A new gating site in human aquaporin-4: Insights from molecular dynamics simulations

Aquaporin-4 (AQP4) is the predominant water channel in different organs and tissues. An alteration of its physiological functioning is responsible for several disorders of water regulation and, thus, is considered an attractive target with a promising therapeutic and diagnostic potential. Molecular dynamics (MD) simulations performed on the AQP4 tetramer embedded in a bilayer of lipid molecules allowed us to analyze the role of spontaneous fluctuations occurring inside the pore. Following the approach by Hashido et al. [Hashido M, Kidera A, Ikeguchi M (2007) Biophys J 93: 373–385], our analysis on 200 ns trajectory discloses three domains inside the pore as key elements for water permeation. Herein, we describe the gating mechanism associated with the well-known selectivity filter on the extracellular side of the pore and the crucial regulation ensured by the NPA motifs (asparagine, proline, alanine). Notably, on the cytoplasmic side, we find a putative gate formed by two residues, namely, a cysteine belonging to the loop D (C178) and a histidine from loop B (H95). We observed that the spontaneous reorientation of the imidazole ring of H95 acts as a molecular switch enabling H-bond interaction with C178. The occurrence of such local interaction seems to be responsible for the narrowing of the pore and thus of a remarkable decrease in water flux rate. Our results are in agreement with recent experimental observations and may represent a promising starting point to pave the way for the discovery of chemical modulators of AQP4 water permeability.     

Structural basis of gating of CNG channels

Cyclic nucleotide-gated (CNG) ion channels, underlying sensory transduction in vertebrate photoreceptors and olfactory sensory neurons, require cyclic nucleotides to open. Here, we present structural models of the tetrameric CNG channel pore from bovine rod in both open and closed states, as obtained by combining homology modeling-based techniques, experimentally derived spatial constraints and structural patterns present in the PDB database. Gating is initiated by an anticlockwise rotation of the N-terminal region of the C-linker, which is then, transmitted through the S6 transmembrane helices to the P-helix, and in turn from this to the pore lumen, which opens up from 2 to 5A thus allowing for ion permeation. The approach, here presented, is expected to provide a general methodology for model ion channels and their gating when structural templates are available and an extensive electrophysiological analysis has been performed.     

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods

The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.     

Exploring gas permeability of cellular membranes and membrane channels with molecular dynamics

Aquaporins are a family of membrane proteins specialized in rapid water conduction across biological membranes. Whether these channels also conduct gas molecules and the physiological significance of this potential function have not been well understood. Here we report 140 ns of molecular dynamics simulations of membrane-embedded AQP1 and of a pure POPE bilayer addressing these questions. The permeability of AQP1 to two types of gas molecules, O2 and CO2, was investigated using two complementary methods, namely, explicit gas diffusion simulation and implicit ligand sampling. The simulations show that the central (tetrameric) pore of AQP1 can be readily used by either gas molecule to permeate the channel. The two approaches produced similar free energy profiles associated with gas permeation through the central pore: a -0.4 to -1.7 kcal/mol energy well in the middle, and a 3.6-4.6 kcal/mol energy barrier in the periplasmic vestibule. The barrier appears to be mainly due to a dense cluster of water molecules anchored in the periplasmic mouth of the central pore by four aspartate residues. Water pores show a very low permeability to O2, but may contribute to the overall permeation of CO2 due to its more hydrophilic nature. Although the central pore of AQP1 is found to be gas permeable, the pure POPE bilayer provides a much larger cross-sectional area, thus exhibiting a much lower free energy barrier for CO2 and O2 permeation. As such, gas conduction through AQP1 may only be physiologically relevant either in membranes of low gas permeability, or in cells where a major fraction of the cellular membrane is occupied by AQPs.     

Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS

The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating. When Asp-62, a negatively charged residue located in the loop that connects the first and second transmembrane helices, was replaced with either a neutral (Cys or Asn) or basic (Arg) amino acid, increases in both the gating threshold and inactivation rate were observed. Similar effects were observed after neutralization or reversal of the charge of either Arg-128 or Arg-131, which are both located near Asp-62 on the upper surface of the cytoplasmic domain. Interestingly, the effects of replacing Asp-62 with arginine were complemented by reversing the charge of Arg-131. Complementation was not observed after simultaneous neutralization of the charge of these residues. These findings suggest that the cytoplasmic domain of MscS affects both the mechanosensitive gating and the channel inactivation rate through the electrostatic interaction between Asp-62 and Arg-131.     

