scholarly journals Structural Basis for pH-Gating of the K+ Channel TWIK1 at the Selectivity Filter

2021 ◽  
Author(s):  
Toby S Turney ◽  
Vivian Li ◽  
Stephen G Brohawn
Keyword(s):  
Conformational Changes ◽  
Pancreatic Beta Cells ◽  
Low Ph ◽  
Selectivity Filter ◽  
Structural Basis ◽  
K Channel ◽  
Open Conformation ◽  
Gating Mechanism ◽  
Lipid Nanodiscs ◽  
Pore Domain

TWIK1 is a widely expressed pH-gated two-pore domain K+ channel (K2P) that contributes to cardiac rhythm generation and insulin release from pancreatic beta cells. TWIK1 displays unique properties among K2Ps including low basal activity and inhibition by extracellular protons through incompletely understood mechanisms. Here, we present cryo-EM structures of TWIK1 in lipid nanodiscs at high and low pH that reveal a novel gating mechanism at the K+ selectivity filter. At high pH, TWIK1 adopts an open conformation. At low pH, protonation of an extracellular histidine results in a cascade of conformational changes that close the channel by sealing the top of the selectivity filter, displacing the helical cap to block extracellular ion access pathways, and opening gaps for lipid block of the intracellular cavity. These data provide a mechanistic understanding for extracellular pH-gating of TWIK1 and show how diverse mechanisms have evolved to gate the selectivity filter of K+ channels.

scholarly journals The gating cycle of a K+ channel at atomic resolution

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

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

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

scholarly journals Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

2019 ◽  
Vol 116 (27) ◽  
pp. 13358-13367 ◽  
Author(s):  
Mette H. Poulsen ◽  
Anahita Poshtiban ◽  
Viktoria Klippenstein ◽  
Valentina Ghisi ◽  
Andrew J. R. Plested
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Uv Light ◽  
Unnatural Amino Acid ◽  
Selectivity Filter ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis ◽  
Mutant Receptors ◽  
Pore Domain

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the vertebrate nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type iGluRs using genetically encoded unnatural amino acid (UAA) photocross-linkers, p-benzoyl-l-phenylalanine (BzF) and p-azido-l-phenylalanine (AzF). We introduced these UAAs at sites throughout the TMD of the GluA2 receptor and characterized the mutants in patch-clamp recordings, exposing them to glutamate and ultraviolet (UV) light. This approach revealed a range of optical effects on the activity of mutant receptors. We found evidence for an interaction between the Pre-M1 and the M4 TMD helix during desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter in the M2 segment, had extraordinarily broad effects on gating and desensitization. This observation suggests coupling to other parts of the receptor and like in other tetrameric ion channels, selectivity filter gating.

scholarly journals Structural basis for adenylation and thioester bond formation in the ubiquitin E1

2019 ◽  
Vol 116 (31) ◽  
pp. 15475-15484 ◽  
Author(s):  
Zachary S. Hann ◽  
Cheng Ji ◽  
Shaun K. Olsen ◽  
Xuequan Lu ◽  
Michaelyn C. Lux ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Biochemical Analysis ◽  
Bond Formation ◽  
Structural Basis ◽  
Closed Conformation ◽  
Open Conformation ◽  
Thioester Bond ◽  
Catalytic Cysteine ◽  
Conjugating Enzymes ◽  
Insight Into

