Molecular dynamics study of the KcsA channel at 2.0-Å resolution: stability and concerted motions within the pore

The stability of the KcsA channel accommodating more than one ion in the pore has been studied with molecular dynamics. We have used the very last X-ray structure of the KcsA channel at 2.0-Å resolution determined by Zhou et al. [Nature 414 (2001) 43]. In this channel, six of the seven experimentally evidenced sites have been considered. We show that the protein remains very stable in the presence of four K⁺ ions (three in the selectivity filter and one in the cavity). The locations and the respective distances of the different K⁺ ions and water molecules (W), calculated within our KWKWKK sequence, also fits well with the experimental observations. The analysis of the K⁺ ions and water molecules displacements shows concerted file motions on the simulated time scale (≈1 ns), which could act as precursor to the diffusion of K⁺ ions inside the channel. A simple one-dimensional dynamical model is used to interpret the concerted motions of the ions and water molecules in the pore leading ultimately to ion transfer.     

Molecular dynamics of the KcsA K(+) channel in a bilayer membrane

Molecular dynamics (MD) simulations of an atomic model of the KcsA K(+) channel embedded in an explicit dipalmitoylphosphatidylcholine (DPPC) phospholipid bilayer solvated by a 150 mM KCl aqueous salt solution are performed and analyzed. The model includes the KcsA K(+) channel, based on the recent crystallographic structure of, Science. 280:69-77), 112 DPPC, K(+) and Cl(-) ions, as well as over 6500 water molecules for a total of more than 40,000 atoms. Three K(+) ions are explicitly included in the pore. Two are positioned in the selectivity filter on the extracellular side and one in the large water-filled cavity. Different starting configurations of the ions and water molecules in the selectivity filter are considered, and MD trajectories are generated for more than 4 ns. The conformation of KcsA is very stable in all of the trajectories, with a global backbone root mean square (RMS) deviation of less than 1.9 A with respect to the crystallographic structure. The RMS atomic fluctuations of the residues surrounding the selectivity filter on the extracellular side of the channel are significantly lower than those on the intracellular side. The motion of the residues with aromatic side chains surrounding the selectivity filter (Trp(67), Trp(68), Tyr(78), and Tyr(82)) is anisotropic with the smallest RMS fluctuations in the direction parallel to the membrane plane. A concerted dynamic transition of the three K(+) ions in the pore is observed, during which the K(+) ion located initially in the cavity moves into the narrow part of the selectivity filter, while the other two K(+) ions move toward the extracellular side. A single water molecule is stabilized between each pair of ions during the transition, suggesting that each K(+) cation translocating through the narrow pore is accompanied by exactly one water molecule, in accord with streaming potential measurements (, Biophys. J. 55:367-371). The displacement of the ions is coupled with the structural fluctuations of Val(76) and Gly(77), in the selectivity filter, as well as the side chains of Glu(71), Asp(80), and Arg(89), near the extracellular side. Thus the mechanical response of the channel structure at distances as large as 10-20 A from the ions in the selectivity filter appears to play an important role in the concerted transition.     

Potassium channel, ions, and water: simulation studies based on the high resolution X-ray structure of KcsA

Interactions of Na(+), K(+), Rb(+), and Cs(+) ions within the selectivity filter of a potassium channel have been investigated via multiple molecular dynamics simulations (total simulation time, 48 ns) based on the high resolution structure of KcsA, embedded in a phospholipid bilayer. As in simulations based on a lower resolution structure of KcsA, concerted motions of ions and water within the filter are seen. Despite the use of a higher resolution structure and the inclusion of four buried water molecules thought to stabilize the filter, this region exhibits a significant degree of flexibility. In particular, pronounced distortion of filter occurs if no ions are present within it. The two most readily permeant ions, K(+) and Rb(+), are similar in their interactions with the selectivity filter. In contrast, Na(+) ions tend to distort the filter by binding to a ring of four carbonyl oxygens. The larger Cs(+) ions result in a small degree of expansion of the filter relative to the x-ray structure. Cs(+) ions also appear to interact differently with the gate region of the channel, showing some tendency to bind within a predominantly hydrophobic pocket. The four water molecules buried between the back of the selectivity filter and the remainder of the protein show comparable mobility to the surrounding protein and do not exchange with water molecules within the filter or the central cavity. A preliminary comparison of the use of particle mesh Ewald versus cutoff protocols for the treatment of long-range electrostatics suggests some difference in the kinetics of ion translocation within the filter.     

