[K+] Induced Conformational Dynamics of the Selectivity Filter of KcsA Monitored by Solid-State NMR

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity 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 exhibits an open pH gate 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 four 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 affects 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.

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Transport-Relevant Protein Conformational Dynamics and Water Dynamics on Multiple Time Scales in an Archetypal Proton Channel: Insights from Solid-State NMR

Journal of the American Chemical Society ◽

10.1021/jacs.7b12464 ◽

2018 ◽

Vol 140 (4) ◽

pp. 1514-1524 ◽

Cited By ~ 17

Author(s):

Venkata S. Mandala ◽

Martin D. Gelenter ◽

Mei Hong

Keyword(s):

Solid State ◽

Time Scales ◽

Solid State Nmr ◽

Conformational Dynamics ◽

Water Dynamics ◽

Proton Channel ◽

Multiple Time Scales ◽

Multiple Time ◽

Protein Conformational Dynamics

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Conformational Dynamics of a Seven Transmembrane Helical Protein Anabaena Sensory Rhodopsin Probed by Solid-State NMR

Journal of the American Chemical Society ◽

10.1021/ja411633w ◽

2014 ◽

Vol 136 (7) ◽

pp. 2833-2842 ◽

Cited By ~ 62

Author(s):

Daryl B. Good ◽

Shenlin Wang ◽

Meaghan E. Ward ◽

Jochem Struppe ◽

Leonid S. Brown ◽

...

Keyword(s):

Solid State ◽

Solid State Nmr ◽

Conformational Dynamics ◽

Sensory Rhodopsin ◽

Helical Protein

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Conformational changes upon gating of KirBac1.1 into an open-activated state revealed by solid-state NMR and functional assays

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915010117 ◽

2020 ◽

Vol 117 (6) ◽

pp. 2938-2947 ◽

Cited By ~ 5

Author(s):

Reza Amani ◽

Collin G. Borcik ◽

Nazmul H. Khan ◽

Derek B. Versteeg ◽

Maryam Yekefallah ◽

...

Keyword(s):

Solid State ◽

Conformational Changes ◽

Solid State Nmr ◽

Lipid Binding ◽

Selectivity Filter ◽

Conformational Exchange ◽

Lipid Mixture ◽

Kir Channels ◽

Activated State ◽

Activation Gate

The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.

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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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