Insights into the binding of GABA to the insect RDL receptor from atomistic simulations: a comparison of models

The resistance to dieldrin (RDL) receptor is an insect pentameric ligand-gated ion channel (pLGIC). It is activated by the neurotransmitter γ-aminobutyric acid (GABA) binding to its extracellular domain; hence elucidating the atomistic details of this interaction is important for understanding how the RDL receptor functions. As no high resolution structures are currently available, we built homology models of the extracellular domain of the RDL receptor using different templates, including the widely used acetylcholine binding protein and two pLGICs, the Erwinia Chrysanthemi ligand-gated ion channel (ELIC) and the more recently resolved GluCl. We then docked GABA into the selected three dimensional structures, which we used as starting points for classical molecular dynamics simulations. This allowed us to analyze in detail the behavior of GABA in the binding sites, including the hydrogen bond and cation-π interaction networks it formed, the conformers it visited and the possible role of water molecules in mediating the interactions; we also estimated the binding free energies. The models were all stable and showed common features, including interactions consistent with experimental data and similar to other pLGICs; differences could be attributed to the quality of the models, which increases with increasing sequence identity, and the use of a pLGIC template. We supplemented the molecular dynamics information with metadynamics, a rare event method, by exploring the free energy landscape of GABA binding to the RDL receptor. Overall, we show that the GluCl template provided the best models. GABA forming direct salt-bridges with Arg211 and Glu204, and cation-π interactions with an aromatic cage including Tyr109, Phe206 and Tyr254, represents a favorable binding arrangement, and the interaction with Glu204 can also be mediated by a water molecule.     

Molecular dynamics of nicotinic acetylcholine receptor correlating biological functions

The nicotinic acetylcholine receptor (nAChR) that mediates fast intercellular communication in response to neurotransmitters is a paradigm of ligand-gated ion channels. Molecular dynamics (MD) simulations are valuable in understanding membrane protein function at atomic level, providing useful clues for further experimental/theoretical studies. In this brief review, recent progress in MD simulations of the nAChR has been illustrated, mainly focusing on the latest simulation of the whole transmembrane domain of the receptor. On the basis of MD simulations, asymmetrical and asynchronous motions of five subunits were observed both in the ligand binding and transmembrane domains; a closed-to-open conformational shift of the gate was captured in different simulation systems; the contributions from the lipid molecules and other transmembrane segments rather than M2 to the gate switch as well as the conformational change of the whole channel were assessed; the dynamic behavior and related physical/chemical properties of the water molecules and cations within the ion channel were examined; and an experimentally comparable single-channel conductance and ion selectivity were obtained.     

Insights into the dynamics of HIV-1 protease: a kinetic network model constructed from atomistic simulations

The conformational dynamics in the flaps of HIV-1 protease plays a crucial role in the mechanism of substrate binding. We develop a kinetic network model, constructed from detailed atomistic simulations, to determine the kinetic mechanisms of the conformational transitions in HIV-1 PR. To overcome the time scale limitation of conventional molecular dynamics (MD) simulations, our method combines replica exchange MD with transition path theory (TPT) to study the diversity and temperature dependence of the pathways connecting functionally important states of the protease. At low temperatures the large-scale flap opening is dominated by a small number of paths; at elevated temperatures the transition occurs through many structurally heterogeneous routes. The expanded conformation in the crystal structure 1TW7 is found to closely mimic a key intermediate in the flap-opening pathways at low temperature. We investigated the different transition mechanisms between the semi-open and closed forms. The calculated relaxation times reveal fast semi-open ? closed transitions, and infrequently the flaps fully open. The ligand binding rate predicted from this kinetic model increases by 38-fold from 285 to 309 K, which is in general agreement with experiments. To our knowledge, this is the first application of a network model constructed from atomistic simulations together with TPT to analyze conformational changes between different functional states of a natively folded protein.     

Silica/Proteoliposomal Nanocomposite as a Potential Platform for Ion Channel Studies

The nanostructuration of solid matrices with lipid nanoparticles containing membrane proteins is a promising tool for the development of high-throughput screening devices. Here, sol-gel silica-derived nanocomposites loaded with liposome-reconstituted KcsA, a prokaryotic potassium channel, have been synthesized. The conformational and functional stability of these lipid nanoparticles before and after sol-gel immobilization have been characterized by using dynamic light scattering, and steady-state and time-resolved fluorescence spectroscopy methods. The lipid-reconstituted KcsA channel entrapped in the sol-gel matrix retained the conformational and stability changes induced by the presence of blocking or permeant cations in the buffer (associated with the conformation of the selectivity filter) or by a drop in the pH (associated with the opening of the activation gate of the protein). Hence, these results indicate that this novel device has the potential to be used as a screening platform to test new modulating drugs of potassium channels.     

