The Experts below are selected from a list of 1689 Experts worldwide ranked by ideXlab platform
Shigetoshi Oiki - One of the best experts on this subject based on the ideXlab platform.
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Queueing arrival and release mechanism for K^+ permeation through a Potassium Channel
The Journal of Physiological Sciences, 2019Co-Authors: Takashi Sumikama, Shigetoshi OikiAbstract:The mechanism underlying ion permeation through Potassium Channels still remains controversial. K^+ ions permeate across a narrow selectivity filter (SF) in a single file. Conventional scenarios assume that K^+ ions are tightly bound in the SF, and, thus, they are displaced from their energy well by ion–ion repulsion with an incoming ion. This tight coupling between entering and exiting ions has been called the “knock-on” mechanism. However, this paradigm is contradicted by experimental data measuring the water–ion flux coupling ratio, demonstrating fewer ion occupancies. Here, the results of molecular dynamics simulations of permeation through the KcsA Potassium Channel revealed an alternative mechanism. In the aligned ions in the SF (an ion queue), the outermost K^+ was readily and spontaneously released toward the extracellular space, and the affinity of the relevant ion was ~ 50 mM. Based on this low-affinity regime, a simple queueing mechanism described by loose coupling of entering and exiting ions is proposed.
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Drop-in-well chamber for droplet interface bilayer with built-in electrodes.
Methods in enzymology, 2019Co-Authors: Kazuhiro Urakubo, Masayuki Iwamoto, Shigetoshi OikiAbstract:Abstract Various methods have been developed for the formation of planar lipid bilayers, and recent techniques using water-in-oil droplets, such as droplet interface bilayer (DIB) and contact bubble bilayer (CBB) methods, allow the ready formation of bilayers with arbitrary lipid compositions. Here, we developed a simple and portable DIB system using drop-in-wells, shaping two merging wells for settling electrolyte droplets. An aliquot of the electrolyte solution (1 μL) is dropped into an organic solvent, and the droplet sinks to the drop-in-well at the bottom, where two monolayer-lined droplets come in contact to form the bilayer. Pre-installed electrodes allow electrophysiological measurements. The detailed drop-in-well method is presented, and some variations of the method, such as the use of microelectrodes and a sheet with a small hole for low-noise recordings, are extended. Examples of single Channel current recordings of the KcsA Potassium Channel are demonstrated.
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Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential
ACS synthetic biology, 2018Co-Authors: Masayuki Iwamoto, Maie A. Elfaramawy, Mariko Yamatake, Tomoaki Matsuura, Shigetoshi OikiAbstract:Processes involved in the functional formation of prokaryotic membrane proteins have remained elusive. Here, we developed a new in vitro membrane protein expression system to detect nascent activities of the KcsA Potassium Channel in lipid bilayers under an applied membrane potential. The Channel was synthesized using a reconstituted Escherichia coli-based in vitro transcription/translation system (IVTT) in a water-in-oil droplet lined by a membrane. The synthesized Channels spontaneously incorporated into the membrane even without the translocon machinery (unassisted pathway) and formed functional Channels with the correct orientation. The single-Channel current of the first appearing nascent Channel was captured, followed by the subsequent appearance of multiple Channels. Notably, the first appearance time shortened substantially as the membrane potential was hyperpolarized. Under a steadily applied membrane potential, this system serves as a production line of membrane proteins via the unassisted pathw...
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Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential
2018Co-Authors: Masayuki Iwamoto, Maie A. Elfaramawy, Mariko Yamatake, Tomoaki Matsuura, Shigetoshi OikiAbstract:Processes involved in the functional formation of prokaryotic membrane proteins have remained elusive. Here, we developed a new in vitro membrane protein expression system to detect nascent activities of the KcsA Potassium Channel in lipid bilayers under an applied membrane potential. The Channel was synthesized using a reconstituted Escherichia coli-based in vitro transcription/translation system (IVTT) in a water-in-oil droplet lined by a membrane. The synthesized Channels spontaneously incorporated into the membrane even without the translocon machinery (unassisted pathway) and formed functional Channels with the correct orientation. The single-Channel current of the first appearing nascent Channel was captured, followed by the subsequent appearance of multiple Channels. Notably, the first appearance time shortened substantially as the membrane potential was hyperpolarized. Under a steadily applied membrane potential, this system serves as a production line of membrane proteins via the unassisted pathway, mimicking the bacterial synthetic membrane
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Oriented Reconstitution of the Full-Length KcsA Potassium Channel in a Lipid Bilayer for AFM Imaging.
