Energetics of ångström-scale conformational changes in an RCK domain of the MthK K+ channel

Large conductance, voltage- and Ca2+-activated K+ (BKCa) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four α subunits of BKCa may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BKCa gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BKCa activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BKCa channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.

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

10.1101/2021.11.09.467928 ◽

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.

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Structure of the RCK Domain from the E. coli K+ Channel and Demonstration of Its Presence in the Human BK Channel

Neuron ◽

10.1016/s0896-6273(01)00236-7 ◽

2001 ◽

Vol 29 (3) ◽

pp. 593-601 ◽

Cited By ~ 219

Author(s):

Youxing Jiang ◽

Alexander Pico ◽

Martine Cadene ◽

Brian T. Chait ◽

Roderick MacKinnon

Keyword(s):

Bk Channel ◽

K Channel ◽

E Coli ◽

Rck Domain

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A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

The Journal of General Physiology ◽

10.1085/jgp.201010455 ◽

2010 ◽

Vol 136 (3) ◽

pp. 311-323 ◽

Cited By ~ 30

Author(s):

Antonella Gradogna ◽

Elena Babini ◽

Alessandra Picollo ◽

Michael Pusch

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Calcium Binding ◽

Chloride Channels ◽

Single Channel ◽

Bartter Syndrome ◽

K Channel ◽

Response Analysis ◽

Triple Mutant ◽

Calcium Binding Site

The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+. A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca2+-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca2+. Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H+ block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H+ -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing conformational changes underlying the complex gating of CLC-K channels.

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Voltage clamp fluorimetry studies of mammalian voltage-gated K+ channel gating

Biochemical Society Transactions ◽

10.1042/bst0351080 ◽

2007 ◽

Vol 35 (5) ◽

pp. 1080-1082 ◽

Cited By ~ 14

Author(s):

T.W. Claydon ◽

D. Fedida

Keyword(s):

Ion Channel ◽

Potassium Channel ◽

Real Time ◽

Voltage Clamp ◽

Conformational Changes ◽

Channel Gating ◽

K Channel ◽

Voltage Gated ◽

Kv Channels ◽

Health And Disease

VCF (voltage clamp fluorimetry) provides a powerful technique to observe real-time conformational changes that are associated with ion channel gating. The present review highlights the insights such experiments have provided in understanding Kv (voltage-gated potassium) channel gating, with particular emphasis on the study of mammalian Kv1 channels. Further applications of VCF that would contribute to our understanding of the modulation of Kv channels in health and disease are also discussed.

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Identification of Divalent Cation Coordinating Residues in a K+ Channel RCK Domain by NMR Spectroscopy

Biophysical Journal ◽

10.1016/j.bpj.2009.12.687 ◽

2010 ◽

Vol 98 (3) ◽

pp. 127a

Author(s):

Karin Abarca-Heidemann ◽

Jens Woehnert ◽

Brad S. Rothberg

Keyword(s):

Nmr Spectroscopy ◽

Divalent Cation ◽

K Channel ◽

Rck Domain

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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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