Effect of external electric fields on the potential energy profile of K+ ions in selective filter of the KcsA potassium channel

In this study, the potential energy profile of potassium ions in the selective filter part of a KcsA channel was investigated via the application of the molecular simulation method. For this purpose, using the molecular dynamics simulation, the effect of an applied electric field, either constant or oscillating, was studied on the dynamics of K ions in the filter. It was found that when the channel is exposed to a constant electric field of strength 0.03 V/nm, the ions experience a hopping motion. Furthermore, it was shown that the application of oscillating electric fields of 1 and 2.5 GHz, can increase the rigidity of the filter atomic bonds. By computing the potential energy of K ion in the filter, it was shown that the depth of the potential wells, corresponding to the filter sites, increased when an alternative field was applied. Therefore, exposing the channel to the GHz oscillating electric field could disturb the passing rate of ions through the filter, which in turn may affect the operation of these kinds of channels.     

Construction of a cyclic nucleotide-gated KcsA K+ channel

The ability of an ion channel to open in response to a defined stimulus is central to its function. In ligand-gated channels, pore opening is conferred through transduction of a conformational change in a gating domain to the helices of the pore. Here, we present the construction of a designed cyclic nucleotide-gated (CNG) channel, named KcsA-CNG, by addition of a prokaryotic cyclic nucleotide-binding domain to a KcsA-derived K+ channel. This channel is functional in lipid bilayers at physiological pH and has the combined properties of both of its parent-derived components. It conducts K+ and is blocked by the K+ channel inhibitors Na+ and agitoxin-2. Channel open times are increased by about two orders of magnitude compared to wild-type KcsA. The average number of open channels increases by approximately 50% upon addition of cAMP. Although the absolute open probabilities are somewhat variable from one channel to the next, the property of cyclic nucleotide sensitivity is very reproducible. An apparent Kd value of approximately 90 nM was estimated. The successful construction of a cyclic nucleotide-gated KcsA K+ channel suggests that it should be possible to produce channels that will respond to novel ligands.     

Simulations of ion current in realistic models of ion channels: the KcsA potassium channel

Realistic studies of ion current in biologic channels present a major challenge for computer simulation approaches. All-atom molecular dynamics simulations involve serious time limitations that prevent their use in direct evaluation of ion current in channels with significant barriers. The alternative use of Brownian dynamics (BD) simulations can provide the current for simplified macroscopic models. However, the time needed for accurate calculations of electrostatic energies can make BD simulations of ion current expensive. The present work develops an approach that overcomes some of the above challenges and allows one to simulate ion currents in models of biologic channels. Our method provides a fast and reliable estimate of the energetics of the system by combining semimacroscopic calculations of the self-energy of each ion and an implicit treatment of the interactions between the ions, as well as the interactions between the ions and the protein-ionizable groups. This treatment involves the use of the semimacroscopic version of the protein dipole Langevin dipole (PDLD/S) model in its linear response approximation (LRA) implementation, which reduces the uncertainties about the value of the protein "dielectric constant." The resulting free energy surface is used to generate the forces for on-the-fly BD simulations of the corresponding ion currents. Our model is examined in a preliminary simulation of the ion current in the KcsA potassium channel. The complete free energy profile for a single ion transport reflects reasonable energetics and captures the effect of the protein-ionized groups. This calculated profile indicates that we are dealing with the channel in its closed state. Reducing the barrier at the gate region allows us to simulate the ion current in a reasonable computational time. Several limiting cases are examined, including those that reproduce the observed current, and the nature of the productive trajectories is considered. The ability to simulate the current in realistic models of ion channels should provide a powerful tool for studies of the biologic function of such systems, including the analysis of the effect of mutations, pH, and electric potentials.     

Mixed modes in opening of KcsA potassium channel from a targeted molecular dynamics simulation

Potassium channels conduct K⁺ flow selectively across the membrane through a central pore. During a process called gating, the potassium channels undergo a conformational change that opens or closes the ion-conducting pore. The potassium channel KcsA has been structurally determined in its closed state. However, the dynamic mechanism of the gating transition of the KcsA channel is still being investigated. Here, a targeted molecular dynamics simulation up to 150 ns is performed to investigate the detailed opening process of the KcsA channel with an open Kv1.2 structure serving as the target. The channel arrived at a self-determined quasi-stable state within 60 ns. The rigid-body and hinge-bending modes are observed mixed together in the remaining 90 ns long quasi-stable state. The mixed-mode movement seems come from the competition between the helix rigidity and the biased-applied gating force.     