The journal of physical chemistry letters, 2017Co-Authors: Ayumi Sumino, Takayuki Uchihashi, Shigetoshi OikiAbstract:Here, we have developed a method of oriented reconstitution of the KcsA Potassium Channel amenable to high-resolution AFM imaging. The solubilized full-length KcsA Channels with histidine-tagged (His-tag) C-terminal ends were attached to a Ni2+-coated mica surface, and then detergent-destabilized liposomes were added to fill the interChannel space. AFM revealed that the membrane-embedded KcsA Channels were oriented with their extracellular faces upward, seen as a tetrameric square shape. This orientation was corroborated by the visible binding of a peptide scorpion toxin, agitoxin-2. To observe the cytoplasmic side of the Channel, a His-tag was inserted into the extracellular loop, and the oppositely oriented Channels provided wholly different images. In either orientation, the Channels were individually dispersed at acidic pH, whereas they were self-assembled at neutral pH, indicating that the oriented Channels are allowed to diffuse in the membrane. This method is readily applicable to membrane proteins...
Shin-ho Chung - One of the best experts on this subject based on the ideXlab platform.
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brownian dynamics theory for predicting internal and external blockages of tetraethylammonium in the KcsA Potassium Channel
Biophysical Journal, 2008Co-Authors: Matthew Hoyles, Vikram Krishnamurthy, May Siksik, Shin-ho ChungAbstract:The theory of Brownian dynamics is used to model permeation and the blocking of KcsA Potassium Channels by tetraethylammonium (TEA). A novel Brownian dynamics simulation algorithm is implemented that comprises two free energy profiles; one profile is seen by the Potassium ions and the other by the TEA molecules whose shape is approximated by a sphere. Our simulations reveal that internally applied TEA blocks the passage of K+ ions by physically occluding the pore. A TEA molecule in the external reservoir encounters an attractive energy-well created by four tyrosine residues at position 82, in addition to all other attractive and repulsive forces impinging on it. Using Brownian dynamics, we investigate how deep the energy-well needs to be to reproduce the experimentally determined inhibitory constant ki for the TEA blockade of KcsA or the mutant Shaker T449Y. The one-dimensional free energy profile obtained from molecular dynamics is first converted into a one-dimensional potential energy profile, and is then transformed into a three-dimensional free energy profile in Brownian dynamics by adding the short-range potential from the Channel walls. When converted, the free energy profile calculated from molecular dynamics gives a well-depth of ∼10 kT. We systematically alter the depths of the profiles, and then use Brownian dynamics simulations to numerically determine the current versus TEA-concentration curves. We show that the sequence of binding and unbinding events of the TEA molecule to the binding pocket can be modeled by a first-order Markov process. The Brownian dynamics simulations also reveal that the probability of a TEA molecule binding to the binding pocket in KcsA Potassium Channels increases exponentially with TEA concentration and depends also on the applied potential and the K+ concentration in the simulation assembly.
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Efficacy of external tetraethylammonium block of the KcsA Potassium Channel: molecular and Brownian dynamics studies.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2008Co-Authors: David Bisset, Shin-ho ChungAbstract:Abstract Blockade of the KcsA Potassium Channel by externally applied tetraethylammonium is investigated using molecular dynamics calculations and Brownian dynamics simulations. In KcsA, the aromatic rings of four tyrosine residues located just external to the selectivity filter create an attractive energy well or a binding cage for a tetraethylammonium molecule. We first investigate the effects of re-orienting the four tyrosine residues such that the centers of the aromatic rings face the tetraethylammonium molecule directly. Then, we systematically move the residues inward in both orientations so that the radius of the binding cage formed by them becomes smaller. For each configuration, we construct a one-dimensional free energy profile by bringing in a tetraethylammonium molecule from the external reservoir toward the selectivity filter. The free energy profile is then converted to a one-dimensional potential energy profile, taking the available space between the tyrosine residues and the tetraethylammonium molecule into account. Incorporating this potential energy profile into the Brownian dynamics algorithm, we determine the conductance properties of the Channel under various conditions, construct the current-tetraethylammonium-concentration curve and compare it with the experimentally determined inhibitory constant k i for externally applied tetraethylammonium. We show that the experimentally determined binding affinity for externally applied tetraethylammonium can be replicated when each of the four tyrosine residues is moved inward by about 0.7 A, irrespective of orientation of their aromatic rings.
