresis in large systems. Therefore, it is important to re-examine the simulation techniques for studying transmembrane permeation mechanism of ions suggested in previous works. In this work, two novel collective variables are designed to acquire a continuous trajectory of the permeation process and small statistical errors through umbrella sampling. A water-bridge mechanism is discussed in detail. In this mechanism, a continuous water chain (or a chain of water molecules and lipid head groups) is formed across the membrane to conduct the transmembrane permeation of charged methyl guanidine. We obtain a continuous transition trajectory by combining the two-dimensional umbrella sampling in the local region of the saddle state and a one-dimensional sampling in the out region. Our free energy analysis shows that, with the presence of the water bridge, the energy barrier of the transmembrane permeation of ions is reduced significantly. Our analysis suggests that the water-bridge mechanism is common for permeation of ions across thick membranes, including palmitoyloleoyl phosphocholine and dipalmitoylphosphatidylcholine membranes.

Ion-Induced Defect Permeation of Lipid Membranes

We have explored the mechanisms of uncatalyzed membrane ion permeation using atomistic simulations and electrophysiological recordings. The solubility-diffusion mechanism of membrane charge transport has prevailed since the 1960s, despite inconsistencies in experimental observations and its lack of consideration for the flexible response of lipid bilayers. We show that direct lipid bilayer translocation of alkali metal cations, Cl–, and a charged arginine side chain analog occurs via an ion-induced defect mechanism. Contrary to some previous suggestions, the arginine analog experiences a large free-energy barrier, very similar to those for Na+, K+, and Cl–. Our simulations reveal that membrane perturbations, due to the movement of an ion, are central for explaining the permeation process, leading to both free-energy and diffusion-coefficient profiles that show little dependence on ion chemistry and charge, despite wide-ranging hydration energies and the membrane’s dipole potential. The results yield membrane permeabilities that are in semiquantitative agreement with experiments in terms of both magnitude and selectivity. We conclude that ion-induced defect-mediated permeation may compete with transient pores as the dominant mechanism of uncatalyzed ion permeation, providing new understanding for the actions of a range of membrane-active peptides and proteins.

Non-Equilibrium Dynamics Contribute to Ion Selectivity in the KcsA Channel

The ability of biological ion channels to conduct selected ions across cell membranes is critical for the survival of both animal and bacterial cells. Numerous investigations of ion selectivity have been conducted over more than 50 years, yet the mechanisms whereby the channels select certain ions and reject others are not well understood. Here we report a new application of Jarzynski’s Equality to investigate the mechanism of ion selectivity using non-equilibrium molecular dynamics simulations of Na+ and K+ ions moving through the KcsA channel. The simulations show that the selectivity filter of KcsA adapts and responds to the presence of the ions with structural rearrangements that are different for Na+ and K+. These structural rearrangements facilitate entry of K+ ions into the selectivity filter and permeation through the channel, and rejection of Na+ ions. A mechanistic model of ion selectivity by this channel based on the results of the simulations relates the structural rearrangement of the selectivity filter to the differential dehydration of ions and multiple-ion occupancy and describes a mechanism to efficiently select and conduct K+. Estimates of the K+/Na+ selectivity ratio and steady state ion conductance for KcsA from the simulations are in good quantitative agreement with experimental measurements. This model also accurately describes experimental observations of channel block by cytoplasmic Na+ ions, the “punch through” relief of channel block by cytoplasmic positive voltages, and is consistent with the knock-on mechanism of ion permeation.

Structural Basis for Ca2+ Selectivity of a Voltage-Gated Calcium Channel

Voltage-gated calcium (CaV) channels catalyze rapid, highly selective influx of Ca2+ into cells despite a 70-fold higher extracellular concentration of Na+. How CaV channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the tetrameric bacterial NaV channel NaVAb. Our results reveal Ca2+ interactions with two high-affinity Ca2+ binding sites followed by a third lower affinity site that Ca2+ would occupy as it moves inward through the pore. At the entry to the selectivity filter, Site 1 is coordinated by a quartet of the acidic residue, D178, which plays a critical role in determining Ca2+ selectivity. In the center of the selectivity filter, Site 2 is constructed of the carboxyl side chains of D177 and the backbone carbonyls of L176, and is targeted by the blocking cations, Cd2+ and Mn2+, with single occupancy. At the intracellular side of the filter, the backbone carbonyls of T175 form the third, lower affinity site for Ca2+, which mediates exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca2+ ions bound in the selectivity filter and supports a "knock-off" mechanism of ion permeation through a stepwise binding process. The multi-ion selectivity filter of our CaV channel model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate CaV channels.

