Insights into the function of ion channels by computational electrophysiology simulations

Ion channels are of universal importance for all cell types and play key roles in cellular physiology and pathology. Increased insight into their functional mechanisms is crucial to enable drug design on this important class of membrane proteins, and to enhance our understanding of some of the fundamental features of cells. This review presents the concepts behind the recently developed simulation protocol Computational Electrophysiology (CompEL), which facilitates the atomistic simulation of ion channels in action. In addition, the review provides guidelines for its application in conjunction with the molecular dynamics software package GROMACS. We first lay out the rationale for designing CompEL as a method that models the driving force for ion permeation through channels the way it is established in cells, i.e., by electrochemical ion gradients across the membrane. This is followed by an outline of its implementation and a description of key settings and parameters helpful to users wishing to set up and conduct such simulations. In recent years, key mechanistic and biophysical insights have been obtained by employing the CompEL protocol to address a wide range of questions on ion channels and permeation. We summarize these recent findings on membrane proteins, which span a spectrum from highly ion-selective, narrow channels to wide diffusion pores. Finally we discuss the future potential of CompEL in light of its limitations and strengths. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov

Direct knock-on of desolvated ions governs strict ion selectivity in K+ channels

The seeming contradiction that K+ channels conduct K+ ions at maximal throughput rates while not permeating slightly smaller Na+ ions has perplexed scientists for decades. Although numerous models have addressed selective permeation in K+ channels, the combination of conduction efficiency and ion selectivity has not yet been linked through a unified functional model. Here, we investigate the mechanism of ion selectivity through atomistic simulations totalling more than 400 μs in length, which include over 7,000 permeation events. Together with free-energy calculations, our simulations show that both rapid permeation of K+ and ion selectivity are ultimately based on a single principle: the direct knock-on of completely desolvated ions in the channels' selectivity filter. Herein, the strong interactions between multiple 'naked' ions in the four filter binding sites give rise to a natural exclusion of any competing ions. Our results are in excellent agreement with experimental selectivity data, measured ion interaction energies and recent two-dimensional infrared spectra of filter ion configurations

Mutations of the H-bond network behind the selectivity filter.

<p>(<b>I</b>) Mutation sites N629Q (pore loop), F617Y, and Y616F (both: pore helix, P). (<b>II</b>) Inactivation properties of wild-type and mutant hERG channels. Inactivation time courses for the different hERG channels were recorded as shown. A conditioning pulse to +20 mV followed by a 100 ms hyperpolarizing pulse to −100 mV preceded various depolarizing pulses from −90 to +40 mV in 10 mV increments as illustrated by the pulse protocol on top. (<b>III</b>) (A) Exemplary wild-type (WT) hERG current traces elicited by 6 s depolarizing voltage steps from −100 to +40 mV followed by a hyperpolarizing pulse to −140 mV. Respective tail currents are shown enlarged at left. (B) Conductance-voltage relations determined from Boltzmann fits to normalized tail current amplitudes for hERG wild-type and Y616F and N629Q mutant channels. (C) Conductance-voltage relation for the mutant hERG channel F617Y. (D) and <i>k</i> parameters obtained under steady state conditions from the Boltzmann fits for wild-type and mutant channels are summarized at the bottom. *p<0.05 versus wild-type. (<b>IV</b>) (A) Deactivation time courses of wild-type and mutant hERG channels. Tail currents were elicited according to the pulse protocol shown on top. (B) Voltage dependence of mean deactivation time constants () (n = 4) for the different channels as indicated.</p

Selectivity filter conformations of hERG simulations and KcsA crystal structures.

<p>For clarity, only two subunits are shown. Snapshots were taken at the end of the simulations with a 5 Å N629–S620 distance (<b>A</b>), and a 10 Å N629–S620 distance (<b>B</b>). A flip of the V625 carbonyl group is seen (black arrows). For comparison, (<b>C</b>) displays the crystal structures of the collapsed (pdb: 1K4D) and (<b>D</b>) the conductive KcsA SF (pdb: 1K4C). (<b>E</b>) Comparison with the non-flipped (pdb: 1ZWI) and (<b>F</b>) flipped SF conformation (pdb: 2ATK) observed in crystal structures of the non-inactivating KcsA mutant E71A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041023#pone.0041023-CorderoMorales1" target="_blank">[8]</a>.</p

Suggested mechanism of action for hERG activators.

