Hydration valve controlled non-selective conduction of Na and K in the NaK channel

The Na⁺ and K⁺ channels are essential to neural signaling, but our current knowledge at the atomic level is mainly limited to the conducting mechanism of K⁺. Unlike a K⁺ channel having four equivalent K⁺-binding sites in its selectivity filter, a NaK channel has a vestibule in the middle part of its selectivity filter, and can conduct both Na⁺ and K⁺ ions. However, the underlying mechanism for non-selective ion conduction in NaK remains elusive. Here we find four small grottos connecting with the vestibule of the NaK selectivity filter, which form a vestibule-grotto complex perpendicular to the filter pore with a few water molecules within it. It is shown that two or more of the water molecules coming to the vestibule to coordinate the cation are necessary for conducting both Na⁺ and K⁺ ions, while only one water molecule in the vestibule will obstruct ion permeation. Thus, the complex with the aid of interior water movement forms a dynamic hydration valve which is flexible in conveying different cations through the vestibule. Similar exquisite hydration valve mechanisms are expected to be utilized by other non-selective cation channels, and the results should shed new light on the importance of water in neural signaling.     

Dynamics, Energetics, and Selectivity of the Low-K KcsA Channel Structure

Potassium channels are a diverse family of integral membrane proteins through which K⁺ can pass selectively. There is ongoing debate about the nature of conformational changes associated with the opening/closing and conductive/nonconductive states of potassium channels. The channels partly exert their function by varying their conductance through a mechanism known as C-type inactivation. Shortly after the activation of K⁺ channels, their selectivity filter stops conducting ions at a rate that depends on various stimuli. The molecular mechanism of C-type inactivation has not been fully understood yet. However, the X-ray structure of the KcsA channel obtained in the presence of low K⁺ concentration is thought to be representative of a K⁺ channel in the C-type inactivated state. Here, extensive, fully atomistic molecular dynamics and free-energy simulations of the low-K⁺ KcsA structure in an explicit lipid bilayer are performed to evaluate the stability of this structure and the selectivity of its binding sites. We find that the low-K⁺ KcsA structure is stable on the timescale of the molecular dynamics simulations performed, and that ions preferably remain in S1 and S4. In the absence of ions, the selectivity filter evolves toward an asymmetric architecture, as already observed in other computations of the high-K⁺ structure of KcsA and KirBac. The low-K⁺ KcsA structure is not permeable by Na⁺, K⁺, or Rb⁺, and the selectivity of its binding sites is different from that of the high-K⁺ structure.     

Non-Equilibrium Dynamics Contribute to Ion Selectivity in the KcsA Channel

The ability of biological ion channels to conduct selected ions across cell membranes is critical for the survival of both animal and bacterial cells. Numerous investigations of ion selectivity have been conducted over more than 50 years, yet the mechanisms whereby the channels select certain ions and reject others are not well understood. Here we report a new application of Jarzynski’s Equality to investigate the mechanism of ion selectivity using non-equilibrium molecular dynamics simulations of Na⁺ and K⁺ ions moving through the KcsA channel. The simulations show that the selectivity filter of KcsA adapts and responds to the presence of the ions with structural rearrangements that are different for Na⁺ and K⁺. These structural rearrangements facilitate entry of K⁺ ions into the selectivity filter and permeation through the channel, and rejection of Na⁺ ions. A mechanistic model of ion selectivity by this channel based on the results of the simulations relates the structural rearrangement of the selectivity filter to the differential dehydration of ions and multiple-ion occupancy and describes a mechanism to efficiently select and conduct K⁺. Estimates of the K⁺/Na⁺ selectivity ratio and steady state ion conductance for KcsA from the simulations are in good quantitative agreement with experimental measurements. This model also accurately describes experimental observations of channel block by cytoplasmic Na⁺ ions, the “punch through” relief of channel block by cytoplasmic positive voltages, and is consistent with the knock-on mechanism of ion permeation.     

