Ion permeation through a narrow channel: using gramicidin to ascertain all-atom molecular dynamics potential of mean force methodology and biomolecular force fields

We investigate methods for extracting the potential of mean force (PMF) governing ion permeation from molecular dynamics simulations (MD) using gramicidin A as a prototypical narrow ion channel. It is possible to obtain well-converged meaningful PMFs using all-atom MD, which predict experimental observables within order-of-magnitude agreement with experimental results. This was possible by careful attention to issues of statistical convergence of the PMF, finite size effects, and lipid hydrocarbon chain polarizability. When comparing the modern all-atom force fields of CHARMM27 and AMBER94, we found that a fairly consistent picture emerges, and that both AMBER94 and CHARMM27 predict observables that are in semiquantitative agreement with both the experimental conductance and dissociation coefficient. Even small changes in the force field, however, result in significant changes in permeation energetics. Furthermore, the full two-dimensional free-energy surface describing permeation reveals the location and magnitude of the central barrier and the location of two binding sites for K(+) ion permeation near the channel entrance--i.e., an inner site on-axis and an outer site off-axis. We conclude that the MD-PMF approach is a powerful tool for understanding and predicting the function of narrow ion channels in a manner that is consistent with the atomic and thermally fluctuating nature of proteins.     

Energetics of ion permeation, rejection, binding, and block in gramicidin A from free energy simulations

The rigid force fields currently used in molecular dynamics (MD) simulations of biomolecules are optimized for globular proteins. Whether they can also be used in MD simulations of membrane proteins is an important issue that needs to be resolved. Here we address this issue using the gramicidin A channel, which provides an ideal test case because of the simplicity of its structure and the availability of a wealth of functional data. Permeation properties of gramicidin A can be summarized as "it conducts monovalent cations, rejects anions, and binds divalent cations." Hence, a comprehensive test should consider the energetics of permeation for all three types of ions. To that end, we construct the potential of mean force for K(+), Cl(-), and Ca(2+) ions along the channel axis. For an independent check of the potential-of-mean-force results, we also calculate the free energy differences for these ions at the channel center and binding sites relative to bulk. We find that "rejection of anions" is satisfied but there are difficulties in accommodating the other two properties using the current MD force fields.     

Molecular dynamics - potential of mean force calculations as a tool for understanding ion permeation and selectivity in narrow channels

Ion channels catalyze the permeation of charged molecules across cell membranes and are essential for many vital physiological functions, including nerve and muscle activity. To understand better the mechanisms underlying ion conduction and valence selectivity of narrow ion channels, we have employed free energy techniques to calculate the potential of mean force (PMF) for ion movement through the prototypical gramicidin A channel. Employing modern all-atom molecular dynamics (MD) force fields with umbrella sampling methods that incorporate one hundred 1-2 ns trajectories, we find that it is possible to achieve semi-quantitative agreement with experimental binding and conductance measurements. We also examine the sensitivity of the MD-PMF results to the choice of MD force field and compare PMFs for potassium, calcium and chloride ions to explore the basis for the valence selectivity of this narrow and uncharged ion channel. A large central barrier is observed for both anions and divalent ions, consistent with lack of experimental conductance. Neither anion or divalent cation is seen to be stabilized inside the channel relative to the bulk electrolyte and each leads to large disruptions to the protein and membrane structure when held deep inside the channel. Weak binding of calcium ions outside the channel corresponds to a free energy well that is too shallow to demonstrate channel blocking. Our findings emphasize the success of the MD-PMF approach and the sensitivity of ion energetics to the choice of biomolecular force field.     

Test of molecular dynamics force fields in gramicidin A

The force fields commonly used in molecular dynamics simulations of proteins are optimized under bulk conditions. Whether the same force fields can be used in simulations of membrane proteins is not well established, although they are increasingly being used for such purposes. Here we consider ion permeation in the gramicidin A channel as a test of the AMBER force field in a membrane environment. The potentials of mean force for potassium ions are calculated along the channel axis and compared with the one deduced from the experimental conductance data. The calculated result indicates a rather large central barrier similar to those obtained from other force fields, which are incompatible with the conductance data. We suggest that lack of polarizability is the most likely cause of this problem, and, therefore, urge development of polarizable force fields for simulations of membrane proteins.     