The ubiquitin (Ub) and Ub-like (Ubl) protein-conjugation cascade is initiated by E1 enzymes that catalyze Ub/Ubl activation through C-terminal adenylation, thioester bond formation with an E1 catalytic cysteine, and thioester bond transfer to Ub/Ubl E2 conjugating enzymes. Each of these reactions is accompanied by conformational changes of the E1 domain that contains the catalytic cysteine (Cys domain). Open conformations of the Cys domain are associated with adenylation and thioester transfer to E2s, while a closed conformation is associated with pyrophosphate release and thioester bond formation. Several structures are available for Ub E1s, but none has been reported in the open state before pyrophosphate release or in the closed state. Here, we describe the structures ofSchizosaccharomyces pombeUb E1 in these two states, captured using semisynthetic Ub probes. In the first, with a Ub-adenylate mimetic (Ub-AMSN) bound, the E1 is in an open conformation before release of pyrophosphate. In the second, with a Ub-vinylsulfonamide (Ub-AVSN) bound covalently to the catalytic cysteine, the E1 is in a closed conformation required for thioester bond formation. These structures provide further insight into Ub E1 adenylation and thioester bond formation. Conformational changes that accompany Cys-domain rotation are conserved for SUMO and Ub E1s, but changes in Ub E1 involve additional surfaces as mutational and biochemical analysis of residues within these surfaces alter Ub E1 activities.

scholarly journals Selectivity filter instability dominates the low intrinsic activity of the TWIK-1 K2P K+ channel

2019 ◽  
Vol 295 (2) ◽  
pp. 610-618
Author(s):  
Ehsan Nematian-Ardestani ◽  
Firdaus Abd-Wahab ◽  
Franck C. Chatelain ◽  
Han Sun ◽  
Marcus Schewe ◽  
...  
Keyword(s):  
Functional Activity ◽  
Selectivity Filter ◽  
Intrinsic Activity ◽  
K Channel ◽  
Open Probability ◽  
Gating Mechanism ◽  
Hydrophobic Barrier ◽  
K2p Channels ◽  
Voltage Dependent ◽  
Low Levels

Two-pore domain K+ (K2P) channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/KCNK1) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by posttranslational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating this further, we observed that the low activity of the SF gate appears to arise from the inefficiency of K+ in stabilizing an active (i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb+, NH4+, and Cs+, strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via an SF-mediated gating mechanism, but we found here that only very strong nonphysiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb+ currents are potently inhibited by intracellular K+ (IC50 = 2.8 mm). We conclude that TWIK-1 displays unique SF gating properties among the family of K2P channels. In particular, the apparent instability of the conductive conformation of the TWIK-1 SF in the presence of K+ appears to dominate the low levels of intrinsic functional activity observed when the channel is expressed at the cell surface.

Na+-induced inward rectification in the two-pore domain K+ channel, TASK-2

2005 ◽  
Vol 288 (1) ◽  
pp. F162-F169 ◽  
Author(s):  
Michael J. Morton ◽  
Sarah Chipperfield ◽  
Abdulrahman Abohamed ◽  
Asipu Sivaprasadarao ◽  
Malcolm Hunter
Keyword(s):  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Selectivity Filter ◽  
Inward Rectification ◽  
K Channel ◽  
Open Probability ◽  
Whole Cell ◽  
Single Channel Currents ◽  
Pore Domain

TASK-2 is a member of the two-pore domain K+ (K2P) channel family that is expressed at high levels in several epithelia, including the proximal tubule. In common with the other TASK channels, TASK-2 is sensitive to changes in extracellular pH. We have expressed human TASK-2 in Chinese hamster ovary cells and studied whole cell and single-channel activity by patch clamp. The open probability of K2P channels is generally independent of voltage, yielding linear current-voltage ( I- V) curves. Despite these properties, we found that these channels showed distinct inward rectification immediately on the establishment of whole cell clamp, which became progressively less pronounced with time. This rectification was due to intracellular Na+ but was unaffected by polyamines or Mg2+ (agents that cause rectification in Kir channels). Rectification was concentration- and voltage-dependent and could be reversibly induced by switching between Na+-rich and Na+-free bath solutions. In excised inside-out patches, Na+ reduced the amplitude of single-channel currents, indicative of rapid block and unblock of the pore. Mutations in the selectivity filter abolished Na+-induced rectification, suggesting that Na+ binds within the selectivity filter in wild-type channels. This sensitivity to intracellular Na+ may be an additional potential regulatory mechanism of TASK-2 channels.

