Mixed modes in opening of KcsA potassium channel from a targeted molecular dynamics simulation

Potassium channels conduct K⁺ flow selectively across the membrane through a central pore. During a process called gating, the potassium channels undergo a conformational change that opens or closes the ion-conducting pore. The potassium channel KcsA has been structurally determined in its closed state. However, the dynamic mechanism of the gating transition of the KcsA channel is still being investigated. Here, a targeted molecular dynamics simulation up to 150 ns is performed to investigate the detailed opening process of the KcsA channel with an open Kv1.2 structure serving as the target. The channel arrived at a self-determined quasi-stable state within 60 ns. The rigid-body and hinge-bending modes are observed mixed together in the remaining 90 ns long quasi-stable state. The mixed-mode movement seems come from the competition between the helix rigidity and the biased-applied gating force.     

Molecular mechanism of voltage sensor movements in a potassium channel

Voltage-gated potassium channels are six-transmembrane (S1-S6) proteins that form a central pore domain (4 x S5-S6) surrounded by four voltage sensor domains (S1-S4), which detect changes in membrane voltage and control pore opening. Upon depolarization, the S4 segments move outward carrying charged residues across the membrane field, thereby leading to the opening of the pore. The mechanism of S4 motion is controversial. We have investigated how S4 moves relative to the pore domain in the prototypical Shaker potassium channel. We introduced pairs of cysteines, one in S4 and the other in S5, and examined proximity changes between each pair of cysteines during activation, using Cd2+ and copper-phenanthroline, which crosslink the cysteines with metal and disulphide bridges, respectively. Modelling of the results suggests a novel mechanism: in the resting state, the top of the S3b-S4 voltage sensor paddle lies close to the top of S5 of the adjacent subunit, but moves towards the top of S5 of its own subunit during depolarization--this motion is accompanied by a reorientation of S4 charges to the extracellular phase.     

Investigating the size and dynamics of voltage-gated sodium channel fenestrations: A molecular dynamics study

Eukaryotic voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in electrically excitable cells, and are important pharmaceutical targets for the treatment of neurological disorders such as epilepsy, cardiac arrhythmias, and chronic pain. Evidence suggests that small, hydrophobic, VGSC-blocking drugs can gain access to binding residues within the central cavity of these channels by passing through lateral, lipid-filled “fenestrations” which run between the exterior of the protein and its central pore. Here, we use molecular dynamics simulations to investigate how the size and shape of fenestrations change over time in several bacterial VGSC models and a homology model of Nav1.4. We show that over the course of the simulations, the size of the fenestrations is primarily influenced by rapid protein motions, such as amino acid side-chain rotation, and highlight that differences between fenestration bottleneck-contributing residues are the primary cause of variations in fenestration size between the 6 bacterial models. In the eukaryotic channel model, 2 fenestrations are wide, but 2 are narrow due to differences in the amino acid sequence in the 4 domains. Lipid molecules are found to influence the size of the fenestrations by protruding acyl chains into the fenestrations and displacing amino acid side-chains. Together, the results suggest that fenestrations provide viable pathways for small, flexible, hydrophobic drugs.     

RT—PCR测定大鼠妊娠早期子宫孕激素受体基因的表达

孕激素在妊娠的建立和维持过程中起着非常重要的作用,但目前为止未见有关大鼠子宫 激素受体（ＰＲ）基因在该过程中表达情况的系统报道。本和反转偶联聚合酶链式反应（ＲＴ－ＰＣＲ）方法测定了大鼠动情周期和妊娠早期子宫孕激素受体基因的转录,结果表明：１．动情周期中,子吕ＰＲｍＲＮＡ水平在动情期最高,在动情后期最低,动情期水平约为动情后期的２倍。２．妊娠开始后,子宫ＰＲｍＲＮＡ水平迅速上地ｄ３－ｄ４（着床前）达     

A comparison between two prokaryotic potassium channels (KirBac1.1 and KcsA) in a molecular dynamics (MD) simulation study

