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.     

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.     

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 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.     

The selectivity of K+ ion channels: testing the hypotheses

How K⁺ channels are able to conduct certain cations yet not others remains an important but unresolved question. The recent elucidation of the structure of NaK, an ion channel that conducts both Na⁺ and K⁺ ions, offers an opportunity to test the various hypotheses that have been put forward to explain the selectivity of K⁺ ion channels. We test the snug-fit, field-strength, and over-coordination hypotheses by comparing their predictions to the results of classical molecular dynamics simulations of the K⁺ selective channel KcsA and the less selective channel NaK embedded in lipid bilayers. Our results are incompatible with the so-called strong variant of the snug-fit hypothesis but are consistent with the over-coordination hypothesis and neither confirm nor refute the field-strength hypothesis. We also find that the ions and waters in the NaK selectivity filter unexpectedly move to a new conformation in seven K⁺ simulations: the two K⁺ ions rapidly move from site S4 to S2 and from the cavity to S4. At the same time, the selectivity filter narrows around sites S1 and S2 and the carbonyl oxygen atoms rotate 20°−40° inwards toward the ion. These motions diminish the large structural differences between the crystallographic structures of the selectivity filters of NaK and KcsA and appear to allow the binding of ions to S2 of NaK at physiological temperature.     

Pore-forming transmembrane domains control ion selectivity and selectivity filter conformation in the KirBac1.1 potassium channel

Potassium (K⁺) channels are membrane proteins with the remarkable ability to very selectively conduct K⁺ ions across the membrane. High-resolution structures have revealed that dehydrated K⁺ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na⁺ flux in the absence of K⁺ and introduce significant proton conductance. Consistent with K⁺ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K⁺ conditions but undergoes transitions to dilated low-FRET states in high-Na⁺/low-K⁺ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K⁺ and Na⁺ dependence of SF dynamics to higher K⁺ and lower Na⁺ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K⁺-induced SF configuration versus nonselective dilated conformations.     

Gating at the selectivity filter of ion channels that conduct Na+ and K+ ions

The NaK channel is a cation selective channel with similar permeability for K⁺ and Na⁺. The available crystallographic structure of wild-type (WT) NaK is usually associated with a conductive state of the channel. Here, potential of mean force for complete conduction events of Na⁺ and K⁺ ions through NaK show that: i), large energy barriers prevent the passage of ions through the WT NaK structure, ii), the barriers are correlated to the presence of a hydrogen bond between Asp-66 and Asn-68, and iii), the structure of NaK mutated to mimic cyclic nucleotide-gated channels conducts Na⁺ and K⁺. These results support the hypothesis that the filter of cation selective channels can adopt at least two different structures: a conductive one, represented by the x-ray structures of the NaK-CNG chimeras, and a closed one, represented by the x-ray structures of the WT NaK.     

Models of the structure and gating mechanisms of the pore domain of the NaChBac ion channel

The NaChBac prokaryotic sodium channel appears to be a descendent of an evolutionary link between voltage-gated K_V and Ca_V channels. Like K_V channels, four identical six-transmembrane subunits comprise the NaChBac channel, but its selectivity filter possesses a signature sequence of eukaryotic Ca_V channels. We developed structural models of the NaChBac channel in closed and open conformations, using K⁺-channel crystal structures as initial templates. Our models were also consistent with numerous experimental results and modeling criteria. This study concerns the pore domain. The major differences between our models and K⁺ crystal structures involve the latter portion of the selectivity filter and the bend region in S6 of the open conformation. These NaChBac models may serve as a stepping stone between K⁺ channels of known structure and Na_V, Ca_V, and TRP channels of unknown structure.     