Mechano-Sensitive Ion Channels (MSCS) Provide Human Breast Cancer Cells with a Sensorium for Mechanical Stress

Mechanical stress is increasingly recognized as a cancerogenic factor in breast cancer. We investigated the biophysical characteristics of mechano-sensitive channels (MSCs) in the breast cancer cell line MCF7 with patch clamp methods.MSCs were present in 132 out of 258 cell-attached membrane patches. MSCs could be activated by negative pressure at the outer side of the membrane in a saturable manner (EP50: 41.2 ± 0.5 mbar (N=13).When K+ was predominantly present in the extracellular pipette solution, single channel conductance exerted to be 25.6± 0.4 pS (N=8). When complete ion selectivity was assessed, conductivity was found to increase in the following order: Li+ < Na+ < K+≈Rb+ ≈ Cs+. Furthermore, conductivity was smaller for divalent cations (100 mmole/L compared to 153 mmole/L) with the order: Ca2+ < Ba2+ < K + ≈ Rb + ≈ Cs +. We compared the biophysical properties of MSCs with a recently discovered mechanosensitive cation channel, the human Piezo1 protein, heterologously overexpressed in HEK293 cells. Single channel conductances were indistinguishable between MSCs from MCF7 and hPiezo1 in HEK293 cells, both when 100% K + and 50% K + / 50% Na + were used as permeant ions.MSCs occur in MCF7 breasts cancer cells, providing a sensorium for mechanical stress. Ion selectivity studies show that divalents like Ca2+ can permeate to a considerable extend. Moreover, permeation of monovalents is apparently restricted by the size of the ions hydrate shell, as indicated by the smaller conductivities for Na + and Li +, respectively, indicating that the ion permeation mechanism through MSCs may be different to the cation channels crystallized so far.This research is supported by grants from the Austrian Research Foundation (FWF22974 (WS); KLIF182 (TB)).

Catalysis and Selectivity of Na+ Permeation in Bacterial Sodium Channel NaVAb

The determination of a high-resolution 3D structure of voltage-gated sodium channel NaVAb opens the way to elucidating the mechanism of ion conductance and selectivity. To examine the selective permeation of Na+ over K+ through the selectivity filter of the channel, we performed large-scale molecular dynamics simulations of NaVAb in an explicit, hydrated lipid bilayer at 0mV successively in 150mM NaCl, 150mM KCl, and a combination of both, for a total simulation time of more than 70 microseconds. Although the cytoplasmic end of the pore is closed, reversible influx and efflux of Na+ and K+ through the selectivity filter occurred spontaneously during simulations, leading to equilibrium movement of these cations between the extracellular medium and the central cavity of the channel. Analysis of Na+ dynamics reveals a knock-on mechanism of ion permeation characterized by alternating occupancy of the channel by 2 and 3 Na+ ions, with a computed rate of translocation of (6±1) × 106 ions per second that is consistent with expectations from electrophysiological studies. The binding of Na+ is intimately coupled to conformational isomerization of the four E177 side chains lining the extracellular end of the selectivity filter. The reciprocal coordination of variable numbers of Na+ ions and carboxylate groups leads to their condensation into ionic clusters of variable charge and spatial arrangement. By stabilizing multiple ionic occupancy states while helping Na+ ions diffuse within the selectivity filter, the conformational flexibility of E177 side chains underpins the knock-on mechanism of Na+ permeation. K+ also forms ionic complexes with E177 but, contrary to Na+, K+ ions pass through the selectivity filter in single file. The analysis of competitive binding of Na+ and K+ in mixed-cation simulations provides detailed mechanistic insight into the molecular basis of ion selectivity in NaVAb.

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.