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Electrostatic Basis of Valence Selectivity in Cationic Channels
Biochimica et biophysica acta, 2005Co-Authors: B Corry, Taira Vora, Shin-ho ChungAbstract:We examine how a variety of cationic Channels discriminate between ions of differing charge. We construct models of the KcsA Potassium Channel, voltage gated sodium Channel and L-type calcium Channel, and show that they all conduct monovalent cations, but that only the calcium Channel conducts divalent cations. In the KcsA and sodium Channels divalent ions block the Channel and prevent any further conduction. We demonstrate that in each case, this discrimination and some of the more complex conductance properties of the Channels is a consequence of the electrostatic interaction of the ions with the charges in the Channel protein. The KcsA and sodium Channels bind divalent ions strongly enough that they cannot be displaced by other ions and thereby block the Channel. On the other hand, the calcium Channel binds them less strongly such that they can be destabilized by the repulsion of another incoming divalent ion, but not by the lesser repulsion from monovalent ions.
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A model of sodium Channels
Biochimica et biophysica acta, 2005Co-Authors: Taira Vora, B Corry, Shin-ho ChungAbstract:We have explored the permeation and blockage of ions in sodium Channels, relating the Channel structure to function using electrostatic profiles and Brownian dynamics simulations. The model used resembles the KcsA Potassium Channel with an added external vestibule and a shorter selectivity filter. The electrostatic energy landscape seen by permeating ions is determined by solving Poisson's equation. The two charged amino acid rings of Glu-Glu-Asp-Asp (EEDD) and Asp-Glu-Lys-Ala (DEKA) around the selectivity filter region are seen to play a crucial role in making the Channel sodium selective, and strongly binding calcium ions such that they block the Channel. Our model closely reproduces a range of experimental data including the current-voltage curves, current-concentration curves and blockage of monovalent ions by divalent ions.
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Ion Channels: recent progress and prospects.
European biophysics journal : EBJ, 2002Co-Authors: Shin-ho Chung, Serdar KuyucakAbstract:Determination of the crystal structure of the KcsA Potassium Channel and its subsequent refinement at 2 A resolution have stimulated much interest in modelling of ion Channels. Here we review the recent developments in ion Channels research, focusing especially on the question of structure-function relationships, and discuss how permeation models based on Brownian and molecular dynamics simulations can be used fruitfully in this endeavour.
Hirofumi Shimizu - One of the best experts on this subject based on the ideXlab platform.
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Surface-enhanced IR absorption spectroscopy of the KcsA Potassium Channel upon application of an electric field
Physical chemistry chemical physics : PCCP, 2015Co-Authors: Akira Yamakata, Hirofumi Shimizu, Shigetoshi OikiAbstract:Surface-enhanced IR absorption spectroscopy (SEIRAS) is a powerful tool for studying the structure of molecules adsorbed on an electrode surface (ATR-SEIRA). Coupled with an electrochemical system, structural changes induced by changes in the electric field can be detected. All the membrane proteins are subjected to the effect of membrane electric field, but conformational changes at different membrane potentials and their functional relevance have not been studied extensively except for Channel proteins. In this contribution, background information of potential-dependent functional and structural changes of a prototypical Channel, the KcsA Channel, is summarized, and SEIRAS applied to the KcsA Channel under the application of the potential is shown. The Potassium Channels allow K+ to permeate selectively through the structural part called the selectivity filter, in which dehydrated K+ ions interact with backbone carbonyls. In the absence of K+, the selectivity filter undergoes conformational changes to the non-conductive collapsed conformation. To apply the electric field, the KcsA Channels were fixed on the gold surface in either upside or reverse orientation. The SEIRA spectrum in K+ or Na+ solution revealed both backbone structural changes and local changes in the OCO-carboxylate groups. Upon application of the negative electric field, the spectrum of OCO was enhanced only in the K+ solution. These results indicate that the negative electric field accumulates local K+ concentration, which turned the collapsed filter to the conductive conformation. ATR-SEIRA serves as an unprecedented experimental system for examining membrane proteins under an electric field.