Effect of channel block on the collective spiking activity of coupled stochastic Hodgkin-Huxley neurons

Toxins, such as tetraethylammonium (TEA) and tetrodotoxin (TTX), can make potassium or sodium ion channels poisoned, respectively, and hence reduce the number of working ion channels and lead to the diminishment of conductance. In this paper, we have studied by numerical simulations the effects of sodium and potassium ion channel poisoning on the collective spiking activity of an array of coupled stochastic Hodgkin-Huxley (HH) neurons. It is found for a given number of neurons sodium or potas- sium ion channel block can either enhance or reduce the collective spiking regularity, depending on the membrane patch size. For a given smaller or larger patch size, potassium and sodium ion channel block can reduce or enhance the collective spiking regularity, but they have different patch size ranges for the transformation. This result shows that sodium or potassium ion channel block might have dif- ferent effects on the collective spiking activity in coupled HH neurons from the effects for a single neuron, which represents the interplay among the diminishment of maximal conductance and the in- crease of channel noise strength due to the channel blocks, as well as the bi-directional coupling be- tween the neurons.     

Conformational dynamics of the nicotinic acetylcholine receptor channel: a 35-ns molecular dynamics simulation study

The nicotinic acetylcholine receptor (AChR) is the paradigm of ligand-gated ion channels, integral membrane proteins that mediate fast intercellular communication in response to neurotransmitters. A 35-ns molecular dynamics simulation has been performed to explore the conformational dynamics of the entire membrane-spanning region, including the ion channel pore of the AChR. In the simulation, the 20 transmembrane (TM) segments that comprise the whole TM domain of the receptor were inserted into a large dipalmitoylphosphatidylcholine (DPPC) bilayer. The dynamic behavior of individual TM segments and their corresponding AChR subunit helix bundles was examined in order to assess the contribution of each to the conformational transitions of the whole channel. Asymmetrical and asynchronous motions of the M1-M3 TM segments of each subunit were revealed. In addition, the outermost ring of five M4 TM helices was found to convey the effects exerted by the lipid molecules to the central channel domain. Remarkably, a closed-to-open conformational shift was found to occur in one of the channel ring positions in the time scale of the present simulations, the possible physiological significance of which is discussed.     

Reversible Ligand-Gated Ion Channel via Interconversion between Hollow Single Helix and Intertwined Double Helix

Ligand responsiveness is one of the typical mechanisms in biological organization to trigger sophisticated channel switching. Here, we report a new type of helical trimer which can undergo transition between a hollow single helix and an intertwined double helix with no cavity by complexation and decomplexation of Cu ions. In addition, the one dimensional (1D) hollow helical tubes spontaneously generated from single helices via π-π interactions embedded into the lipid bilayers and displayed satisfactory channel stability and efficiency. With the addition of Cu^I ions and further extraction with ammonia, the disassembly and reassembly of 1D hollow helical tubes gave rise to the reversible switching of channel activity in situ inside the bilayers. The synthetic helical system provides the first model of reversible ligand-gated ion channel by means of dynamic transition between single and double helices, which will be available for developing intelligent artificial nanochannels for potential biological and medicinal applications.     

Molecular dynamics and brownian dynamics investigation of ion permeation and anesthetic halothane effects on a proton-gated ion channel

Bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC) is activated to cation permeation upon lowering the solution pH. Its function can be modulated by anesthetic halothane. In the present work, we integrate molecular dynamics (MD) and Brownian dynamics (BD) simulations to elucidate the ion conduction, charge selectivity, and halothane modulation mechanisms in GLIC, based on recently resolved X-ray crystal structures of the open-channel GLIC. MD calculations of the potential of mean force (PMF) for a Na(+) revealed two energy barriers in the extracellular domain (R109 and K38) and at the hydrophobic gate of transmembrane domain (I233), respectively. An energy well for Na(+) was near the intracellular entrance: the depth of this energy well was modulated strongly by the protonation state of E222. The energy barrier for Cl(-) was found to be 3-4 times higher than that for Na(+). Ion permeation characteristics were determined through BD simulations using a hybrid MD/continuum electrostatics approach to evaluate the energy profiles governing the ion movement. The resultant channel conductance and a near-zero permeability ratio (P(Cl)/P(Na)) were comparable to experimental data. On the basis of these calculations, we suggest that a ring of five E222 residues may act as an electrostatic gate. In addition, the hydrophobic gate region may play a role in charge selectivity due to a higher dehydration energy barrier for Cl(-) ions. The effect of halothane on the Na(+) PMF was also evaluated. Halothane was found to perturb salt bridges in GLIC that may be crucial for channel gating and open-channel stability, but had no significant impact on the single ion PMF profiles.     