Diffusion constant of K+ inside Gramicidin A: a comparative study of four computational methods

The local diffusion constant of K(+) inside the Gramicidin A (GA) channel has been calculated using four computational methods based on molecular dynamics (MD) simulations, specifically: Mean Square Displacement (MSD), Velocity Autocorrelation Function (VACF), Second Fluctuation Dissipation Theorem (SFDT) and analysis of the Generalized Langevin Equation for a Harmonic Oscillator (GLE-HO). All methods were first tested and compared for K(+) in bulk water--all predicted the correct diffusion constant. Inside GA, MSD and VACF methods were found to be unreliable because they are biased by the systematic force exerted by the membrane-channel system on the ion. SFDT and GLE-HO techniques properly unbias the influence of the systematic force on the diffusion properties and predicted a similar diffusion constant of K(+) inside GA, namely, ca. 10 times smaller than in the bulk. It was found that both SFDT and GLE-HO methods require extensive MD sampling on the order of tens of nanoseconds to predict a reliable diffusion constant of K(+) inside GA.     

Gramicidin A directly inhibits mammalian Na+/K+-ATPase

The linear pentadecapeptide gramicidin A forms an ion channel in the lipid bilayer to selectively transport monovalent cations. Nevertheless, we have surprisingly found that gramicidin A directly inhibits mammalian Na(+)/K(+)-ATPase. Gramicidin A inhibited ATP hydrolysis by Na(+)/K(+)-ATPase from porcine cerebral cortex at the IC(50) value of 8.1 microM, while gramicidin S was approximately fivefold less active. The synthetic gramicidin A analog lacking N-terminal formylation and C-terminal ethanolamine exhibited a weaker inhibitory effect on the ATP-hydrolyzing activity of Na(+)/K(+)-ATPase than gramicidin A, indicating that these end modifications are necessary for gramicidin A to inhibit Na(+)/K(+)-ATPase activity. Moreover, Lineweaver-Burk analysis showed that gramicidin A exhibits a mixed type of inhibition. In addition to the most well-studied ionophore activity, our present study has disclosed a novel biological function of gramicidin A as a direct inhibitor of mammalian Na(+)/K(+)-ATPase activity.     

Molecular dynamics computations and solid state nuclear magnetic resonance of the gramicidin cation channel.

This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other. When applied in a coordinated manner they provide a powerful approach to structure determination in molecular systems not readily amenable to x-ray diffraction.     

Molecular dynamics simulations of gramicidin A in a lipid bilayer: from structure-function relations to force fields

Molecular dynamics simulations of membrane proteins have become a popular tool for studying their dynamic features, which are not easily accessible by experiments. Whether the force fields developed for globular proteins are adequate this purpose is an important question that is often glossed over. Here we determine the permeation properties of potassium ions in the gramicidin A channel in a lipid bilayer from free energy simulations, and compare the results to experimental data. In particular, we check the dependence of the free energy barriers ions face at the channel center on the membrane size. The results indicate that there is a serious problem with the current rigid force fields independent of the membrane size, and new, possibly polarizable, force fields need to be developed to resolve this problem.     

