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

2020 ◽  
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.

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

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.

scholarly journals Regulation of Kir Channels by Intracellular pH and Extracellular K+

2004 ◽  
Vol 123 (4) ◽  
pp. 441-454 ◽  
Author(s):  
Anke Dahlmann ◽  
Min Li ◽  
ZhongHua Gao ◽  
Deirdre McGarrigle ◽  
Henry Sackin ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Ph Sensor ◽  
Outer Part ◽  
Selectivity Filter ◽  
Inward Rectification ◽  
Ion Binding ◽  
Enhanced Sensitivity ◽  
Internal Ph ◽  
Additional Channel

ROMK channels are regulated by internal pH (pHi) and extracellular K+ (K+o). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H+, increasing the apparent pKa for inhibition by ∼0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K+o, consistent with coupling between the responses to pHi and K+o. The ion selectivity sequence of the activation of the channel by cations was K+ ≅ Rb+ > NH4+ >> Na+, similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K+ from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K+o. We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K+o between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K+ to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba2+ block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H+. We conclude that decreasing pHI increases the sensitivity of ROMK2 channels to K+o by altering the properties of the selectivity filter.

scholarly journals Selectivity filter ion binding affinity determines inactivation in a potassium channel

2020 ◽  
Vol 117 (47) ◽  
pp. 29968-29978
Author(s):  
Céline Boiteux ◽  
David J. Posson ◽  
Toby W. Allen ◽  
Crina M. Nimigean
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Bk Channels ◽  
Selectivity Filter ◽  
Ion Binding ◽  
X Ray Crystallography ◽  
Extracellular Side ◽  
Opening And Closing ◽  
Low K ◽  
Conductive State

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.

scholarly journals Mechanism of Ion Permeation in Skeletal Muscle Chloride Channels

1997 ◽  
Vol 110 (5) ◽  
pp. 551-564 ◽  
Author(s):  
Christoph Fahlke ◽  
Christine Dürr ◽  
Alfred L. George
Keyword(s):  
Skeletal Muscle ◽  
Conformational Changes ◽  
Binding Sites ◽  
Chloride Channels ◽  
Ion Selectivity ◽  
Ion Binding ◽  
Ion Permeation ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Ion Binding Sites

Voltage-gated Cl− channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl− channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I− and related anions. Extracellular and intracellular I− exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br−) and polyatomic (SCN−, NO3−, CH3SO3−) anions. In addition, I− block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I− causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I− slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined KD values for I− block in three distinct kinetic states and found that binding of I− to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I− binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.

scholarly journals A structural framework for unidirectional transport by a bacterial ABC exporter

2020 ◽  
Vol 117 (32) ◽  
pp. 19228-19236
Author(s):  
Chengcheng Fan ◽  
Jens T. Kaiser ◽  
Douglas C. Rees
Keyword(s):  
Conformational Changes ◽  
Iron Homeostasis ◽  
Atp Hydrolysis ◽  
Transmembrane Helix ◽  
Reverse Direction ◽  
Conformational States ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Structural Framework ◽  
Glutathione Derivatives

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

scholarly journals Identifying dynamic, partially occupied residues using anomalous scattering

2019 ◽  
Author(s):  
Serena Rocchio ◽  
Ramona Duman ◽  
Kamel El Omari ◽  
Vitaliy Mykhaylyk ◽  
Zhen Yan ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Structural Changes ◽  
Structural Data ◽  
Anomalous Scattering ◽  
Long Wavelength ◽  
X Ray Crystallography ◽  
Structural Ensembles ◽  
Analysis Strategy ◽  
Chain Conformations

AbstractX-ray crystallography is generally used to take single snapshots of a protein’s conformation. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes Residual Anomalous and Electron Density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data collection and analysis strategy for partially occupied iodine anomalous signals. Using the long wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with as low as ~12% occupancy is demonstrated. The number and position of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly dynamic sections of crystal structures.SynopsisStructural studies on partially occupied, dynamic protein systems by crystallography are difficult. We present methods here for detecting these states in crystals.

scholarly journals Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

2015 ◽  
Vol 112 (50) ◽  
pp. 15366-15371 ◽  
Author(s):  
Joshua B. Brettmann ◽  
Darya Urusova ◽  
Marco Tonelli ◽  
Jonathan R. Silva ◽  
Katherine A. Henzler-Wildman
Keyword(s):  
Ion Selectivity ◽  
Selectivity Filter ◽  
Structural Basis ◽  
Functional Coupling ◽  
Dynamic Coupling ◽  
Allosteric Coupling ◽  
Allosteric Communication ◽  
Dependent Inactivation ◽  
Ion Binding Sites ◽  
Essential Connection

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.

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

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.

scholarly journals Pore-forming transmembrane domains control ion selectivity and selectivity filter conformation in the KirBac1.1 potassium channel

2021 ◽  
Vol 153 (5) ◽  
Author(s):  
Marcos Matamoros ◽  
Colin G. Nichols
Keyword(s):  
Single Molecule ◽  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Selectivity Filter ◽  
K Channel ◽  
Physical Interaction ◽  
Conformationally Constrained ◽  
Single Molecule Fret ◽  
High K ◽  
Low K

Potassium (K+) channels are membrane proteins with the remarkable ability to very selectively conduct K+ ions across the membrane. High-resolution structures have revealed that dehydrated K+ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na+ flux in the absence of K+ and introduce significant proton conductance. Consistent with K+ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K+ conditions but undergoes transitions to dilated low-FRET states in high-Na+/low-K+ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K+ and Na+ dependence of SF dynamics to higher K+ and lower Na+ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K+-induced SF configuration versus nonselective dilated conformations.

scholarly journals Structural Plasticity of the Selectivity Filter in Cation Channels

2021 ◽  
Vol 12 ◽  
Author(s):  
Kitty Hendriks ◽  
Carl Öster ◽  
Adam Lange
Keyword(s):  
Structural Plasticity ◽  
Selectivity Filter ◽  
Cation Channels ◽  
Ion Binding ◽  
Atomic Structures ◽  
Conserved Sequence ◽  
Molecular Determinant ◽  
A Cell ◽  
Ion Binding Sites ◽  
Dynamic States

Ion channels allow for the passage of ions across biological membranes, which is essential for the functioning of a cell. In pore loop channels the selectivity filter (SF) is a conserved sequence that forms a constriction with multiple ion binding sites. It is becoming increasingly clear that there are several conformations and dynamic states of the SF in cation channels. Here we outline specific modes of structural plasticity observed in the SFs of various pore loop channels: disorder, asymmetry, and collapse. We summarize the multiple atomic structures with varying SF conformations as well as asymmetric and more dynamic states that were discovered recently using structural biology, spectroscopic, and computational methods. Overall, we discuss here that structural plasticity within the SF is a key molecular determinant of ion channel gating behavior.

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