Shifts in the selectivity filter dynamics cause modal gating in k+ channels

ABSTRACT Spontaneous activity shifts at constant experimental conditions represent a widespread regulatory mechanism in ion channels. The molecular origins of these modal gating shifts are

poorly understood. In the K+ channel KcsA, a multitude of fast activity shifts that emulate the native modal gating behaviour can be triggered by point-mutations in the hydrogen bonding

network that controls the selectivity filter. Using solid-state NMR and molecular dynamics simulations in a variety of KcsA mutants, here we show that modal gating shifts in K+ channels are

associated with important changes in the channel dynamics that strongly perturb the selectivity filter equilibrium conformation. Furthermore, our study reveals a drastically different

motional and conformational selectivity filter landscape in a mutant that mimics voltage-gated K+ channels, which provides a foundation for an improved understanding of eukaryotic K+

channels. Altogether, our results provide a high-resolution perspective on some of the complex functional behaviour of K+ channels. SIMILAR CONTENT BEING VIEWED BY OTHERS CONFORMATIONAL

PLASTICITY OF NAK2K AND TREK2 POTASSIUM CHANNEL SELECTIVITY FILTERS Article Open access 06 January 2023 A DISTINCT MECHANISM OF C-TYPE INACTIVATION IN THE KV-LIKE KCSA MUTANT E71V Article

Open access 23 March 2022 ION-DEPENDENT STRUCTURE, DYNAMICS, AND ALLOSTERIC COUPLING IN A NON-SELECTIVE CATION CHANNEL Article Open access 28 October 2021 INTRODUCTION Potassium (K+)

channels are of fundamental importance for the functioning of excitable cells1. They allow selective and rapid flux of K+ across the cell membrane through a central pore, which is regulated

by the interplay between a cytoplasmic activation gate and an extracellular C-type inactivation gate known as selectivity filter. The selectivity filter sequence TVGYG is highly conserved,

and its backbone carbonyl-groups together with the threonine hydroxyl group line up to form the five K+ coordination sites (S0–S4)2,3. Extensive crystallographic studies in the well-accepted

model K+ channel KcsA showed that C-type inactivation is governed by a complex hydrogen bond network behind the selectivity filter4,5. Residue E71 is at the centre of this network, and

modulates the selectivity filter by coordinating to the backbone of Y78 and, mediated via a water molecule, the D80 as well as the W67 side chains (Fig. 1a). While W67 and D80 are highly

conserved in K+ channels, E71 is commonly replaced by a valine or isoleucine in eukaryotes (Fig. 1b), which is assumed to critically modulate selectivity filter gating. Indeed,

electrophysiological measurements showed that point-mutations at E71 lock the KcsA channel into different, natively occurring gating modes, which are best represented by a high-open

probability (E71A), a low-open probability (E71I), and a high-frequency flicker (E71Q) mode6. Random shifts between such gating modes, known as modal gating shifts, were observed in various

eukaryotic and prokaryotic K+ channels, and are a widespread regulatory mechanism of channel activity4,7,8,9,10,11. Yet, despite their broad functional importance, the structural correlates

and triggers of modal gating shifts are unknown. Modal gating shifts were suggested to relate to selectivity filter rearrangements; however, a series of X-ray structures of E71X mutants

showed no changes in the filter (RMSD relative to WT KcsA is <0.25 Å)6 despite the marked functional heterogeneity of these mutants. Curiously, for the E71A mutant, a well-established

model to study K+ channel gating12,13, a second, strongly different filter conformation of uncertain functional relevance was crystallised4,14. Besides the lack of clarity on the selectivity

filter conformation, it was further assumed that changes in the filter dynamics could cause modal gating shifts6. However, also here, whether the selectivity filter dynamics change in

reference to the gating mode is unknown, and experimental data are scarce to resolve this question. Altogether, there is a fundamental lack of knowledge on how the hydrogen bond network

surrounding the selectivity filter modulates its gating, which critically limits our understanding of modal gating shifts of Kv channels. Here, we use modern proton-detected

(1H-detected)15,16,17,18,19,20 solid-state NMR (ssNMR) in native-like membranes to compare the selectivity filter in WT KcsA and the three mutants (E71A, E71I, E71Q) that are best

representatives of modal gating. We show that E71 point-mutations cause marked changes in the selectivity filter conformational dynamics, in contrast to previous crystallographic studies

that revealed virtually no changes in structure6. By combining ssNMR with molecular dynamics (MD) simulations, we demonstrate that altered structural dynamics in E71X mutants drive the

selectivity filter into new conformational equilibria that represent the molecular origin of modal gating. Furthermore, we show that modal gating goes hand in hand with fluctuations in the

hydrogen bonding and water network behind the filter, which are triggers of sudden mode shifts. Altogether, these results provide a high-resolution perspective on the complex kinetic

behaviour of the selectivity filter of K+ channels. Importantly, the pronounced conformational and motional changes that we observe in E71I KcsA provide a foundation for future elucidation

of the selectivity filter of eukaryotic K+ channels. RESULTS NMR ASSIGNMENTS OF THE CHANNELS AT NEAR-NATIVE CONDITIONS We assigned the 1H, 13C, and 15N ssNMR chemical shifts of WT KcsA and

the E71A, E71I, E71Q mutants in order to analyse their conformational dynamics in membranes. First, we prepared uniformly [13C,15N]-labelled inversely Fractionally Deuterated17 channels in

liposomes composed of _Escherichia coli_ lipids. Samples were prepared in buffer conditions (pH 7.4, 100 mM K+) at which WT KcsA is in the closed-conductive state21, i.e., a state with a

closed activation gate and a conductive selectivity filter. De novo backbone chemical shift assignments were obtained for mutant E71A using a set of four dipolar-based three-dimensional (3D)

1H-detected ssNMR experiments (CANH, CONH, CAcoNH, COcaNH) (Fig. 1c and Supplementary Figure 1). The high spectral quality enabled us to almost fully assign residues L41–W87, which include

the complete selectivity filter and pore helix, the pore loop, larger parts of the outer transmembrane 1 (TM1) helix, and a few residues of the inner transmembrane 2 (TM2) helix. Since we

used dipolar-based magnetisation transfer steps that decrease in efficiency with increasing molecular mobility, the cytoplasmic domain (F125-R160) and the membrane-associated M0 helix

(M1-H20) were not detectable at our experimental temperatures of 300–310 K22. Assignments were then transferred to KcsA mutants E71I and E71Q and confirmed with a reduced set of 3D ssNMR

experiments (CANH, CONH). The reduced set was also used to complement our previous WT KcsA assignments17. For the flicker mutant E71Q, spectral sensitivity was significantly lower,

indicative of increased mobility, which averages dipolar magnetisation transfer efficiency. Furthermore, for all mutants and WT KcsA, we acquired two-dimensional (2D) 13C–13C experiments.