Chloride ion conduction without water coordination in the pore of ClC protein

In the present work, we have found by an atomistic molecular dynamics simulation that hydrogen atoms originating from the residues of a prokaryotic ClC protein (EcClC) stabilize the chloride ion without water molecules in the pore of ClC protein. When the chloride ion conduction is simulated by pulling a chloride ion along the pore axis, the free energy barrier for chloride ion conduction is calculated to be low (4 kcal/mol), although the chloride ion is stripped of its hydration shell as it passes through the dehydrated pore region. The calculation of the number of hydrogen atoms surrounding the chloride ion reveals that water molecules hydrating the chloride ion are replaced by polar and non‐polar hydrogen atoms protruding from the protein residues. From the analysis of the pair interaction energy between the chloride ion and these hydrogen atoms, it is realized that the hydrogen atoms from the protein residues stabilize the chloride ion at the dehydrated region instead of water molecules, by which the energetic penalty for detaching water molecules from the permeating ion is compensated. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

A parallel finite element simulator for ion transport through three‐dimensional ion channel systems

A parallel finite element simulator, ichannel, is developed for ion transport through three‐dimensional ion channel systems that consist of protein and membrane. The coordinates of heavy atoms of the protein are taken from the Protein Data Bank and the membrane is represented as a slab. The simulator contains two components: a parallel adaptive finite element solver for a set of Poisson–Nernst–Planck (PNP) equations that describe the electrodiffusion process of ion transport, and a mesh generation tool chain for ion channel systems, which is an essential component for the finite element computations. The finite element method has advantages in modeling irregular geometries and complex boundary conditions. We have built a tool chain to get the surface and volume mesh for ion channel systems, which consists of a set of mesh generation tools. The adaptive finite element solver in our simulator is implemented using the parallel adaptive finite element package Parallel Hierarchical Grid (PHG) developed by one of the authors, which provides the capability of doing large scale parallel computations with high parallel efficiency and the flexibility of choosing high order elements to achieve high order accuracy. The simulator is applied to a real transmembrane protein, the gramicidin A (gA) channel protein, to calculate the electrostatic potential, ion concentrations and I – V curve, with which both primitive and transformed PNP equations are studied and their numerical performances are compared. To further validate the method, we also apply the simulator to two other ion channel systems, the voltage dependent anion channel (VDAC) and α‐Hemolysin (α‐HL). The simulation results agree well with Brownian dynamics (BD) simulation results and experimental results. Moreover, because ionic finite size effects can be included in PNP model now, we also perform simulations using a size‐modified PNP (SMPNP) model on VDAC and α‐HL. It is shown that the size effects in SMPNP can effectively lead to reduced current in the channel, and the results are closer to BD simulation results. © 2013 Wiley Periodicals, Inc.     

自组装杂化离子通道膜的构筑及阳离子传输机制

以3-异氰酸丙基三乙氧基硅烷和对甲氧基苯胺为原料合成了一种可以自组装形成有机-无机杂化材料的化合物--3-(脲基-4-甲氧基苯基)丙基三乙氧基硅烷. 采用FT-IR, ¹H NMR, DSC 和XRD 分析方法对该化合物的结构以及结晶性进行了表征. 将该化合物与聚乙烯醇(PVA)共混, 利用化合物的自组装性质构筑结构均一且致密无孔的离子通道杂化膜, 通过自制的膜运输实验装置测定膜对阳离子的传输性能并提出了相应的传输机制. SEM 照片显示, 自组装杂化膜致密无缺陷, 膜厚度为8 μm. 选择5 种阳离子进行运输实验测试, 结果表明, 自组装杂化离子通道膜对一价的碱金属离子Li⁺, Na⁺和K⁺有很好的传输功能, 这要归功于杂化材料中甲氧基苯基与碱金属阳离子形成的阳离子-π相互作用力. 碱金属阳离子在膜中的扩散过程可由溶解-扩散机制来解释, 结果显示, Li⁺, Na⁺和K⁺在杂化膜中传输的渗透率大小为: P_Na⁺ > P_K⁺ > P_Li⁺ , 说明本研究中的的自组装杂化离子通道膜对Na⁺有优先选择性. 杂化离子通道膜对二价的Ca²⁺和Mg²⁺没有传输作用, 此结果给一二价阳离子的分离带来很好的研究思路.     