Preferential binding of K+ ions in the selectivity filter at equilibrium explains high selectivity of K+ channels

K⁺ channels exhibit strong selectivity for K⁺ ions over Na⁺ ions based on electrophysiology experiments that measure ions competing for passage through the channel. During this conduction process, multiple ions interact within the region of the channel called the selectivity filter. Ion selectivity may arise from an equilibrium preference for K⁺ ions within the selectivity filter or from a kinetic mechanism whereby Na⁺ ions are precluded from entering the selectivity filter. Here, we measure the equilibrium affinity and selectivity of K⁺ and Na⁺ ions binding to two different K⁺ channels, KcsA and MthK, using isothermal titration calorimetry. Both channels exhibit a large preference for K⁺ over Na⁺ ions at equilibrium, in line with electrophysiology recordings of reversal potentials and Ba²⁺ block experiments used to measure the selectivity of the external-most ion-binding sites. These results suggest that the high selectivity observed during ion conduction can originate from a strong equilibrium preference for K⁺ ions in the selectivity filter, and that K⁺ selectivity is an intrinsic property of the filter. We hypothesize that the equilibrium preference for K⁺ ions originates in part through the optimal spacing between sites to accommodate multiple K⁺ ions within the selectivity filter.     

Sodium Ions Do Not Stabilize the Selectivity Filter of a Potassium Channel

Ion conduction is an essential function for electrical activity in all organisms. The non-selective ion channel NaK was previously shown to adopt two stable conformations of the selectivity filter. Here, we present solid-state NMR measurements of NaK demonstrating a population shift between these conformations induced by changing the ions in the sample while the overall structure of NaK is not affected. We show that two K⁺-selective mutants (NaK2K and NaK2K-Y66F) suffer a complete loss of selectivity filter stability under Na⁺ conditions, but do not collapse into a defined structure. Widespread chemical shift perturbations are seen between the Na⁺ and K⁺ states of the K⁺-selective mutants in the region of the pore helix indicating structural changes. We conclude that the stronger link between the selectivity filter and the pore helix in the K⁺-selective mutants, compared to the non-selective wild-type NaK channel, reduces the ion-dependent conformational flexibility of the selectivity filter.     

Conduction of Na+ and K+ through the NaK channel: molecular and Brownian dynamics studies

Conduction of ions through the NaK channel, with M0 helix removed, was studied using both Brownian dynamics and molecular dynamics. Brownian dynamics simulations predict that the truncated NaK has approximately a third of the conductance of the related KcsA K⁺ channel, is outwardly rectifying, and has a Michaelis-Menten current-concentration relationship. Current magnitude increases when the glutamine residue located near the intracellular gate is replaced with a glutamate residue. The channel is blocked by extracellular Ca²⁺. Molecular dynamics simulations show that, under the influence of a strong applied potential, both Na⁺ and K⁺ move across the selectivity filter, although conduction rates for Na⁺ ions are somewhat lower. The mechanism of conduction of Na⁺ differs significantly from that of K⁺ in that Na⁺ is preferentially coordinated by single planes of pore-lining carbonyl oxygens, instead of two planes as in the usual K⁺ binding sites. The water-containing filter pocket resulting from a single change in the selectivity filter sequence (compared to potassium channels) disrupts several of the planes of carbonyl oxygens, and thus reduces the filter's ability to discriminate against sodium.     

Ion–protein interactions of a potassium ion channel studied by attenuated total reflection Fourier transform infrared spectroscopy

An understanding of ion–protein interactions is key to a better understanding of the molecular mechanisms of proteins, such as enzymes, ion channels, and ion pumps. A potassium ion channel, KcsA, has been extensively studied in terms of ion selectivity. Alkali metal cations in the selectivity filter were visualized by X-ray crystallography. Infrared spectroscopy has an intrinsically higher structural sensitivity due to frequency changes in molecular vibrations interacting with different ions. In this review article, I attempt to summarize ion-exchange-induced differences in Fourier transform infrared spectroscopy, as applied to KcsA, to explain how this method can be utilized to study ion–protein interactions in the KcsA selectivity filter. A band at 1680 cm^?1 in the amide I region would be a marker band for the ion occupancy of K⁺, Rb⁺, and Cs⁺ in the filter. The band at 1627 cm^?1 observed in both Na⁺ and Li⁺ conditions suggests that the selectivity filter similarly interacts with these ions. In addition to the structural information, the results show that the titration of K⁺ ions provides quantitative information on the ion affinity of the selectivity filter.     

