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.     

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.     

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.     

Filter flexibility and distortion in a bacterial inward rectifier K+ channel: simulation studies of KirBac1.1

The bacterial channel KirBac1.1 provides a structural homolog of mammalian inward rectifier potassium (Kir) channels. The conformational dynamics of the selectivity filter of Kir channels are of some interest in the context of possible permeation and gating mechanisms for this channel. Molecular dynamics simulations of KirBac have been performed on a 10-ns timescale, i.e., comparable to that of ion permeation. The results of five simulations (total simulation time 50 ns) based on three different initial ion configurations and two different model membranes are reported. These simulation data provide evidence for limited (<0.1 nm) filter flexibility during the concerted motion of ions and water molecules within the filter, such local changes in conformation occurring on an approximately 1-ns timescale. In the absence of K(+) ions, the KirBac selectivity filter undergoes more substantial distortions. These resemble those seen in comparable simulations of other channels (e.g., KcsA and KcsA-based homology models) and are likely to lead to functional closure of the channel. This suggests filter distortions may provide a mechanism of K-channel gating in addition to changes in the hydrophobic gate formed at the intracellular crossing point of the M2 helices. The simulation data also provide evidence for interactions of the "slide" (pre-M1) helix of KirBac with phospholipid headgroups.     

Hierarchical approach to predicting permeation in ion channels

A hierarchical computational strategy combining molecular modeling, electrostatics calculations, molecular dynamics, and Brownian dynamics simulations is developed and implemented to compute electrophysiologically measurable properties of the KcsA potassium channel. Models for a series of channels with different pore sizes are developed from the known x-ray structure, using insights into the gating conformational changes as suggested by a variety of published experiments. Information on the pH dependence of the channel gating is incorporated into the calculation of potential profiles for K(+) ions inside the channel, which are then combined with K(+) ion mobilities inside the channel, as computed by molecular dynamics simulations, to provide inputs into Brownian dynamics simulations for computing ion fluxes. The open model structure has a conductance of approximately 110 pS under symmetric 250 mM K(+) conditions, in reasonable agreement with experiments for the largest conducting substate. The dimensions of this channel are consistent with electrophysiologically determined size dependence of quaternary ammonium ion blocking from the intracellular end of this channel as well as with direct structural evidence that tetrabutylammonium ions can enter into the interior cavity of the channel. Realistic values of Ussing flux ratio exponents, distribution of ions within the channel, and shapes of the current-voltage and current-concentration curves are obtained. The Brownian dynamics calculations suggest passage of ions through the selectivity filter proceeds by a "knock-off" mechanism involving three ions, as has been previously inferred from functional and structural studies of barium ion blocking. These results suggest that the present calculations capture the essential nature of K(+) ion permeation in the KcsA channel and provide a proof-of-concept for the integrated microscopic/mesoscopic multitiered approach for predicting ion channel function from structure, which can be applied to other channel structures.     

A computational study of ion binding and protonation states in the KcsA potassium channel

We report results from microscopic molecular dynamics and free energy perturbation simulations of the KcsA potassium channel based on its experimental atomic structure. Conformational properties of selected amino acid residues as well as equilibrium positions of K(+) ions inside the selectivity filter and the internal water cavity are examined. Positions three and four (counting from the extracellular site) in the experimental structure correspond to distinctly separate binding sites for K(+) ions inside the selectivity filter. The protonation states of Glu71 and Asp80, which are close to each other and to the selectivity filter, as well as K(+) binding energies are determined using free energy perturbation calculations. The Glu71 residue which is buried inside a protein cavity is found to be most stable in the neutral form while the solvent exposed Asp80 is ionized. The channel altogether exothermically binds up to three ions, where two of them are located inside the selectivity filter and one in the internal water cavity. Ion permeation mechanisms are discussed in relation to these results.     

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.     