Moreover, we acquired dipolar 2D 15N–1H ssNMR spectra, in which each signal relates to one 15N–1H backbone or side chain unit, and which represent spectral fingerprints. MODAL GATING RELATES

TO CHANGES IN THE FILTER CONFORMATION NMR chemical shifts are sensitive reporters of protein conformation. Therefore, comparing the chemical shifts of the mutants to WT KcsA enables the

analysis of the structural impact of the substitutions at E71. The 2D 15N–1H and 13C–13C spectra of all E71X mutants superimposed very well onto WT KcsA, demonstrating that the global

protein fold is conserved (Supplementary Figure 2). However, for all mutants, we observed remarkably large 1H and 15N (HN) as well as 13C chemical shift perturbations (CSPs) across the

selectivity filter (Fig. 1d, e and Supplementary Figure 3). For all mutants, we observed very important HN CSPs for the essential residues W67, Y78, and D80 behind the filter, demonstrating

changes in their hydrogen bond interactions. At the same time, the large Cα and CO CSPs that we observed for residues G77, Y78, and D80 imply pronounced conformational filter backbone

changes, which likely have implications for the K+ occupancy. Intriguingly, all mutants also exhibited a large CSP for V76CO, which is immediately involved in K+ binding and key residue for

C-type inactivation (Fig. 1e)3. Mutants E71A and E71I showed a strikingly similar CSP pattern. Here, the HN and 13C signal shifts of W67, V76, Y78, and D80 are all similar in magnitude and

in the same direction. This similarity appears plausible, given that neither alanine nor isoleucine is able to mimic the hydrogen bonding capacities of the E71 carboxyl group. However,

although the CSP pattern is similar, E71I shows a much larger Y78Cα CSP than E71A, which implies conformational differences. While we also observed CSP maxima at W67, G77, Y78, and D80 in

E71Q, these signal shifts were partially in opposite direction relative to E71A and E71I, and less strong. In general, among the three mutants, the chemical shifts of E71Q deviated the least

from WT KcsA. Considering that a glutamine can partly substitute for some of the hydrogen bonds of a glutamate, it is likely that the E71Q filter conformation is relatively close to WT

KcsA. Indeed, the D80 side chain and the Y78 backbone showed by far the smallest CSPs for E71Q, which strongly suggests that Q71–D80 and Q71–Y78 maintain interactions analogous to WT KcsA,

while these interactions are lost in E71A and E71I (Figs. 2a and 1e). Next to conformational changes in the filter, the mutants showed moderate to larger CSPs around residues W67–V70. These

changes presumably relate to modulations in the aromatic belt W67, W68, and Y78 that surrounds the filter, and to the loss or modulation of interaction E71–D80, which acts as a molecular

spring that couples the pore helix to the pore mouth in WT KcsA (Fig. 2b)5. Our data show that the pore helices in all mutant channels exhibit, compared to WT KcsA, local changes (Fig. 2d)

that modulate the residues T74–T75–V76–G77 at the N-terminal end of the selectivity filter, which are directly coordinated to the pore helix by hydrogen bonds. Indeed, all mutants show clear

perturbations at V76 and G77 (Fig. 1d, e). We observed the largest CSPs in the E71I pore helix, which correlates with strong signal shifts of the T74 side chain. Since conformational

changes of the T74 side chain relate to C-type inactivation23,24, this perturbation could be functionally important for E71I, which favours transitions to a non-conductive state6. This

assumption is corroborated by 2D CC spectra, which show that the T74 side chain conformation in E71I (pH 7.4, 100 mM K+) and in the inactivated filter of WT KcsA (pH 4, 0 mM K+) are close to

each other (Fig. 2e, f). Furthermore, we observed remarkable conformational changes in the turret G53–T61. The turret is an important drug binding site25 and responds to gating changes26;

however, the structural underpinning is unclear and inaccessible from X-ray structures because of intense interactions between turret and Fab fragments4. In our ssNMR experiments, the CSPs

in the turret are small. However, to our surprise, residues P55, G56, and A57 in E71I disappeared or showed signal splittings, strongly indicative of stark structural heterogeneity (Fig. 

2c). This implies that the replacement of E71 by isoleucine causes long-range effect that are felt ~2 nm away from the mutation site. Note that CSPs for the TM1 and TM2 helices, as well as

for un-annotated TM residues, were very small, confirming that the global WT KcsA fold is conserved in the mutants. CHANGES IN THE FILTER DYNAMICS AND MODAL GATING SHIFTS Historically, the

selectivity filter was thought to form a stiff framework in order to allow fast conduction of K+ together with high selectivity over Na+2. More recent studies point to a more dynamic

filter27, and hypothesise that modal gating shifts relate to changes in the motional behaviour of the filter6. However, quantitative experimental data on selectivity filter dynamics are not

available in membranes, critically limiting our understanding of K+ channel function. Here we probe the filter dynamics in reference to the gating mode with ssNMR relaxation, which is an

ideal approach to measure the internal dynamics of membrane proteins at native conditions17,28. Site-resolved ssNMR relaxation can be probed with 2D 15N–1H experiments that include a

relaxation element that is sensitive to dynamics on a certain time-scale. A series of spectra is then acquired with increasing duration of the relaxation element, and the signal sensitivity

decreases according to the motion for a given residue. The signal decay is then converted into a relaxation rate _R_, and higher rates indicate enhanced dynamics. We probed the 15N slow

rotating-frame relaxation (_R_1rho) for WT KcsA and the mutants (Fig. 3) using extensive series of 1H-detected 2D 15N–1H experiments29. 15N _R_1rho relaxation is sensitive to dynamics in the

nanosecond–millisecond range but dominated by motion with slow correlation times in the microsecond range30. The high sensitivity with 1H-detection enabled us to measure relaxation rates