Efficacy of external tetraethylammonium block of the KcsA potassium channel: Molecular and Brownian dynamics studies

Blockade of the KcsA potassium channel by externally applied tetraethylammonium is investigated using molecular dynamics calculations and Brownian dynamics simulations. In KcsA, the aromatic rings of four tyrosine residues located just external to the selectivity filter create an attractive energy well or a binding cage for a tetraethylammonium molecule. We first investigate the effects of re-orienting the four tyrosine residues such that the centers of the aromatic rings face the tetraethylammonium molecule directly. Then, we systematically move the residues inward in both orientations so that the radius of the binding cage formed by them becomes smaller. For each configuration, we construct a one-dimensional free energy profile by bringing in a tetraethylammonium molecule from the external reservoir toward the selectivity filter. The free energy profile is then converted to a one-dimensional potential energy profile, taking the available space between the tyrosine residues and the tetraethylammonium molecule into account. Incorporating this potential energy profile into the Brownian dynamics algorithm, we determine the conductance properties of the channel under various conditions, construct the current-tetraethylammonium-concentration curve and compare it with the experimentally determined inhibitory constant k_i for externally applied tetraethylammonium. We show that the experimentally determined binding affinity for externally applied tetraethylammonium can be replicated when each of the four tyrosine residues is moved inward by about 0.7 Å, irrespective of orientation of their aromatic rings.     

Simulation of the effect of an external GHz electric field on the potential energy profile of Ca2+ ions in the selectivity filter of the CaVAb channel

Ca_V channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca²⁺ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion‐selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the Ca_VAb channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca²⁺ ions in the Ca_VAb channels in presence and absence of an external field. Exposing Ca_VAb channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca²⁺ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca²⁺ ions binding in the selectivity filter may interrupt the mechanism of Ca²⁺ ion conduction, and leads to a reduction of Ca²⁺ ion permeation through the Ca_VAb channel.     

Permeation of water through the KcsA K+ channel

Previous studies have reported that the KcsA potassium channel has an osmotic permeability coefficient of 4.8 x 10(-12) cm3/s, giving it a significantly higher osmotic permeability coefficient than that of some membrane channels specialized in water transport. This high osmotic permeability is proposed to occur when the channel is depleted of potassium ions, the presence of which slow down the water permeation process. The atomic structure of the potassium-depleted KcsA channel and the mechanisms of water permeation have not been well characterized so far. Here, all-atom molecular dynamics simulations, in conjunction with an umbrella sampling strategy and a nonequilibrium approach to simulate pressure gradients are employed to illustrate the permeation of water in the absence of ions through the KcsA K+ channel. Equilibrium molecular dynamics simulations (95 ns combined total length) identified a possible structure of the potassium-depleted KcsA channel, and umbrella sampling calculations (160 ns combined total length) revealed that this structure is not permeable by water molecules moving along the channel axis. The simulation of a pressure gradient across the channel (30 ns combined total length) identified an alternative permeation pathway with a computed osmotic permeability of approximately (2.7 +/- 0.9) x 10(-13) cm3/s. Water fluxes along this pathway did not proceed through collective water motions or transitions to vapor state. All of the major results of this study were robust against variations in a wide set of simulation parameters (force field, water model, membrane model, and channel conformation).     

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.     

Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA

Here we present functional evidence for involvement of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) in ion conduction and selection at the intracellular side of the Streptomyces lividans potassium channel, KcsA. At < or = 25 degrees C, KcsA forms channels in planar bilayers that display signal characteristics of PHB/polyP channels at the intracellular side; i.e., a preference for divalent Mg(2+) cations at pH 7.2, and a preference for monovalent K+ cations at pH 6.8. Between 25 and 26 degrees C, KcsA undergoes a transition to a new conformation in which the channel exhibits high selectivity for K+, regardless of solution pH. This suggests that basic residues of the C-terminal polypeptides have moved closer to the polyP end unit, reducing its negative charge. The data support a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K+ is governed by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands.     

The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates

Potassium ions diffuse across the cell membrane in a single file through the narrow selectivity filter of potassium channels. The crystal structure of the KcsA K+ channel revealed the chemical structure of the selectivity filter, which contains four binding sites for K+. In this study, we used Tl+ in place of K+ to address the question of how many ions bind within the filter at a given time, i.e. what is the absolute ion occupancy? By refining the Tl+ structure against data to 1.9A resolution with an anomalous signal, we determined the absolute occupancy of Tl+. Then, by comparing the electron density of Tl+ with that of K+, Rb+ and Cs+, we estimated the absolute occupancy of these three ions. We further analyzed how the ion occupancy affects the conformation of the selectivity filter by analyzing the structure of KcsA at different concentrations of Tl+. Our results indicate that the average occupancy for each site in the selectivity filter is about 0.63 for Tl+ and 0.53 for K+. For K+, Rb+ and Cs+, the total number of ions contained within four sites in the selectivity filter is about two. At low concentrations of permeant ion, the number of ions drops to one in association with a conformational change in the selectivity filter. We conclude that electrostatic balance and coupling of ion binding to a protein conformational change underlie high conduction rates in the setting of high selectivity.     