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 solvation and structural stability in a sodium channel investigated by molecular dynamics calculations

The stability and ion binding properties of the homo-tetrameric pore domain of a prokaryotic, voltage-gated sodium channel are studied by extensive all-atom molecular dynamics simulations, with the channel protein being embedded in a fully hydrated lipid bilayer. It is found that Na(+) ion presents in a mostly hydrated state inside the wide pore of the selectivity filter of the sodium channel, in sharp contrast to the nearly fully dehydrated state for K(+) ions in potassium channels. Our results also indicate that Na(+) ions make contact with only one or two out of the four polypeptide chains forming the selectivity filter, and surprisingly, the selectivity filter exhibits robust stability for various initial ion configurations even in the absence of ions. These findings are quite different from those in potassium channels. Furthermore, an electric field above 0.5V/nm is suggested to be able to induce Na(+) permeation through the selectivity filter.     

Molecular dynamics study of the KcsA channel at 2.0-A 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-A 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 (approximately 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.     

Conducting-state properties of the KcsA potassium channel from molecular and Brownian dynamics simulations.

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.     

Dynamics of K+ ion conduction through Kv1.2

The crystallographic structure of a potassium channel, Kv1.2, in an open state makes it feasible to simulate entire K(+) ion permeation events driven by a voltage bias and, thereby, elucidate the mechanism underlying ion conduction and selectivity of this type of channel. This Letter demonstrates that molecular dynamics simulations can provide movies of the overall conduction of K(+) ions through Kv1.2. As suggested earlier, the conduction is concerted in the selectivity filter, involving 2-3 ions residing mainly at sites identified previously by crystallography and modeling. The simulations reveal, however, the jumps of ions between these sites and identify the sequence of multi-ion configurations involved in permeation.     

Modeling permeation energetics in the KcsA potassium channel

The thermodynamics of cation permeation through the KcsA K(+) channel selectivity filter is studied from the perspective of a physically transparent semimicroscopic model using Monte Carlo free energy integration. The computational approach chosen permits dissection of the separate contributions to ionic stabilization arising from different parts of the channel (selectivity filter carbonyls, single-file water, cavity water, reaction field of bulk water, inner helices, ionizable residues). All features play important roles; their relative significance varies with the ion's position in the filter. The cavity appears to act as an electrostatic buffer, shielding filter ions from structural changes in the inner pore. The model exhibits K(+) vs. Na(+) selectivity, and roughly isoenergetic profiles for K(+) and Rb(+), and discriminates against Cs(+), all in agreement with experimental data. It also indicates that Ba(2+) and Na(+) compete effectively with permeant ions at a site near the boundary between the filter and the cavity, in the vicinity of the barium blocker site.     

Filter flexibility in a mammalian K channel: models and simulations of Kir6.2 mutants

The single-channel conductance varies significantly between different members of the inward rectifier (Kir) family of potassium channels. Mutations at three sites in Kir6.2 have been shown to produce channels with reduced single-channel conductance, the largest reduction (to 40% of wild-type) being for V127T. We have used homology modeling (based on a KcsA template) combined with molecular dynamics simulations in a phosphatidycholine bilayer to explore whether changes in structural dynamics of the filter were induced by three such mutations: V127T, M137C, and G135F. Overall, 12 simulations of Kir6.2 models, corresponding to a total simulation time of 27 ns, have been performed. In these simulations we focused on distortions of the selectivity filter, and on the presence/absence of water molecules lying behind the filter, which form interactions with the filter and the remainder of the protein. Relative to the wild-type simulation, the V127T mutant showed significant distortion of the filter such that approximately 50% of the simulation time was spent in a closed conformation. While in this conformation, translocation of K(+) ions between sites S1 and S2 was blocked. The distorted filter conformation resembles that of the bacterial channel KcsA when crystallized in the presence of a low [K(+)]. This suggests filter distortion may be a possible general model for determining the conductance of K channels.     

Electrostatic tuning of ion conductance in potassium channels

Members of the K(+) channel family display remarkable conservation of sequence and structure of the ion selectivity filter, whereas the rates of K(+) turnover vary widely within the family. Here we show that channel conductance is strongly influenced by charge at the channel's intracellular mouth. Introduction of a ring of negative charges at this position in KcsA, a bacterial K(+) channel, augments the conductance in a pH-dependent manner. These results are explained by a simple electrostatic effect based on known channel structures, where the negative charges serve to alter the electrical potential at the inner mouth and, thus, to increase the local K(+) concentration. In addition, removal of the conserved negative charges at equivalent positions in a high-conductance eukaryotic K(+) channel leads to a decrease in conductance.     