with high accuracy. In all investigated channels, our study unravelled strikingly different filter dynamics that clearly correlate with the CSP maxima, thereby linking conformational and

motional changes. In WT KcsA, the filter (~15 ms−1 _R_1rho) is the most dynamic membrane-embedded region, with G79 as a distinct local maximum. The dynamics of the pore helix is slightly

lower, TM1 residues show the least dynamics, while the extracellular turret is by far the most mobile region. These results agree with our previous relaxation studies in WT KcsA17. The

global dynamics of mutant E71I, which mimics certain Kv channels, is particularly interesting. Surprisingly, compared to WT KcsA, we measured strongly enhanced dynamics at the filter

entrance Y78-D80 (~25 ms−1 _R_1rho). These residues also showed the largest CSPs, demonstrating again that local conformational and motional changes correlate. Furthermore, we observed

sizeable stiffening at G77 in the middle of the E71I filter. Intriguingly, the selectivity filter dynamics is very different in the E71A mutant, in which the middle and upper filter regions

(V76–D80) drastically rigidified (~8 ms−1 _R_1rho). This means that E71A and E71I exhibit clearly different filter dynamics in spite of similar CSP patterns. It is easy to imagine that these

differential dynamics are at the origin of some of the heterogeneous gating kinetics observed in KcsA and other K+ channels. As for the CSPs, the flicker mutant E71Q behaved very

differently and featured substantially and globally enhanced _R_1rho values with a maximum at V76. This means that the entire channel undergoes pronounced large-scale slow motions, which

explains the strongly reduced sensitivity in our dipolar experiments with E71Q. Altogether, our data hence strongly suggest that the rapid flickering between open and closed states in KcsA,

and observed in all K+ channels6,10, relates to large-scale dynamics of the pore domain. Another noteworthy observation was the stark change in the dynamics of the turret, which rigidified

in E71A and E71I. As we concluded above (Fig. 2c), this implies that the loss of the E71–D80 interaction causes allosteric changes 2 nm distal from the mutation site, presumably by

successive changes of hydrogen bonding partners. A likely starting point for this chain reaction could be D80, which adopts a clearly different conformation in E71A and E71I compared to WT

KcsA (Fig. 2a). FLUCTUATIONS IN A CRITICAL WATER CAVITY TRIGGER MODAL GATING The strongly different filter dynamics in the E71X mutants raise the question which molecular events could cause

spontaneous mode shifts. One potential trigger could be buried water, localised in a cavity behind the filter, which is a decisive gating determinant in K+ channels, relating to C-type

inactivation and recovery22,31,32. Previous X-ray studies suggested that changes in the size of the water cavity could trigger modal gating shifts. In these and other previous studies, two

water molecules were resolved behind the filter of E71I and E71A6,12, while the presence of buried water molecules in E71Q was unclear due to insufficient resolution. Here, we revisit the

water distribution behind the selectivity filter using H/D exchange ssNMR17,33,34, for which we acquired 2D 15N–1H spectra in fully deuterated buffers. At these conditions, exchange with

deuterons strongly attenuates signals of water-exposed amino-protons, which provides high-resolution information on the water cavity size22. The channels were incubated in deuterated buffers

for 2 days, and the completeness of the exchange was confirmed on the fully water-exposed turret, which entirely disappeared from the 2D NH spectra (Fig. 4a, right panel). In WT KcsA, G79

is the only filter residue that disappeared in deuterated buffers while Y78–T74 showed no signs of H/D exchange (Fig. 4a, b). The lack of exchange for Y78 in WT KcsA is astonishing, given

that a water molecule is in direct proximity in the X-ray structure (Fig. 1a), and strongly suggests that a tight hydrogen bond with E71 protects Y78 from H/D exchange. In line with this

conclusion, Y78 showed attenuated intensity in both E71I (−33%) and E71A (−81%) (Fig. 4b, c), in which the X71–Y78 hydrogen bond is lost. Intriguingly, the much faster exchange of Y78 in

E71A implies a larger water cavity, which agrees with the smaller size of alanine relative to isoleucine. The widened water cavity is also corroborated by the high rigidity of E71A (Fig. 3),

which renders enhanced molecular fluctuations an unlikely cause for increased H/D exchange. Strikingly, in the flickery E71Q channel, G79 did not exchange (Fig. 4a). Similarly, L81 did not

exchange in E71Q, while it disappeared in E71A, E71I, and WT KcsA, which confirms that the water cavity is smaller or fully absent behind the E71Q filter. Altogether, our NMR data

demonstrate that, in membranes, E71X point-mutations change the size of the water cavity in reference to the gating mode, analogous to the crucial change of the water cavity during C-type

inactivation31. Note that long MD simulations, which are discussed in detail in the following sections, also show widened water cavities and higher exchange-rates with bulk water for E71A

and E71I (Supplementary Figure 4). SHIFTS IN THE EQUILIBRIUM STRUCTURE OF THE FILTER Our ssNMR data demonstrate that E71 point-mutations cause large CSPs in the selectivity filter, with

maxima at Y78Cα and V76CO; the latter forming the S2 and S3 sites that are critically involved in C-type inactivation3. Such large perturbations in the heart of the filter are astonishing,

given that E71A and E71I show a sharply reduced extent of C-type inactivation6. To gain a structural understanding of the ssNMR CSPs, we performed a series of 1-μs-long MD simulations for

each WT KcsA and the mutants (Fig. 5a and Supplementary Figure 5). For WT KcsA, simulations show that the selectivity filter samples two conformations in which V76CO points either towards

(inwards conformation) or away (outwards) from the filter pore35. The equilibrium between these two, most likely rapidly converting, conformations was recently confirmed by 2D IR data that

showed a 60:40 ratio between inwards:outwards conformations. Remarkably, simulations also indicate that E71X mutations perturb this equilibrium, stabilising the V76CO inwards conformation in

E71I and E71A, while the outwards state is favoured in E71Q. To probe if the V76CO CSPs could be explained by a change in the average conformation of the filter, we back-calculated36 the

13C ssNMR chemical shifts over the MD simulations (Fig. 5b, c). These calculations yielded a much lower V76CO chemical shift in the outwards conformation (predicted ΔV76COinwards–outwards = 