A comparison between two prokaryotic potassium channels (KirBac1.1 and KcsA) in a molecular dynamics (MD) simulation study

The two potassium ion channels KirBac1.1 and KcsA are compared in a Molecular Dynamics (MD) simulation study. The location and motion of the potassium ions observed in the simulations are compared to those in the X-ray structures and previous simulations. In our simulations several of the crystallography resolved ion sites in KirBac1.1 are occupied by ions. In addition to this, two in KirBac1.1 unresolved sites where occupied by ions at sites that are in close correspondence to sites found in KcsA. There is every reason to believe that the conserved alignment of the selectivity filter in the potassium ion channel family corresponds to a very similar mechanism for ion transport across the filter. The gate residues, Phe146 in KirBac1.1 and Ala111 in KcsA acted in the simulations as effective barriers which never were passed by ions nor water molecules.     

The barium site in a potassium channel by x-ray crystallography

X-ray diffraction data were collected from frozen crystals (100 degrees K) of the KcsA K(+) channel equilibrated with solutions containing barium chloride. Difference electron density maps (F(barium) - F(native), 5.0 A resolution) show that Ba(2+) resides at a single location within the selectivity filter. The Ba(2+) blocking site corresponds to the internal aspect (adjacent to the central cavity) of the "inner ion" position where an alkali metal cation is found in the absence of the blocking Ba(2+) ion. The location of Ba(2+) with respect to Rb(+) ions in the pore is in good agreement with the findings on the functional interaction of Ba(2+) with K(+) (and Rb(+)) in Ca(2+)-activated K(+) channels (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-567). Taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side.     

The influence of a membrane environment on the structure and stability of a prokaryotic potassium channel, KcsA

The lack of a membrane environment in membrane protein crystals is considered one of the major limiting factors to fully imply X-ray structural data to explain functional properties of ion channels [Gulbis, J.M. and Doyle, D. (2004) Curr. Opin. Struct. Biol. 14, 440-446]. Here, we provide infrared spectroscopic evidence that the structure and stability of the potassium channel KcsA and its chymotryptic derivative 1-125 KcsA reconstituted into native-like membranes differ from those exhibited by these proteins in detergent solution, the latter taken as an approximation of the mixed detergent-protein crystal conditions.     

Extracellular blockade of K(+) channels by TEA: results from molecular dynamics simulations of the KcsA channel

TEA is a classical blocker of K(+) channels. From mutagenesis studies, it has been shown that external blockade by TEA is strongly dependent upon the presence of aromatic residue at Shaker position 449 which is located near the extracellular entrance to the pore (Heginbotham, L., and R. MacKinnon. 1992. Neuron. 8:483-491). The data suggest that TEA interacts simultaneously with the aromatic residues of the four monomers. The determination of the 3-D structure of the KcsA channel using X-ray crystallography (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) has raised some issues that remain currently unresolved concerning the interpretation of these observations. In particular, the center of the Tyr82 side chains in KcsA (corresponding to position 449 in Shaker) forms a square of 11.8-A side, a distance which is too large to allow simultaneous interactions of a TEA molecule with the four aromatic side chains. In this paper, the external blockade by TEA is explored by molecular dynamics simulations of an atomic model of KcsA in an explicit phospholipid bilayer with aqueous salt solution. It is observed, in qualitative accord with the experimental results, that TEA is stable when bound to the external side of the wild-type KcsA channel (with Tyr82), but is unstable when bound to a mutant channel in which the tyrosine residue has been substituted by a threonine. The free energy profile of TEA relative to the pore is calculated using umbrella sampling simulations to characterize quantitatively the extracellular blockade. It is found, in remarkable agreement with the experiment, that the TEA is more stably bound by 2.3 kcal/mol to the channel with four tyrosine residues. In the case of the wild-type KcsA channel, TEA (which has the shape of a flattened oblate spheroid) acts as an ideal plug blocking the pore. In contrast, it is considerably more off-centered and tilted in the case of the mutant channel. The enhanced stability conferred by the tyrosine residues does not arise from Pi-cation interactions, but appears to be due to differences in the hydration structure of the TEA. Finally, it is shown that the experimentally observed voltage dependence of TEA block, which is traditionally interpreted in terms of the physical position of the TEA along the axis of the pore, must arise indirectly via coupling with the ions in the pore.     