The two potassium ion channels KirBac1.1 and KcsA are compared in a Molecular Dynamics (MD) simulation study. The location and motion of the potassium ions observed in the simulations are compared to those in the X-ray structures and previous simulations. In our simulations several of the crystallography resolved ion sites in KirBac1.1 are occupied by ions. In addition to this, two in KirBac1.1 unresolved sites where occupied by ions at sites that are in close correspondence to sites found in KcsA. There is every reason to believe that the conserved alignment of the selectivity filter in the potassium ion channel family corresponds to a very similar mechanism for ion transport across the filter. The gate residues, Phe146 in KirBac1.1 and Ala111 in KcsA acted in the simulations as effective barriers which never were passed by ions nor water molecules.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations: From tension sensing to channel opening

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

Drug block of the hERG potassium channel: insight from modeling

Many commonly used, structurally diverse, drugs block the human ether-a-go-go-related gene (hERG) K(+) channel to cause acquired long QT syndrome, which can lead to sudden death via lethal cardiac arrhythmias. This undesirable side effect is a major hurdle in the development of safe drugs. To gain insight about the structure of hERG and the nature of drug block we have produced structural models of the channel pore domain, into each of which we have docked a set of 20 hERG blockers. In the absence of an experimentally determined three-dimensional structure of hERG, each of the models was validated against site-directed mutagenesis data. First, hERG models were produced of the open and closed channel states, based on homology with the prokaryotic K(+) channel crystal structures. The modeled complexes were in partial agreement with the mutagenesis data. To improve agreement with mutagenesis data, a KcsA-based model was refined by rotating the four copies of the S6 transmembrane helix half a residue position toward the C-terminus, so as to place all residues known to be involved in drug binding in positions lining the central cavity. This model produces complexes that are consistent with mutagenesis data for smaller, but not larger, ligands. Larger ligands could be accommodated following refinement of this model by enlarging the cavity using the inherent flexibility about the glycine hinge (Gly648) in S6, to produce results consistent with the experimental data for the majority of ligands tested.     

Interaction between dihydropyridines and phospholipid bilayers: a molecular dynamics simulation

Interaction of the calcium-channel antagonist dihydropyridines (DHPs), lacidipine and nifedipine, with a phospholipid bilayer was studied using 600 ps molecular dynamic simulations. We have constructed a double layer membrane model composed of 42 dimirystoyl-phosphatidylcholine molecules. The DHP molecules locate at about 7 Å from the centre of the membrane, inducing an asymmetry in the bilayer. While lacidipine did not induce significant local perturbations as judged by the gauche-trans isomerisation rate, nifedipine significantly decreased this rate, probably by producing a local rigidity of the membrane in the vicinity of the DHP.     

Investigating the size and dynamics of voltage-gated sodium channel fenestrations

Eukaryotic voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in electrically excitable cells, and are important pharmaceutical targets for the treatment of neurological disorders such as epilepsy, cardiac arrhythmias, and chronic pain. Evidence suggests that small, hydrophobic, VGSC-blocking drugs can gain access to binding residues within the central cavity of these channels by passing through lateral, lipid-filled “fenestrations” which run between the exterior of the protein and its central pore. Here, we use molecular dynamics simulations to investigate how the size and shape of fenestrations change over time in several bacterial VGSC models and a homology model of Nav1.4. We show that over the course of the simulations, the size of the fenestrations is primarily influenced by rapid protein motions, such as amino acid side-chain rotation, and highlight that differences between fenestration bottleneck-contributing residues are the primary cause of variations in fenestration size between the 6 bacterial models. In the eukaryotic channel model, 2 fenestrations are wide, but 2 are narrow due to differences in the amino acid sequence in the 4 domains. Lipid molecules are found to influence the size of the fenestrations by protruding acyl chains into the fenestrations and displacing amino acid side-chains. Together, the results suggest that fenestrations provide viable pathways for small, flexible, hydrophobic drugs.     

A BEST example of channel structure annotation by molecular simulation

An increasing number of ion channel structures are being determined. This generates a need for computational tools to enable functional annotation of channel structures. However, several studies of ion channel and model pores have indicated that the physical dimensions of a pore are not always a reliable indicator of its conductive status. This is due to the unusual behavior of water within nano-confined spaces, resulting in a phenomenon referred to as “hydrophobic gating”. We have recently demonstrated how simulating the behavior of water within an ion channel pore can be used to predict its conductive status. In this addendum to our study, we apply this method to compare the recently solved structure of a mutant of the bestrophin chloride channel BEST1 with that of the wild-type channel. Our results support the hypothesis of a hydrophobic gate within the narrow neck of BEST1. This provides further validation that this simulation approach provides the basis for an accurate and computationally efficient tool for the functional annotation of ion channel structures.     