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ATR-FTIR Spectroscopy Revealing the Different Vibrational Modes of the Selectivity Filter Interacting with K+ and Na+ in the Open and Collapsed Conformations of the KcsA Potassium Channel
2015Co-Authors: Yuji Furutani, Shigetoshi Oiki, Hirofumi Shimizu, Yusuke Asai, Tetsuya Fukuda, Hideki KandoriAbstract:The Potassium Channel is highly selective for K+ over Na+, and the selectivity filter binds multiple dehydrated K+ ions upon permeation. Here, we applied attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to extract ion-binding-induced signals of the KcsA Potassium Channel at neutral pH. Shifts in the peak of the amide-I signal towards lower vibrational frequencies were observed as K+ was replaced with Na+. These ion species-specific shifts deduced the selectivity filter as the source of the signal, which was supported by the spectra of a mutant for the selectivity filter (Y78F). The difference FTIR spectra between the solution containing various concentrations of K+ and that containing pure Na+ demonstrated two types of peak shifts of the amide-I vibration in response to the K+ concentration. These signals represent the binding of K+ ions to the different sites in the selectivity filter with different dissociation constants (KD = 9 or 18 mM)
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pH-dependent promotion of phospholipid flip-flop by the KcsA Potassium Channel.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015Co-Authors: Hiroyuki Nakao, Shigetoshi Oiki, Masayuki Iwamoto, Hirofumi Shimizu, Keisuke Ikeda, Yasushi Ishihama, Minoru NakanoAbstract:Abstract KcsA is a pH-dependent Potassium Channel that is activated at acidic pH. The Channel undergoes global conformational changes upon activation. We hypothesized that the open–close conformational changes of the transmembrane region could promote the flip-flop of phospholipids. Based on this hypothesis, we measured the flip-flop of NBD-labeled phospholipids in KcsA-incorporated proteoliposomes. Both flip and flop rates of C6NBD-PC were significantly enhanced in the presence of KcsA and were several times higher at pH 4.0 than at pH 7.4, suggesting that KcsA promotes the phospholipid flip in a conformation-dependent manner. Phospholipids were non-selectively flipped with respect to the glycerophospholipid structure. In the active state of KcsA Channel, tetrabutylammonium locks the Channel in the open conformation at acidic pH; however, it did not alter the flip rate of C6NBD-PC. Thus, the open–close transition of the transmembrane region did not affect the flip-flop of phospholipids. In addition, the KcsA mutant that lacked an N-terminal amphipathic helix (M0-helix) was found to show reduced ability to flip C6NBD-phospholipids at acidic pH. The closed conformation is stabilized in the absence of M0-helix, and thus the attenuated flip could be explained by the reduced prevalence of the open conformation. These results suggest that the open conformation of KcsA can disturb the bilayer integrity and facilitate the flip-flop of phospholipids.
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Structural changes of the KcsA Potassium Channel upon application of the electrode potential studied by surface-enhanced IR absorption spectroscopy
Chemical Physics, 2013Co-Authors: Akira Yamakata, Hirofumi Shimizu, Masatoshi Osawa, Shigetoshi OikiAbstract:Abstract Structural changes of the KcsA Potassium Channel fixed on gold electrode surface in the upright orientation were studied by surface-enhanced IR absorption spectroscopy (SEIRAS). Measurements were performed at neutral pH, where the activation gate is kept closed. Band intensities were enhanced for the asymmetric (1565 cm−1) and symmetric (1405 cm−1) OCO-carboxylate groups at negative electrode potentials in the K+ solution, but not in the Na+ solution. Even for the reverse-oriented Channel, the enhanced OCO-carboxylate band was evident at negative potential. When TBA was loaded in the central cavity, the K+-specific OCO band was not elicited. These results indicate that the negative electrode potential renders the local K+ concentration accumulated at the vicinity of the electrode surface, and the KcsA Channel bathed in high K+ changes conformation of the selectivity filter from the collapsed to the open, and OCO-carboxylate groups (D80 and E71) in the back of the filter were rearranged.
