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 fromMethanobacterium 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.

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K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions

10.1101/2020.03.20.000893 ◽

2020 ◽

Cited By ~ 3

Author(s):

Marco Lolicato ◽

Andrew M. Natale ◽

Fayal Abderemane-Ali ◽

David Crottès ◽

Sara Capponi ◽

...

Keyword(s):

Conformational Changes ◽

Ion Selectivity ◽

Transmembrane Helix ◽

Selectivity Filter ◽

Ion Binding ◽

Anomalous Scattering ◽

X Ray Crystallography ◽

Functional Studies ◽

Ion Binding Sites ◽

Physical And Chemical

K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action of their selectivity filter (C-type) gate3-6. Although structural studies show K2P conformations that impact activity7-13, no selectivity filter conformational changes have been observed. Here, combining K2P2.1 (TREK-1) X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and functional studies, we uncover the unprecedented, asymmetric, potassium-dependent conformational changes underlying K2P C-type gating. Low potassium concentrations evoke conformational changes in selectivity filter strand 1 (SF1), selectivity filter strand 2 (SF2), and the SF2-transmembrane helix 4 loop (SF2-M4 loop) that destroy the S1 and S2 ion binding sites and are suppressed by C-type gate activator ML335. Shortening the uniquely long SF2-M4 loop to match the canonical length found in other potassium channels or disrupting the conserved Glu234 hydrogen bond network supporting this loop blunts C-type gate response to various physical and chemical stimuli. Glu234 network destabilization also compromises ion selectivity, but can be reversed by channel activation, indicating that the ion binding site loss reduces selectivity similar to other channels14. Together, our data establish that C-type gating occurs through potassium-dependent order-disorder transitions in the selectivity filter and adjacent loops that respond to gating cues relayed through the SF2-M4 loop. These findings underscore the potential for targeting the SF2-M4 loop for the development of new, selective K2P channel modulators.

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The gating cycle of a K+ channel at atomic resolution

eLife ◽

10.7554/elife.28032 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 39

Author(s):

Luis G Cuello ◽

D Marien Cortes ◽

Eduardo Perozo

Keyword(s):

Conformational Changes ◽

Selectivity Filter ◽

Atomic Resolution ◽

K Channel ◽

Allosteric Interactions ◽

Inactivation Gating ◽

Pore Domain ◽

Fine Tune ◽

Conductive State

C-type inactivation in potassium channels helps fine-tune long-term channel activity through conformational changes at the selectivity filter. Here, through the use of cross-linked constitutively open constructs, we determined the structures of KcsA’s mutants that stabilize the selectivity filter in its conductive (E71A, at 2.25 Å) and deep C-type inactivated (Y82A at 2.4 Å) conformations. These structural snapshots represent KcsA’s transient open-conductive (O/O) and the stable open deep C-type inactivated states (O/I), respectively. The present structures provide an unprecedented view of the selectivity filter backbone in its collapsed deep C-type inactivated conformation, highlighting the close interactions with structural waters and the local allosteric interactions that couple activation and inactivation gating. Together with the structures associated with the closed-inactivated state (C/I) and in the well-known closed conductive state (C/O), this work recapitulates, at atomic resolution, the key conformational changes of a potassium channel pore domain as it progresses along its gating cycle.

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Mechanism of activation at the selectivity filter of the KcsA K+ channel

eLife ◽

10.7554/elife.25844 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 23

Author(s):

Florian T Heer ◽

David J Posson ◽

Wojciech Wojtas-Niziurski ◽

Crina M Nimigean ◽

Simon Bernèche

Keyword(s):

Conformational Changes ◽

Selectivity Filter ◽

K Channel ◽

Ion Permeation ◽

K Channels ◽

Narrow Region ◽

Gating Mechanism ◽

Structural Fluctuations ◽

Extracellular Side ◽

Dynamics Simulations

Potassium channels are opened by ligands and/or membrane potential. In voltage-gated K+ channels and the prokaryotic KcsA channel, conduction is believed to result from opening of an intracellular constriction that prevents ion entry into the pore. On the other hand, numerous ligand-gated K+ channels lack such gate, suggesting that they may be activated by a change within the selectivity filter, a narrow region at the extracellular side of the pore. Using molecular dynamics simulations and electrophysiology measurements, we show that ligand-induced conformational changes in the KcsA channel removes steric restraints at the selectivity filter, thus resulting in structural fluctuations, reduced K+ affinity, and increased ion permeation. Such activation of the selectivity filter may be a universal gating mechanism within K+ channels. The occlusion of the pore at the level of the intracellular gate appears to be secondary.

