Calcium-gated potassium channel blockade via membrane-facing fenestrations.

Quaternary ammonium blockers were previously shown to bind in the pore to block both open and closed conformations of large-conductance calcium-activated potassium (BK and MthK) channels. Because blocker entry was assumed through the intracellular entryway (bundle crossing), closed-pore access suggested that the gate was not at the bundle crossing. Structures of closed MthK, a Methanobacterium thermoautotrophicum homolog of BK channels, revealed a tightly constricted intracellular gate, leading us to investigate the membrane-facing fenestrations as alternative pathways for blocker access directly from the membrane. Atomistic free energy simulations showed that intracellular blockers indeed access the pore through the fenestrations, and a mutant channel with narrower fenestrations displayed no closed-state TPeA block at concentrations that blocked the wild-type channel. Apo BK channels display similar fenestrations, suggesting that blockers may use them as access paths into closed channels. Thus, membrane fenestrations represent a non-canonical pathway for selective targeting of specific channel conformations, opening novel ways to selectively drug BK channels.

Ball-and-chain inactivation in a calcium-gated potassium channel.

Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a 'ball-and-chain' mechanism3-7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum-a purely calcium-gated and inactivating channel-in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism.

Selectivity filter ion binding affinity determines inactivation in a potassium channel.

Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca2+-activated K+ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K+ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF's four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K+ affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K+, similar to KcsA, but that even a single K+ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K+ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK's S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.

Atomistic Simulations of Membrane Ion Channel Conduction, Gating, and Modulation.

Membrane ion channels are the fundamental electrical components in the nervous system. Recent developments in X-ray crystallography and cryo-EM microscopy have revealed what these proteins look like in atomic detail but do not tell us how they function. Molecular dynamics simulations have progressed to the point that we can now simulate realistic molecular assemblies to produce quantitative calculations of the thermodynamic and kinetic quantities that control function. In this review, we summarize the state of atomistic simulation methods for ion channels to understand their conduction, activation, and drug modulation mechanisms. We are at a crossroads in atomistic simulation, where long time scale observation can provide unbiased exploration of mechanisms, supplemented by biased free energy methodologies. We illustrate the use of these approaches to describe ion conduction and selectivity in voltage-gated sodium and acid-sensing ion channels. Studies of channel gating present a significant challenge, as activation occurs on longer time scales. Enhanced sampling approaches can ensure convergence on minimum free energy pathways for activation, as illustrated here for pentameric ligand-gated ion channels that are principal to nervous system function and the actions of general anesthetics. We also examine recent studies of local anesthetic and antiepileptic drug binding to a sodium channel, revealing sites and pathways that may offer new targets for drug development. Modern simulations thus offer a range of molecular-level insights into ion channel function and modulation as a learning platform for mechanistic discovery and drug development.

Cholesterol depletion inhibits Na+,K+-ATPase activity in a near-native membrane environment.

Cholesterol's effects on Na+,K+-ATPase reconstituted in phospholipid vesicles have been extensively studied. However, previous studies have reported both cholesterol-mediated stimulation and inhibition of Na+,K+-ATPase activity. Here, using partial reaction kinetics determined via stopped-flow experiments, we studied cholesterol's effect on Na+,K+-ATPase in a near-native environment in which purified membrane fragments were depleted of cholesterol with methyl-ß-cyclodextrin (mßCD). The mßCD-treated Na+,K+-ATPase had significantly reduced overall activity and exhibited decreased observed rate constants for ATP phosphorylation (ENa3+ → E2P, i.e. phosphorylation by ATP and Na+ occlusion from the cytoplasm) and K+ deocclusion with subsequent intracellular Na+ binding (E2K2+ → E1Na3+). However, cholesterol depletion did not affect the observed rate constant for K+ occlusion by phosphorylated Na+,K+-ATPase on the extracellular face and subsequent dephosphorylation (E2P → E2K2+). Thus, partial reactions involving cation binding and release at the protein's intracellular side were most dependent on cholesterol. Fluorescence measurements with the probe eosin indicated that cholesterol depletion stabilizes the unphosphorylated E2 state relative to E1, and the cholesterol depletion-induced slowing of ATP phosphorylation kinetics was consistent with partial conversion of Na+,K+-ATPase into the E2 state, requiring a slow E2 → E1 transition before the phosphorylation. Molecular dynamics simulations of Na+,K+-ATPase in membranes with 40 mol % cholesterol revealed cholesterol interaction sites that differ markedly among protein conformations. They further indicated state-dependent effects on membrane shape, with the E2 state being likely disfavored in cholesterol-rich bilayers relative to the E1P state because of a greater hydrophobic mismatch. In summary, cholesterol extraction from membranes significantly decreases Na+,K+-ATPase steady-state activity.

