Werner Treptow

Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations

The response of a membrane-bound Kv1.2 ion channel to an applied transmembrane potential has been studied using molecular dynamics simulations. Channel deactivation is shown to involve three intermediate states of the voltage sensor domain (VSD), and concomitant movement of helix S4 charges 10–15 Å along the bilayer normal; the latter being enabled by zipper-like sequential pairing of S4 basic residues with neighboring VSD acidic residues and membrane-lipid head groups. During the observed sequential transitions S4 basic residues pass through the recently discovered charge transfer center with its conserved phenylalanine residue, F233. Analysis indicates that the local electric field within the VSD is focused near the F233 residue and that it remains essentially unaltered during the entire process. Overall, the present computations provide an atomistic description of VSD response to hyperpolarization, add support to the sliding helix model, and capture essential features inferred from a variety of recent experiments.

Modeling Membranes under a Transmembrane Potential

Accurate modeling of ion transport through synthetic and biological transmembrane channels has been so far a challenging problem. We introduce here a new method that allows one to study such transport under realistic biological conditions. We present results from molecular dynamics simulations of an ion channel formed by a peptide nanotube, embedded in a lipid bilayer, and subject to transmembrane potentials generated by asymmetric distributions of ions on both sides of the membrane. We show that the method is efficient for generating ionic currents and allows us to estimate the intrinsic conductance of the channel.

Gating motions in voltage-gated potassium channels revealed by coarse-grained molecular dynamics simulations

Voltage-gated potassium (Kv) channels are ubiquitous transmembrane proteins involved in electric signaling of excitable tissues. A fundamental property of these channels is the ability to open or close in response to changes in the membrane potential. To date, their structure-based activation mechanism remains unclear, and there is a large controversy on how these gates function at the molecular level, in particular, how movements of the voltage sensor domain are coupled to channel gating. So far, all mechanisms proposed for this coupling are based on the crystal structure of the open voltage-gated Kv1.2 channel and structural models of the closed form based on electrophysiology experiments. Here, we use coarse-grain (CG) molecular dynamics simulations that allow conformational changes from the open to the closed form of the channel (embedded in its membrane environment) to be followed. Despite the low specificity of the CG force field, the obtained closed structure satisfies several experimental constraints. The overall results suggest a gating mechanism in which a lateral displacement the S4-S5 linker leads to a closing of the gate. Only a small up-down movement of the S4 helices is noticed. Additionally, the study suggests a peculiar upward motion of the intracellular tetramerization domain of the channel, hence providing a molecular view on how this domain may further regulate conduction in Kv channels.

Conduction in a biological sodium selective channel.

The crystal structure of NavAb, a bacterial voltage gated Na(+) channel, exhibits a selectivity filter (SF) wider than that of K(+) channels. This new structure provides the opportunity to explore the mechanism of conduction and help rationalize its selectivity for sodium. Recent molecular dynamics (MD) simulations of single- and two-ion permeation processes have revealed that a partially hydrated Na(+) permeates the channel by exploring three SF binding sites while being loosely coupled to other ions and/or water molecules; a finding that differs significantly from the behavior of K(+) selective channels. Herein, we present results derived from a combination of metadynamics and voltage-biased MD simulations that throws more light on the nature of the Na(+) conduction mechanism. Conduction under 0 mV bias explores several distinct pathways involving the binding of two ions to three possible SF sites. While these pathways are very similar to those observed in the presence of a negative potential (inward conduction), a completely different mechanism operates for outward conduction at positive potentials.

Affinity of C60 Neat Fullerenes with Membrane Proteins: A Computational Study on Potassium Channels

Most studies of the interactions of neat and functionalized fullerenes with cells have focused so far on their ability to cross the cell membrane envelopes. Membranes are, however, also host to a large number of proteins responsible for various cellular functions. Among these, ion channels are prominent components of the nervous system. Recently, it was shown that fullerenes may act as blockers or modulators of a variety of K+ channels. Here we use computer simulations to investigate the propensity of such nanocompounds to bind to K+ channels. Our results based on extensive atomistic molecular dynamics simulations reveal a variety of specific binding sites depending on the structure and properties of the channel. The corresponding binding free energies and putative mechanisms suggest that C60 may indeed effectively hinder the function of K+ channels and hence induce toxicity.

