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.     

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.     

KcsA: it's a potassium channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) > Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) > Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.     

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.     

Potassium-selective block of barium permeation through single KcsA channels

Ba(2+), a doubly charged analogue of K(+), specifically blocks K(+) channels by virtue of electrostatic stabilization in the permeation pathway. Ba(2+) block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K(+), the block-time distribution is double exponential, marking at least two Ba(2+) sites in the selectivity filter, in accord with a Ba(2+)-containing crystal structure of KcsA. By analyzing block as a function of extracellular K(+), we determined the equilibrium dissociation constant of K(+) and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb(+) (3 μM) > Cs(+) (23 μM) > K(+) (29 μM) > NH(4)(+) (440 μM) > Na(+) and Li(+) (>1 M). This represents an unusually high selectivity for K(+) over Na(+), with |ΔΔG(0)| of at least 7 kcal mol(-1). These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions.     

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.     

Ion binding affinity in the cavity of the KcsA potassium channel

The hydrophobic cell membrane interior presents a large energy barrier for ions to permeate. Potassium channels reduce this barrier by creating a water-filled cavity at the middle of their ion conduction pore to allow ion hydration and by directing the C-terminal "end charge" of four alpha-helices toward the water-filled cavity. Here we have studied the interaction of monovalent cations with the cavity of the KcsA K(+) channel using X-ray crystallography. In these studies, Tl(+) was used as an analogue for K(+) and the total ion-stabilization energy for Tl(+) in the cavity was estimated by measuring its binding affinity. Binding affinity for the Na(+) ion was also measured, revealing a weak selectivity ( approximately 7-fold) favoring Tl(+) over Na(+). The structures of the cavity containing Na(+), K(+), Tl(+), Rb(+), and Cs(+) are compared. These results are consistent with a fairly large (more negative than -100 mV) electrostatic potential inside the cavity, and they also imply the presence of a weak nonelectrostatic component to a cation's interaction with the cavity.     

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.     

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.     

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.     

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.     

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.     

Na+ block and permeation in a K+ channel of known structure

The effects of intracellular Na(+) were studied on K(+) and Rb(+) currents through single KcsA channels. At low voltage, Na(+) produces voltage-dependent block, which becomes relieved at high voltage by a "punchthrough" mechanism representing Na(+) escaping from its blocking site through the selectivity filter. The Na(+) blocking site is located in the wide, hydrated vestibule, and it displays unexpected selectivity for K(+) and Rb(+) against Na(+). The voltage dependence of Na(+) block reflects coordinated movements of the blocker with permeant ions in the selectivity filter.     

Stable cation coordination at a single outer pore residue defines permeation properties in Kir channels

In epithelial Kir7.1 channels a non-conserved methionine in the outer pore region adjacent to the G-Y-G selectivity filter (position +2) was found to determine unique properties for permeant and blocking ions characteristic of a K(+) channel in a single-occupancy state. The monovalent cation permeability sequence of Kir7.1 channels expressed in Xenopus oocytes was Tl(+)>K(+)>Rb(+)NH(4)(+)>Cs(+)>Na(+)>Li(+), but the macroscopic conductance for Rb(+) was approximately 8-fold larger than for the smaller K(+) ions, and decreased approximately 40-fold with the conserved arginine at the +2 position (Kir7.1M125R). Moreover, in Kir7.1 Rb(+) restored the typical permeation properties of other multi-ion channels indicating that a stable coordination of permeant ions at the +2 position defines the initial step in the conduction pathway of Kir channels.     

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.     

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.     

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.     

Sodium, an Obligate Growth Requirement for Predominant Rumen Bacteria

Sodium is an obligate growth requirement for most currently recognized predominant species of rumen bacteria. The isoosmotic deletion of Na(+) from a nutritionally adequate defined medium completely eliminated growth of most species. Growth yields and rates were both a function of Na(+) concentration for Na(+)-requiring species, and Na(+) could not be replaced by Rb(+), Li(+), or Cs(+) when these ions were substituted for Na(+) at a concentration equivalent to an Na(+) concentration that supported abundant growth. Li(+), Cs(+), or Rb(+) was toxic at an Na(+)-replacing concentration (15 mM) but not at a K(+)-replacing concentration (0.65 mM). K(+) was also an obligate growth requirement for rumen bacteria in media containing Na(+) and K(+) as major monovalent cations, but K(+) could be replaced, for most species, by Rb(+). The quantities of Na(+) that support rapid and abundant growth of Na(+)-requiring rumen bacteria show that these organisms are slight halophiles. A growth requirement for Na(+) appears more frequent among nonmarine bacteria than has been previously believed.     

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.