The Gating Mechanism of the Human Aquaporin 5 Revealed by Molecular Dynamics Simulations

Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5) shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value.     

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

Nitric oxide conduction by the brain aquaporin AQP4

Involvement of aquaporins in gas conduction across the membrane and the physiological significance of this process have attracted marked attention from both experimental and theoretical studies. Previous work demonstrated that AQP1 is permeable to both CO₂ and O₂. Here we employ various simulation techniques to examine the permeability of the brain aquaporin AQP4 to NO and O₂ and to describe energetics and pathways associated with these phenomena. The energy barrier to NO and O₂ permeation through AQP4 central pore is found to be only ～3 kcal mol^?1. The results suggest that the central pore of AQP4, similar to that of AQP1, can indeed conduct gas molecules. Interestingly, despite a longer and narrower central pore, AQP4 appears to provide an energetically more favorable permeation pathway for gas molecules than AQP1, mainly due to the different orientation of its charged residues near the pore entrance. Although the low barrier against gas permeation through AQP4 indicates that it can participate in gas conduction across the cellular membrane, physiological relevance of the phenomenon remains to be established experimentally, particularly since pure lipid bilayers appear to present a more favorable pathway for gas conduction across the membrane. With an energy well of ?1.8 kcal mol^?1, the central pore of AQP4 may also act as a reservoir for NO molecules to accumulate in the membrane. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Role of the juxtamembrane region of cytoplasmic loop 3 in the gating and conductance of the cystic fibrosis transmembrane conductance regulator chloride channel

Opening and closing of the cystic fibrosis transmembrane conductance regulator chloride channel are controlled by interactions of ATP with its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected to the transmembrane pore via four cytoplasmic loops. These loops have been suggested to play roles both in channel gating and in forming a cytoplasmic extension of the channel pore. To investigate the structure and function of one of these cytoplasmic loops, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced into loop 3. We find that methanethiosulfonate (MTS) reagents modify cysteines introduced at 14 of 16 sites studied in the juxtamembrane region of loop 3, in all cases leading to inhibition of channel function. In most cases, both the functional effects of modification and the rate of modification were similar for negatively and positively charged MTS reagents. Single-channel recordings indicated that, at all sites, inhibition was the result of an MTS reagent-induced decrease in channel open probability; in no case was the Cl(-) conductance of open channels altered by modification. These results indicate that loop 3 is readily accessible to the cytoplasm and support the involvement of this region in the control of channel gating. However, our results do not support the hypothesis that this region is close enough to the Cl(-) permeation pathway to exert any influence on permeating Cl(-) ions. We propose that either the cytoplasmic pore is very wide or cytoplasmic Cl(-) ions use other routes to access the transmembrane pore.     

Structural Determinants of Skeletal Muscle Ryanodine Receptor Gating

Ryanodine receptor type 1 (RyR1) releases Ca²⁺ from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca²⁺. However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912–Glu-4948) and a linker helix (Val-4830–Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.     

Molecular Mechanisms of How Mercury Inhibits Water Permeation through Aquaporin-1: Understanding by Molecular Dynamics Simulation

Aquaporin (AQP) functions as a water-conducting pore. Mercury inhibits the water permeation through AQP. Although site-directed mutagenesis has shown that mercury binds to Cys¹⁸⁹ during the inhibition process, it is not fully understood how this inhibits the water permeation through AQP1. We carried out 40 ns molecular dynamics simulations of bovine AQP1 tetramer with mercury (Hg-AQP1) or without mercury (Free AQP1). In Hg-AQP1, Cys¹⁹¹ (Cys¹⁸⁹ in human AQP1) is converted to Cys-SHg⁺ in each monomer. During each last 10 ns, we observed water permeation events occurred 23 times in Free AQP1 and never in Hg-AQP1. Mercury binding did not influence the whole structure, but did induce a collapse in the orientation of several residues at the ar/R region. In Free AQP1, backbone oxygen atoms of Gly¹⁹⁰, Cys¹⁹¹, and Gly¹⁹² lined, and were oriented to, the surface of the water pore channel. In Hg-AQP1, however, the SHg⁺ of Cys191-SHg⁺ was oriented toward the outside of the water pore. As a result, the backbone oxygen atoms of Gly¹⁹⁰, Cys¹⁹¹, and Gly¹⁹² became disorganized and the ar/R region collapsed, thereby obstructing the permeation of water. We suggest that mercury disrupts the water pore of AQP1 through local conformational changes in the ar/R region.     