Independence and cooperativity in rearrangements of a potassium channel voltage sensor revealed by single subunit fluorescence

Voltage-gated potassium channels are composed of four subunits. Voltage-dependent activation of these channels consists of a depolarization-triggered series of charge-carrying steps that occur in each subunit. These major charge-carrying steps are followed by cooperative step(s) that lead to channel opening. Unlike the late cooperative steps, the major charge-carrying steps have been proposed to occur independently in each of the channel subunits. In this paper, we examine this further. We showed earlier that the two major charge-carrying steps are associated with two sequential outward transmembrane movements of the charged S4 segment. We now use voltage clamp fluorometry to monitor these S4 movements in individual subunits of heterotetrameric channels. In this way, we estimate the influence of one subunit's S4 movement on another's when the energetics of their transmembrane movements differ. Our results show that the first S4 movement occurs independently in each subunit, while the second occurs cooperatively. At least part of the cooperativity appears to be intrinsic to the second S4 charge-carrying rearrangement. Such cooperativity in gating of voltage-dependent channels has great physiological relevance since it can affect both action potential threshold and rate of propagation.     

Molecular movement of the voltage sensor in a K channel

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 A) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 A) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model.     

Force-induced unfolding of human telomeric G-quadruplex: A steered molecular dynamics simulation study

We study the unfolding of a parallel G-quadruplex from human telomeric DNA by mechanical stretching using steered molecular dynamics (MD) simulation. We find that the force curves and unfolding processes strongly depend on the pulling sites. With pulling sites located on the sugar-phosphate backbone, the force-extension curve shows a single peak and the unfolding proceeds sequentially. Pulling sites located on the terminal nucleobases lead to a force-extension curve with two peaks and the unfolding is more cooperative. Simulations of the refolding of partially unfolded quadruplexes show very different behavior for the two different pulling modalities. In particular, starting from an unfolded state prepared by nucleobase pulling leads to a long-lived intermediate state whose existence is also corroborated by the free energy profile computed with the Jarzynski equation. Based on this observation, we propose a novel folding pathway for parallel G-quadruplexes with the human telomere sequence.     

Refinement of homology-based protein structures by molecular dynamics simulation techniques

The use of classical molecular dynamics simulations, performed in explicit water, for the refinement of structural models of proteins generated ab initio or based on homology has been investigated. The study involved a test set of 15 proteins that were previously used by Baker and coworkers to assess the efficiency of the ROSETTA method for ab initio protein structure prediction. For each protein, four models generated using the ROSETTA procedure were simulated for periods of between 5 and 400 nsec in explicit solvent, under identical conditions. In addition, the experimentally determined structure and the experimentally derived structure in which the side chains of all residues had been deleted and then regenerated using the WHATIF program were simulated and used as controls. A significant improvement in the deviation of the model structures from the experimentally determined structures was observed in several cases. In addition, it was found that in certain cases in which the experimental structure deviated rapidly from the initial structure in the simulations, indicating internal strain, the structures were more stable after regenerating the side-chain positions. Overall, the results indicate that molecular dynamics simulations on a tens to hundreds of nanoseconds time scale are useful for the refinement of homology or ab initio models of small to medium-size proteins.     

Homology modeling and molecular dynamics simulations of the alpha1 glycine receptor reveals different states of the channel

Homology modeling is used to build initial models of the transmembrane domain of the human alpha1 glycine receptor (GlyR) based on the most recently published refined structure of nAChR (PDB ID: 2BG9). Six preliminary GlyR models are constructed using two different approaches. In one approach, five different homopentamers are built by symmetric assembly of alpha1 GlyR subunits using only one of the five unique chains of nAChR as a template. In a second approach, each nAChR subunit serves as a template for an alpha1 GlyR subunit. All six initial GlyR constructs are then embedded into a hydrated POPC lipid bilayer and subjected to molecular dynamics simulation for at least six nanoseconds. Each model is stable throughout the simulation, and the final models fall into three distinct categories. Homopentameric GlyR bundles using a single alpha nAChR subunit as a template appear to be in an open conformation. Under an applied external potential, permeation of Cl(-) ions is observed within several ns in a channel built on an alpha chain. Model channels built on non-alpha chains have a constriction either near the intracellular mouth or more centrally located in the pore domain, both of which may be narrow enough to close the channel and whose locations correspond to putative gates observed in nicotinicoid receptors. The differences between these three general models suggest that channel closure may be effected by either rotation or tangential tilting of TM2.     