The conserved potassium channel filter can have distinct ion binding profiles: Structural analysis of rubidium,cesium, and barium binding in NaK2K

Potassium channels are highly selective for K⁺ over the smaller Na⁺. Intriguingly, they are permeable to larger monovalent cations such as Rb⁺ and Cs⁺ but are specifically blocked by the similarly sized Ba²⁺. In this study, we used structural analysis to determine the binding profiles for these permeant and blocking ions in the selectivity filter of the potassium-selective NaK channel mutant NaK2K and also performed permeation experiments using single-channel recordings. Our data revealed that some ion binding properties of NaK2K are distinct from those of the canonical K⁺ channels KcsA and MthK. Rb⁺ bound at sites 1, 3, and 4 in NaK2K, as it does in KcsA. Cs⁺, however, bound predominantly at sites 1 and 3 in NaK2K, whereas it binds at sites 1, 3, and 4 in KcsA. Moreover, Ba²⁺ binding in NaK2K was distinct from that which has been observed in KcsA and MthK, even though all of these channels show similar Ba²⁺ block. In the presence of K⁺, Ba²⁺ bound to the NaK2K channel at site 3 in conjunction with a K⁺ at site 1; this led to a prolonged block of the channel (the external K⁺-dependent Ba²⁺ lock-in state). In the absence of K⁺, however, Ba²⁺ acts as a permeating blocker. We found that, under these conditions, Ba²⁺ bound at sites 1 or 0 as well as site 3, allowing it to enter the filter from the intracellular side and exit from the extracellular side. The difference in the Ba²⁺ binding profile in the presence and absence of K⁺ thus provides a structural explanation for the short and prolonged Ba²⁺ block observed in NaK2K.     

K Conduction in the Selectivity Filter of Potassium Channels Is Monitored by the Charge Distribution along Their Sequence

Potassium channels display a high conservation of sequence of the selectivity filter (SF), yet nature has designed a variety of channels that present a wide range of absolute rates of K⁺ permeation. In KcsA, the structural archetype for K channels, under physiological concentrations, two K⁺ ions reside in the SF in configurations 1,3 (up state) and 2,4 (down state) and ion conduction is believed to follow a throughput cycle involving a transition between these states. Using free-energy calculations of KcsA, Kv1.2, and mutant channels, we show that this transition is characterized by a channel-dependent energy barrier. This barrier is strongly influenced by the charges partitioned along the sequence of each channel. These results unveil therefore how, for similar structures of the SF, the rate of K⁺ turnover may be fine-tuned within the family of potassium channels.     

Sodium permeability and sensitivity induced by mutations in the selectivity filter of the KcsA channel towards Kir channels

The bacterial potassium (K⁺) channel KcsA provides an attractive model system to study ion permeation behavior in a selective K⁺-channel. We changed residue at the N-terminal end of the selectivity filter of KcsA (T74V) to its counterpart in inwardly rectifying K⁺-channels (Kir). The tetramer was found to be stable as unmodified KcsA. Under symmetrical and asymmetrical conditions, Na⁺ increased the inward current in the virtual absence of K⁺ however outward currents were nearly abolished which could be recovered upon internal K⁺ addition. Na⁺ also drastically increased the channel open time either in the presence or virtual absence of K⁺. Furthermore, the T74V mutation decreased the internal Ba²⁺ affinity of the channel possibly by binding to a K⁺ site in the pore. In additional experiments, another point mutation V76I in T74V mutant was carried out thus the selectivity filter resembled more the selectivity filter of Kir channels. The mutant tetramer was converted into monomers as determined by conventional gel electrophoresis. However, native like gel electrophoresis, Trp fluorescence and acrylamide quenching experiments indicated that this mutant still formed a tetramer and apparently adopted similar folding properties as unmodified KcsA. Single-channel experiments further demonstrated that the channel was selective for K⁺ over Na⁺ as Na⁺ blocked channel currents. These data suggest that single point mutation T74V alters the selectivity filter and allows simultaneous occupancy and conduction of K⁺ and Na⁺ probably via ion–ion interaction in the pore. In contrast, both mutations (T74V and V76I) in the same molecule seem to reorganize the pore conformation which controls the overall stability of a selective K⁺-channel.     