NMR study of the tetrameric KcsA potassium channel in detergent micelles

Nuclear magnetic resonance (NMR) studies of large membrane-associated proteins are limited by the difficulties in preparation of stable protein-detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68-kDa homotetrameric KcsA, a thermostable N-terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C-terminal cytoplasmic domain, absent in the original structure, contains a 14-residue helix that could participate in tetramerization by forming an intersubunit four-helix bundle. A quantitative estimate of cross- relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS-KcsA mixed micelles form an oblate spheroid with approximately 180 SDS molecules per channel. K(+) ions bind to the micelle-solubilized channel with a K(D) of 3 +/- 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH-induced changes in chemical shift along the "outer" transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH-gating of the potassium channel.     

Molecular dynamics simulations of the calmodulin-induced α-helix in the SK2 calcium-gated potassium ion channel

The family of small-conductance Ca²⁺-activated potassium ion channels (SK channels) is composed of four members (SK1, SK2, SK3, and SK4) involved in neuron-firing regulation. The gating of these channels depends on the intracellular Ca²⁺ concentration, and their sensitivity to this ion is provided by calmodulin (CaM). This protein binds to a specific region in SK channels known as the calmodulin-binding domain (CaMBD), an event which is essential for their gating. While CaMBDs are typically disordered in the absence of CaM, the SK2 channel subtype displays a small prefolded α-helical region in its CaMBD even if CaM is not present. This small helix is known to turn into a full α-helix upon CaM binding, although the molecular-level details for this conversion are not fully understood yet. In this work, we offer new insights on this physiologically relevant process by means of enhanced sampling, atomistic Hamiltonian replica exchange molecular dynamics simulations, providing a more detailed understanding of CaM binding to this target. Our results show that CaM is necessary for inducing a full α-helix along the SK2 CaMBD through hydrophobic interactions with V426 and L427. However, it is also necessary that W431 does not compete for these interactions; the role of the small prefolded α-helix in the SK2 CaMBD would be to stabilize W431 so that this is the case. In conclusion, our findings provide further insight into a key interaction between CaM and SK channels that is important for channel sensitivity to Ca²⁺.     

Ion channel enzyme in an oscillating electric field

To explain the electrical activation of several membrane ATPases, an electroconformational coupling (ECC) model has previously been proposed. The model explained many features of experimental data but failed to reproduce a window of the field intensity for the stimulated activity. It is shown here that if the affinities of the ion for the two conformational states of the transporter (one with binding site on the left side and the other on the right side of the membrane) are dependent on the electric field, the field-dependent transport can exhibit the observed window. The transporter may be described as a channel enzyme which opens to one side of the membrane at a time. It retains the energy-transducing ability of the earlier ECC models. Analysis of the channel enzyme in terms of the Michaelis-Menten kinetics has been done. The model reproduced the amplitude window for the electric field-induced cation pumping by (Na,K)-ATPase.     

Probing the pH-dependent structural features of alpha-KTx12.1, a potassium channel blocker from the scorpion Tityus serrulatus

Potassium channels are widespread in living cells and are involved in many diseases. The scorpion toxin alpha-KTx(12.1) interacts with various K(+) channels, suggesting its capacity to match diverse channel pores. It is recognized that tissue injuries may affect the pH at toxins site of action, thereby modulating both protein conformation and activity. To better understand its molecular mechanism of action, we studied alpha-KTx(12.1) using pH as a tool to explore its plasticity and NMR in combination with MD calculations to detect it. The toxin solution structure consists of an alpha-helix and a triple-stranded beta-sheet stabilized by four disulfide bridges. The NMR results show, in addition, that His28 possesses an unusually low pK(a) of 5.2. The best set of protein conformers is obtained at pH 4.5, while at pH 7.0, the reduced number of NOEs resulting from a faster hydrogen exchange does not allow to reach a good structural convergence. Nonetheless, MD calculations show that the toxin structure does not vary significantly in that pH range, while conformational changes and modifications of the surface charge distribution occur when His28 is fully protonated. Moreover, essential dynamics analysis reveals variations in the toxin's coherent motions. In conclusion, His28, with its low pK(a) value, provides alpha-KTx(12.1) with the ability to preserve its active conformation over a wide pH interval, thus expanding the range of cellular conditions where the toxin can fully exhibit its activity. Overall, the results further underline the role of histidine as a natural controller of proteins' functionality.