Ion selectivity of the Kat1 K+ channel pore

Kat1 is a highly selective inward-rectifying K⁺ channel that opens for extended periods under conditions of extreme hyperpolarization. Over 200 point mutants in the pore region of the Kat1 K⁺ channel were generated and examined in the yeast Saccharomyces cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity. Substitutions at the tyrosine of the signature sequence G-Y-G resulted in the most significant alterations in ion selectivity, consistent with its role in the selectivity filter. However, greater than 80% of the mutations throughout the greater pore region also conferred a defect in selectivity demonstrating that the entire pore of Kat1 contributes to the ion selectivity of this channel. Surprisingly, we identified a novel class of mutant channel that conferred enhanced selectivity of K⁺ over Na⁺. Mutants of this class frequently displayed sensitivity to the competing ion Cs⁺. This finding has led us to speculate that the Kat1 channel pore has evolved to balance not only K⁺/Na⁺ selectivity, but selectivity over Cs⁺, and possibly a wide spectrum of potential competing ions.     

Sodium Blocking Induced by a Point Mutation at the C-Terminal End of the Pore Helix of the KAT1 Channel

A plant hyperpolarization-activating K⁺ channel, KAT1, is highly selective for K⁺ over Na⁺ and is little affected by external Na⁺, which is crucial to take up K⁺ effectively in a Na⁺-containing environment. It has been shown that a mutation at the location (Thr256) preceding the selectivity signature sequence dramatically enhanced the sensitivity of the KAT1 channel to external Na⁺. We report here electrophysiological experiments for the mechanism of action of external Na⁺ on KAT1 channels. The Thr256 residue was substituted with either glutamine (Q) or glutamate (E). The wild-type channel was insensitive to external Na⁺. However, the activity of both mutant channels was significantly depressed by Na⁺ with apparent dissociation constants of 6.7 mm and 11.3 mm for T256Q and T256E, respectively. The instantaneous current-voltage relationships revealed distinct blocking mechanisms for these mutants. For T256Q a typical voltage-dependent fast blocking was shown. On the other hand, the blocking for the T256E mutant was voltage-independent at low Na⁺ concentrations and became voltage-dependent at higher concentrations. At extreme hyperpolarization the blocking was relieved significantly. These data strongly suggest that the mutation at the end of the pore helix rearranged the selectivity filter and allows Na⁺ to penetrate into the pore. Received: 16 October 2000/Revised: 20 February 2001     

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.     

Energy distribution and ion selectivity of the bacterial potassium channel

The ion selectivity of the bacterial potassium channel K_CSA is explained upon comparing the energy characteristics of the interaction of cations (Li⁺, Na⁺, K⁺) with atoms of the selectivity filter of the protein pore. Quantum-chemical calculations reveal a deeper potential well for potassium ions, which accounts for preferred K⁺ permeation. It is shown that the conventional methods with AMBER, CHARMM, OPLS force fields in standard parametrization as well as partial re-parametrization give incorrect estimates of ion energy distribution in the channel.     

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.     

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.     

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.     

Using mutagenesis to study potassium channel mechanisms

The voltage-activated K⁺ channels are members of an ion channel family that includes the voltage-activated Na⁺ and Ca²⁺ channels. These ion channels mediate the transmembrane ionic currents that are responsible for the electrical signals produced by cells. The recent cloning of numerous voltage-activated K⁺ channels has made it possible to combine molecular-genetic and biophysical methods to study K⁺ channel mechanisms. These mutagenesis-function studies are beginning to provide new information about the architecture of K⁺ channel proteins and how they form a voltage-gated, K⁺-selective pore.     

Ion behavior in the selectivity filter of HCN1 channels

Hyperpolarization-activated cyclic-nucleotide gated channels (HCNs) are responsible for the generation of pacemaker currents (I_f or I_h) in cardiac and neuronal cells. Despite the overall structural similarity to voltage-gated potassium (Kv) channels, HCNs show much lower selectivity for K⁺ over Na⁺ ions. This increased permeability to Na⁺ is critical to their role in membrane depolarization. HCNs can also select between Na⁺ and Li⁺ ions. Here, we investigate the unique ion selectivity properties of HCNs using molecular-dynamics simulations. Our simulations suggest that the HCN1 pore is flexible and dilated compared with Kv channels with only one stable ion binding site within the selectivity filter. We also observe that ion coordination and hydration differ within the HCN1 selectivity filter compared with those in Kv and cyclic-nucleotide gated channels. Additionally, the C358T mutation further stabilizes the symmetry of the binding site and provides a more fit space for ion coordination, particularly for Li⁺.     