+ 2 to + 3 ppm) (Fig. 5d); a result that agrees with the much lower chemical shift of V76CO at low [K+] in the collapsed filter with a flipped V76–G77 plane (experimental ΔV76COconductive

filter–collapsed filter = + 3.3 ppm)21,37]. Hence, these results show that the V76CO inwards conformation is strongly stabilised in E71A (V76CO CSP = + 0.94 ppm relative to WT KcsA) and E71I

(+0.77 ppm), while the sampling of the outwards conformation is enhanced in E71Q (−0.55 ppm) (Fig. 5e). This conclusion also agrees with our relaxation data, which show reduced dynamics for

V76 and G77 in E71A and E71I, whereas both residues show much higher mobility in E71Q. Notably, the stabilisation of the V76CO inwards state correlates with the decrease of C-type

inactivation in E71A and E71I6. Similarly, we could derive a structural understanding of the stark Y78Cα CSPs. In MD simulations, WT KcsA exclusively adopts the Y78CO inwards conformation

that is stabilised by the E71–Y78 hydrogen bond. However, in E71I, the Y78 backbone is no longer stabilised and we observe sizeable sampling of an outwards conformation (Fig. 5a). Here

again, back-calculated chemical shifts show a much lower Y78Cα signal in the Y78CO outwards conformation (predicted ΔY78Cαinwards–outwards = + 2 ppm) (Fig. 5c, d). These results imply that a

Y78CO outwards state is frequently sampled in E71I (Y78Cα CSP = −1.94 ppm relative to WT KcsA) and less frequently or only partially in E71A (Y78Cα CSP = −0.84 ppm). Note that we could not

observe a Y78CO outwards state in E71A simulations, presumably due to insufficient sampling. DESTABILISATION OF THE CRITICAL INTERACTION D80–W67 An especially remarkable finding in our study

is the drastically increased filter dynamics in E71I compared to E71A despite similar nonpolar E71 substitutions, and this surge in flexibility most likely causes increased sampling of the

Y78CO outwards state in E71I. We used MD simulations to gain a molecular understanding of the enhanced E71I filter dynamics. In our simulations, the conformational space of the D80 side

chain is an event of particular interest (Supplementary Figure 6). In WT KcsA, the interaction with E71 locks D80 in a down conformation (Fig. 6a), and only this conformation enables the

W67–D80 interaction, which is critical for gating in Kv channels38,39. The down conformation prevails in E71A, enabling a steady W67–D80 interaction (Fig. 6b, d). However, in E71I

simulations, the bulky isoleucine sterically destabilises the W67–D80 interaction and causes D80 to increasingly sample middle and up conformations (Fig. 6c). We validated the loss of the

W67–D80 interaction in E71I with ssNMR 15N _T_1 relaxation experiments that are sensitive to the fast pico-to-nanosecond motion of unbound side chains. These experiments clearly show

markedly enhanced W67 side chain dynamics in E71I, confirming an unstable W67–D80 interaction in this mutant (Fig. 6d, e). To compensate, D80 increasingly engages in interactions with the

functionally important residues R64 and Y824,31 that are hardly sampled in WT KcsA. The D80 promiscuity is pronounced in MD simulations of E71I and E71Q and agrees with the enhanced ssNMR

_R_1rho dynamics at the filter entrance of E71I and E71Q (Fig. 3). In E71A, however, a steady D80–W67 interaction stabilises the filter, as demonstrated by strongly reduced _R_1rho dynamics,

and this stabilisation is most likely the reason why the Y78CO outwards state is much less frequently sampled in E71A than in E71I. Interestingly, our extensive MD analysis also

demonstrates that the E71 point-mutations and the D80 promiscuity cause long-range modulations in the turret (Supplementary Figure 6), which presumably relate to the turret heterogeneity in

the mutants (Figs. 2c and 3). DISCUSSION Modal gating shifts at constant experimental conditions have been observed in K+4,6,7,8,9,10, Na+40, Ca2+41,42, and other ion channels43,44, and are

considered a widespread regulatory mechanism11,45, potentially to achieve intermediate activity levels. While known for a long-time, the molecular underpinning of modal gating behaviour is

poorly understood. In KcsA, E71 point-mutations emulate modal gating shifts; however, X-ray structures of E71X mutants showed no differences compared to WT KcsA6. These seemingly disparate

perspectives of functional heterogeneity and of structural similarity raise critical problems for our understanding of modal gating and also of Kv channel function (Fig. 1b). Our ssNMR study

in native-like conditions paints a strikingly different picture, demonstrating that E71 substitutions lock the selectivity filter in characteristic conformational and motional landscapes

that markedly diverge from WT KcsA. These landscapes strongly depend on the nature of residue 71 and directly relate to the heterogeneous functional behaviour observed in K+ channels6. By

integrating ssNMR and MD simulations, we show that E71 point-mutations rearrange the network behind the filter and perturb the K+ binding sites V76 and Y78 (Fig. 1a, e). Thereby, we show

that E71X mutations change the equilibrium between intrinsically sampled filter states (Fig. 5e), which agrees with 2D IR data35 and the so-called ‘flipped’ E71A structure that points

towards a complex selectivity filter landscape that includes dynamical flips of K+ coordinating peptide planes35. Our data demonstrate a stabilisation of the V76CO inwards state in E71A and

E71I relative to WT KcsA, which correlates with a sharply reduced entry into the C-type inactivated state. Furthermore, our data clearly show Y78 conformational perturbations in E71I and

E71A. Such perturbations could not be observed in WT KcsA X-ray structures3; however, there is strong evidence from previous ssNMR studies that Y78 modulations can accompany filter gating.