Simulations of ion permeation through a potassium channel: molecular dynamics of KcsA in a phospholipid bilayer

Potassium channels enable K(+) ions to move passively across biological membranes. Multiple nanosecond-duration molecular dynamics simulations (total simulation time 5 ns) of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal motions of ions, water, and protein. Comparison of simulations with and without K(+) ions indicate that the absence of ions destabilizes the structure of the selectivity filter. Within the selectivity filter, K(+) ions interact with the backbone (carbonyl) oxygens, and with the side-chain oxygen of T75. Concerted single-file motions of water molecules and K(+) ions within the selectivity filter of the channel occur on a 100-ps time scale. In a simulation with three K(+) ions (initially two in the filter and one in the cavity), the ion within the central cavity leaves the channel via its intracellular mouth after approximately 900 ps; within the cavity this ion interacts with the Ogamma atoms of two T107 side chains, revealing a favorable site within the otherwise hydrophobically lined cavity. Exit of this ion from the channel is enabled by a transient increase in the diameter of the intracellular mouth. Such "breathing" motions may form the molecular basis of channel gating.     

Homology modeling and molecular dynamics simulation studies of an inward rectifier potassium channel

A homology model has been generated for the pore-forming domain of Kir6.2, a component of an ATP-sensitive K channel, based on the x-ray structure of the bacterial channel KcsA. Analysis of the lipid-exposed and pore-lining surfaces of the model reveals them to be compatible with the known features of membrane proteins and Kir channels, respectively. The Kir6.2 homology model was used as the starting point for nanosecond-duration molecular dynamics simulations in a solvated phospholipid bilayer. The overall drift from the model structure was comparable to that seen for KcsA in previous similar simulations. Preliminary analysis of the interactions of the Kir6.2 channel model with K(+) ions and water molecules during these simulations suggests that concerted single-file motion of K(+) ions and water through the selectivity filter occurs. This is similar to such motion observed in simulations of KcsA. This suggests that a single-filing mechanism is conserved between different K channel structures and may be robust to changes in simulation details. Comparison of Kir6.2 and KcsA suggests some degree of flexibility in the filter, thus complicating models of ion selectivity based upon a rigid filter.     

Potassium and sodium binding to the outer mouth of the K+ channel.

Molecular dynamics simulations of the K+ channel from Streptomyces lividans (KcsA channel) were performed in a membrane-mimetic environment with Na+ and K+ in different initial locations. The structure of the channel remained stable and well preserved for simulations lasting up to 1.5 ns. Salt bridges between Asp80 and Arg89 of neighboring subunits, not detected in the X-ray structure, enhanced the stability of the tetrameric structure. Na+ or K+ ions located in the channel vestibule lost part of their hydration shell and diffused into the channel inner pore in less than a few hundred picoseconds. This powerful catalytic action was caused by strong electrostatic interactions with Asp80 and Glu71. The hydration state of the metal ions turned out to depend significantly on the conformational flexibility of the channel. Furthermore, Na+ entered the channel inner pore bound to more water molecules than K+. The different hydration state of the two ions may be a determinant factor in the ion selectivity of the channel.     

K(+) versus Na(+) ions in a K channel selectivity filter: a simulation study

Molecular dynamics simulations of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal significant differences in interactions of the selectivity filter with K(+) compared with Na(+) ions. K(+) ions and water molecules within the filter undergo concerted single-file motion in which they translocate between adjacent sites within the filter on a nanosecond timescale. In contrast, Na(+) ions remain bound to sites within the filter and do not exhibit translocation on a nanosecond timescale. Furthermore, entry of a K(+) ion into the filter from the extracellular mouth is observed, whereas this does not occur for a Na(+) ion. Whereas K(+) ions prefer to sit within a cage of eight oxygen atoms of the filter, Na(+) ions prefer to interact with a ring of four oxygen atoms plus two water molecules. These differences in interactions in the selectivity filter may contribute to the selectivity of KcsA for K(+) ions (in addition to the differences in dehydration energy between K(+) and Na(+)) and the block of KcsA by internal Na(+) ions. In our simulations the selectivity filter exhibits significant flexibility in response to changes in ion/protein interactions, with a somewhat greater distortion induced by Na(+) than by K(+) ions.     