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Closed state-coupled C-type inactivation in BK channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1607584113 ◽

2016 ◽

Vol 113 (25) ◽

pp. 6991-6996 ◽

Cited By ~ 2

Author(s):

Jiusheng Yan ◽

Qin Li ◽

Richard W. Aldrich

Keyword(s):

Conformational Changes ◽

Bk Channels ◽

Bk Channel ◽

Membrane Potentials ◽

Selectivity Filter ◽

Open Probability ◽

Closed State ◽

State Dependent ◽

Channel Opening ◽

Activation Gate

Ion channels regulate ion flow by opening and closing their pore gates. K+ channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel’s activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K+ together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K+ channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca2+]i) and recovered with depolarized membrane potentials or elevated [Ca2+]i. Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel’s normal closing may represent an early conformational stage of C-type inactivation.

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Structural basis for C-type inactivation in a Shaker family voltage gated K+ channel

10.1101/2021.10.01.462615 ◽

2021 ◽

Author(s):

Ravikumar Reddi ◽

Kimberly Matulef ◽

Erika A. Riederer ◽

Matthew R. Whorton ◽

Francis I. Valiyaveetil

Keyword(s):

Crystal Structure ◽

Binding Sites ◽

Structural Changes ◽

Selectivity Filter ◽

Structural Basis ◽

K Channel ◽

Ion Binding ◽

Channel Pore ◽

Voltage Gated ◽

Ion Binding Sites

AbstractC-type inactivation is a process by which ion flux through a voltage-gated K+ (Kv) channel is regulated at the selectivity filter. While prior studies have indicated that C-type inactivation involves structural changes at the selectivity filter, the nature of the changes have not been resolved. Here we report the crystal structure of the Kv1.2 channel in a C-type inactivated state. The structure shows that C-type inactivation involves changes in the selectivity filter that disrupt the outer two ion binding sites in the filter. The changes at the selectivity filter propagate to the extracellular mouth and the turret regions of the channel pore. The structural changes observed are consistent with the functional hallmarks of C-type inactivation. This study highlights the intricate interplay between K+ occupancy at the ion binding sites and the interactions of the selectivity filter in determining the balance between the conductive and the inactivated conformations of the filter.

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K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions

Science Advances ◽

10.1126/sciadv.abc9174 ◽

2020 ◽

Vol 6 (44) ◽

pp. eabc9174

Author(s):

Marco Lolicato ◽

Andrew M. Natale ◽

Fayal Abderemane-Ali ◽

David Crottès ◽

Sara Capponi ◽

...

Keyword(s):

Potassium Channels ◽

Conformational Changes ◽

Structural Changes ◽

Selectivity Filter ◽

Anomalous Scattering ◽

X Ray Crystallography ◽

Gating Mechanisms ◽

Cellular Excitability ◽

Ion Binding Sites ◽

Channel Modulator

K2P potassium channels regulate cellular excitability using their selectivity filter (C-type) gate. C-type gating mechanisms, best characterized in homotetrameric potassium channels, remain controversial and are attributed to selectivity filter pinching, dilation, or subtle structural changes. The extent to which such mechanisms control C-type gating of innately heterodimeric K2Ps is unknown. Here, combining K2P2.1 (TREK-1) x-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and electrophysiology, we uncover unprecedented, asymmetric, potassium-dependent conformational changes that underlie K2P C-type gating. These asymmetric order-disorder transitions, enabled by the K2P heterodimeric architecture, encompass pinching and dilation, disrupt the S1 and S2 ion binding sites, require the uniquely long K2P SF2-M4 loop and conserved “M3 glutamate network,” and are suppressed by the K2P C-type gate activator ML335. These findings demonstrate that two distinct C-type gating mechanisms can operate in one channel and underscore the SF2-M4 loop as a target for K2P channel modulator development.

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Ion-binding properties of a K+ channel selectivity filter in different conformations

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1510526112 ◽

2015 ◽

Vol 112 (49) ◽

pp. 15096-15100 ◽

Cited By ~ 24

Author(s):

Shian Liu ◽

Paul J. Focke ◽

Kimberly Matulef ◽

Xuelin Bian ◽

Pierre Moënne-Loccoz ◽

...

Keyword(s):

Lipid Bilayers ◽

Selectivity Filter ◽

Titration Calorimetry ◽

K Channel ◽

Ion Binding ◽

Binding Properties ◽

Channel Opening ◽

Channel Selectivity ◽

Inactivation Process ◽

Low K

K+ channels are membrane proteins that selectively conduct K+ ions across lipid bilayers. Many voltage-gated K+ (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K+ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na+ or low [K+]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K+ ion binding to channels with an inactivated or conductive selectivity filter is different from K+ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.

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Accessibility of Cations to the Selectivity Filter of KcsA in the Inactivated State: An Equilibrium Binding Study

International Journal of Molecular Sciences ◽

10.3390/ijms20030689 ◽

2019 ◽

Vol 20 (3) ◽

pp. 689 ◽

Cited By ~ 2

Author(s):

Ana Giudici ◽

Maria Renart ◽

Clara Díaz-García ◽

Andrés Morales ◽

José Poveda ◽

...