K(+)/Na(+) selectivity of the KcsA potassium channel from microscopic free energy perturbation calculations

Microscopic molecular dynamics free energy perturbation calculations of the K(+)/Na(+) selectivity in the KcsA potassium channel, based on its experimental three-dimensional structure, are reported. The relative binding free energies for K(+) and Na(+) in the most relevant ion occupancy states of the four-site selectivity filter are calculated. The previously proposed mechanism for ion permeation through the KcsA channel is predicted, in agreement with available experimental data, to have a significant selectivity for K(+) over Na(+). The calculations also show that the individual 'binding site' selectivities are generally not additive and the doubly loaded states of the filter thus display cooperative effects. The only site that is not K(+) selective is that which is located at the entrance to the internal water cavity, suggesting the possibility that internal Na(+) could block outward currents.     

The barium site in a potassium channel by x-ray crystallography

X-ray diffraction data were collected from frozen crystals (100 degrees K) of the KcsA K(+) channel equilibrated with solutions containing barium chloride. Difference electron density maps (F(barium) - F(native), 5.0 A resolution) show that Ba(2+) resides at a single location within the selectivity filter. The Ba(2+) blocking site corresponds to the internal aspect (adjacent to the central cavity) of the "inner ion" position where an alkali metal cation is found in the absence of the blocking Ba(2+) ion. The location of Ba(2+) with respect to Rb(+) ions in the pore is in good agreement with the findings on the functional interaction of Ba(2+) with K(+) (and Rb(+)) in Ca(2+)-activated K(+) channels (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-567). Taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side.     

Current and selectivity in a model sodium channel under physiological conditions: Dynamic Monte Carlo simulations

A reduced model of a sodium channel is analyzed using Dynamic Monte Carlo simulations. These include the first simulations of ionic current under approximately physiological ionic conditions through a model sodium channel and an analysis of how mutations of the sodium channel's DEKA selectivity filter motif transform the channel from being Na(+) selective to being Ca(2+) selective. Even though the model of the pore, amino acids, and permeant ions is simplified, the model reproduces the fundamental properties of a sodium channel (e.g., 10 to 1 Na(+) over K(+) selectivity, Ca(2+) exclusion, and Ca(2+) selectivity after several point mutations). In this model pore, ions move through the pore one at a time by simple diffusion and Na(+) versus K(+) selectivity is due to both the larger K(+) not fitting well into the selectivity filter that contains amino acid terminal groups and K(+) moving more slowly (compared to Na(+)) when it is in the selectivity filter.     

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.     

Ion selectivity in potassium channels

Potassium channels are tetrameric membrane-spanning proteins that provide a selective pore for the conduction of K(+) across the cell membranes. One of the main physiological functions of potassium channels is efficient and very selective transport of K(+) ions through the membrane to the cell. Classical views of ion selectivity are summarized within a historical perspective, and contrasted with the molecular dynamics (MD) simulations free energy perturbation (FEP) performed on the basis of the crystallographic structure of the KcsA phospholipid membrane. The results show that the KcsA channel does not select for K(+) ions by providing a binding site of an appropriate (fixed) cavity size. Rather, selectivity for K(+) arises directly from the intrinsic local physical properties of the ligands coordinating the cation in the binding site, and is a robust feature of a pore symmetrically lined by backbone carbonyl groups. Further analysis reveals that it is the interplay between the attractive ion-ligand (favoring smaller cation) and repulsive ligand-ligand interactions (favoring larger cations) that is the basic element governing Na(+)/K(+) selectivity in flexible protein binding sites. Because the number and the type of ligands coordinating an ion directly modulate such local interactions, this provides a potent molecular mechanism to achieve and maintain a high selectivity in protein binding sites despite a significant conformational flexibility.