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.     

Interaction of local anesthetics with the K+ channel pore domain

Local anesthetics and related drugs block ionic currents of Na⁺, K⁺ and Ca²⁺ conducted across the cell membrane by voltage-dependent ion channels. Many of these drugs bind in the permeation pathway, occlude the pore and stop ion movement. However channel-blocking drugs have also been associated with decreased membrane stability of certain tetrameric K⁺ channels, similar to the destabilization of channel function observed at low extracellular K⁺ concentration. Such drug-dependent stability may result from electrostatic repulsion of K⁺ from the selectivity filter by a cationic drug molecule bound in the central cavity of the channel. In this study we used the pore domain of the KcsA K⁺ channel protein to test this hypothesis experimentally with a biochemical assay of tetramer stability and theoretically by computational simulation of local anesthetic docking to the central cavity. We find that two common local anesthetics, lidocaine and tetracaine, promote thermal dissociation of the KcsA tetramer in a K⁺-dependent fashion. Docking simulations of these drugs with open, open-inactivated and closed crystal structures of KcsA yield many energetically favorable drug-channel complexes characterized by nonbonded attraction to pore-lining residues and electrostatic repulsion of K⁺. The results suggest that binding of cationic drugs to the inner cavity can reduce tetramer stability of K⁺ channels.     

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.     

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.     

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.     

Mutations in the K+-Channel KcsA Toward Kir Channels Alter Salt-Induced Clusterization and Blockade by Quaternary Alkylammonium Ions

Protein aggregation is a result of malfunction in protein folding, assembly, and transport, caused by protein mutation and/or changes in the cell environment, thus triggering many human diseases. We have shown that bacterial K⁺-channel KcsA, which acts as a representative model for ion channels, forms salt-induced large conductive complexes in a particular environment. In the present study, we investigated the effects of point mutations in the selectivity filter of KcsA on intrinsic stability, aggregation, and channel blocking behavior. First, we found that a low sodium chloride concentration in potassium-containing media induced fast transfer of single channels to a planar lipid bilayer. Second, increasing the sodium chloride concentration drastically increased the total channel current, indicating enhanced vesicle fusion and transfer of multiple channels to a planar lipid bilayer. However, such complexes exhibited high conductance as well as higher open probability compared to the unmodified KcsA behavior shown previously. Interestingly, the affinity of aggregated complexes for larger symmetric quaternary alkylammonium ions (QAs) was found to be much higher than that for tetraethylammonium, a classical blocker of the K⁺ channel. Based on these findings, we propose that mutant channel complexes exhibit larger pore dimensions, thus resembling more the topological properties of voltage-gated and inwardly rectifying K⁺ channels.     

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.     

Phosphatidic acid plays a special role in stabilizing and folding of the tetrameric potassium channel KcsA

In this study, we investigated how the presence of anionic lipids influenced the stability and folding properties of the potassium channel KcsA. By using a combination of gel electrophoresis, tryptophan fluorescence and acrylamide quenching experiments, we found that the presence of the anionic lipid phosphatidylglycerol (PG) in a phosphatidylcholine (PC) bilayer slightly stabilized the tetramer and protected it from trifluoroethanol-induced dissociation. Surprisingly, the presence of phosphatidic acid (PA) had a much larger effect on the stability of KcsA and this lipid, in addition, significantly influenced the folding properties of the protein. The data indicate that PA creates some specificity over PG, and that it most likely stabilizes the tetramer via both electrostatic and hydrogen bond interactions.     

Dissimilarity in the channel intrinsic stability among the bacterial KcsA and the inwardly rectifying potassium channel ROMK1

In this study, we compared the channel intrinsic stability of the bacterial K⁺-channel KcsA and the inwardly rectifying potassium channel (Kir) ROMK1. ROMK1 was successfully cloned, expressed and purified from Saccharomyces cerevisae. By conventional gel electrophoresis, ROMK1 was detected in monomeric form running exclusively at ～45 kDa either in its oxidized or reduced form. By perfluoro-octanoic acid (PFO)-PAGE, the reduced ROMK1 was identified as tetrameric as well as oligomeric complex. However, in its oxidized form ROMK1 was exclusively detected in oligomeric form thus indicating the role of intrinsic cysteine residues and formation of disulfide bonds in stabilizing the oligomeric ROMK1. On the other hand, KcsA purified from E. coli was detected as an extremely stable tetramer both in its oxidized or reduced forms as determined by conventional or PFO-PAGE. Furthermore, in planar lipid bilayer ROMK1 exhibited prominent inward rectification, low single channel conductance and high channel open probability as compared to the KcsA channel which showed typically slight outward rectification and low open probability under similar conditions. Our experiments clearly indicate that KcsA and ROMK1 channels differ with regard to their intrinsic stability which might be related to their structural and functional differences.     

Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel

KcsA is a proton-activated K⁺ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K⁺, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K⁺ selectivity, and permitted Na⁺ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.     

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.     

Separation of heteromeric potassium channel Kcv towards probing subunit composition-regulated ion permeation and gating

The chlorella virus-encoded Kcv can form a homo-tetrameric potassium channel in lipid membranes. This miniature peptide can be synthesized in vitro, and the tetramer purified from the SDS-polyacrylamide gel retains the K⁺ channel functionality. Combining this capability with the mass-tagging method, we propose a simple, straightforward approach that can generically manipulate individual subunits in the tetramer, thereby enabling the detection of contribution from individual subunits to the channel functions. Using this approach, we showed that the structural change in the selectivity filter from only one subunit is sufficient to cause permanent channel inactivation (“all-or-none” mechanism), whereas the mutation near the extracellular entrance additively modifies the ion permeation with the number of mutant subunits in the tetramer (“additive” mechanism).     

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.     

Coupling of activation and inactivation gate in a K+‐channel: potassium and ligand sensitivity

Potassium (K⁺)‐channel gating is choreographed by a complex interplay between external stimuli, K⁺ concentration and lipidic environment. We combined solid‐state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K⁺, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH‐induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K⁺ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K⁺ ion on the inactivation gate modulate activation‐gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K⁺‐channel pore.     

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Ion conduction across the cellular membrane requires the simultaneous opening of activation and inactivation gates of the K⁺ channel pore. The bacterial KcsA channel has served as a powerful system for dissecting the structural changes that are related to four major functional states associated with K⁺ gating. Yet, the direct observation of the full gating cycle of KcsA has remained structurally elusive, and crystal structures mimicking these gating events require mutations in or stabilization of functionally relevant channel segments. Here, we found that changes in lipid composition strongly increased the KcsA open probability. This enabled us to probe all four major gating states in native-like membranes by combining electrophysiological and solid-state NMR experiments. In contrast to previous crystallographic views, we found that the selectivity filter and turret region, coupled to the surrounding bilayer, were actively involved in channel gating. The increase in overall steady-state open probability was accompanied by a reduction in activation-gate opening, underscoring the important role of the surrounding lipid bilayer in the delicate conformational coupling of the inactivation and activation gates.     