Initial Response of the Potassium Channel Voltage Sensor to a Transmembrane Potential

Early transition events of the voltage sensor (VS) of Kv1.2 potassium channel embedded in a lipid membrane are triggered using full atomistic molecular dynamics (MD) simulations. When subject to an applied hyperpolarized transmembrane (TM) voltage, the VS undergoes conformational changes and reaches a stable kinetic intermediate state, beta, within 20 ns. The gating charge ( approximately 2e) associated with this fast transition results mainly from salt-bridge rearrangements involving negative charges in S2 and S3 and all but the two top residues R(294) and R(297) of S4. Interactions of the latter with phosphomoieties of the lipid head groups appear to stabilize the kinetic state beta.

Effect of Sensor Domain Mutations on the Properties of Voltage-Gated Ion Channels: Molecular Dynamics Studies of the Potassium Channel Kv1.2

The effects on the structural and functional properties of the Kv1.2 voltage-gated ion channel, caused by selective mutation of voltage sensor domain residues, have been investigated using classical molecular dynamics simulations. Following experiments that have identified mutations of voltage-gated ion channels involved in state-dependent omega currents, we observe for both the open and closed conformations of the Kv1.2 that specific mutations of S4 gating-charge residues destabilize the electrostatic network between helices of the voltage sensor domain, resulting in the formation of hydrophilic pathways linking the intra- and extracellular media. When such mutant channels are subject to transmembrane potentials, they conduct cations via these so-called omega pores. This study provides therefore further insight into the molecular mechanisms that lead to omega currents, which have been linked to certain channelopathies.

Pore waters regulate ion permeation in a calcium release-activated calcium channel

Significance Calcium channel dysfunction is implicated in cardiac arrhythmias and immunodeficiency disorders. The recent publication of the crystal structure of a calcium ion channel denoted CRAC has allowed us to use atomic-level computer simulation to begin to understand the mechanism of ion permeation through this ubiquitous and vital cell entry pathway. Our computations identify pore water molecules as a crucial contributor in governing the channel conductance characteristics not only of the wild-type protein but also a disease-related mutant. The present study should be helpful for research designed to improve treatment of immune diseases caused by disrupted calcium signaling. The recent crystal structure of Orai, the pore unit of a calcium release-activated calcium (CRAC) channel, is used as the starting point for molecular dynamics and free-energy calculations designed to probe this channel’s conduction properties. In free molecular dynamics simulations, cations localize preferentially at the extracellular channel entrance near the ring of Glu residues identified in the crystal structure, whereas anions localize in the basic intracellular half of the pore. To begin to understand ion permeation, the potential of mean force (PMF) was calculated for displacing a single Na+ ion along the pore of the CRAC channel. The computed PMF indicates that the central hydrophobic region provides the major hindrance for ion diffusion along the permeation pathway, thereby illustrating the nonconducting nature of the crystal structure conformation. Strikingly, further PMF calculations demonstrate that the mutation V174A decreases the free energy barrier for conduction, rendering the channel effectively open. This seemingly dramatic effect of mutating a nonpolar residue for a smaller nonpolar residue in the pore hydrophobic region suggests an important role for the latter in conduction. Indeed, our computations show that even without significant channel-gating motions, a subtle change in the number of pore waters is sufficient to reshape the local electrostatic field and modulate the energetics of conduction, a result that rationalizes recent experimental findings. The present work suggests the activation mechanism for the wild-type CRAC channel is likely regulated by the number of pore waters and hence pore hydration governs the conductance.

Molecular Mapping of General Anesthetic Sites in a Voltage-Gated Ion Channel

Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channels activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and inxa0silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces.

The membrane-bound state of K2P potassium channels.

Potassium channel subunits composed of two-pore domains arranged in tandem (K(2P)) are of paramount importance for neural function. A variety of stimuli, such as membrane depolarization and tension, acidification, and anesthetic action, activate K(2P) channels. Most of the channel sensitivity is attributed to its intracellular C-terminal moiety, which works as a sensor domain required for proper integration of the electrical, chemical, and mechanical signals into channel activity. Herein, the structure of K(2P) in a membrane environment has been studied using molecular dynamics (MD). Two distinct fully atomistic models for the most studied K(2P) channel, namely, the TWIK-related (TREK)-1 channel have been built. These constructs were then inserted into a fully hydrated zwitterionic lipid bilayer, and each relaxed by means of MD simulations spanning approximately 0.3 micros. Both simulated TREK-1 structures converged to a final conformation characterized by a closed pore and a C-terminal domain adsorbed onto the lipid bilayer surface. The C-terminus, which is physically linked to the pore and energetically coupled to the bilayer, is poised to gate the channel in response to membrane stimulation. The present study indicates the nature of the direct coupling between the C-terminal domain and the membrane, which is a key structural feature underlying K(2P) channel function.