Crystallographic study of the tetrabutylammonium block to the KcsA K+ channel

K(+) channels play essential roles in regulating membrane excitability of many diverse cell types by selectively conducting K(+) ions through their pores. Many diverse molecules can plug the pore and modulate the K(+) current. Quaternary ammonium (QA) ions are a class of pore blockers that have been used for decades by biophysicists to probe the pore, leading to important insights into the structure-function relation of K(+) channels. However, many key aspects of the QA-blocking mechanisms remain unclear to date, and understanding these questions requires high resolution structural information. Here, we address the question of whether intracellular QA blockade causes conformational changes of the K(+) channel selectivity filter. We have solved the structures of the KcsA K(+) channel in complex with tetrabutylammonium (TBA) and tetrabutylantimony (TBSb) under various ionic conditions. Our results demonstrate that binding of TBA or TBSb causes no significant change in the KcsA structure at high concentrations of permeant ions. We did observe the expected conformational change of the filter at low concentration of K(+), but this change appears to be independent of TBA or TBSb blockade.     

Permeation of water through the KcsA K+ channel

Previous studies have reported that the KcsA potassium channel has an osmotic permeability coefficient of 4.8 x 10(-12) cm3/s, giving it a significantly higher osmotic permeability coefficient than that of some membrane channels specialized in water transport. This high osmotic permeability is proposed to occur when the channel is depleted of potassium ions, the presence of which slow down the water permeation process. The atomic structure of the potassium-depleted KcsA channel and the mechanisms of water permeation have not been well characterized so far. Here, all-atom molecular dynamics simulations, in conjunction with an umbrella sampling strategy and a nonequilibrium approach to simulate pressure gradients are employed to illustrate the permeation of water in the absence of ions through the KcsA K+ channel. Equilibrium molecular dynamics simulations (95 ns combined total length) identified a possible structure of the potassium-depleted KcsA channel, and umbrella sampling calculations (160 ns combined total length) revealed that this structure is not permeable by water molecules moving along the channel axis. The simulation of a pressure gradient across the channel (30 ns combined total length) identified an alternative permeation pathway with a computed osmotic permeability of approximately (2.7 +/- 0.9) x 10(-13) cm3/s. Water fluxes along this pathway did not proceed through collective water motions or transitions to vapor state. All of the major results of this study were robust against variations in a wide set of simulation parameters (force field, water model, membrane model, and channel conformation).     

Exploring the open pore of the potassium channel from Streptomyces lividans

The tetrameric potassium channel from Streptomyces lividans (KcsA) embedded in planar bilayers exhibits the following electrophysiological characteristics: (i) K+ ions can cross the pore in a highly hydrated state (nH2O > or = 6), (ii) the selectivity for K+ exceeds that for Na+ ions by 11 times, and both Ca2+ and Mg2+ are permeant, (iii) the internal side is blocked by Ba2+ ions in a voltage-dependent manner, (iv) intrinsic rectification is due to gating, depending on the direction of the electric field, (v) the internal side is pH-sensitive, and (vi) the open pore has a diameter of approximately 5.8 A. In conclusion, our results show that ion conduction and selectivity of KcsA cannot easily be reconciled with the properties deduced from the rigid crystal structure [Doyle et al., Science 280 (1998) 69-77], which must be concluded to have the pore trapped in its closed state.     

Water and potassium dynamics inside the KcsA K(+) channel

Molecular dynamics simulations and electrostatic modeling are used to investigate structural and dynamical properties of the potassium ions and of water molecules inside the KcsA channel immersed in a membrane-mimetic environment. Two potassium ions, initially located in the selectivity filter binding sites, maintain their position during 2 ns of dynamics. A third potassium ion is very mobile in the water-filled cavity. The protein appears engineered so as to polarize water molecules inside the channel cavity. The resulting water induced dipole and the positively charged potassium ion within the cavity are the key ingredients for stabilizing the two K(+) ions in the binding sites. These two ions experience single file movements upon removal of the potassium in the cavity, confirming the role of the latter in ion transport through the channel.     