Ion binding properties and structure stability of the NaK channel

Ion distribution in the selectivity filter and ion-water and ion-protein interactions of NaK channel are systematically investigated by all-atom molecular dynamics simulations, with the tetramer channel protein being embedded in a solvated phospholipid bilayer. Analysis of the simulation results indicates that K⁺ ions prefer to bind within the sites formed by two adjacent planes of oxygen atoms from the selectivity filter, while Na⁺ ions are inclined to bind to a single plane of four oxygen atoms. At the same time, both K⁺ and Na⁺ ions can diffuse in the vestibule, accompanying with movements of the water molecules confined in a complex formed by the vestibule together with four small grottos connecting to it. As a result, K⁺ ions show a wide range of coordination numbers (6-8), while Na⁺ ions display a constant coordination number of ∼ 6 in the selectivity filter, which may result in the loss of selectivity of NaK. It is also found that a Ca²⁺ can bind at the extracellular site as reported in the crystal structure in a partially hydrated state, or at a higher site in a full hydration state. Furthermore, the carbonyl group of Asp66 can reorient to point towards the center pore when an ion exists in the vestibule, while that of Gly65 always aligns tangentially to the channel axis, as in the crystallographic structures.     

Nonselective Conduction in a Mutated NaK Channel with Three Cation-Binding Sites

The NaK channel is a cation-selective protein with similar permeability for K⁺ and Na⁺ ions. Crystallographic structures are available for the wild-type and mutated NaK channels with different numbers of cation-binding sites. We have performed a comparison between the potentials of mean force governing the translocation of K⁺ ions and mixtures of one Na⁺ and three K⁺ ions in a mutated NaK channel with only three cation-binding sites (NaK-CNG). Since NaK-CNG is not selective for K⁺ over Na⁺, analysis of its multi-ion potential energy surfaces can provide clues about how selectivity originates. Comparison of the potentials of mean force of NaK-CNG and K⁺-selective channels yields observations that strongly suggest that the number of contiguous ion binding sites in a single-file mechanism is the key determinant of the channel’s selectivity properties, as already proposed by experimental studies. We conclude that the presence of four binding sites in K⁺-selective channels is essential for highly selective and efficient permeation of K⁺ ions, and that a key difference between K⁺-selective and nonselective channels is the absence/presence of a binding site for Na⁺ ions at the boundary between S2 and S3 in the context of multi-ion permeation events.     

Potassium and sodium ions in a potassium channel studied by molecular dynamics simulations

We have performed simulations of both a single potassium ion and a single sodium ion within the pore of the bacterial potassium channel KcsA. For both ions there is a dehydration energy barrier at the cytoplasmic mouth suggesting that the crystal structure is a closed conformation of the channel. There is a potential energy barrier for a sodium ion in the selectivity filter that is not seen for potassium. Radial distribution functions for both ions with the carbonyl oxygens of the selectivity filter indicate that sodium may interact more tightly with the filter than does potassium. This suggests that the key to the ion selectivity of KcsA is the greater dehydration energy of Na⁺ ions, and helps to explain the block of KcsA by internal Na⁺ ions.     

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.     

Structures of Gating Intermediates in a K+ channel

Regulation of ion conduction through the pore of a K⁺ channel takes place through the coordinated action of the activation gate at the bundle crossing of the inner helices and the inactivation gate located at the selectivity filter. The mechanism of allosteric coupling of these gates is of key interest. Here we report new insights into this allosteric coupling mechanism from studies on a W67F mutant of the KcsA channel. W67 is in the pore helix and is highly conserved in K⁺ channels. The KcsA W67F channel shows severely reduced inactivation and an enhanced rate of activation. We use continuous wave EPR spectroscopy to establish that the KcsA W67F channel shows an altered pH dependence of activation. Structural studies on the W67F channel provide the structures of two intermediate states: a pre- open state and a pre-inactivated state of the KcsA channel. These structures highlight key nodes in the allosteric pathway. The structure of the KcsA W67F channel with the activation gate open shows altered ion occupancy at the second ion binding site (S2) in the selectivity filter. This finding in combination with previous studies strongly support a requirement for ion occupancy at the S2 site for the channel to inactivate.     