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ATR-FTIR Spectroscopy Revealing the Different Vibrational Modes of the Selectivity Filter Interacting with K(+) and Na(+) in the Open and Collapsed Conformations of the KcsA Potassium Channel.
The journal of physical chemistry letters, 2012Co-Authors: Yuji Furutani, Shigetoshi Oiki, Hirofumi Shimizu, Yusuke Asai, Tetsuya Fukuda, Hideki KandoriAbstract:The Potassium Channel is highly selective for K(+) over Na(+), and the selectivity filter binds multiple dehydrated K(+) ions upon permeation. Here, we applied attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to extract ion-binding-induced signals of the KcsA Potassium Channel at neutral pH. Shifts in the peak of the amide-I signal towards lower vibrational frequencies were observed as K(+) was replaced with Na(+). These ion species-specific shifts deduced the selectivity filter as the source of the signal, which was supported by the spectra of a mutant for the selectivity filter (Y78F). The difference FTIR spectra between the solution containing various concentrations of K(+) and that containing pure Na(+) demonstrated two types of peak shifts of the amide-I vibration in response to the K(+) concentration. These signals represent the binding of K(+) ions to the different sites in the selectivity filter with different dissociation constants (KD = 9 or 18 mM).
Masayuki Iwamoto - One of the best experts on this subject based on the ideXlab platform.
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Drop-in-well chamber for droplet interface bilayer with built-in electrodes.
Methods in enzymology, 2019Co-Authors: Kazuhiro Urakubo, Masayuki Iwamoto, Shigetoshi OikiAbstract:Abstract Various methods have been developed for the formation of planar lipid bilayers, and recent techniques using water-in-oil droplets, such as droplet interface bilayer (DIB) and contact bubble bilayer (CBB) methods, allow the ready formation of bilayers with arbitrary lipid compositions. Here, we developed a simple and portable DIB system using drop-in-wells, shaping two merging wells for settling electrolyte droplets. An aliquot of the electrolyte solution (1 μL) is dropped into an organic solvent, and the droplet sinks to the drop-in-well at the bottom, where two monolayer-lined droplets come in contact to form the bilayer. Pre-installed electrodes allow electrophysiological measurements. The detailed drop-in-well method is presented, and some variations of the method, such as the use of microelectrodes and a sheet with a small hole for low-noise recordings, are extended. Examples of single Channel current recordings of the KcsA Potassium Channel are demonstrated.
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Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential
ACS synthetic biology, 2018Co-Authors: Masayuki Iwamoto, Maie A. Elfaramawy, Mariko Yamatake, Tomoaki Matsuura, Shigetoshi OikiAbstract:Processes involved in the functional formation of prokaryotic membrane proteins have remained elusive. Here, we developed a new in vitro membrane protein expression system to detect nascent activities of the KcsA Potassium Channel in lipid bilayers under an applied membrane potential. The Channel was synthesized using a reconstituted Escherichia coli-based in vitro transcription/translation system (IVTT) in a water-in-oil droplet lined by a membrane. The synthesized Channels spontaneously incorporated into the membrane even without the translocon machinery (unassisted pathway) and formed functional Channels with the correct orientation. The single-Channel current of the first appearing nascent Channel was captured, followed by the subsequent appearance of multiple Channels. Notably, the first appearance time shortened substantially as the membrane potential was hyperpolarized. Under a steadily applied membrane potential, this system serves as a production line of membrane proteins via the unassisted pathw...