Gating mechanisms in Cys-loop receptors

The Cys-loop receptor superfamily of ligand-gated ion channels has a prominent role in neuronal signalling. These receptors are pentamers, each subunit containing ten β-strands in the extracellular domain and four α-helical transmembrane domains (M1–M4). The M2 domain of each subunit lines the intrinsic ion channel pore and residues within the extracellular domain form ligand binding sites. Ligand binding initiates a conformational change that opens the ion-selective pore. The coupling between ligand binding in the extracellular domain and opening of the intrinsic ion channel pore located in the membrane is not fully understood. Several loop structures, such as loop 2, the Cys-loop, the pre-M1 region and the M2–M3 loop have been implicated in receptor activation. The current “conformational change wave” hypothesis suggests that binding of a ligand initiates a rotation of the β-sheets around an axis that passes through the Cys-loop. Due to this rotation, the Cys-loop and loop 2 are displaced. Movement of the M2–M3 loop then twists the M2 domain leading to a separation of the helices and opening of the pore. The publication of a crystal structure of an acetylcholine binding protein and the refined structure of the Torpedo marmorata acetylcholine receptor have improved the understanding of the mechanisms and structures involved in coupling ligand binding to channel gating. In this review, the most recent findings on some of these loop structures will be reported and discussed in view of their role in the gating mechanism.     

Contributions of a negatively charged residue in the hydrophobic domain of the IRK1 inwardly rectifying K+ channel to K(+)-selective permeation.

Inwardly rectifying K+ channels are highly selective for K+ ions and show strong interaction with ions in the pore. Both features are important for the physiological functions of these channels and pose intriguing mechanistic questions of ion permeation. The aspartate residue in the second putative transmembrane segment of the IRK1 inwardly rectifying K+ channel, previously implicated in inward rectification gating due to cytoplasmic Mg2+ and polyamine block, is found in this study to be crucial for the channel's ability to distinguish between K+ and Rb+ ions. Mutation of this residue also perturbs the interaction between the channel pore and the Sr2+ blocking ion. Our studies suggest that this aspartate residue contributes to a selectivity filter near the cytoplasmic end of the pore.     

Insights into the biophysical properties of GABA(A) ion channels: modulation of ion permeation by drugs and protein interactions

The fundamental properties of ion channels assure their selectivity for a particular ion, its rapid permeation through a central pore and that such electrical activity is modulated by factors that control the opening and closing (gating) of the channel. All cell types possess ion channels and their regulated flux of ions across the membrane play critical roles in all steps of life. An ion channel does not act alone to control cell excitability but rather forms part of larger protein complexes. The identification of protein interaction partners of ion channels and their influence on both the fundamental biophysical properties of the channel and its expression in the membrane are revealing the many ways in which electrical activity may be regulated. Highlighted here is the novel use of the patch clamp method to dissect out the influence of protein interactions on the activity of individual GABA(A) receptors. The studies demonstrate that ion conduction is a dynamic property of a channel and that protein interactions in a cytoplasmic domain underlie the channel's ability to alter ion permeation. A structural model describing a reorganisation of the conserved cytoplasmic gondola domain and the influence of drugs on this process are presented.     

Global twisting motion of single molecular KcsA potassium channel upon gating

Ion channels are signal transduction molecules that switch ion permeation pathways on and off (gating). Crystal structures of several kinds of potassium channels have revealed open and closed conformations, which provide static pictures of gating status. Here we studied KcsA potassium channels undergoing conformational changes at the single-molecule level. A KcsA channel with a gold nanocrystal attached was irradiated by white X-rays and motions of the diffraction spot from the nanocrystal were tracked in real time. Upon gating, the KcsA channels twisted around the axis of the pore. These conformational changes were prevented by an open-channel blocker, tetrabuthylammonium. Random clockwise and counterclockwise twisting in the range of several tens of degrees originated in the transmembrane domain and was transmitted to the cytoplasmic domain. This coupling suggests a mechanical interplay between the transmembrane and cytoplasmic domains.     