A marriage of convenience: beta-subunits and voltage-dependent K+ channels

The movement of ions across cell membranes is essential for a wide variety of fundamental physiological processes, including secretion, muscle contraction, and neuronal excitation. This movement is possible because of the presence in the cell membrane of a class of integral membrane proteins dubbed ion channels. Ion channels, thanks to the presence of aqueous pores in their structure, catalyze the passage of ions across the otherwise ion-impermeable lipid bilayer. Ion conduction across ion channels is highly regulated, and in the case of voltage-dependent K(+) channels, the molecular foundations of the voltage-dependent conformational changes leading to the their open (conducting) configuration have provided most of the driving force for research in ion channel biophysics since the pioneering work of Hodgkin and Huxley (Hodgkin, A. L., and Huxley, A. F. (1952) J. Physiol. 117, 500-544). The voltage-dependent K(+) channels are the prototypical voltage-gated channels and govern the resting membrane potential. They are responsible for returning the membrane potential to its resting state at the termination of each action potential in excitable membranes. The pore-forming subunits (alpha) of many voltage-dependent K(+) channels and modulatory beta-subunits exist in the membrane as one component of macromolecular complexes, able to integrate a myriad of cellular signals that regulate ion channel behavior. In this review, we have focused on the modulatory effects of beta-subunits on the voltage-dependent K(+) (Kv) channel and on the large conductance Ca(2+)- and voltage-dependent (BK(Ca)) channel.     

In silico analysis of conformational changes induced by mutation of aromatic binding residues: consequences for drug binding in the hERG K+ channel

Pharmacological inhibition of cardiac hERG K(+) channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652) substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences.     

Hierarchical approach to predicting permeation in ion channels

A hierarchical computational strategy combining molecular modeling, electrostatics calculations, molecular dynamics, and Brownian dynamics simulations is developed and implemented to compute electrophysiologically measurable properties of the KcsA potassium channel. Models for a series of channels with different pore sizes are developed from the known x-ray structure, using insights into the gating conformational changes as suggested by a variety of published experiments. Information on the pH dependence of the channel gating is incorporated into the calculation of potential profiles for K(+) ions inside the channel, which are then combined with K(+) ion mobilities inside the channel, as computed by molecular dynamics simulations, to provide inputs into Brownian dynamics simulations for computing ion fluxes. The open model structure has a conductance of approximately 110 pS under symmetric 250 mM K(+) conditions, in reasonable agreement with experiments for the largest conducting substate. The dimensions of this channel are consistent with electrophysiologically determined size dependence of quaternary ammonium ion blocking from the intracellular end of this channel as well as with direct structural evidence that tetrabutylammonium ions can enter into the interior cavity of the channel. Realistic values of Ussing flux ratio exponents, distribution of ions within the channel, and shapes of the current-voltage and current-concentration curves are obtained. The Brownian dynamics calculations suggest passage of ions through the selectivity filter proceeds by a "knock-off" mechanism involving three ions, as has been previously inferred from functional and structural studies of barium ion blocking. These results suggest that the present calculations capture the essential nature of K(+) ion permeation in the KcsA channel and provide a proof-of-concept for the integrated microscopic/mesoscopic multitiered approach for predicting ion channel function from structure, which can be applied to other channel structures.     

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.     

Computational studies of gramicidin permeation: An entryway sulfonate enhances cation occupancy at entry sites

The impact on the cation-transport free-energy profile of replacing the C-terminal ethanolamine in the gramicidin A channel with a taurine residue is studied using molecular dynamics simulations of gramicidin A (1JNO) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl saline solution. The potential of mean force for ion transport is obtained by umbrella sampling. The presence of a negatively charged sulfonate group at the entrance of the gramicidin channel affects the depth and the location of the binding sites, producing a strong attraction for the cations in the bulk. The potential of mean force by the sulfonate acting directly through electrostatics and van der Waals interactions on the test ion is highly modulated by indirect effects (i.e., sulfonate effects on other components of the system that, in turn, affect the ion free-energy profile in the channel). Because the “entry” sites are located symmetrically at both entry and exit of the channel, the deeper free-energy wells should inhibit exit. Given that the channel has increased conductance experimentally, the simulation results suggest that the channel conductance is normally entry limited.     