<p>(<b>A</b>) Binding pocket for activators, shown here located between the pore helices of two adjacent subunits (orange surface). (<b>B</b>) The experimentally determined binding pocket for PD-118057 and ICA-105574 is located around residue F619 and extends to residue L622 (secondary subunit contacts are marked with a prime symbol). (<b>C</b>) A cascade of conformational changes triggered by collapse of the SF leads to constriction of the binding pocket (orange lines) and rearrangement of L622. (<b>D</b>) The cavity is large enough to accommodate PD-118057. All residues known to affect PD-118057 binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041023#pone.0041023-Perry1" target="_blank">[23]</a> line the pocket (yellow). (<b>E</b>) The activator molecule ICA-105574, shown docked to the side-pocket with residues known to influence binding in yellow.</p

Hydrogen bonds formed by N629 at different S620–N629 distances.

<p>For each S620–N629 C distance (right), the number of hydrogen bonds formed by the N629 side-chain to the same subunit (blue area, mainly S620) and to neighboring subunits (green area, mainly G628) is shown over simulation time. A steady rise in the proportion of inter-subunit hydrogen bonds can be seen with an increase in S-N distance.</p

Model structure of the hERG channel and switch behind the selectivity filter.

<p>(<b>A</b>) Model of the hERG channel, lined by the S6 helices (green), and including the K⁺ selectivity filter (SF, red), pore helices (P, blue), internal cavity and outer pore loop. As structural information on the turret loops is sparse and modeling according to homology is not possible in this region, the loops were modeled as in KvAP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041023#pone.0041023-Stary1" target="_blank">[28]</a>. (A, inset) Scan of H-bonds between N629 and S620. (<b>B</b>) Dependence of the backbone carbonyl fluctuation (RMSF) of SF residues S624–F627 on the distance between S620 and N629. (<b>C</b>) Small separations between S620 and N629 (5 Å, blue curve) promote SF collapse (1K4D, upper red bar), while larger separations (10 Å, green curve) stabilize its conductive state (1K4C, lower red bar). For an equally short separation (5 Å, magenta curve) the S620T mutant displays a marked deviation from the WT. Each time trace represents the mean of four independent simulations with their standard error (shaded area) (<b>D</b>) Direct dependence of the extent of SF collapse on the interaction between N629 and S620 (blue line). The formation of a stable inter-chain H-bond to G628 stabilizes the conductive SF (green circle). The non-inactivating mutant S620T does not reach a fully collapsed state even at a T620-N629 distance of 5 Å (magenta circle), while the double mutation G628C/S631C precludes a close contact between N629 and S620 and hence a transition to the collapsed state (red circle).</p

A molecular switch driving inactivation in the cardiac k(+) channel HERG

Contains fulltext : 103432.pdf (publisher's version ) (Open Access)K(+) channels control transmembrane action potentials by gating open or closed in response to external stimuli. Inactivation gating, involving a conformational change at the K(+) selectivity filter, has recently been recognized as a major K(+) channel regulatory mechanism. In the K(+) channel hERG, inactivation controls the length of the human cardiac action potential. Mutations impairing hERG inactivation cause life-threatening cardiac arrhythmia, which also occur as undesired side effects of drugs. In this paper, we report atomistic molecular dynamics simulations, complemented by mutational and electrophysiological studies, which suggest that the selectivity filter adopts a collapsed conformation in the inactivated state of hERG. The selectivity filter is gated by an intricate hydrogen bond network around residues S620 and N629. Mutations of this hydrogen bond network are shown to cause inactivation deficiency in electrophysiological measurements. In addition, drug-related conformational changes around the central cavity and pore helix provide a functional mechanism for newly discovered hERG activators