Effects of the Protonation State of the EEEE Motif of a Bacterial Na-channel on Conduction and Pore Structure

A distinctive feature of prokaryotic Na⁺-channels is the presence of four glutamate residues in their selectivity filter. In this study, how the structure of the selectivity filter, and the free-energy profile of permeating Na⁺ ions are altered by the protonation state of Glu177 are analyzed. It was found that protonation of a single glutamate residue was enough to modify the conformation of the selectivity filter and its conduction properties. Molecular dynamics simulations revealed that Glu177 residues may adopt two conformations, with the side chain directed toward the extracellular entrance of the channel or the intracellular cavity. The likelihood of the inwardly directed arrangement increases when Glu177 residues are protonated. The presence of one glutamate residue with its chain directed toward the intracellular cavity increases the energy barrier for translocation of Na⁺ ions. These higher-energy barriers preclude Na⁺ ions to permeate the selectivity filter of prokaryotic Na⁺-channels when one or more Glu177 residues are protonated.     

The Mechanism of Na+/K+ Selectivity in Mammalian Voltage-Gated Sodium Channels Based on Molecular Dynamics Simulation

Voltage-gated sodium (Na_v) channels and their Na⁺/K⁺ selectivity are of great importance in the mammalian neuronal signaling. According to mutational analysis, the Na⁺/K⁺ selectivity in mammalian Na_v channels is mainly determined by the Lys and Asp/Glu residues located at the constriction site within the selectivity filter. Despite successful molecular dynamics simulations conducted on the prokaryotic Na_v channels, the lack of Lys at the constriction site of prokaryotic Na_v channels limits how much can be learned about the Na⁺/K⁺ selectivity in mammalian Na_v channels. In this work, we modeled the mammalian Na_v channel by mutating the key residues at the constriction site in a prokaryotic Na_v channel (Na_vRh) to its mammalian counterpart. By simulating the mutant structure, we found that the Na⁺ preference in mammalian Na_v channels is collaboratively achieved by the deselection from Lys and the selection from Asp/Glu within the constriction site.     

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.     

On conduction in a bacterial sodium channel

Voltage-gated Na⁺-channels are transmembrane proteins that are responsible for the fast depolarizing phase of the action potential in nerve and muscular cells. Selective permeability of Na⁺ over Ca²⁺ or K⁺ ions is essential for the biological function of Na⁺-channels. After the emergence of the first high-resolution structure of a Na⁺-channel, an anionic coordination site was proposed to confer Na⁺ selectivity through partial dehydration of Na⁺ via its direct interaction with conserved glutamate side chains. By combining molecular dynamics simulations and free-energy calculations, a low-energy permeation pathway for Na⁺ ion translocation through the selectivity filter of the recently determined crystal structure of a prokaryotic sodium channel from Arcobacter butzleri is characterised. The picture that emerges is that of a pore preferentially occupied by two ions, which can switch between different configurations by crossing low free-energy barriers. In contrast to K⁺-channels, the movements of the ions appear to be weakly coupled in Na⁺-channels. When the free-energy maps for Na⁺ and K⁺ ions are compared, a selective site is characterised in the narrowest region of the filter, where a hydrated Na⁺ ion, and not a hydrated K⁺ ion, is energetically stable.     

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.     

Molecular Dynamics Simulations Elucidate the Mechanism of Proton Transport in the Glutamate Transporter EAAT3

The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na⁺ ions and the countertransport of a K⁺ ion. In this study, we use an EAAT3 homology model to calculate the pK_a of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pK_a of this residue and favors the release of the proton to solution. Removing the remaining Na⁺ ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pK_a of E374 with a K⁺ ion bound in three possible sites, we show that binding of the K⁺ ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K⁺ ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs.     

Molecular Dynamics Simulations Elucidate the Mechanism of Proton Transport in the Glutamate Transporter EAAT3

The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na⁺ ions and the countertransport of a K⁺ ion. In this study, we use an EAAT3 homology model to calculate the pK_a of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pK_a of this residue and favors the release of the proton to solution. Removing the remaining Na⁺ ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pK_a of E374 with a K⁺ ion bound in three possible sites, we show that binding of the K⁺ ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K⁺ ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs.     

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.     

Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

NaChBac is a bacterial voltage-gated sodium (Na_v) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Na_v channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of Na_vRh, a marine bacterial NaChBac ortholog. Two Na⁺ binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na⁺ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca²⁺ ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na⁺ and Ca²⁺ to the vestibule of the SF. In addition, Na⁺ permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Na_v orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na⁺ over Ca²⁺ in mammalian Na_v channels.     