1P152 細菌べん毛モーター固定子複合体のイオン透過メカニズム(11. 分子モーター,ポスター,第52回日本生物物理学会年会(2014年度))

1P152 細菌べん毛モーター固定子複合体のイオン透過メカニズム(11. 分子モーター,ポスター,第52回日本生物物理学会年会(2014年度))

The Dipolar Solvent Model and Its Applications to the Structural Analysis of Low-(SAXS) and High-(CRYSTALLOGRAPHY) Resolution X-Ray Data

The Poisson-Boltzmann (PB) formalism is one of the most popular approaches to modeling the solvation of molecules. It assumes a continuum model for water, leading to a dielectric permittivity that depends on regions of space (inside or outside the solute). In contrast, the dipolar Poisson-Boltzmann-Langevin (DPBL) formalism follows a grid-based approach where the solvent is represented as a collection of freely orientable dipoles with nonuniform concentration [1]. This leads to a nonlinear permittivity function that depends both on the position and on the local electric field at that position. In practice, solving the modified PB equation in the DPBL formalism yields density maps for water (dipoles) and salt (ions). We illustrate here how these maps can be used to help in the analysis of both low- and high-resolution structural X-ray data. As for the former, we implemented a program, AquaSAXS [2], available as a web server, which uses the solvent density map to represent the hydration layer of a given protein atomic model. This approach improves the model where the hydration layer is defined as a constant layer on the surface. As for the latter, we will present how this approach is suited for electrostatics studies of solvation in confined geometries. In particular, its application to the study of pentameric ligand-gated ion channels helped proposing a plausible ion permeation mechanism [3].[1] Koehl and Delarue (2010) J Chem Phys 132(6) - 064101.[2] Poitevin et al (2011) NAR 39 - W184-9.[3] Sauguet, Poitevin, Murail et al (2013) EMBO J 32(5) - 728-41.

2P072 変異体を用いた分子動力学シミュレーションによる電位依存性カリウムチャネルのイオン透過機構の解析(01D. 蛋白質:機能,ポスター,第52回日本生物物理学会年会(2014年度))

2P072 変異体を用いた分子動力学シミュレーションによる電位依存性カリウムチャネルのイオン透過機構の解析(01D. 蛋白質:機能,ポスター,第52回日本生物物理学会年会(2014年度))

Structural basis for Ca2+ selectivity of a voltage-gated calcium channel

Voltage-gated calcium (CaV) channels catalyze rapid, highly selective influx of Ca2+ into cells despite 70-fold higher extracellular concentration of Na+. How CaV channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial NaV channel NaVAb. Our results reveal interactions of hydrated Ca2+ with two high-affinity Ca2+-binding sites followed by a third lower-affinity site that would coordinate Ca2+ as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side-chains, which play a critical role in determining Ca2+ selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations, Cd2+ and Mn2+, with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca2+ ions bound in the selectivity filter and supports a “knock-off” mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our CaVAb model establishes a structural framework for understanding mechanisms of ion selectivity and conductance by vertebrate CaV channels.

Mechanism of ion permeation through a model channel: Roles of energetic and entropic contributions

Mechanism of ion permeation through an anion-doped carbon nanotube (ANT), a model of ion channel, is investigated. Using this model system, many trajectory calculations are performed to obtain the potential energy profile, in addition to the free energy profile, that enables to separate the energy and the entropic contributions, along the ion permeation. It is found that the mechanism of the transport is governed by the interplay between the energetic and the entropic forces. The rate of the ion permeation can be controlled by changing the balance between these contributions with altering, for example, the charge and/or the length of ANT, which increases the rate of the ion permeation by nearly two orders of magnitude. The dominant free energy barrier at the entrance of ANT is found to be caused by the entropy bottleneck due to the narrow phase space for the exchange of a water molecule and an incoming ion.

Pore waters regulate ion permeation in a calcium release-activated calcium channel

The recent crystal structure of Orai, the pore unit of a calcium release-activated calcium (CRAC) channel, is used as the starting point for molecular dynamics and free-energy calculations designed to probe this channel's conduction properties. In free molecular dynamics simulations, cations localize preferentially at the extracellular channel entrance near the ring of Glu residues identified in the crystal structure, whereas anions localize in the basic intracellular half of the pore. To begin to understand ion permeation, the potential of mean force (PMF) was calculated for displacing a single Na(+) ion along the pore of the CRAC channel. The computed PMF indicates that the central hydrophobic region provides the major hindrance for ion diffusion along the permeation pathway, thereby illustrating the nonconducting nature of the crystal structure conformation. Strikingly, further PMF calculations demonstrate that the mutation V174A decreases the free energy barrier for conduction, rendering the channel effectively open. This seemingly dramatic effect of mutating a nonpolar residue for a smaller nonpolar residue in the pore hydrophobic region suggests an important role for the latter in conduction. Indeed, our computations show that even without significant channel-gating motions, a subtle change in the number of pore waters is sufficient to reshape the local electrostatic field and modulate the energetics of conduction, a result that rationalizes recent experimental findings. The present work suggests the activation mechanism for the wild-type CRAC channel is likely regulated by the number of pore waters and hence pore hydration governs the conductance.