Ion binding to KcsA: implications in ion selectivity and channel gating

Binding of K+ and Na+ to the potassium channel KcsA has been characterized from the stabilization observed in the heat-induced denaturation of the protein as the ion concentration is increased. KcsA thermal denaturation is known to include (i) dissociation of the homotetrameric channel into its constituent subunits and (ii) protein unfolding. The ion concentration-dependent changes in the thermal stability of the protein, evaluated as the Tm value for thermal-induced denaturation of the protein, may suggest the existence of both high- and low-affinity K+ binding sites of KcsA, which lend support to the tenet that channel gating may be governed by K+ concentration-dependent transitions between different affinity states of the channel selectivity filter. We also found that Na+ binds to KcsA with a KD similar to that estimated electrophysiologically from channel blockade. Therefore, our findings on ion binding to KcsA partly account for K+ over Na+ selectivity and Na+ blockade and argue against the strict “snug fit” hypothesis used initially to explain ion selectivity from the X-ray channel structure. Furthermore, the remarkable effects of increasing the ion concentration, K+ in particular, on the Tm of the denaturation process evidence that synergistic effects of the metal-mediated intersubunit interactions at the channel selectivity filter are a major contributor to the stability of the tetrameric protein. This observation substantiates the notion of a role for ions as structural “effectors” of ion channels.     

Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA

Here we present functional evidence for involvement of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) in ion conduction and selection at the intracellular side of the Streptomyces lividans potassium channel, KcsA. At < or = 25 degrees C, KcsA forms channels in planar bilayers that display signal characteristics of PHB/polyP channels at the intracellular side; i.e., a preference for divalent Mg(2+) cations at pH 7.2, and a preference for monovalent K+ cations at pH 6.8. Between 25 and 26 degrees C, KcsA undergoes a transition to a new conformation in which the channel exhibits high selectivity for K+, regardless of solution pH. This suggests that basic residues of the C-terminal polypeptides have moved closer to the polyP end unit, reducing its negative charge. The data support a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K+ is governed by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands.     

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.     

Absence of ion-binding affinity in the putatively inactivated low-[K+] structure of the KcsA potassium channel

Potassium channels are membrane proteins that selectively conduct K(+) across cellular membranes. The narrowest part of their pore, the selectivity filter, is responsible for distinguishing K(+) from Na(+), and can also act as a gate through a mechanism known as C-type inactivation. It has been proposed that a conformation of the KcsA channel obtained by crystallization in presence of low concentration of K(+) (PDB 1K4D) could correspond to the C-type inactivated state. Here, we show using molecular mechanics simulations that such conformation has little ion-binding affinity and that ions do not contribute to its stability. The simulations suggest that, in this conformation, the selectivity filter is mostly occupied by water molecules. Whether such ion-free state of the KcsA channel is physiologically accessible and representative of the inactivated state of eukaryotic channels remains unclear.     

Construction of a cyclic nucleotide-gated KcsA K+ channel

The ability of an ion channel to open in response to a defined stimulus is central to its function. In ligand-gated channels, pore opening is conferred through transduction of a conformational change in a gating domain to the helices of the pore. Here, we present the construction of a designed cyclic nucleotide-gated (CNG) channel, named KcsA-CNG, by addition of a prokaryotic cyclic nucleotide-binding domain to a KcsA-derived K+ channel. This channel is functional in lipid bilayers at physiological pH and has the combined properties of both of its parent-derived components. It conducts K+ and is blocked by the K+ channel inhibitors Na+ and agitoxin-2. Channel open times are increased by about two orders of magnitude compared to wild-type KcsA. The average number of open channels increases by approximately 50% upon addition of cAMP. Although the absolute open probabilities are somewhat variable from one channel to the next, the property of cyclic nucleotide sensitivity is very reproducible. An apparent Kd value of approximately 90 nM was estimated. The successful construction of a cyclic nucleotide-gated KcsA K+ channel suggests that it should be possible to produce channels that will respond to novel ligands.     