Simulating ion channel activation mechanisms using swarms of trajectories.

Atomic-level studies of protein activity represent a significant challenge as a result of the complexity of conformational changes occurring on wide-ranging timescales, often greatly exceeding that of even the longest simulations. A prime example is the elucidation of protein allosteric mechanisms, where localized perturbations transmit throughout a large macromolecule to generate a response signal. For example, the conversion of chemical to electrical signals during synaptic neurotransmission in the brain is achieved by specialized membrane proteins called pentameric ligand-gated ion channels. Here, the binding of a neurotransmitter results in a global conformational change to open an ion-conducting pore across the nerve cell membrane. X-ray crystallography has produced static structures of the open and closed states of the proton-gated GLIC pentameric ligand-gated ion channel protein, allowing for atomistic simulations that can uncover changes related to activation. We discuss a range of enhanced sampling approaches that could be used to explore activation mechanisms. In particular, we describe recent application of an atomistic string method, based on Roux's "swarms of trajectories" approach, to elucidate the sequence and interdependence of conformational changes during activation. We illustrate how this can be combined with transition analysis and Brownian dynamics to extract thermodynamic and kinetic information, leading to understanding of what controls ion channel function. © 2019 Wiley Periodicals, Inc.

String method solution of the gating pathways for a pentameric ligand-gated ion channel.

Pentameric ligand-gated ion channels control synaptic neurotransmission by converting chemical signals into electrical signals. Agonist binding leads to rapid signal transduction via an allosteric mechanism, where global protein conformational changes open a pore across the nerve cell membrane. We use all-atom molecular dynamics with a swarm-based string method to solve for the minimum free-energy gating pathways of the proton-activated bacterial GLIC channel. We describe stable wetted/open and dewetted/closed states, and uncover conformational changes in the agonist-binding extracellular domain, ion-conducting transmembrane domain, and gating interface that control communication between these domains. Transition analysis is used to compute free-energy surfaces that suggest allosteric pathways; stabilization with pH; and intermediates, including states that facilitate channel closing in the presence of an agonist. We describe a switching mechanism that senses proton binding by marked reorganization of subunit interface, altering the packing of ß-sheets to induce changes that lead to asynchronous pore-lining M2 helix movements. These results provide molecular details of GLIC gating and insight into the allosteric mechanisms for the superfamily of pentameric ligand-gated channels.

Selective ion permeation involves complexation with carboxylates and lysine in a model human sodium channel.

Bacterial and human voltage-gated sodium channels (Navs) exhibit similar cation selectivity, despite their distinct EEEE and DEKA selectivity filter signature sequences. Recent high-resolution structures for bacterial Navs have allowed us to learn about ion conduction mechanisms in these simpler homo-tetrameric channels, but our understanding of the function of their mammalian counterparts remains limited. To probe these conduction mechanisms, a model of the human Nav1.2 channel has been constructed by grafting residues of its selectivity filter and external vestibular region onto the bacterial NavRh channel with atomic-resolution structure. Multi-µs fully atomistic simulations capture long time-scale ion and protein movements associated with the permeation of Na+ and K+ ions, and their differences. We observe a Na+ ion knock-on conduction mechanism facilitated by low energy multi-carboxylate/multi-Na+ complexes, akin to the bacterial channels. These complexes involve both the DEKA and vestibular EEDD rings, acting to draw multiple Na+ into the selectivity filter and promote permeation. When the DEKA ring lysine is protonated, we observe that its ammonium group is actively participating in Na+ permeation, presuming the role of another ion. It participates in the formation of a stable complex involving carboxylates that collectively bind both Na+ and the Lys ammonium group in a high-field strength site, permitting pass-by translocation of Na+. In contrast, multiple K+ ion complexes with the DEKA and EEDD rings are disfavored by up to 8.3 kcal/mol, with the K+-lysine-carboxylate complex non-existent. As a result, lysine acts as an electrostatic plug that partially blocks the flow of K+ ions, which must instead wait for isomerization of lysine downward to clear the path for K+ passage. These distinct mechanisms give us insight into the nature of ion conduction and selectivity in human Nav channels, while uncovering high field strength carboxylate binding complexes that define the more general phenomenon of Na+-selective ion transport in nature.