Catalysis of Na + permeation in the bacterial sodium channel Na V Ab

Determination of a high-resolution 3D structure of voltage-gated sodium channel Na(V)Ab opens the way to elucidating the mechanism of ion conductance and selectivity. To examine permeation of Na(+) through the selectivity filter of the channel, we performed large-scale molecular dynamics simulations of Na(V)Ab in an explicit, hydrated lipid bilayer at 0 mV in 150 mM NaCl, for a total simulation time of 21.6 μs. Although the cytoplasmic end of the pore is closed, reversible influx and efflux of Na(+) through the selectivity filter occurred spontaneously during simulations, leading to equilibrium movement of Na(+) between the extracellular medium and the central cavity of the channel. Analysis of Na(+) dynamics reveals a knock-on mechanism of ion permeation characterized by alternating occupancy of the channel by 2 and 3 Na(+) ions, with a computed rate of translocation of (6 ± 1) × 10(6) ions⋅s(-1) that is consistent with expectations from electrophysiological studies. The binding of Na(+) is intimately coupled to conformational isomerization of the four E177 side chains lining the extracellular end of the selectivity filter. The reciprocal coordination of variable numbers of Na(+) ions and carboxylate groups leads to their condensation into ionic clusters of variable charge and spatial arrangement. Structural fluctuations of these ionic clusters result in a myriad of ion binding modes and foster a highly degenerate, liquid-like energy landscape propitious to Na(+) diffusion. By stabilizing multiple ionic occupancy states while helping Na(+) ions diffuse within the selectivity filter, the conformational flexibility of E177 side chains underpins the knock-on mechanism of Na(+) permeation.

Interactions of Drugs and Toxins with Permeant Ions in Potassium, Sodium, and Calcium Channels

Ion channels in cell membranes are targets for a multitude of ligands including naturally occurring toxins, illicit drugs, and medications used to manage pain and treat cardiovascular, neurological, autoimmune, and other health disorders. In the past decade, the x-ray crystallography revealed 3D structures of several ion channels in their open, closed, and inactivated states, shedding light on mechanisms of channel gating, ion permeation and selectivity. However, atomistic mechanisms of the channel modulation by ligands are poorly understood. Increasing evidence suggest that cationophilic groups in ion channels and in some ligands may simultaneously coordinate permeant cations, which form indispensible (but underappreciated) components of respective receptors. This review describes ternary ligand-metal-channel complexes predicted by means of computer-based molecular modeling. The models rationalize a large body of experimental data including paradoxes in structure-activity relationships, effects of mutations on the ligand action, sensitivity of the ligand action to the nature of current-carrying cations, and action of ligands that bind in the ion-permeation pathway but increase rather than decrease the current. Recent mutational and ligand-binding experiments designed to test the models have confirmed the ternary-complex concept providing new knowledge on physiological roles of metal ions and atomistic mechanisms of action of ion channel ligands.

Structural basis for ion permeation mechanism in pentameric ligand-gated ion channels

To understand the molecular mechanism of ion permeation in pentameric ligand-gated ion channels (pLGIC), we solved the structure of an open form of GLIC, a prokaryotic pLGIC, at 2.4 Å. Anomalous diffraction data were used to place bound anions and cations. This reveals ordered water molecules at the level of two rings of hydroxylated residues (named Ser6' and Thr2') that contribute to the ion selectivity filter. Two water pentagons are observed, a self-stabilized ice-like water pentagon and a second wider water pentagon, with one sodium ion between them. Single-channel electrophysiology shows that the side-chain hydroxyl of Ser6' is crucial for ion translocation. Simulations and electrostatics calculations complemented the description of hydration in the pore and suggest that the water pentagons observed in the crystal are important for the ion to cross hydrophobic constriction barriers. Simulations that pull a cation through the pore reveal that residue Ser6' actively contributes to ion translocation by reorienting its side chain when the ion is going through the pore. Generalization of these findings to the pLGIC family is proposed.