Role of hydrophobic residues in the voltage sensors of the voltage-gated sodium channel

The role of hydrophobic residues in voltage sensors S4 of voltage-sensitive ion channels is less documented than that of charged residues. We performed alanine-substitution of branched-sidechain residues contiguous to the third, fourth and fifth positively charged residues in S4s of the first three domains of the sodium channel expressed in HEK cells. These locations were selected because they are close to the arginines and lysines important in gating. Mutations in the first two domains (DIS4 and DIIS4) altered steady-state activation curves. In DIIIS4, the mutation L1131A next to the third arginine greatly slowed inactivation in a manner similar to that for substitutions of charged residues in DIVS4, whereas the mutation L1137A next to the fifth arginine preserved wild-type behaviour. Homology models of domain III, based on the structure of a crystallized mammalian potassium channel, shows that L1131 is located at the interface between S3 and S4 helices, whereas L1137, on the opposite side of S4, does not interact with the voltage sensor. The two mutated residues are closer to each other in domains I and II than in domain III, as may be corroborated by their different electrophysiological effects.     

Toward elucidating the heat activation mechanism of the TRPV1 channel gating by molecular dynamics simulation

As a key cellular sensor, the TRPV1 cation channel undergoes a gating transition from a closed state to an open state in response to various physical and chemical stimuli including noxious heat. Despite years of study, the heat activation mechanism of TRPV1 gating remains enigmatic at the molecular level. Toward elucidating the structural and energetic basis of TRPV1 gating, we have performed molecular dynamics (MD) simulations (with cumulative simulation time of 3 μs), starting from the high‐resolution closed and open structures of TRPV1 solved by cryo‐electron microscopy. In the closed‐state simulations at 30°C, we observed a stably closed channel constricted at the lower gate (near residue I679), while the upper gate (near residues G643 and M644) is dynamic and undergoes flickery opening/closing. In the open‐state simulations at 60°C, we found higher conformational variation consistent with a large entropy increase required for the heat activation, and both the lower and upper gates are dynamic with transient opening/closing. Through ensemble‐based structural analyses of the closed state versus the open state, we revealed pronounced closed‐to‐open conformational changes involving the membrane proximal domain (MPD) linker, the outer pore, and the TRP helix, which are accompanied by breaking/forming of a network of closed/open‐state specific hydrogen bonds. By comparing the closed‐state simulations at 30°C and 60°C, we observed heat‐activated conformational changes in the MPD linker, the outer pore, and the TRP helix that resemble the closed‐to‐open conformational changes, along with partial formation of the open‐state specific hydrogen bonds. Some of the residues involved in the above key hydrogen bonds were validated by previous mutational studies. Taken together, our MD simulations have offered rich structural and dynamic details beyond the static structures of TRPV1, and promising targets for future mutagenesis and functional studies of the TRPV1 channel. Proteins 2016; 84:1938–1949. © 2016 Wiley Periodicals, Inc.     

Arrangement and mobility of the voltage sensor domain in prokaryotic voltage-gated sodium channels

Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.     

Gating and regulation of the calcium release-activated calcium channel: Recent progress from experiments and molecular modeling

Calcium release-activated calcium (CRAC) channels are highly calcium ion (Ca²⁺)-selective channels in the plasma membrane. The transient drop of endoplasmic reticulum Ca²⁺ level activates its calcium sensor stromal interaction molecule (STIM) and then triggers the gating of the CRAC channel pore unit Orai. This process involves a variety of activities of the immune system. Therefore, understanding how the activation and regulation of the CRAC channel can be accomplished is essential. Here we briefly summarize the recent progress on Orai gating and its regulation by 2-aminoethoxydiphenylborate (2-APB) obtained from structural biology studies, biochemical and electrophysiological measurements, as well as molecular modeling. Indeed, integration between experiments and computations has further deepened our understanding of the channel gating and regulation.