The voltage-sensitive dye RH421 detects a Na+,K+-ATPase conformational change at the membrane surface.

RH421 is a voltage-sensitive fluorescent styrylpyridinium dye which has often been used to probe the kinetics of Na+,K+-ATPase partial reactions. The origin of the dye's response has up to now been unclear. Here we show that RH421 responds to phosphorylation of the Na+,K+-ATPase by inorganic phosphate with a fluorescence increase. Analysis of the kinetics of the fluorescence response indicates that the probe is not detecting phosphorylation itself but rather a shift in the protein's E1/E2 conformational equilibrium induced by preferential phosphate binding to and phosphorylation of enzyme in the E2 conformation. Molecular dynamics simulations of crystal structures in lipid bilayers indicate some change in the protein's hydrophobic thickness during the E1-E2 transition, which may influence the dye response. However, the transition is known to involve significant rearrangement of the protein's highly charged lysine-rich cytoplasmic N-terminal sequence. Using poly-l-lysine as a model of the N-terminus, we show that an analogous response of RH421 to the E1→E2P conformational change is produced by poly-l-lysine binding to the surface of the Na+,K+-ATPase-containing membrane fragments. Thus, it seems that the prime origin of the RH421 fluorescence response is a change in the interaction of the protein's N-terminus with the surrounding membrane. Quantum mechanical calculations of the dye's visible absorption spectrum give further support to this conclusion. The results obtained indicate that membrane binding and release of the N-terminus of the Na+,K+-ATPase α-subunit are intimately involved in the protein's catalytic cycle and could represent an effective site of regulation.

Ion conduction and conformational flexibility of a bacterial voltage-gated sodium channel.

Voltage-gated Na(+) channels play an essential role in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. The recent solution of X-ray structures for the bacterial channel NavAb has provided an opportunity to study functional mechanisms at the atomic level. This channel's selectivity filter exhibits an EEEE ring sequence, characteristic of mammalian Ca(2+), not Na(+), channels. This raises the fundamentally important question: just what makes a Na(+) channel conduct Na(+) ions? Here we explore ion permeation on multimicrosecond timescales using the purpose-built Anton supercomputer. We isolate the likely protonation states of the EEEE ring and observe a striking flexibility of the filter that demonstrates the necessity for extended simulations to study conduction in this channel. We construct free energy maps to reveal complex multi-ion conduction via knock-on and "pass-by" mechanisms, involving concerted ion and glutamate side chain movements. Simulations in mixed ionic solutions reveal relative energetics for Na(+), K(+), and Ca(2+) within the pore that are consistent with the modest selectivity seen experimentally. We have observed conformational changes in the pore domain leading to asymmetrical collapses of the activation gate, similar to proposed inactivated structures of NavAb, with helix bending involving conserved residues that are critical for slow inactivation. These structural changes are shown to regulate access to fenestrations suggested to be pathways for lipophilic drugs and provide deeper insight into the molecular mechanisms connecting drug activity and slow inactivation.

Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel.

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the "hinged lid"-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water-protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors.

Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory.

Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.

Ion-induced defect permeation of lipid membranes.