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Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential
2018Co-Authors: Masayuki Iwamoto, Maie A. Elfaramawy, Mariko Yamatake, Tomoaki Matsuura, Shigetoshi OikiAbstract:Processes involved in the functional formation of prokaryotic membrane proteins have remained elusive. Here, we developed a new in vitro membrane protein expression system to detect nascent activities of the KcsA Potassium Channel in lipid bilayers under an applied membrane potential. The Channel was synthesized using a reconstituted Escherichia coli-based in vitro transcription/translation system (IVTT) in a water-in-oil droplet lined by a membrane. The synthesized Channels spontaneously incorporated into the membrane even without the translocon machinery (unassisted pathway) and formed functional Channels with the correct orientation. The single-Channel current of the first appearing nascent Channel was captured, followed by the subsequent appearance of multiple Channels. Notably, the first appearance time shortened substantially as the membrane potential was hyperpolarized. Under a steadily applied membrane potential, this system serves as a production line of membrane proteins via the unassisted pathway, mimicking the bacterial synthetic membrane
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pH-dependent promotion of phospholipid flip-flop by the KcsA Potassium Channel.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015Co-Authors: Hiroyuki Nakao, Shigetoshi Oiki, Masayuki Iwamoto, Hirofumi Shimizu, Keisuke Ikeda, Yasushi Ishihama, Minoru NakanoAbstract:Abstract KcsA is a pH-dependent Potassium Channel that is activated at acidic pH. The Channel undergoes global conformational changes upon activation. We hypothesized that the open–close conformational changes of the transmembrane region could promote the flip-flop of phospholipids. Based on this hypothesis, we measured the flip-flop of NBD-labeled phospholipids in KcsA-incorporated proteoliposomes. Both flip and flop rates of C6NBD-PC were significantly enhanced in the presence of KcsA and were several times higher at pH 4.0 than at pH 7.4, suggesting that KcsA promotes the phospholipid flip in a conformation-dependent manner. Phospholipids were non-selectively flipped with respect to the glycerophospholipid structure. In the active state of KcsA Channel, tetrabutylammonium locks the Channel in the open conformation at acidic pH; however, it did not alter the flip rate of C6NBD-PC. Thus, the open–close transition of the transmembrane region did not affect the flip-flop of phospholipids. In addition, the KcsA mutant that lacked an N-terminal amphipathic helix (M0-helix) was found to show reduced ability to flip C6NBD-phospholipids at acidic pH. The closed conformation is stabilized in the absence of M0-helix, and thus the attenuated flip could be explained by the reduced prevalence of the open conformation. These results suggest that the open conformation of KcsA can disturb the bilayer integrity and facilitate the flip-flop of phospholipids.
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Gating-Associated Clustering–Dispersion Dynamics of the KcsA Potassium Channel in a Lipid Membrane
2015Co-Authors: Ayumi Sumino, Masayuki Iwamoto, Takehisa Dewa, Daisuke Yamamoto, Shigetoshi OikiAbstract:The KcsA Potassium Channel is a prototypical Channel of bacterial origin, and the mechanism underlying the pH-dependent gating has been studied extensively. With the high-resolution atomic force microscopy (AFM), we have resolved functional open and closed gates of the KcsA Channel under the membrane-embedded condition. Here we surprisingly found that the pH-dependent gating of the KcsA Channels was associated with clustering–dispersion dynamics. At neutral pH, the resting, closed Channels were coalesced, forming nanoclusters. At acidic pH, the open-gated Channels were dispersed as singly isolated Channels. Time-lapse AFM revealed reversible clustering–dispersion transitions upon pH changes. At acidic equilibrium, a small fraction of the Channels was nanoclustered, in which the gate was apparently closed. Thus, it is suggested that opening of the gate and the dispersion are tightly linked. The interplay between the intramolecular conformational change and the supramolecular clustering–dispersion dynamics provides insights into understanding of unprecedented functional cooperativity of Channels
Serdar Kuyucak - One of the best experts on this subject based on the ideXlab platform.
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Comparative study of the energetics of ion permeation in Kv1.2 and KcsA Potassium Channels.
Biophysical journal, 2011Co-Authors: Turgut Baştuğ, Serdar KuyucakAbstract:Biological ion Channels rely on a multi-ion transport mechanism for fast yet selective permeation of ions. The crystal structure of the KcsA Potassium Channel provided the first microscopic picture of this process. A similar mechanism is assumed to operate in all Potassium Channels, but the validity of this assumption has not been well investigated. Here, we examine the energetics of ion permeation in Shaker Kv1.2 and KcsA Channels, which exemplify the six-transmembrane voltage-gated and two-transmembrane inward-rectifier Channels. We study the feasibility of binding a third ion to the filter and the concerted motion of ions in the Channel by constructing the potential of mean force for K+ ions in various configurations. For both Channels, we find that a pair of K+ ions can move almost freely within the filter, but a relatively large free-energy barrier hinders the K+ ion from stepping outside the filter. We discuss the effect of the CMAP dihedral energy correction that was recently incorporated into the CHARMM force field on ion permeation dynamics.