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Potassium channels are tetrameric proteins that mediate K⁺-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (∼40 kDa) on SDS-PAGE. Addition of Ba²⁺ (K_1/2 ∼ 50 μm) prior to loading results in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue located near the expected Ba²⁺-binding site, decreased tetramer stabilization by Ba²⁺ (K_1/2 ∼ 300 μm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba²⁺. Neither mutation affected Ba²⁺ block of channel activity (using ⁸⁶Rb⁺ flux assay). In contrast to Ba²⁺, Mg²⁺ had no effect on tetramer stability (even though Mg²⁺ was a potent blocker). Many studies have shown Cd²⁺ block of K⁺ channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd²⁺ on KirBac1.1 with cysteine substitutions in M2. Cd²⁺ block potency followed an α-helical pattern consistent with the crystal structure. Significantly, Cd²⁺ strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba²⁺, consistent with both ions simultaneously occupying the channel: Ba²⁺ at the selectivity filter entrance and Cd²⁺ coordinated by I138C side chains in the inner cavity.Potassium channels are expressed in many cell types and are key players in a wide range of physiological processes. One subset of potassium channels, the inward-rectifying potassium (Kir) channels, are functionally blocked by cytosolic cations such as Mg²⁺ and polyamines and contribute to the regulation of membrane excitability, cardiac rhythm, vascular tone, insulin release, and salt flow across epithelia (1–3). There are seven subfamilies of eukaryotic Kir channel genes. Among them, Kir1 encodes weak rectifiers, whereas Kir2 and Kir5 encode strong rectifiers; Kir3 encodes G-protein-regulated channels; and Kir6 encodes ATP-sensitive channels (4). Recently, a related bacterial family of genes (KirBac) has been identified (5, 6), and in 2003, the first member (KirBac1.1) was crystallized (7), providing a structural model for eukaryotic channels.The crystal structure of KirBac1.1 revealed a tetrameric pore structure similar to that seen in KcsA and a novel cytoplasmic domain (7, 8). The selectivity filter of both KirBac1.1 and KcsA consists of an extremely conserved pore loop followed by a central cavity, forming a transmembrane ion-selective permeation pore (7, 8). The linear arrangement of five oxygen rings (four from carbonyl oxygens and one from a Thr side chain) in the selectivity filter coordinates with ions, compensating for the energy barrier caused by K⁺ dehydration, thereby facilitating the rapid diffusion of K⁺ across the membrane (8–12). Two-thirds of the KirBac1.1 amino acid residues constitute the cytosolic domain that is highly conserved among the Kir subfamilies and form the cytosolic vestibule (13–16), which, together with the transmembrane pore, generates an 88-Å-long ion conduction pore (7).The prototypic potassium channel KcsA exists very stably as a tetramer, even in the harsh conditions of SDS-PAGE (17). In addition to protein-protein interaction between monomers, protein-lipid and protein-ion interactions play important roles in stabilizing the KcsA tetramer (17–20). The selectivity filter of KcsA, coordinated with K⁺ ions, can serve as a bridge between the four monomers to maintain the structure of the selectivity filter and the tetrameric architecture of the channel as a whole (11, 21). Blocking ions, such as Ba²⁺, also act as strong stabilizers (17). In the crystal structure of KcsA, Ba²⁺ occupies a site equivalent to the S4 K⁺-binding site within the selectivity filter (22). Other permeant ions (Rb⁺, Cs⁺, Tl⁺, and NH⁺₄) and strong blockers (Sr²⁺) can also contribute to the thermostability of the KcsA tetramer in SDS-PAGE (17). In contrast, impermeant ions such as Na⁺ and Li⁺ or weak blockers such as Mg²⁺ tend to destabilize the KcsA tetramer (17, 19).Like KcsA, KirBac1.1 purified using decylmaltoside or tridecylmaltoside is active and presumably stable as a tetramer in mild detergent solutions. However, in SDS-PAGE, KirBac1.1 migrates exclusively as a monomer (23). Because KcsA and KirBac1.1 are structurally similar in the transmembrane region of the pore, we hypothesized that permeant and blocking ions would also affect KirBac1.1 tetramer stability in SDS-PAGE. In the present work, the effects of blocking ions such as Ba²⁺ and Mg²⁺ on KirBac1.1 tetramer stability were examined to provide insight to the physical nature of their interaction with KirBac1.1, particularly in the selectivity filter and TM2 cavity. The data reveal important differences in the nature of the interaction of Mg²⁺ and Ba²⁺ with the channel as well as provide previously unavailable evidence for the nature of Cd²⁺ coordination within the channel.     

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K⁺ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K⁺ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.     

Molecular Docking of the Scorpion Toxin Tc1 to the Structural Model of the Voltage-gated Potassium Channel Kv1.1 from Human Homo sapiens

Abstract

In this study, structural model of the pore loop region of the voltage-gated potassium channel Kv1.1 from human Homo sapiens was constructed based on the crystallographic structure of KcsA by structural homology. The pore loop region of Kv1.1 exhibits similar folds as that of KcsA. The structural feature of the selectivity filter of Kv1.1 is nearly identical to that of KcsA, whereas most of the structural variations occur in the turret as well as in the inner and outer helices. Molecular docking experiments of the scorpion toxin Tc1 from Tityus cambridgei to the outer vestibule of KcsA as well as Kv1.1 were subsequently performed with various initial Tc1 orientations. Tc1 was found to form the most stable complexes with these two K⁺ channels when the side chain of Lys14 occupies the pore of the selectivity filter through electrostatic interaction. Tc1 binds preferentially towards Kv1.1 than KcsA due to stronger hydrophobic and electrostatic interactions formed between the toxin and the selectivity filter and outer vestibule of Kv1.1. Furthermore, surface complementarity of the outer vestibules of the channels to the Tc1 spatial conformations also plays an important role in stabilizing both the Tc1/KcsA and Tc1/Kv1.1 complexes.     

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.