We have explored the mechanisms of uncatalyzed membrane ion permeation using atomistic simulations and electrophysiological recordings. The solubility-diffusion mechanism of membrane charge transport has prevailed since the 1960s, despite inconsistencies in experimental observations and its lack of consideration for the flexible response of lipid bilayers. We show that direct lipid bilayer translocation of alkali metal cations, Cl(-), and a charged arginine side chain analog occurs via an ion-induced defect mechanism. Contrary to some previous suggestions, the arginine analog experiences a large free-energy barrier, very similar to those for Na(+), K(+), and Cl(-). Our simulations reveal that membrane perturbations, due to the movement of an ion, are central for explaining the permeation process, leading to both free-energy and diffusion-coefficient profiles that show little dependence on ion chemistry and charge, despite wide-ranging hydration energies and the membrane's dipole potential. The results yield membrane permeabilities that are in semiquantitative agreement with experiments in terms of both magnitude and selectivity. We conclude that ion-induced defect-mediated permeation may compete with transient pores as the dominant mechanism of uncatalyzed ion permeation, providing new understanding for the actions of a range of membrane-active peptides and proteins.

Identification of electric-field-dependent steps in the Na(+),K(+)-pump cycle.

The charge-transporting activity of the Na(+),K(+)-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme's reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na(+),K(+)-ATPase's transport sites in competition with Na(+) and K(+), but is not occluded within the protein. We find that only the occludable ions Na(+), K(+), Rb(+), and Cs(+) cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na(+) or K(+) binding in a high field access channel is a major electrogenic reaction of the Na(+),K(+)-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.

The determinants of hydrophobic mismatch response for transmembrane helices.

Hydrophobic mismatch arises from a difference in the hydrophobic thickness of a lipid membrane and a transmembrane protein segment, and is thought to play an important role in the folding, stability and function of membrane proteins. We have investigated the possible adaptations that lipid bilayers and transmembrane α-helices undergo in response to mismatch, using fully-atomistic molecular dynamics simulations totaling 1.4 µs. We have created 25 different tryptophan-alanine-leucine transmembrane α-helical peptide systems, each composed of a hydrophobic alanine-leucine stretch, flanked by 1-4 tryptophan side chains, as well as the ß-helical peptide dimer, gramicidin A. Membrane responses to mismatch include changes in local bilayer thickness and lipid order, varying systematically with peptide length. Adding more flanking tryptophan side chains led to an increase in bilayer thinning for negatively mismatched peptides, though it was also associated with a spreading of the bilayer interface. Peptide tilting, bending and stretching were systematic, with tilting dominating the responses, with values of up to ~45° for the most positively mismatched peptides. Peptide responses were modulated by the number of tryptophan side chains due to their anchoring roles and distributions around the helices. Potential of mean force calculations for local membrane thickness changes, helix tilting, bending and stretching revealed that membrane deformation is the least energetically costly of all mismatch responses, except for positively mismatched peptides where helix tilting also contributes substantially. This comparison of energetic driving forces of mismatch responses allows for deeper insight into protein stability and conformational changes in lipid membranes.

The role of tryptophan side chains in membrane protein anchoring and hydrophobic mismatch.

Tryptophan (Trp) is abundant in membrane proteins, preferentially residing near the lipid-water interface where it is thought to play a significant anchoring role. Using a total of 3 µs of molecular dynamics simulations for a library of hydrophobic WALP-like peptides, a long poly-Leu α-helix, and the methyl-indole analog, we explore the thermodynamics of the Trp movement in membranes that governs the stability and orientation of transmembrane protein segments. We examine the dominant hydrogen-bonding interactions between the Trp and lipid carbonyl and phosphate moieties, cation-π interactions to lipid choline moieties, and elucidate the contributions to the thermodynamics that serve to localize the Trp, by ~4 kcal/mol, near the membrane glycerol backbone region. We show a striking similarity between the free energy to move an isolated Trp side chain to that found from a wide range of WALP peptides, suggesting that the location of this side chain is nearly independent of the host transmembrane segment. Our calculations provide quantitative measures that explain Trp's role as a modulator of responses to hydrophobic mismatch, providing a deeper understanding of how lipid composition may control a range of membrane active peptides and proteins.

On the selective ion binding hypothesis for potassium channels.