Water channel structures analysed by electron crystallography

Background

The mechanisms underlying water transport through aquaporin (AQP) have been debated for two decades. The water permeation phenomenon of AQP seems inexplicable because the Grotthuss mechanism does not allow for simultaneous fast water permeability and inhibition of proton transfer through the hydrogen bonds of water molecules.

Scope of review

The AQP1 structure determined by electron crystallography provided the first insights into the proton exclusion mechanism despite fast water permeation. Although several studies have provided clues about the mechanism based on the AQP structure, each proposed mechanism remains incomplete. The present review is focused on AQP function and structure solved by electron crystallography in an attempt to fill the gaps between the findings in the absence and presence of lipids.

Major conclusions

Many AQP structures can be superimposed regardless of the determination method. The AQP fold is preserved even under conditions lacking lipids, but the water arrangement in the channel pore differs. The differences might be explained by dipole moments formed by the two short helices in the lipid bilayer. In addition, structure analyses of double-layered two-dimensional crystals of AQP suggest an array formation and cell adhesive function.

General significance

Electron crystallography findings not only have contributed to resolve some of the water permeation mechanisms, but have also elucidated the multiple functions of AQPs in the membrane. The roles of AQPs in the brain remain obscure, but their multiple activities might be important in the regulation of brain and other biological functions. This article is part of a Special Issue entitled Aquaporins.     

Carboxy-terminal truncations modify the outer pore vestibule of muscle chloride channels

Mammalian ClC-type chloride channels have large cytoplasmic carboxy-terminal domains whose function is still insufficiently understood. We investigated the role of the distal part of the carboxy-terminus of the muscle isoform ClC-1 by constructing and functionally evaluating two truncation mutants, R894X and K875X. Truncated channels exhibit normal unitary conductances and anion selectivities but altered apparent anion binding affinities in the open and in the closed state. Since voltage-dependent gating is strictly coupled to ion permeation in ClC-1 channels, the changed pore properties result in different fast and slow gating. Full length and truncated channels also differed in methanethiosulphonate (MTS) modification rate constants of an engineered cysteine at position 231 near the selectivity filter. Our data demonstrate that the carboxy-terminus of ClC channels modifies the conformation of the outer pore vestibule.     

Mechanism of voltage-dependent gating in skeletal muscle chloride channels.

Voltage-dependent gating was investigated in a recombinant human skeletal muscle Cl- channel, hCIC-1, heterologously expressed in human embryonic kidney (HEK-293) cells. Gating was found to be mediated by two qualitatively distinct processes. One gating step operates on a microsecond time scale and involves the rapid rearrangement of two identical intramembranous voltage sensors, each consisting of a single titratable residue. The second process occurs on a millisecond time scale and is due to a blocking-unblocking reaction mediated by a cytoplasmic gate that interacts with the ion pore of the channel. These results illustrate a rather simple structural basis for voltage sensing that has evolved in skeletal muscle Cl- channels and provides evidence for the existence of a cytoplasmic gating mechanism in an anion channel analogous to the "ball and chain" mechanism of voltage-gated cation channels.     

Structural Determinants of Oligomerization of the Aquaporin-4 Channel

The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family.     

Emerging models of glutamate receptor ion channel structure and function

Excitatory synaptic transmission in the brain is mediated by ligand-gated ion channels (iGluRs) activated by glutamate. Distinct from other neurotransmitter receptors, the extracellular domains of iGluRs are loosely packed assemblies with two clearly distinct layers, each of which has both local and global 2-fold axes of symmetry. By contrast, the iGluR transmembrane segments have 4-fold symmetry and share a conserved pore loop architecture found in tetrameric voltage-gated ion channels. The striking layered architecture of iGluRs revealed by the 3.6?? resolution structure of an AMPA receptor homotetramer likely arose from gene fusion events that occurred early in evolution. Although this modular design has greatly facilitated biophysical and structural studies on individual iGluR domains, and suggested conserved mechanisms for iGluR gating, recent work is beginning to reveal unanticipated diversity in the structure, allosteric regulation, and assembly of iGluR subtypes.