Keyword(s):

Potassium Channel ◽

Binding Sites ◽

Cation Binding ◽

Selectivity Filter ◽

Ion Binding ◽

Channel State ◽

Equilibrium Conditions ◽

Equilibrium Binding ◽

Potassium Channel Kcsa ◽

Ion Binding Sites

Cation binding under equilibrium conditions has been used as a tool to explore the accessibility of permeant and nonpermeant cations to the selectivity filter in three different inactivated models of the potassium channel KcsA. The results show that the stack of ion binding sites (S1 to S4) in the inactivated filter models remain accessible to cations as they are in the resting channel state. The inactivated state of the selectivity filter is therefore “resting-like” under such equilibrium conditions. Nonetheless, quantitative differences in the apparent KD’s of the binding processes reveal that the affinity for the binding of permeant cations to the inactivated channel models, mainly K+, decreases considerably with respect to the resting channel. This is likely to cause a loss of K+ from the inactivated filter and consequently, to promote nonconductive conformations. The most affected site by the affinity loss seems to be S4, which is interesting because S4 is the first site to accommodate K+ coming from the channel vestibule when K+ exits the cell. Moreover, binding of the nonpermeant species, Na+, is not substantially affected by inactivation, meaning that the inactivated channels are also less selective for permeant versus nonpermeant cations under equilibrium conditions.

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Energetics and dynamics of a light-driven sodium-pumping rhodopsin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1703625114 ◽

2017 ◽

Vol 114 (27) ◽

pp. 7043-7048 ◽

Cited By ~ 38

Author(s):

Carl-Mikael Suomivuori ◽

Ana P. Gamiz-Hernandez ◽

Dage Sundholm ◽

Ville R. I. Kaila

Keyword(s):

Conformational Changes ◽

Binding Sites ◽

Rational Design ◽

Md Simulations ◽

Energy Transduction ◽

Quantum Molecular Dynamics ◽

Ion Pumps ◽

Ion Translocation ◽

Extracellular Side ◽

Key Residues

The conversion of light energy into ion gradients across biological membranes is one of the most fundamental reactions in primary biological energy transduction. Recently, the structure of the first light-activated Na+ pump, Krokinobacter eikastus rhodopsin 2 (KR2), was resolved at atomic resolution [Kato HE, et al. (2015) Nature 521:48–53]. To elucidate its molecular mechanism for Na+ pumping, we perform here extensive classical and quantum molecular dynamics (MD) simulations of transient photocycle states. Our simulations show how the dynamics of key residues regulate water and ion access between the bulk and the buried light-triggered retinal site. We identify putative Na+ binding sites and show how protonation and conformational changes gate the ion through these sites toward the extracellular side. We further show by correlated ab initio quantum chemical calculations that the obtained putative photocycle intermediates are in close agreement with experimental transient optical spectroscopic data. The combined results of the ion translocation and gating mechanisms in KR2 may provide a basis for the rational design of novel light-driven ion pumps with optogenetic applications.

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Relationship between Pore Occupancy and Gating in BK Potassium Channels

The Journal of General Physiology ◽

10.1085/jgp.200509482 ◽

2006 ◽

Vol 127 (5) ◽

pp. 557-576 ◽

Cited By ~ 37

Author(s):

Rebecca A. Piskorowski ◽

Richard W. Aldrich

Keyword(s):

Potassium Channels ◽

Conformational Changes ◽

Single Channel ◽

Bk Channels ◽

Ion Concentration ◽

Open Time ◽

Voltage Curve ◽

Activation Pathways ◽

Opening And Closing ◽

Kcsa Potassium Channel

Permeant ions can have significant effects on ion channel conformational changes. To further understand the relationship between ion occupancy and gating conformational changes, we have studied macroscopic and single-channel gating of BK potassium channels with different permeant monovalent cations. While the slopes of the conductance–voltage curve were reduced with respect to potassium for all permeant ions, BK channels required stronger depolarization to open only when thallium was the permeant ion. Thallium also slowed the activation and deactivation kinetics. Both the change in kinetics and the shift in the GV curve were dependent on the thallium passing through the permeation pathway, as well as on the concentration of thallium. There was a decrease in the mean open time and an increase in the number of short flicker closing events with thallium as the permeating ion. Mean closed durations were unaffected. Application of previously established allosteric gating models indicated that thallium specifically alters the opening and closing transition of the channel and does not alter the calcium activation or voltage activation pathways. Addition of a closed flicker state into the allosteric model can account for the effect of thallium on gating. Consideration of the thallium concentration dependence of the gating effects suggests that the flicker state may correspond to the collapsed selectivity filter seen in crystal structures of the KcsA potassium channel under the condition of low permeant ion concentration.

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