Insight into the mechanism of inactivation and pH sensitivity in potassium channels from molecular dynamics simulations

Potassium (K (+)) channels can regulate ionic conduction through their pore by a mechanism, involving the selectivity filter, known as C-type inactivation. This process is rapid in the hERG K (+) channel and is fundamental to its physiological role. Although mutations within hERG are known to remove this process, a structural basis for the inactivation mechanism has yet to be characterized. Using MD simulations based on homology modeling, we observe that the carbonyl of the filter aromatic, Phe627, forming the S 0 K (+) binding site, swiftly rotates away from the conduction axis in the wild-type channel. In contrast, in well-characterized non-inactivating mutant channels, this conformational change occurs less frequently. In the non-inactivating channels, interactions with a water molecule located behind the selectivity filter are critical to the enhanced stability of the conducting state. We observe comparable conformational changes in the acid sensitive TASK-1 channel and propose a common mechanism in these channels for regulating efflux of K (+) ions through the selectivity filter.     

Ion selectivity in potassium channels

Potassium channels are tetrameric membrane-spanning proteins that provide a selective pore for the conduction of K(+) across the cell membranes. One of the main physiological functions of potassium channels is efficient and very selective transport of K(+) ions through the membrane to the cell. Classical views of ion selectivity are summarized within a historical perspective, and contrasted with the molecular dynamics (MD) simulations free energy perturbation (FEP) performed on the basis of the crystallographic structure of the KcsA phospholipid membrane. The results show that the KcsA channel does not select for K(+) ions by providing a binding site of an appropriate (fixed) cavity size. Rather, selectivity for K(+) arises directly from the intrinsic local physical properties of the ligands coordinating the cation in the binding site, and is a robust feature of a pore symmetrically lined by backbone carbonyl groups. Further analysis reveals that it is the interplay between the attractive ion-ligand (favoring smaller cation) and repulsive ligand-ligand interactions (favoring larger cations) that is the basic element governing Na(+)/K(+) selectivity in flexible protein binding sites. Because the number and the type of ligands coordinating an ion directly modulate such local interactions, this provides a potent molecular mechanism to achieve and maintain a high selectivity in protein binding sites despite a significant conformational flexibility.     

Ion channels of glutamate receptors: structural modeling

Ionotropic glutamate receptors belong to the superfamily of P-loop channels as well as K(+), Na(+), and Ca(2+) channels. However, the structural similarity between ion channels of the glutamate receptors and K(+) channels is a matter of discussion. The aim of this study was to analyze differences between the structures of K(+) channels and glutamate receptor channels. For this purpose, homology models of NMDA and AMPA receptor channels (M2 and M3 segments) were built using X-ray structures of K(+) channels as templates. The models were optimized and used to reproduce specific data on the structure of glutamate receptor channels. Particular attention was paid to the data of the binding of channel blockers and to the results of scanning mutagenesis. The modeling demonstrates that properties of glutamate receptor channel can be reproduced assuming only local structural deformations of the K(+) channel templates. The most valuable differences were found in the selectivity-filter region, whereas helical parts of M2 and M3 segments could have similar spatial organization with homologous segments in K(+) channels. It is concluded that the current experimental data on glutamate receptor channels does not reveal global structural differences with K(+) channels.     

Electric field effects on membranes: gramicidin A as a test ground

Electric fields due to transmembrane potential differences or ionic gradients across the membrane are presumably crucial for many reactions across membranes or close to membranes like signal transduction, control of ion channels or the generation of neural impulses. Molecular dynamics simulations have been used to study the influence of external electric fields on a mixed gramicidin/phospholipid bilayer system. At high field strengths, formation of membrane electropores occurred both close and distal to the gramicidin. Gramicidin was found to stabilize the membrane adjacent to the protein but also at larger distances of up to 2-3 nm. As a result, membrane pore formation was found to be significantly suppressed for the mixed gramicidin/DMPC system. Moderate field strengths only weakly affected the structure and dynamics of the gramicidin. Spontaneous potassium passage events in external electric fields were observed for both the head-to-head helical conformation as well as for the double helical conformation of gramicidin A. The double-helical conformation was found to facilitate ion passage compared to the head-to-head helical dimer.