Indeed, ssNMR data show that Y78Cα exhibits a drastically lower signal in the open-collapsed state (experimental ΔY78Cαconductive filter–collapsed filter = +4.3 ppm)37, which unambiguously

argues that the Y78 conformation can change in reference to the filter mode. This notion is also corroborated by the ‘flipped’ E71A X-ray structure (2ATK), which also features a Y78CO

partial outward state4,46, and it is in line with a recent cryo-EM structure of the hERG channel47. While the exact role in KcsA is unclear, we surmise that Y78 backbone modulations may

relate to a non-conductive state that is favoured by E71I, which agree with the strongly reduced K+ occupancy at S0–S2 in E71I6. Importantly, the marked conformational changes in the E71I

selectivity filter, and especially the destabilisation of the W67–D80 interaction (Fig. 6d, e), suggest to be of broad relevance for Kv channels (Fig. 1b) where the W67–D80 interaction plays

a defining role in the inactivation process. Mutational studies showed that the inability to establish this highly conserved interaction entails severe functional perturbations for KcsA,

Shaker, and Kv1.238,39. Moreover, in line with the effect of the weakening of the W67–D80 interaction in E71I, the destabilisation of the analogous interaction W434–E447 in Shaker modulates

the equilibrium between conducting and non-conducting filter states48. On the basis of our set of results, we show that modal gating shifts in K+ channels relate to changes in the

statistical weighting of pre-existing selectivity filter states which are triggered by fluctuations in the hydrogen bonding (Fig. 6) and water network (Fig. 4). Notably, we show that

modulations in this network cause changes in the turret over more than 2 nm (Figs. 2c, 3), which opens a pathway how turret-binding drugs, lipids, or other proteins can allosterically

modulate the filter21,25,49,50,51. The question arises why most of these conformational subtypes could not be crystallised. Here, the reason is most likely the interaction with Fab fragments

that act as crystallographic chaperons and attach to KcsA X-ray structures. In agreement with electrophysiological measurements4, these artificial Fab interactions lock the selectivity

filter in a specific conformation, and thereby hinder the capturing of transient configurations, masking the effect of E71 point-mutations. Interestingly, the lack of non-canonical filter

conformations in X-ray structures was mirrored by additional torsional or position restraints in MD simulations to stabilise a conductive filter conformation52,53. At least for KcsA, such

potentials could mask the physiological filter plasticity, as demonstrated in this study and previously with 2D IR35. In conclusion, our work establishes the shifts in the conformational

dynamics of the selectivity filter as the key physiological determinant of modal gating behaviour. At the same time, our work provides a long-needed quantitative description of the

selectivity filter dynamics in a native environment, which is of fundamental importance to understand ion channel function54. Given that the here-described filter dynamics are strongly

different in mutant E71I, our study may ultimately help to better understand eukaryotic Kv channels. Finally, we like to emphasise that further experiments with open channels under

inactivating conditions will be critically required to fully comprehend how E71X mutations modulate channel open probability and C-type inactivation. METHODS SAMPLE PREPARATION WT KcsA and

E71X mutant channels were expressed in _E. coli_ M15 cells (Qiagen) using standard H2O-based M9 medium supplemented with 0.5 g/L 15NH4Cl and 2 g/L d-glucose-13C6-d7 in order to improve the

spectral resolution in 1H-detected ssNMR experiments17. Cells were subsequently harvested, treated with lysozyme, and lysed via French press. The membranes containing the KcsA channels were

collected by centrifugation (100,000×_g_) and proteins were extracted with 40 mM DM (Anatrace)26. KcsA channels were purified using Ni-NTA agarose beads (Qiagen), resulting in a final yield

of 10 mg/L for WT KcsA and 5 mg/L for the E71X KcsA mutants. Liposome reconstitution was performed using _E. coli_ polar lipids (Avanti) at 1:100 protein:lipid molar ratio, in which the

detergent was removed using polystyrene beads (Bio-Beads SM-2)26. Before the ssNMR measurements, reconstituted samples were suspended in fully protonated phosphate buffer (pH 7.4, 100 mM 

K+). For spectral assignments, a fully protonated buffer was used in order to observe the entire channel in 1H-detected ssNMR experiments. For proton/deuterium (H/D) exchange ssNMR

spectroscopy, ion channels were incubated in fully deuterated buffers (pH 7.4, 100 mM K+) for a total of 2 days prior to the measurements. SOLID-STATE NMR SPECTROSCOPY 3D ssNMR experiments

for sequential backbone chemical shift assignments were performed at 800 MHz (1H-frequency) using 60 kHz magic angle spinning (MAS) frequency and a real temperature of approximately 305 K.

In total, we ran ten dipolar-based 3D ssNMR experiments to assign the three mutant channels and WT KcsA. The pulse sequences and experimental setups were performed as previously described17.

2D 13C–13C PARISxy55 (_N_ = ½, _m_ = 1) experiments for side chain chemical shift assignments were performed at 700 MHz using 42 kHz MAS and a 13C–13C mixing time of 110 ms. 15N _T_1rho

relaxation experiments were performed as descripted for the water-inaccessible part of KcsA and measured at 700 MHz and 58 kHz MAS using a 15N spin lock amplitude of 17.5 kHz17. We used

1H-detected 2D experiments together with relaxation increments of 0, 5, 10, 20, 40, and 80 ms. For the much faster relaxing flicker E71Q channel, we used increments of 0, 5, 10, 20, 40, and

60 ms. 15N _T_1 measurements were performed at 950 MHz and 60 kHz MAS using relaxation elements of 0, 2, 4, 10, and 20 s. The W67 side chain is spectrally isolated in E71A and E71I

(Supplementary Figure 2) and could be readily analysed in a series of 1D experiments. For the analysis of H/D exchange data from 2D NH spectra acquired in protonated and deuterated buffers,

we used the signal intensities, which were normalised to the water-inaccessible residues S69 and V70 that are not subjected to H/D exchange. PISSARRO56 decoupling was used as decoupling

method in all direct and indirect dimensions. Chemical shifts were back-calculated from MD simulations with the SPARTA+ program36. Channels structures were extracted from MD simulations with

a time-increment of 10 ns, yielding a total of 400 monomer structures. Compared to our experimental data, the SPARTA+ predictions give systematically lower chemical shifts for the V76

backbone carbonyl carbon, which we aligned by adding 2.5 ppm to the predicted carbonyl chemical shifts for all channels. MD SIMULATIONS Atomic models of KcsA were constructed based on

crystal structures (1K4C3 and 3OR66) that represent a structural state with closed inner gate. Considering the high similarity between crystal structures of WT (1K4C), E71A (1ZWI), and E71I

(3OR7), the simulation systems for E71A and E71I are built based on the fully equilibrated WT system by introducing respective single mutation. With more substantial difference compared with