Free energy simulations of ligand binding to the aspartate transporter Glt(Ph)

Glutamate/Aspartate transporters cotransport three Na⁺ and one H⁺ ions with the substrate and countertransport one K⁺ ion. The binding sites for the substrate and two Na⁺ ions have been observed in the crystal structure of the archeal homolog Glt_Ph, while the binding site for the third Na⁺ ion has been proposed from computational studies and confirmed by experiments. Here we perform detailed free energy simulations of Glt_Ph, giving a comprehensive characterization of the substrate and ion binding sites, and calculating their binding free energies in various configurations. Our results show unequivocally that the substrate binds after the binding of two Na⁺ ions. They also shed light into Asp/Glu selectivity of Glt_Ph, which is not observed in eukaryotic glutamate transporters.     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

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.     

Identification and characterization of inward K+ -channels in plasma membranes ofArabidopsis root cortex cells

Patch clamping whole-cell recording techniques were applied to study the inward K⁺ -channels inArabidopsis root cortex cells. The inward K⁺ -channels in the plasma membranes of the root cortex cell protoplasts were activated by hyperpolarized membrane potentials. The channels were highly selective for K⁺ ions over Na⁺ ions. The channel activity was significantly inhibited by the external TEA⁺ or Ba²⁺. The changes in cytoplasmic Ca²⁺ concentrations did not affect the whole-cell inward K⁺ -currents. The possible association between the channel selectivity to K⁺ and Na⁺ ions and plant salt-tolerance was also discussed.     

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.     

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.     

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.     

Quantum calculations on water in the KcsA channel cavity with permeant and non-permeant ions

Different ions in the pore of the KcsA channel behave differently, and we relate this to their solvation. We show that the selectivity is dependent, in part, on the solvation in the cavity (sometimes referred to as the vestibule, it is the region containing water molecules between the intracellular gate and the selectivity filter at the extracellular end of the pore). We have shown earlier that potassium is more dependent at the upper end of the cavity region on solvation by the threonines there, while sodium ion has more water molecules as ligands. In addition, sodium ion is placed asymmetrically, while potassium is nearly exactly symmetric with respect to the four-fold symmetry of the channel. We have now extended these calculations to rubidium and cesium ions, and find that rubidium solvation resembles that of potassium (and both are permeant ions), while cesium resembles sodium (and both are non-permeant), in terms of the geometry of up to eight hydrating, and four non-hydrating, water molecules. In each case, a maximum of 12 water molecules are relevant to the calculation. The placement of the water molecules in the two cases is essentially the same as found from the electron density in the X-ray structure of Zhou and MacKinnon. For Na⁺ and K⁺, we show that energy decreases from bulk to the cavity to the lowest position in the selectivity filter (accurate energy could not be calculated for the heavier ions). A separate calculation shows that fixing the Na⁺ ion at the position of the K⁺ minimum, followed by re-optimization produced a significantly modified system, not something that could be produced by thermal fluctuations. Moving the K⁺ into the Na⁺ position in the upper cavity led to a small increase in energy, ≈ 3 k_BT, but was accompanied by large shifts in the positions of hydrating waters, which would create a major kinetic barrier. Therefore, thermal fluctuations could not invalidate the conclusions of the main calculations.     

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.     

Probing ion binding in the selectivity filter of the Cav1.1 channel with molecular dynamics

Ca_v1.1 is the voltage-gated calcium channel essential for the contraction of skeletal muscles upon membrane potential changes. Structural determination of the Ca_v1.1 channel opens the avenue toward understanding of the structure-function relationship of voltage-gated calcium channels. Here, we show that there exist two Ca²⁺-binding sites, termed S1 and S2, within the selectivity filter of Ca_v1.1 through extensive molecular dynamics simulations on various initial ion arrangement configurations. The formation of both binding sites is associated with the four Glu residues (Glu292/614/1014/1323) that constitute the so-called EEEE locus. At the S1 site near the extracellular side, the Ca²⁺ ion is coordinated with the negatively charged carboxylic groups of these Glu residues and of the Asp615 residue either in a direct way or via an intermediate water molecule. At the S2 site, Ca²⁺ binding shows two distinct states: an upper state involving two out of the four Glu residues in the EEEE locus and a lower state involving only one Glu residue. In addition, there exist two recruitment sites for Ca²⁺ above the entrance of the filter. These findings promote the understanding of mechanism for ion permeation and selectivity in calcium channels.