Ion mobility mass spectrometry of two tetrameric membrane protein complexes reveals compact structures and differences in stability and packing

Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for the ion channel are in accord with a compact configuration of subunits, suggesting that the native-like structure can be preserved under the harsh activation conditions required to release it from the detergent micelle into the gas phase. We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable once stripped of detergents and bulk water. These results highlight the potential of ion mobility mass spectrometry for characterizing the overall topologies of membrane protein complexes and the structural changes associated with nucleotide, lipid, and drug binding.     

Insertion and assembly of membrane proteins via simulation

Interactions of lipids are central to the folding and stability of membrane proteins. Coarse-grained molecular dynamics simulations have been used to reveal the mechanisms of self-assembly of protein/membrane and protein/detergent complexes for representatives of two classes of membrane protein, namely, glycophorin (a simple alpha-helical bundle) and OmpA (a beta-barrel). The accuracy of the coarse-grained simulations is established via comparison with the equivalent atomistic simulations of self-assembly of protein/detergent micelles. The simulation of OmpA/bilayer self-assembly reveals how a folded outer membrane protein can be inserted in a bilayer. The glycophorin/bilayer simulation supports the two-state model of membrane folding, in which transmembrane helix insertion precedes dimer self-assembly within a bilayer. The simulations also suggest that a dynamic equilibrium exists between the glycophorin helix monomer and dimer within a bilayer. The simulated glycophorin helix dimer is remarkably close in structure to that revealed by NMR. Thus, coarse-grained methods may help to define mechanisms of membrane protein (re)folding and will prove suitable for simulation of larger scale dynamic rearrangements of biological membranes.     

Gating motions in voltage-gated potassium channels revealed by coarse-grained molecular dynamics simulations

Voltage-gated potassium (Kv) channels are ubiquitous transmembrane proteins involved in electric signaling of excitable tissues. A fundamental property of these channels is the ability to open or close in response to changes in the membrane potential. To date, their structure-based activation mechanism remains unclear, and there is a large controversy on how these gates function at the molecular level, in particular, how movements of the voltage sensor domain are coupled to channel gating. So far, all mechanisms proposed for this coupling are based on the crystal structure of the open voltage-gated Kv1.2 channel and structural models of the closed form based on electrophysiology experiments. Here, we use coarse-grain (CG) molecular dynamics simulations that allow conformational changes from the open to the closed form of the channel (embedded in its membrane environment) to be followed. Despite the low specificity of the CG force field, the obtained closed structure satisfies several experimental constraints. The overall results suggest a gating mechanism in which a lateral displacement the S4-S5 linker leads to a closing of the gate. Only a small up-down movement of the S4 helices is noticed. Additionally, the study suggests a peculiar upward motion of the intracellular tetramerization domain of the channel, hence providing a molecular view on how this domain may further regulate conduction in Kv channels.     

Ion channel screening

Ion channels are attractive targets for drug discovery with recent estimates indicating that voltage and ligand-gated channels account for the third and fourth largest gene families represented in company portfolios after the G protein coupled and nuclear hormone receptor families. A historical limitation on ion channel targeted drug discovery in the form of the extremely low throughput nature of the gold standard assay for assessing functional activity, patch clamp electrophysiology in mammalian cells, has been overcome by the implementation of multi-well plate format cell-based screening strategies for ion channels. These have taken advantage of various approaches to monitor ion flux or membrane potential using radioactive, non-radioactive, spectroscopic and fluorescence measurements and have significantly impacted both high-throughput screening and lead optimization efforts. In addition, major advances have been made in the development of automated electrophysiological platforms to increase capacity for cell-based screening using formats aimed at recapitulating the gold standard assay. This review addresses the options available for cell-based screening of ion channels with examples of their utility and presents case studies on the successful implementation of high-throughput screening campaigns for a ligand-gated ion channel using a fluorescent calcium indicator, and a voltage-gated ion channel using a fluorescent membrane potential sensitive dye.     

