potassium channel kcsa
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2021 ◽  
Vol 118 (46) ◽  
pp. e2113229118
Author(s):  
Radda Rusinova ◽  
Changhao He ◽  
Olaf S. Andersen

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. Drug regulation of membrane protein function thus may be mediated by both direct interactions with the protein and drug-induced alterations of bilayer properties, in which the latter will alter the energetics of protein conformational changes. To tease apart these mechanisms, we examine how the prototypical, proton-gated bacterial potassium channel KcsA is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). All tested drugs inhibited KcsA activity, and the changes in the different gating steps varied with bilayer thickness, suggesting a coupling to the bilayer. Examining the correlations between changes in KcsA gating steps and bilayer properties reveals that drug-induced regulation of membrane protein function indeed involves bilayer-mediated mechanisms. Both direct, either specific or nonspecific, binding and bilayer-mediated mechanisms therefore are likely to be important whenever there is overlap between the concentration ranges at which a drug alters membrane protein function and bilayer properties. Because changes in bilayer properties will impact many diverse membrane proteins, they may cause indiscriminate changes in protein function.



2021 ◽  
Vol 14 ◽  
Author(s):  
Masayuki Iwamoto ◽  
Shigetoshi Oiki

Once membrane potential changes or ligand binding activates the ion channel, the activity of the channel is finely modulated by the fluctuating membrane environment, involving local lipid composition and membrane tension. In the age of post-structural biology, the factors in the membrane that affect the ion channel function and how they affect it are a central concern among ion channel researchers. This review presents our strategies for elucidating the molecular mechanism of membrane effects on ion channel activity. The membrane’s diverse and intricate effects consist of chemical and physical processes. These elements can be quantified separately using lipid bilayer methods, in which a membrane is reconstructed only from the components of interest. In our advanced lipid bilayer platform (contact bubble bilayer, CBB), physical features of the membrane, such as tension, are freely controlled. We have elucidated how the specific lipid or membrane tension modulates the gating of a prototypical potassium channel, KcsA, embedded in the lipid bilayer. Our results reveal the molecular mechanism of the channel for sensing and responding to the membrane environment.



2020 ◽  
Vol 117 (13) ◽  
pp. 7171-7175
Author(s):  
Zhiyu Sun ◽  
Yunyao Xu ◽  
Dongyu Zhang ◽  
Ann E. McDermott

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.



Author(s):  
Anna Olerinyova ◽  
Adar Sonn Segev ◽  
Joseph Gault ◽  
Cedric Eichmann ◽  
Johannes Schimpf ◽  
...  

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid - like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane mimetic systems at the single particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterising the particle size, sample purity and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high - resolution studies of membrane protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene - maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers - in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non - functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.



2019 ◽  
Vol 1861 (10) ◽  
pp. 183029 ◽  
Author(s):  
José A. Poveda ◽  
A. Marcela Giudici ◽  
M. Lourdes Renart ◽  
Oscar Millet ◽  
Andrés Morales ◽  
...  


2019 ◽  
Vol 3 ◽  
pp. 100009 ◽  
Author(s):  
Yunyao Xu ◽  
Ann E. McDermott


2019 ◽  
Vol 20 (3) ◽  
pp. 689 ◽  
Author(s):  
Ana Giudici ◽  
Maria Renart ◽  
Clara Díaz-García ◽  
Andrés Morales ◽  
José Poveda ◽  
...  

Cation binding under equilibrium conditions has been used as a tool to explore the accessibility of permeant and nonpermeant cations to the selectivity filter in three different inactivated models of the potassium channel KcsA. The results show that the stack of ion binding sites (S1 to S4) in the inactivated filter models remain accessible to cations as they are in the resting channel state. The inactivated state of the selectivity filter is therefore “resting-like” under such equilibrium conditions. Nonetheless, quantitative differences in the apparent KD’s of the binding processes reveal that the affinity for the binding of permeant cations to the inactivated channel models, mainly K+, decreases considerably with respect to the resting channel. This is likely to cause a loss of K+ from the inactivated filter and consequently, to promote nonconductive conformations. The most affected site by the affinity loss seems to be S4, which is interesting because S4 is the first site to accommodate K+ coming from the channel vestibule when K+ exits the cell. Moreover, binding of the nonpermeant species, Na+, is not substantially affected by inactivation, meaning that the inactivated channels are also less selective for permeant versus nonpermeant cations under equilibrium conditions.



2018 ◽  
Vol 115 (51) ◽  
pp. 13117-13122 ◽  
Author(s):  
Masayuki Iwamoto ◽  
Shigetoshi Oiki

Molecular mechanisms underlying channel-membrane interplay have been extensively studied. Cholesterol, as a major component of the cell membrane, participates either in specific binding to channels or via modification of membrane physical features. Here, we examined the action of various sterols (cholesterol, epicholesterol, etc.) on a prototypical potassium channel (KcsA). Single-channel current recordings of the KcsA channel were performed in a water-in-oil droplet bilayer (contact bubble bilayer) with a mixed phospholipid composition (azolectin). Upon membrane perfusion of sterols, the activated gate at acidic pH closed immediately, irrespective of the sterol species. During perfusion, we found that the contacting bubbles changed their shapes, indicating alterations in membrane physical features. Absolute bilayer tension was measured according to the principle of surface chemistry, and inherent bilayer tension was ∼5 mN/m. All tested sterols decreased the tension, and the nonspecific sterol action to the channel was likely mediated by the bilayer tension. Purely mechanical manipulation that reduced bilayer tension also closed the gate, whereas the resting channel at neutral pH never activated upon increased tension. Thus, rather than conventional stretch activation, the channel, once ready to activate by acidic pH, changes the open probability through the action of bilayer tension. This constitutes a channel regulating modality by two successive stimuli. In the contact bubble bilayer, inherent bilayer tension was high, and the channel remained boosted. In the cell membrane, resting tension is low, and it is anticipated that the ready-to-activate channel remains closed until bilayer tension reaches a few millinewton/meter during physiological and pathological cellular activities.



2018 ◽  
Vol 556 ◽  
pp. 70-77 ◽  
Author(s):  
Amit Vaish ◽  
Shaobin Guo ◽  
Richard M. Murray ◽  
Peter J. Grandsard ◽  
Qing Chen


2016 ◽  
Vol 148 (2) ◽  
pp. 119-132 ◽  
Author(s):  
Dorothy M. Kim ◽  
Igor Dikiy ◽  
Vikrant Upadhyay ◽  
David J. Posson ◽  
David Eliezer ◽  
...  

The process of ion channel gating—opening and closing—involves local and global structural changes in the channel in response to external stimuli. Conformational changes depend on the energetic landscape that underlies the transition between closed and open states, which plays a key role in ion channel gating. For the prokaryotic, pH-gated potassium channel KcsA, closed and open states have been extensively studied using structural and functional methods, but the dynamics within each of these functional states as well as the transition between them is not as well understood. In this study, we used solution nuclear magnetic resonance (NMR) spectroscopy to investigate the conformational transitions within specific functional states of KcsA. We incorporated KcsA channels into lipid bicelles and stabilized them into a closed state by using either phosphatidylcholine lipids, known to favor the closed channel, or mutations designed to trap the channel shut by disulfide cross-linking. A distinct state, consistent with an open channel, was uncovered by the addition of cardiolipin lipids. Using selective amino acid labeling at locations within the channel that are known to move during gating, we observed at least two different slowly interconverting conformational states for both closed and open channels. The pH dependence of these conformations and the predictable disruptions to this dependence observed in mutant channels with altered pH sensing highlight the importance of conformational heterogeneity for KcsA gating.



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