Diverse gating in K channels: Differential role of the pore-helix glutamate in stabilizing the channel pore

The selectivity filter and adjacent regions in the bacterial KcsA and inwardly rectifying K⁺ (Kir) channels reveal significant conformational changes that cause the channel pore to transition from an activated to inactive state (C-type inactivation) once the channel is open. The meshwork of residues stabilizing the pore of KcsA involves Glu71–Asp80 carboxyl–carboxylate interaction ‘behind’ the selectivity filter. Interestingly, the Kir channels do not have this exact interaction, but instead have a Glu–Arg salt bridge where the Glu is in the same position but the Arg is one position N-terminal compared to the Asp in KcsA. Also, the Kir channels lack the Trp that hydrogen bonds to Asp80 in KcsA. Here, the sequence and structural information are combined to understand the dissimilarity in the role of the pore-helix Glu in stabilizing the pore structure in KcsA and Kir channels. This review illustrates that although Glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other; thereby suggesting a unique phenomenon of diverse gating patterns in K⁺ channels.     

K+-Dependent Selectivity and External Ca2+ Block of Shab K+ Channels

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na_o/K_i or Na_o/Rb_i, conditions. However, when internal K⁺ is replaced by Cs⁺ (Na_o/Cs_i), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²⁺ ions, and compare the effect that internal K⁺ replacement exerts on both Ca²⁺ and TEA block. Our observations indicate that Ca²⁺ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²⁺ is probably coordinated by main chain carbonyls of the pore´s first K⁺-binding site.     

KcsA 通道对Na+、K+及Rb+离子选择性的统计热力学研究

钾离子的通透率至少比钠离子的通透率大10000倍,这个问题至今没有很好地解决.为了在分子水平阐释钾离子通道的选择性机制,以KcsA钾通道X射线衍射结构为基础,采用密度泛函理论计算了不同离子在离子通道中的位能.计算结果表明,Rb+离子具有与K+离子相类似的位能曲线,但是其在通透过程遇到的位垒要比K+离子的位垒高,因而所对应的通透率也就小于钾离子的通透率,而钠离子的的通透率仅仅是钾离子通透率的0.0067%.文中所涉及的系统仅仅包含269个原子,而用分子动力学虽然也可以得到相近的结果,但是它的系统大小为41 000个原子.     

Coordination Numbers of K and Na Ions Inside the Selectivity Filter of the KcsA Potassium Channel: Insights from First Principles Molecular Dynamics

Quantum mechanics/molecular mechanics (QM/MM) Car-Parrinello simulations were performed to estimate the coordination numbers of K⁺ and Na⁺ ions in the selectivity filter of the KcsA channel, and in water. At the DFT/BLYP level, K⁺ ions were found to display an average coordination number of 6.6 in the filter, and 6.2 in water. Na⁺ ions displayed an average coordination number of 5.2 in the filter, and 5.0 in water. A comparison was made with the average coordination numbers obtained from using classical molecular dynamics (6.7 for K⁺ in the filter, 6.6 for K⁺ in water, 6.0 for Na⁺ in the filter, and 5.2 for Na⁺ in water). The observation that different coordination numbers were displayed by the ions in QM/MM simulations and in classical molecular dynamics is relevant to the discussion of selectivity in K-channels.     

Changing Val-76 towards Kir channels drastically influences the folding and gating properties of the bacterial potassium channel KcsA

All K⁺-channels are stabilized by K⁺-ions in the selectivity filter. However, they differ from each other with regard to their selectivity filter. In this study, we changed specific residue Val-76 in the selectivity filter of KcsA to its counterpart Ile in inwardly rectifying K⁺-channels (Kir). The tetramer was exclusively converted into monomers as determined by conventional gel electrophoresis. However, by perfluoro-octanoic acid (PFO) gel electrophoresis mutant channel was mostly detected as tetramer. Tryptophan fluorescence and acrylamide quenching experiments demonstrated significant alteration in channel folding properties via increase in hydrophilicity of local environment. Furthermore, in planar lipid bilayer experiments V76I exhibited drastically lower conductance and decreased channel open time as compared to the unmodified KcsA. These studies suggest that V76I might contribute to determine the stabilizing, folding and channel gating properties in a selective K⁺-channel.