scholarly journals Structural basis for pH gating of the two-pore domain K+ channel TASK2

Nature ◽  
2020 ◽  
Vol 586 (7829) ◽  
pp. 457-462
Author(s):  
Baobin Li ◽  
Robert A. Rietmeijer ◽  
Stephen G. Brohawn
Keyword(s):  
Structural Basis ◽  
K Channel ◽  
Pore Domain

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 Structural basis of ion permeation gating in Slo2.1 K+ channels

2013 ◽  
Vol 142 (5) ◽  
pp. 523-542 ◽  
Author(s):  
Priyanka Garg ◽  
Alison Gardner ◽  
Vivek Garg ◽  
Michael C. Sanguinetti
Keyword(s):  
Selectivity Filter ◽  
Structural Basis ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Ion Permeation ◽  
Channel Activation ◽  
K Channels ◽  
Voltage Gated ◽  
Channel Function ◽  
Activation Gate

The activation gate of ion channels controls the transmembrane flux of permeant ions. In voltage-gated K+ channels, the aperture formed by the S6 bundle crossing can widen to open or narrow to close the ion permeation pathway, whereas the selectivity filter gates ion flux in cyclic-nucleotide gated (CNG) and Slo1 channels. Here we explore the structural basis of the activation gate for Slo2.1, a weakly voltage-dependent K+ channel that is activated by intracellular Na+ and Cl−. Slo2.1 channels were heterologously expressed in Xenopus laevis oocytes and activated by elevated [NaCl]i or extracellular application of niflumic acid. In contrast to other voltage-gated channels, Slo2.1 was blocked by verapamil in an activation-independent manner, implying that the S6 bundle crossing does not gate the access of verapamil to its central cavity binding site. The structural basis of Slo2.1 activation was probed by Ala scanning mutagenesis of the S6 segment and by mutation of selected residues in the pore helix and S5 segment. Mutation to Ala of three S6 residues caused reduced trafficking of channels to the cell surface and partial (K256A, I263A, Q273A) or complete loss (E275A) of channel function. P271A Slo2.1 channels trafficked normally, but were nonfunctional. Further mutagenesis and intragenic rescue by second site mutations suggest that Pro271 and Glu275 maintain the inner pore in an open configuration by preventing formation of a tight S6 bundle crossing. Mutation of several residues in S6 and S5 predicted by homology modeling to contact residues in the pore helix induced a gain of channel function. Substitution of the pore helix residue Phe240 with polar residues induced constitutive channel activation. Together these findings suggest that (1) the selectivity filter and not the bundle crossing gates ion permeation and (2) dynamic coupling between the pore helix and the S5 and S6 segments mediates Slo2.1 channel activation.

scholarly journals Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

2018 ◽  
Author(s):  
Mette H. Poulsen ◽  
Anahita Poshtiban ◽  
Viktoria Klippenstein ◽  
Valentina Ghisi ◽  
Plested Andrew
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Uv Light ◽  
Unnatural Amino Acid ◽  
Selectivity Filter ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis ◽  
Mutant Receptors ◽  
Pore Domain

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of AMPA-type iGluRs using genetically-encoded unnatural amino acid (UAA) photo-crosslinkers, p-benzoyl-L-phenylalanine (BzF) and p-azido-L-phenylalanine (AzF). We introduced UAAs at sites throughout the TMD of the GluA2 receptor and characterized these mutants in patch-clamp recordings, exposing them to glutamate and UV light. This approach revealed a range of optical effects on the activity of mutant receptors. We found evidence that an interaction between the Pre-M1 and the M4 TMD helix was essential for normal activation and desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter, had extraordinarily broad effects on gating and desensitization. This observation suggests coupling to other parts of the receptor and like in other tetrameric channels, selectivity filter gating.

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