Assessing atomistic and coarse-grained force fields for protein-lipid interactions: the formidable challenge of an ionizable side chain in a membrane

Ionizable amino acid side chains play important roles in membrane protein structure and function, including the activation of voltage-gated ion channels, where it has been previously suggested that charged side chains may move through the hydrocarbon core of the membrane. However, all-atom molecular dynamics simulations have demonstrated large free energy barriers for such lipid-exposed motions. These simulations have also revealed that the membrane will deform due to the presence of a charged side chain, leading to a complex solvation microenvironment for which empirical force fields were not specifically parametrized. We have tested the ability of the all-atom CHARMM, Drude polarizable CHARMM, and a recent implementation of a coarse-grained force field to measure the thermodynamics of arginine-membrane interactions as a function of protonation state. We have employed model systems to attempt to match experimental bulk partitioning and quantum mechanical interactions within the membrane and found that free energy profiles from nonpolarizable and polarizable CHARMM simulations are accurate to within 1-2 kcal/mol. In contrast, the coarse-grained simulations failed to reproduce the same membrane deformations, exhibit interactions that are an order of magnitude too small, and thus, have incorrect free energy profiles. These results illustrate the need for careful parametrization of coarse-grained force fields and demonstrate the utility of atomistic molecular dynamics for providing quantitative thermodynamic and mechanistic analysis of protein-lipid interactions.     

充蜡石墨支撑的季铵离子/己二酸混合双层膜上钙离子诱发的离子通道行为

以充蜡石墨电极作为新型支撑体,成功制备了一种季铵离子为内层（包括四丁 基铵TBA,十六烷基三甲基铵CTrMA）,己二酸（HDA）为外层的新型自组装混合双 层膜,以循环伏安和电化学交流阻抗方法研究了膜的离子通道行为。该膜能够接受 Ca~(2+)的刺激作用而打开[Fe(CN)_6]~(3-/4-)电极氧化还原的离子通道,撤走该 刺激离子则通道关闭。提出了混合双层膜的结构和离子通道作用的模型,指出外层 膜HDA分子可能具有V型和W型两种结构。     

Photomodification of Cardiac Membrane: Chaotic Currents and High Conductance States in Isolated Patches

We have demonstrated previously that photomodification permeabilizes cardiac cells as evidenced by activation of a whole-cell leak current. In this paper we report that photomodification induces in cell-attached and inside-out cardiac membrane patches a chaotic current. Unlike current recordings from many protein ion channels that show stepwise amplitude changes associated with open and closed states of the channel, the chaotic current consists of variable amplitude spike-like transitions. The amplitudes of these spikes can vary from tenths to tens of picoamperes at a constant transmembrane potential. We provide evidence that the chaotic current is transmembrane rather than trans-seal and has a voltage dependency expected for current flow through nonspecific conductance pathways. Photomodification can also induce high conductance states (greater than 500 pS) in cell-attached and inside-out patches. We present evidence that the high conductance state is also not related to seal breakdown. Our results suggest that both the chaotic current activity in and high conductance state of photo-modified cardiac membrane patches result from the opening of many small conductance, nonspecific pathways through the membrane.     

Computational Insights into Allosteric Conformational Modulation of P-Glycoprotein by Substrate and Inhibitor Binding

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a physiologically essential membrane protein that protects many tissues against xenobiotic molecules, but limits the access of chemotherapeutics into tumor cells, thus contributing to multidrug resistance. The atomic-level mechanism of how substrates and inhibitors differentially affect the ATP hydrolysis by P-gp remains to be elucidated. In this work, atomistic molecular dynamics simulations in an explicit membrane/water environment were performed to explore the effects of substrate and inhibitor binding on the conformational dynamics of P-gp. Distinct differences in conformational changes that mainly occurred in the nucleotide-binding domains (NBDs) were observed from the substrate- and inhibitor-bound simulations. The binding of rhodamine-123 can increase the probability of the formation of an intermediate conformation, in which the NBDs were closer and better aligned, suggesting that substrate binding may prime the transporter for ATP hydrolysis. By contrast, the inhibitor QZ-Leu stabilized NBDs in a much more separated and misaligned conformation, which may result in the deficiency of ATP hydrolysis. The significant differences in conformational modulation of P-gp by substrate and inhibitor binding provided a molecular explanation of how these small molecules exert opposite effects on the ATPase activity. A further structural analysis suggested that the allosteric communication between transmembrane domains (TMDs) and NBDs was primarily mediated by two intracellular coupling helices. Our computational simulations provide not only valuable insights into the transport mechanism of P-gp substrates, but also for the molecular design of P-gp inhibitors.     

Natural products as tools for studies of ligand-gated ion channels

Ligand-gated ion channels, or ionotropic receptors, constitute a group of membrane-bound proteins that regulate the flux of ions across the cell membrane. In the brain, ligand-gated ion channels mediate fast neurotransmission. They are crucial for normal brain function and involved in many diseases in the brain. Historically, natural products have been used extensively in biomedical studies and ultimately as drugs or leads for drug design. In studies of ligand-gated ion channels, natural products have been essential for the understanding of their structure and function. In the following a short survey of natural products and their use in studies of ligand-gated ion channels is given.