Contribution of ion binding affinity to ion selectivity and permeation in KcsA, a model potassium channel

Ion permeation and selectivity, key features in ion channel function, are believed to arise from a complex ensemble of energetic and kinetic variables. Here we evaluate the contribution of pore cation binding to ion permeation and selectivity features of KcsA, a model potassium channel. For this, we used E71A and M96V KcsA mutants in which the equilibrium between conductive and nonconductive conformations of the channel is differently shifted. E71A KcsA is a noninactivating channel mutant. Binding of K(+) to this mutant reveals a single set of low-affinity K(+) binding sites, similar to that seen in the binding of K(+) to wild-type KcsA that produces a conductive, low-affinity complex. This seems consistent with the observed K(+) permeation in E71A. Nonetheless, the E71A mutant retains K(+) selectivity, which cannot be explained on the basis of just its low affinity for this ion. At variance, M96V KcsA is a rapidly inactivating mutant that has lost selectivity for K(+) and also conducts Na(+). Here, low-affinity binding and high-affinity binding of both cations are detected, seemingly in agreement with both being permeating species in this mutant channel. In conclusion, binding of the ion to the channel protein seemingly explains certain gating, ion selectivity, and permeation properties. Ion binding stabilizes greatly the channel and, depending upon ion type and concentration, leads to different conformations and ion binding affinities. High-affinity states guarantee binding of specific ions and mediate ion selectivity but are nonconductive. Conversely, low-affinity states would not discriminate well among different ions but allow permeation to occur.     

Influence of interionic interactions on functional state and blocker binding of voltage-gated potassium channels

A mechanism of ion conduction of a voltage-gated potassium channel KcsA was investigated in full-atomic approximation at a trajectory length of 100 ns using the Lomonosov supercomputer. Methods of molecular dynamics were employed. A structure of the KcsA channel in the open state obtained by X-ray structure analysis (PDB ID 3fb7) was used. Free energy profiles of the KcsA pore occupied with either one or three potassium ions were calculated. It was shown that, under physiological conditions, ions pass through the channel pore cooperatively and the mechanism most probably includes three ions permeating in concert. Interactions of the mammalian voltage-gated channel Kv1.2 with neurotoxin were investigated. It was demonstrated that the effect of interionic interactions on binding of a blocker is rather insufficient.     

The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates

Potassium ions diffuse across the cell membrane in a single file through the narrow selectivity filter of potassium channels. The crystal structure of the KcsA K+ channel revealed the chemical structure of the selectivity filter, which contains four binding sites for K+. In this study, we used Tl+ in place of K+ to address the question of how many ions bind within the filter at a given time, i.e. what is the absolute ion occupancy? By refining the Tl+ structure against data to 1.9A resolution with an anomalous signal, we determined the absolute occupancy of Tl+. Then, by comparing the electron density of Tl+ with that of K+, Rb+ and Cs+, we estimated the absolute occupancy of these three ions. We further analyzed how the ion occupancy affects the conformation of the selectivity filter by analyzing the structure of KcsA at different concentrations of Tl+. Our results indicate that the average occupancy for each site in the selectivity filter is about 0.63 for Tl+ and 0.53 for K+. For K+, Rb+ and Cs+, the total number of ions contained within four sites in the selectivity filter is about two. At low concentrations of permeant ion, the number of ions drops to one in association with a conformational change in the selectivity filter. We conclude that electrostatic balance and coupling of ion binding to a protein conformational change underlie high conduction rates in the setting of high selectivity.     

Modelling of ion permeation in K+ channels by nonequilibrium molecular dynamics simulations: I. Permeation energetics and structure stability.

Because of the great importance of physiological and pathophysiological processes in which ion channels are involved and because their operation is described by physicochemical laws, there have been many attempts to develop physical models able to describe the membrane permeability and also the structural and functional properties of the channel protein structures. In this study (in two parts) we present a series of simulations on a K+ channel model (KcsA) using Nonequilibrium Molecular Dynamics simulations (NEMD), in order to follow structure stability, permeation energetics and the possibility of obtaining quantitative information about the permeation process using the Linear Response Theory (LRT). On K+ ions were applied external forces to determine them to pass through the channel in a relatively small amount of time, accessible computationally. We ascertained a high resistance of the protein to deformation even in conditions when great forces were applied on ions (the system was far from equilibrium). The estimation of energy profiles in the course of ions passage through the channel demonstrates that these proteins create a conductivity pathway with no energetic barriers for ions movement across the channel (which could be present due to ions dehydration). The dynamic model used demonstrates (as proposed before in the literature after the examination of the static KcsA structure obtained by X-Ray crystallography) that this is due to the interaction of ions with the negatively charged carbonyl oxygens of the main polypeptide chain in the selectivity filter region.