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Molecular dynamics and continuum electrostatics studies of inactivation in the HERG Potassium Channel.
The journal of physical chemistry. B, 2007Co-Authors: Ramzi Kutteh, Jamie I. Vandenberg, Serdar KuyucakAbstract:Fast inactivation of the HERG Potassium Channel plays a critical role in normal cardiac function. Malfunction of these Channels due to either genetic mutations or blockade by drugs leads to cardiac arrhythmias. An unusually long S5-P linker in the outer mouth of HERG is implicated in the fast inactivation mechanism. To examine the role of the S5-P linker in this inactivation mechanism, we study the permeation properties of the open and inactive states of a recent homology model of HERG. This model was constructed using the KcsA Potassium Channel as a template and contains specific conformations of the S5-P linker in the open and inactive states. We perform molecular dynamics simulations on the HERG model, followed by free energy, structural, and continuum electrostatics calculations. Our free energy calculations lead to selectivity results of the model Channel (K+ over Na+) that are different in some respects from those of other Potassium Channels but consistent with experimental observations. Our structu...
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Ion Channels: recent progress and prospects.
European biophysics journal : EBJ, 2002Co-Authors: Shin-ho Chung, Serdar KuyucakAbstract:Determination of the crystal structure of the KcsA Potassium Channel and its subsequent refinement at 2 A resolution have stimulated much interest in modelling of ion Channels. Here we review the recent developments in ion Channels research, focusing especially on the question of structure-function relationships, and discuss how permeation models based on Brownian and molecular dynamics simulations can be used fruitfully in this endeavour.
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Conducting-State Properties of the KcsA Potassium Channel from Molecular and Brownian Dynamics Simulations
Biophysical journal, 2002Co-Authors: Shin-ho Chung, Toby W. Allen, Serdar KuyucakAbstract:The mechanisms underlying transport of ions across the Potassium Channel are examined using electrostatic calculations and three-dimensional Brownian dynamics simulations. We first build open-state configurations of the Channel with molecular dynamics simulations, by pulling the transmembrane helices outward until the Channel attains the desired interior radius. To gain insights into ion permeation, we construct potential energy profiles experienced by an ion traversing the Channel in the presence of other resident ions. These profiles reveal that in the absence of an applied field the Channel accommodates three Potassium ions in a stable equilibrium, two in the selectivity filter and one in the central cavity. In the presence of a driving potential, this three-ion state becomes unstable, and ion permeation across the Channel is observed. These qualitative explanations are confirmed by the results of three-dimensional Brownian dynamics simulations. We find that the Channel conducts when the ionizable residues near the extracellular entrance are fully charged and those near the intracellular side are partially charged. The conductance increases steeply as the radius of the intracellular mouth of the Channel is increased from 2 A to 5 A. Our simulation results reproduce several experimental observations, including the current-voltage curves, conductance-concentration relationships, and outward rectification of currents.
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Molecular dynamics estimates of ion diffusion in model hydrophobic and KcsA Potassium Channels.
Biophysical chemistry, 2000Co-Authors: Toby W. Allen, Serdar Kuyucak, Shin-ho ChungAbstract:Abstract Molecular dynamics simulations are carried out to obtain estimates of diffusion coefficients of biologically important Na + , K + , Ca 2+ and Cl − ions in hydrophobic cylindrical Channels with varying radii and large reservoirs. Calculations for the cylindrical Channels are compared to those for the KcsA Potassium Channel, for which the protein structure has recently been determined from X-ray diffraction experiments. Our results show that ion diffusion is maintained at reasonably high levels even within narrow Channels, and does not support the very small diffusion coefficients used in some continuum models in order to fit experimental data. The present estimates of ion diffusion coefficients are useful in the calculation of Channel conductance using the Poisson–Nernst–Planck theory, or Brownian dynamics.