Allosteric Coupling of the Inner Activation Gate to the Outer Pore of a Potassium Channel

The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation pKa of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.

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Rapid outer pore movements after opening in a KV1 potassium channel are revealed by TMRM fluorescence from the S3-S4 linker, and modulated by extracellular potassium

Channels ◽

10.4161/chan.3.1.7369 ◽

2009 ◽

Vol 3 (1) ◽

pp. 3-5 ◽

Cited By ~ 4

Author(s):

Moninder Vaid ◽

Andrew Horne ◽

Thomas Claydon ◽

David Fedida

Keyword(s):

Potassium Channel ◽

Extracellular Potassium ◽

Outer Pore

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Outer Pore Residues Control the H+ and K+ Sensitivity of the Arabidopsis Potassium Channel AKT3

The Plant Cell ◽

10.1105/tpc.003244 ◽

2002 ◽

Vol 14 (8) ◽

pp. 1859-1868 ◽

Cited By ~ 33

Author(s):

Dietmar Geiger ◽

Dirk Becker ◽

Benoit Lacombe ◽

Rainer Hedrich

Keyword(s):

Potassium Channel ◽

Outer Pore

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Interactions of Extracellular Potassium, Calcium, Magnesium and Hydrogen with the outer Pore of the Cardiac Potassium Channel hERG

Biophysical Journal ◽

10.1016/j.bpj.2012.11.1499 ◽

2013 ◽

Vol 104 (2) ◽

pp. 267a

Author(s):

Shaun Rafael ◽

Eric Lau ◽

Ryan Fraiser ◽

Vincent Carbone ◽

Kevin Ha ◽

...

Keyword(s):

Potassium Channel ◽

Extracellular Potassium ◽

Outer Pore ◽

Calcium Magnesium

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Mechanism of C-type inactivation in the hERG potassium channel

Science Advances ◽

10.1126/sciadv.abd6203 ◽

2021 ◽

Vol 7 (5) ◽

pp. eabd6203

Author(s):

Jing Li ◽

Rong Shen ◽

Bharat Reddy ◽

Eduardo Perozo ◽

Benoît Roux

Keyword(s):

Potassium Channel ◽

Critical Role ◽

Cardiac Cells ◽

Side Chain ◽

Missense Mutations ◽

Nonspecific Binding ◽

Allosteric Coupling ◽

Naturally Occurring ◽

Long Time ◽

Dynamics Simulations

The fast C-type inactivation displayed by the voltage-activated potassium channel hERG plays a critical role in the repolarization of cardiac cells, and malfunction caused by nonspecific binding of drugs or naturally occurring missense mutations affecting inactivation can lead to pathologies. Because of its impact on human health, understanding the molecular mechanism of C-type inactivation in hERG represents an advance of paramount importance. Here, long–time scale molecular dynamics simulations, free energy landscape calculations, and electrophysiological experiments are combined to address the structural and functional impacts of several disease-associated mutations. Results suggest that C-type inactivation in hERG is associated with an asymmetrical constricted-like conformation of the selectivity filter, identifying F627 side-chain rotation and the hydrogen bond between Y616 and N629 as key determinants. Comparison of hERG with other K+ channels suggests that C-type inactivation depends on the degree of opening of the intracellular gate via the filter-gate allosteric coupling.

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Divalent Ions and H+ Block the Cardiac Potassium Channel HERG at an Outer Pore Site

Biophysical Journal ◽

10.1016/j.bpj.2017.11.1674 ◽

2018 ◽

Vol 114 (3) ◽

pp. 293a

Author(s):

Gagandeep Singh ◽

Kavaldeep Singh ◽

Souad Hamade ◽

Alan Miller

Keyword(s):

Potassium Channel ◽

Divalent Ions ◽

Outer Pore

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The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating

The Journal of General Physiology ◽

10.1085/jgp.200509321 ◽

2005 ◽

Vol 126 (3) ◽

pp. 227-241 ◽

Cited By ~ 23

Author(s):

Gayathri Krishnamoorthy ◽

Jingyi Shi ◽

David Sept ◽

Jianmin Cui

Keyword(s):

Conformational Changes ◽

K Channel ◽

Bkca Channel ◽

Bkca Channels ◽

Allosteric Coupling ◽

Channel Opening ◽

Activation Gate ◽

Rck Domain ◽

Dual Activation ◽

Α Subunits

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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Interactions of Extracellular Potassium, Calcium and Hydrogen with the Outer Pore of the Cardiac Potassium Channel Herg

Biophysical Journal ◽

10.1016/j.bpj.2011.11.1807 ◽

2012 ◽

Vol 102 (3) ◽

pp. 330a

Author(s):

Kevin Ha ◽

Steve Tseng ◽

Kristeen Pareja ◽

Vikram Makhijani ◽

Attilio Macrito ◽

...

Keyword(s):

Potassium Channel ◽

Extracellular Potassium ◽

Outer Pore

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Probing Allosteric Coupling of a Constitutively Open Mutant of the Ion Channel KcsA using Solid State NMR

10.1101/567024 ◽

2019 ◽

Author(s):

Zhiyu Sun ◽

Yunyao Xu ◽

Dongyu Zhang ◽

Ann E McDermott

Keyword(s):

Solid State ◽

Binding Affinity ◽

Solid State Nmr ◽

Selectivity Filter ◽

Wild Type ◽

Allosteric Coupling ◽

Channel Inactivation ◽

Activation Gate ◽

Inactivation Process ◽

Potassium Binding

AbstractTransmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the mean open time of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that has the pH gate open across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about 4 orders of magnitude weaker than that of wild type KcsA at neutral pH and is comparable to the value for wild type KcsA at low pH (pH ∼ 3.5). This result strongly supports our assertion that the open pH gate allosterically effects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.Significance statementInactivation of potassium channels controls mean open times and provides exquisite control over biological processes. In the highly conserved C-type inactivation process, opening of the activation gate causes subsequent inactivation. We test whether the open state of the channel simply has a poor ability to bind the K+ ion. Previously, activated and inactivated states were stabilized using truncations or a significant pH drop. Here, we use the H25R/E118A constitutively open mutant of KcsA and also observe a large drop in potassium binding affinity. This provides strong evidence that channel opening causes an allosteric loss of ion affinity, and that the central feature of this universal channel inactivation process is loss of ion affinity at the selectivity filter.

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