WT, the crystal structure for E71Q (3OR6) was used to build its simulation system. For all MD simulations, the channel was embedded in a bilayer of 3:1 POPC:POPG lipids and solvated in 150 

mM or 200 mM KCl using the web service CHARMM-GUI57,58. Most residues were assigned their standard protonation state at pH 7. The total number of atoms in the MD systems is on the order of

~49,000. The CHARMM force field PARAM36 for protein59, lipids60, and ions61 was used. Explicit water was described with the TIP3P model. In WT, the residue Glu71 is protonated to form a key

hydrogen bond with Asp8039,62. The models of KcsA were refined using energy minimization for at least 2000 steps, and the ions and non-filter backbone atoms were kept fixed throughout the

minimization procedure. After energy minimisation, the conductive filter was restrained for 10–20 ns to relax any unfavourable contacts destabilising the filter. All the simulations were

performed under constant NPT conditions at 310 K and 1 atmosphere, and periodic boundary conditions with electrostatic interactions were treated by the particle-mesh Ewald (PME) method and a

real-space cutoff of 12 Å. The simulations use a time step of 2 fs. After minimization and equilibration with harmonic positional restraints on all of the C atoms, MD simulations were

performed for 1 μs for wild type and all mutants, by using either NAMD version 2.1163, or on the special purpose computer Anton (Pittsburgh Supercomputer Center)64. REPORTING SUMMARY Further

information on experimental design is available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY Data supporting the findings of this manuscript are

available from the corresponding author upon reasonable request. The solid-state NMR assignments have been deposited in the BMRB (accession number 27676 for WT KcsA, 27678 for E71A KcsA,

27679 for E71I KcsA, and 27680 for E71Q KcsA). The Source Data underlying Figs. 1d, 3a, 5b, 6d, and Supplementary Figure 3 are provided as a Source Data file. REFERENCES * Yellen, G. The

voltage-gated potassium channels and their relatives. _Nature_ 419, 35–42 (2002). Article  ADS  CAS  Google Scholar  * Doyle, D. A. et al. The structure of the potassium channel: molecular

basis of K+ conduction and selectivity. _Science_ 280, 69–77 (1998). Article  ADS  CAS  Google Scholar  * Zhou, Y. F., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion

coordination and hydration revealed by a K+ channel-Fab complex at 2.0 angstrom resolution. _Nature_ 414, 43–48 (2001). Article  ADS  CAS  Google Scholar  * Cordero-Morales, J. F. et al.

Molecular determinants of gating at the potassium-channel selectivity filter. _Nat. Struct. Mol. Biol._ 13, 311–318 (2006). Article  CAS  Google Scholar  * Cordero-Morales, J. F. et al.

Molecular driving forces determining potassium channel slow inactivation. _Nat. Struct. Mol. Biol._ 14, 1062–1069 (2007). Article  CAS  Google Scholar  * Chakrapani, S. et al. On the

structural basis of modal gating behavior in K+ channels. _Nat. Struct. Mol. Biol._ 18, 67–74 (2011). Article  CAS  Google Scholar  * Zagotta, W. N., Hoshi, T. & Aldrich, R. W. Shaker

potassium channel gating. III: Evaluation of kinetic models for activation. _J. Gen. Physiol._ 103, 321–362 (1994). Article  CAS  Google Scholar  * Zheng, J. & Sigworth, F. J.

Selectivity changes during activation of mutant Shaker potassium channels. _J. Gen. Physiol._ 110, 101–117 (1997). Article  CAS  Google Scholar  * Schoppa, N. E. & Sigworth, F. J.

Activation of shaker potassium channels. I. Characterization of voltage-dependent transitions. _J. Gen. Physiol._ 111, 271–294 (1998). Article  CAS  Google Scholar  * Chakrapani, S.,

Cordero-Morales, J. F. & Perozo, E. A quantitative description of KcsA gating II: single-channel currents. _J. Gen. Physiol._ 130, 479–496 (2007). Article  CAS  Google Scholar  *

Bicknell, B. A. & Goodhill, G. J. Emergence of ion channel modal gating from independent subunit kinetics. _Proc. Natl Acad. Sci. USA_ 113, E5288–E5297 (2016). Article  CAS  Google

Scholar  * Cuello, L. G., Cortes, D. M. & Perozo, E. The gating cycle of a K+ channel at atomic resolution. _eLife_ 6, https://doi.org/10.7554/eLife.28032 (2017). * Heer, F. T., Posson,

D. J., Wojtas-Niziurski, W., Nimigean, C. M. & Berneche, S. Mechanism of activation at the selectivity filter of the KcsA K + channel. _eLife_ 6, https://doi.org/10.7554/eLife.25844.001

(2017). * Cheng, W. W. L., Mccoy, J. G., Thompson, A. N., Nichols, C. G. & Nimigean, C. M. Mechanism for selectivity-inactivation coupling in KcsA potassium channels. _Proc. Natl Acad.

Sci. USA_ 108, 5272–5277 (2011). Article  ADS  CAS  Google Scholar  * Chevelkov, V., Rehbein, K., Diehl, A. & Reif, B. Ultrahigh resolution in proton solid-state NMR spectroscopy at high

levels of deuteration. _Angew. Chem. Int. Ed. Engl._ 45, 3878–3881 (2006). Article  CAS  Google Scholar  * Agarwal, V. et al. De novo 3D structure determination from sub-milligram protein

samples by solid-state 100 kHz MAS NMR spectroscopy. _Angew. Chem. Int. Ed. Engl._ 53, 12253–12256 (2014). Article  CAS  Google Scholar  * Medeiros-Silva, J. et al. 1 H detected solid-state

NMR studies of water inaccessible proteins in vitro and in situ. _Angew. Chem. Int. Ed. Engl_. 55, 13606–13610 (2016). * Andreas, L. B. et al. Structure of fully protonated proteins by

proton-detected magic-angle spinning NMR. _Proc. Natl Acad. Sci. USA_ 113, 9187–9192 (2016). Article  CAS  Google Scholar  * Retel, J. S. et al. Structure of outer membrane protein G in

lipid bilayers. _Nat. Commun_. 8, https://doi.org/10.1038/s41467-017-02228-2 (2017). * Medeiros-Silva, J. et al. High-resolution NMR studies of antibiotics in cellular membranes. _Nat.