The mechanism by which K(+) channels select for K(+) over Na(+) ions has been debated for the better part of a century. The prevailing view is that K(+) channels contain highly conserved sites that selectively bind K(+) over Na(+) ions through optimal coordination. We demonstrate that a series of alternating sites within the KcsA channel selectivity filter exists, which are thermodynamically selective for either K(+) (cage made from two planes of oxygen atoms) or Na(+) ions (a single plane of four oxygen atoms). By combining Bennett free energy perturbation calculations with umbrella sampling, we show that when K(+) and Na(+) are both permitted to move into their preferred positions, the thermodynamic preference for K(+) over Na(+) is significantly reduced throughout the entire selectivity filter. We offer a rationale for experimental measures of thermodynamic preference for K(+) over Na(+) from Ba(2+) blocking data, by demonstrating that the presence of Ba(2+) ions exaggerates K(+) over Na(+) thermodynamic stability due to the different binding locations of these ions. These studies reveal that K(+) channel selectivity may not be associated with the thermodynamics of ions in crystallographic K(+) binding sites, but requires consideration of the kinetic barriers associated with the different multi-ion permeation mechanisms.

Computational Models for Predictive Cardiac Ion Channel Pharmacology.

A wealth of experimental data exists describing the elementary building blocks of complex physiological systems. However, it is increasingly apparent in the biomedical sciences that mechanisms of biological function cannot be observed or readily predicted via study of constituent elements alone. This is especially clear in the longstanding failures in prediction of effects of drug treatment for heart rhythm disturbances. These failures stem in part from classical assumptions that have been made in cardiac antiarrhythmic drug development - that a drug operates by one mechanism via one target receptor that arises from one gene.

The Membrane Dipole Potential and the Roles of Interfacial Water and Lipid Hydrocarbon Chains.

Understanding membrane charge transport processes, including the actions of ion channels, pumps, carriers, and membrane-active peptides, requires a description of the electrostatics of the lipid bilayer. We have simulated a library of different lipid chemistries to reveal the impact of the headgroup, glycerol backbone, and hydrocarbon chains on the membrane dipole potential. We found a strong dependence of the potential on lipid packing, but this was not caused by the packing of lipid polar components, due to cancellation of their electric fields by electrolyte. In contrast, lipid tail contributions were determined by area per lipid, arising from two countering effects. Increased area per lipid leads to chain tilting that increases methylene dipole projections to strengthen the electric field within the bilayer, while at the same time decreasing the electric field from terminal methyl groups. Moreover, electric fields from some nonterminal groups and the terminal methyl group can extend beyond the bilayer center and be canceled by the opposing leaflet. This interleaflet field annulment explains the experimental reduction in dipole potential for unsaturated and branched lipid bilayers, by as much as ∼200 mV, as well as experiments that substitute chain carbons with sulfur. Replacing ester with ether groups (eliminating two carbonyl groups) causes a significant reduction in potential, also by ∼200 mV, in agreement with experiment. We show that the effect can be largely attributed to the loss of aligned water molecules in the glycerol backbone region, lowering the potential inside the bilayer core. When only one of the two carbonyls is removed (using a hybrid ester-ether lipid or a single-chain lipid), most of this reduction in potential was lost, with the single carbonyl group able to maintain full hydration in the interfacial region. While headgroup chemistry can have a major effect (by as much as ±100 mV relative to phosphatidylcholine), anionic headgroups either decrease or increase the dipole potential, with the variation involving perturbation in hydrogen-bonded water molecules and changes in packing of lipid tails. Overall, these results suggest that membrane electrostatics are dominated by aligned water molecules at the polar-hydrocarbon interface and, surprisingly, by the charge distribution of the nonpolar lipid tails, and not the packing of headgroup and glycerol carbonyl dipoles.

Potassium dependent structural changes in the selectivity filter of HERG potassium channels.

The fine tuning of biological electrical signaling is mediated by variations in the rates of opening and closing of gates that control ion flux through different ion channels. Human ether-a-go-go related gene (HERG) potassium channels have uniquely rapid inactivation kinetics which are critical to the role they play in regulating cardiac electrical activity. Here, we exploit the K+ sensitivity of HERG inactivation to determine structures of both a conductive and non-conductive selectivity filter structure of HERG. The conductive state has a canonical cylindrical shaped selectivity filter. The non-conductive state is characterized by flipping of the selectivity filter valine backbone carbonyls to point away from the central axis. The side chain of S620 on the pore helix plays a central role in this process, by coordinating distinct sets of interactions in the conductive, non-conductive, and transition states. Our model represents a distinct mechanism by which ion channels fine tune their activity and could explain the uniquely rapid inactivation kinetics of HERG.