Determinants of cation transport selectivity: Equilibrium binding and transport kinetics

ConclusionThe equilibrium ion-binding properties of ion channels and transporters can be difficult to discern from crystal structures alone, as proteins often adopt different lowest energy states depending on the ions bound. In cases where transport is slow, their inherent ion-binding preferences can be used to infer their transport preferences. However, in cases where transport is fast, the transport selectivity can hide their equilibrium preferences by accentuating the kinetics of ions hopping through a channel over its inherent ion-binding preferences. Thus, depending on the arrangement of ion-binding sites in a channel’s selectivity filter, one can achieve either selective or nonselective ion transport.The equilibrium K⁺ selectivity of some nonselective channels suggests a potential mechanism whereby they could evolve into a fast K⁺-selective channel. K⁺ channels and nonselective channels like CNG and HCN are related to one another in both sequence and structure, suggesting an evolutionary link between them. Swap experiments show that only a few mutations separate a nonselective channel from a K⁺-selective channel. One might imagine an evolutionary path between these channels in which the equilibrium preference for a K⁺ ion in a nonselective channel evolves into a K⁺-selective channel through these few mutations to create the selective ion queue. Alternatively, a slow single-ion channel with an equilibrium and transport preference for K⁺ ions could be transformed into a fast multi-ion channel through mutations that create a queue of K⁺-selective ion-binding sites, as is seen in most K⁺ channels studied to date.In the case of multi-ion selectivity filters, such as those found in K⁺ channels, the selectivity filter can be viewed as the active site that interacts with different queues of ions and water molecules. At least three properties emerge from multi-ion queues: (1) high conductance by reducing the affinity of multiple bound ions versus single ions; (2) high selectivity by allowing disfavored ions time to dissociate back into solution; and, consequently, (3) robust selectivity in an environment where ion concentrations can change. For transporters and carriers, the equilibrium preference and slow transport naturally create robust selectivity. In all these cases, equilibrium-based ion selectivity is achieved by slowing transport enough so that the disfavored ion is able to dissociate back into solution before transport takes place.     

The external pore loop interacts with S6 and S3-S4 linker in domain 4 to assume an essential role in gating control and anticonvulsant action in the Na+ channel

Carbamazepine, phenytoin, and lamotrigine are widely prescribed anticonvulsants in neurological clinics. These drugs bind to the same receptor site, probably with the diphenyl motif in their structure, to inhibit the Na⁺ channel. However, the location of the drug receptor remains controversial. In this study, we demonstrate close proximity and potential interaction between an external aromatic residue (W1716 in the external pore loop) and an internal aromatic residue (F1764 in the pore-lining part of the sixth transmembrane segment, S6) of domain 4 (D4), both being closely related to anticonvulsant and/or local anesthetic binding to the Na⁺ channel. Double-mutant cycle analysis reveals significant cooperativity between the two phenyl residues for anticonvulsant binding. Concomitant F1764C mutation evidently decreases the susceptibility of W1716C to external Cd²⁺ and membrane-impermeable methanethiosulfonate reagents. Also, the W1716E/F1764R and G1715E/F1764R double mutations significantly alter the selectivity for Na⁺ over K⁺ and markedly shift the activation curve, respectively. W1716 and F1764 therefore very likely form a link connecting the outer and inner compartments of the Na⁺ channel pore (in addition to the selectivity filter). Anticonvulsants and local anesthetics may well traverse this “S6 recess” without trespassing on the selectivity filter. Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na⁺ channel. The effect of Y1618K mutation, however, is abolished by concomitant point mutations in the vicinity of Y1618, but not by those in the internally located inactivation machinery, supporting a direct local rather than a long-range allosteric action. Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action. We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na⁺ channel.     

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.