Commun._ 9, 3963 (2018). Article  ADS  Google Scholar  * Ader, C. et al. A structural link between inactivation and block of a K+ channel. _Nat. Struct. Mol. Biol._ 15, 605–612 (2008).

Article  CAS  Google Scholar  * Weingarth, M. et al. Quantitative analysis of the water occupancy around the selectivity filter of a K+ channel in different gating modes. _J. Am. Chem. Soc._

136, 2000–2007 (2014). Article  CAS  Google Scholar  * Cuello, L. G. et al. Structural basis for the coupling between activation and inactivation gates in K+ channels. _Nature_ 466, 272–275

(2010). Article  ADS  CAS  Google Scholar  * Pan, A. C., Cuello, L. G., Perozo, E. & Roux, B. Thermodynamic coupling between activation and inactivation gating in potassium channels

revealed by free energy molecular dynamics simulations. _J. Gen. Physiol._ 138, 571–580 (2011). Article  CAS  Google Scholar  * Lange, A. et al. Toxin-induced conformational changes in a

potassium channel revealed by solid-state NMR. _Nature_ 440, 959–962 (2006). Article  ADS  CAS  Google Scholar  * van der Cruijsen, E. A. et al. Importance of lipid-pore loop interface for

potassium channel structure and function. _Proc. Natl Acad. Sci. USA_ 110, 13008–13013 (2013). Article  ADS  Google Scholar  * Baker, K. A., Tzitzilonis, C., Kwiatkowski, W., Choe, S. &

Riek, R. Conformational dynamics of the KcsA potassium channel governs gating properties. _Nat. Struct. Mol. Biol._ 14, 1089–1095 (2007). Article  CAS  Google Scholar  * Good, D. B. et al.

Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR. _J. Am. Chem. Soc._ 136, 2833–2842 (2014). Article  CAS  Google Scholar

  * Lewandowski, J. R., Sass, H. J., Grzesiek, S., Blackledge, M. & Emsley, L. Site-specific measurement of slow motions in proteins. _J. Am. Chem. Soc._ 133, 16762–16765 (2011). Article

  CAS  Google Scholar  * Lewandowski, J. R. Advances in solid-state relaxation methodology for probing site-specific protein dynamics. _Acc. Chem. Res._ 46, 2018–2027 (2013). Article  CAS 

Google Scholar  * Ostmeyer, J., Chakrapani, S., Pan, A. C., Perozo, E. & Roux, B. Recovery from slow inactivation in K + channels is controlled by water molecules. _Nature_ 501, 121–124

(2013). Article  ADS  CAS  Google Scholar  * Li, J. et al. Chemical substitutions in the selectivity filter of potassium channels do not rule out constricted-like conformations for C-type

inactivation. _Proc. Natl Acad. Sci. USA_ 114, 11145–11150 (2017). Article  CAS  Google Scholar  * Shi, L., Kawamura, I., Jung, K. H., Brown, L. S. & Ladizhansky, V. Conformation of a

seven-helical transmembrane photosensor in the lipid environment. _Angew. Chem. Int. Ed. Engl._ 50, 1302–1305 (2011). Article  CAS  Google Scholar  * Medeiros-Silva, J., Jekhmane, S.,

Baldus, M. & Weingarth, M. Hydrogen bond strength in membrane proteins probed by time-resolved (1)H-detected solid-state NMR and MD simulations. _Solid State Nucl. Magn. Reson._ 87,

80–85 (2017). Article  CAS  Google Scholar  * Kratochvil, H. T. et al. Instantaneous ion configurations in the K+ ion channel selectivity filter revealed by 2D IR spectroscopy. _Science_

353, 1040–1044 (2016). Article  ADS  CAS  Google Scholar  * Shen, Y. & Bax, A. SPARTA+: a modest improvement in empirical NMR chemical shift prediction by means of an artificial neural

network. _J. Biomol. NMR_ 48, 13–22 (2010). Article  CAS  Google Scholar  * Wylie, B. J., Bhate, M. P. & McDermott, A. E. Transmembrane allosteric coupling of the gates in a potassium

channel. _Proc. Natl Acad. Sci. USA_ 111, 185–190 (2014). Article  ADS  CAS  Google Scholar  * Perozo, E., Mackinnon, R., Bezanilla, F. & Stefani, E. Gating currents from a nonconducting

mutant reveal open-closed conformations in shaker K+ channels. _Neuron_ 11, 353–358 (1993). Article  CAS  Google Scholar  * Cordero-Morales, J. F., Jogini, V., Chakrapani, S. & Perozo,

E. A multipoint hydrogen-bond network underlying KcsA C-type inactivation. _Biophys. J._ 100, 2387–2393 (2011). Article  ADS  CAS  Google Scholar  * Howe, J. R. & Ritchie, J. M. Multiple

kinetic components of sodium-channel inactivation in rabbit schwann-cells. _J. Physiol._ 455, 529–566 (1992). Article  CAS  Google Scholar  * Delcour, A. H. & Tsien, R. W. Altered

prevalence of gating modes in neurotransmitter inhibition of N-type calcium channels. _Science_ 259, 980–984 (1993). Article  ADS  CAS  Google Scholar  * Ionescu, L. et al. Mode switching is

the major mechanism of ligand regulation of InsP(3) receptor calcium release channels. _J. Gen. Physiol._ 130, 631–645 (2007). Article  CAS  Google Scholar  * Popescu, G. & Auerbach, A.

Modal gating of NMDA receptors and the shape of their synaptic response. _Nat. Neurosci._ 6, 476–483 (2003). Article  CAS  Google Scholar  * Popescu, G., Robert, A., Howe, J. R. &

Auerbach, A. Reaction mechanism determines NMDA receptor response to repetitive stimulation. _Nature_ 430, 790–793 (2004). Article  ADS  CAS  Google Scholar  * Geng, Y. Y. & Magleby, K.

L. Modal gating of endplate acetylcholine receptors: a proposed mechanism. _J. Gen. Physiol._ 146, 435–439 (2015). Article  CAS  Google Scholar  * McCoy, J. G., Cheng, W., Thompson, A. N.,

Nimigean, C. M. & Nichols, C. G. Mechanism for selectivity-inactivation coupling in KcsA potassium channels. _Biophys. J._ 100, 565–565 (2011). Article  ADS  Google Scholar  * Wang, W.

W. & MacKinnon, R. Cryo-EM structure of the open human ether-a-go-go-related K + channel hERG. _Cell_ 169, 422–430.e10 (2017). Article  CAS  Google Scholar  * Lueck, J. D. et al. Atomic

mutagenesis in ion channels with engineered stoichiometry. _eLife_ 5, https://doi.org/10.7554/eLife.18976 (2016). * Weingarth, M. et al. Structural determinants of specific lipid binding to

potassium channels. _J. Am. Chem. Soc._ 135, 3983–3988 (2013). Article  CAS  Google Scholar  * Bohlen, C. J. et al. A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by

targeting the outer pore domain. _Cell_ 141, 834–845 (2010). Article  CAS  Google Scholar  * Visscher, K. M. et al. Supramolecular organization and functional implications of K(+) channel

clusters in membranes. _Angew. Chem. Int. Ed. Engl._ 56, 13222–13227 (2017). Article  CAS  Google Scholar  * Jensen, M. O. et al. Mechanism of voltage gating in potassium channels. _Science_

336, 229–233 (2012). Article  ADS  CAS  Google Scholar  * Kopfer, D. A. et al. Ion permeation in K(+) channels occurs by direct Coulomb knock-on. _Science_ 346, 352–355 (2014). Article  ADS

  Google Scholar  * Shi, C. et al. A single NaK channel conformation is not enough for non-selective ion conduction. _Nat. Commun._ 9, 717 (2018). Article  ADS  Google Scholar  * Weingarth,

M., Bodenhausen, G. & Tekely, P. Broadband magnetization transfer using moderate radio-frequency fields for NMR with very high static fields and spinning speeds. _Chem. Phys. Lett._ 488,

10–16 (2010). Article  ADS  CAS  Google Scholar  * Weingarth, M., Bodenhausen, G. & Tekely, P. Low-power decoupling at high spinning frequencies in high static fields. _J. Magn. Reson._

199, 238–241 (2009). Article  ADS  CAS  Google Scholar  * Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. _J. Comput. Chem._ 29,

1859–1865 (2008). Article  CAS  Google Scholar  * Jo, S., Lim, J. B., Klauda, J. B. & Im, W. CHARMM-GUI membrane builder for mixed bilayers and its application to yeast membranes.

_Biophys. J._ 97, 50–58 (2009). Article  ADS  CAS  Google Scholar  * Best, R. B. et al. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the

backbone phi, psi and side-chain chi(1) and chi(2) dihedral angles. _J. Chem. Theory Comput._ 8, 3257–3273 (2012). Article  CAS  Google Scholar  * Klauda, J. B. et al. Update of the CHARMM

all-atom additive force field for lipids: validation on six lipid types. _J. Phys. Chem. B_ 114, 7830–7843 (2010). Article  CAS  Google Scholar  * Beglov, D. & Roux, B. Finite

representation of an infinite bulk system—solvent boundary potential for computer-simulations. _J. Chem. Phys._ 100, 9050–9063 (1994). Article  ADS  CAS  Google Scholar  * Bhate, M. P. &

McDermott, A. E. Protonation state of E71 in KcsA and its role for channel collapse and inactivation. _Proc. Natl Acad. Sci. USA_ 109, 15265–15270 (2012). Article  ADS  CAS  Google Scholar

  * Phillips, J. C. et al. Scalable molecular dynamics with NAMD. _J. Comput. Chem._ 26, 1781–1802 (2005). Article  CAS  Google Scholar  * Shaw, D. E. et. al. Millisecond-scale molecular

dynamics simulations on Anton. In _Proc. Conference on High Performance Computing, Networking, Storage And Analysis_, (Association for Computing Machinery, New York)1–11 (2009). Download

references ACKNOWLEDGEMENTS This work was funded in part by the National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) through grants R01-GM062342 and 

U54-GM087519, and the Netherlands Science Organisation for Scientific Research (NWO, grant numbers 723.014.003 & 711.018.001 to M.W.; 700.26.121 to M.B.). Experiments at the 950 MHz

instrument were supported by uNMR-NL, an NWO-funded Roadmap NMR Facility (no. 184.032.207). AUTHOR INFORMATION Author notes * These authors contributed equally: Shehrazade Jekhmane, João

Medeiros-Silva. AUTHORS AND AFFILIATIONS * NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584, CH

Utrecht, The Netherlands Shehrazade Jekhmane, João Medeiros-Silva, Felix Kümmerer, Christoph Müller-Hermes, Marc Baldus & Markus Weingarth * Department of Biochemistry and Molecular

Biology, The University of Chicago, 929 E57th Street, Chicago, IL, 60637, USA Jing Li & Benoît Roux Authors * Shehrazade Jekhmane View author publications You can also search for this

author inPubMed Google Scholar * João Medeiros-Silva View author publications You can also search for this author inPubMed Google Scholar * Jing Li View author publications You can also

search for this author inPubMed Google Scholar * Felix Kümmerer View author publications You can also search for this author inPubMed Google Scholar * Christoph Müller-Hermes View author

publications You can also search for this author inPubMed Google Scholar * Marc Baldus View author publications You can also search for this author inPubMed Google Scholar * Benoît Roux View

author publications You can also search for this author inPubMed Google Scholar * Markus Weingarth View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS S.J., J.M.S., and F.K. acquired the ssNMR data. J.L. and B.R. performed the MD simulations. All authors contributed to data analysis and drafting of the manuscript. All authors

critically reviewed the paper. CORRESPONDING AUTHOR Correspondence to Markus Weingarth. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL

INFORMATION JOURNAL PEER REVIEW INFORMATION: _Nature Communications_ thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are

available. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SOURCE DATA

SUPPLEMENTARY INFORMATION PEER REVIEW FILE REPORTING SUMMARY RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International License, which

permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to

the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless

indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or

exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints

and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Jekhmane, S., Medeiros-Silva, J., Li, J. _et al._ Shifts in the selectivity filter dynamics cause modal gating in K+ channels. _Nat

Commun_ 10, 123 (2019). https://doi.org/10.1038/s41467-018-07973-6 Download citation * Received: 01 October 2018 * Accepted: 07 December 2018 * Published: 10 January 2019 * DOI:

https://doi.org/10.1038/s41467-018-07973-6 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative