Pocket delipidation induced by membrane tension or modification leads to a structurally analogous mechanosensitive channel state

Mechanosensitive channels are detected in all cells and are speculated to play a key role in many functions including osmoregulation, growth, hearing, balance, and touch. In prokaryotic cells, a direct gating of mechanosensitive channels by membrane tension was clearly demonstrated because the purified channels could be functionally reconstituted in a lipid bilayer. No such evidence has been presented yet in the case of mechanosensitive channels from animal cells. TREK-1, a two-pore domain K+ channel, was the first animal mechanosensitive channel identified at the molecular level. It is the target of a large variety of agents such as volatile anesthetics, neuroprotective agents, and antidepressants. We have produced the mouse TREK-1 in yeast, purified it, and reconstituted the protein in giant liposomes amenable to patch clamp recording. The protein exhibited the expected electrophysiological properties in terms of kinetics, selectivity, and pharmacology. Negative pressure (suction) applied through the pipette had no effect on the channel, but positive pressure could completely and reversibly close the channel. Our interpretation of these data is that the intrinsic tension in the lipid bilayer is sufficient to maximally activate the channel, which can be closed upon modification of the tension. These results indicate that TREK-1 is directly sensitive to membrane tension.

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Connecting the Dots between Mechanosensitive Channel Abundance, Osmotic Shock, and Survival at Single-Cell Resolution

Journal of Bacteriology ◽

10.1128/jb.00460-18 ◽

2018 ◽

Vol 200 (23) ◽

Cited By ~ 1

Author(s):

Griffin Chure ◽

Heun Jin Lee ◽

Akiko Rasmussen ◽

Rob Phillips

Keyword(s):

Cell Survival ◽

Single Cell ◽

Osmotic Shock ◽

Single Cells ◽

Mechanosensitive Channel ◽

Membrane Tension ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Structural Methods

ABSTRACTRapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks,Escherichia coliand other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. InE. coli, one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single-cell resolution using quantitative fluorescence microscopy. We conducted these experiments in anE. colistrain which is lacking all mechanosensitive channel genes save for MscL, whose expression was tuned across 3 orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-typeE. colicells through proteomic studies and quantitative Western blotting. Furthermore, we observed zero survival events in cells with fewer than ∼100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival, as well as regulation of their activity.IMPORTANCEMechanosensitive (MS) channels are transmembrane protein complexes which open and close in response to changes in membrane tension as a result of osmotic shock. Despite extensive biophysical characterization, the contribution of these channels to cell survival remains largely unknown. In this work, we used quantitative video microscopy to measure the abundance of a single species of MS channel in single cells, followed by their survival after a large osmotic shock. We observed total death of the population with fewer than ∼100 channels per cell and determined that approximately 500 to 700 channels were needed for 80% survival. The number of channels we found to confer nearly full survival is consistent with the counts of the numbers of channels in wild-type cells in several earlier studies. These results prompt further studies to dissect the contribution of other channel species to survival.

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The cytoplasmic cage domain of the mechanosensitive channel MscS is a sensor of macromolecular crowding

The Journal of General Physiology ◽

10.1085/jgp.201311114 ◽

2014 ◽

Vol 143 (5) ◽

pp. 543-557 ◽

Cited By ~ 30

Author(s):

Ian Rowe ◽

Andriy Anishkin ◽

Kishore Kamaraju ◽

Kenjiro Yoshimura ◽

Sergei Sukharev

Keyword(s):

Transmembrane Domain ◽

Feedback Mechanism ◽

Axial Position ◽

Mechanosensitive Channel ◽

Membrane Tension ◽

The Core ◽

Specific Feedback ◽

Dynamics Simulations ◽

Association Result ◽

Small Conductance

Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1–TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1–TM2 shaft and the state of the associated hollow cytoplasmic domain (“cage”). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the β domain of the cage. We found that mutations destabilizing the TM3b–β interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.

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Curcumin activation of a bacterial mechanosensitive channel underlies its membrane permeability and adjuvant properties

PLoS Pathogens ◽

10.1371/journal.ppat.1010198 ◽

2021 ◽

Vol 17 (12) ◽

pp. e1010198

Author(s):

Robin Wray ◽

Irene Iscla ◽

Paul Blount

Keyword(s):

Membrane Permeability ◽

Natural Compound ◽

Bacterial Species ◽

Antibacterial Properties ◽

Cell Lysis ◽

Membrane Permeabilization ◽

Mechanosensitive Channel ◽

Bacterial Membrane ◽

Physiological Effects ◽

Membrane Tension

Curcumin, a natural compound isolated from the rhizome of turmeric, has been shown to have antibacterial properties. It has several physiological effects on bacteria including an apoptosis-like response involving RecA, membrane permeabilization, inhibiting septation, and it can also work synergistically with other antibiotics. The mechanism by which curcumin permeabilizes the bacterial membrane has been unclear. Most bacterial species contain a Mechanosensitive channel of large conductance, MscL, which serves the function of a biological emergency release valve; these large-pore channels open in response to membrane tension from osmotic shifts and, to avoid cell lysis, allow the release of solutes from the cytoplasm. Here we show that the MscL channel underlies the membrane permeabilization by curcumin as well as its synergistic properties with other antibiotics, by allowing access of antibiotics to the cytoplasm; MscL also appears to have an inhibitory role in septation, which is enhanced when activated by curcumin.

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Sensing bilayer tension: bacterial mechanosensitive channels and their gating mechanisms

Biochemical Society Transactions ◽

10.1042/bst0390733 ◽

2011 ◽

Vol 39 (3) ◽

pp. 733-740 ◽

Cited By ~ 20

Author(s):

Ian R. Booth ◽

Tim Rasmussen ◽

Michelle D. Edwards ◽

Susan Black ◽

Akiko Rasmussen ◽

...

Keyword(s):

Pore Diameter ◽

Ionic Composition ◽

Unique Property ◽

Mechanosensitive Channel ◽

Mechanosensitive Channels ◽

Membrane Tension ◽

Cellular Integrity ◽

Sense And Respond ◽

Cellular Behaviour ◽

Lipid Interaction

Mechanosensitive channels sense and respond to changes in bilayer tension. In many respects, this is a unique property: the changes in membrane tension gate the channel, leading to the transient formation of open non-selective pores. Pore diameter is also high for the bacterial channels studied, MscS and MscL. Consequently, in cells, gating has severe consequences for energetics and homoeostasis, since membrane depolarization and modification of cytoplasmic ionic composition is an immediate consequence. Protection against disruption of cellular integrity, which is the function of the major channels, provides a strong evolutionary rationale for possession of such disruptive channels. The elegant crystal structures for these channels has opened the way to detailed investigations that combine molecular genetics with electrophysiology and studies of cellular behaviour. In the present article, the focus is primarily on the structure of MscS, the small mechanosensitive channel. The description of the structure is accompanied by discussion of the major sites of channel–lipid interaction and reasoned, but limited, speculation on the potential mechanisms of tension sensing leading to gating.

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Gain-of-function Mutations Reveal Expanded Intermediate States and a Sequential Action of Two Gates in MscL

The Journal of General Physiology ◽

10.1085/jgp.200409118 ◽

2005 ◽

Vol 125 (2) ◽

pp. 155-170 ◽

Cited By ~ 61

Author(s):

Andriy Anishkin ◽

Chien-Sung Chiang ◽

Sergei Sukharev

Keyword(s):

Mechanosensitive Channel ◽

Membrane Tension ◽

Wild Type ◽

Gain Of Function ◽

Sequential Action ◽

Intermediate States ◽

A Minor ◽

Open States ◽

Rate Limiting ◽

S States

The tension-driven gating transition in the large mechanosensitive channel MscL proceeds through detectable states of intermediate conductance. Gain-of-function (GOF) mutants with polar or charged substitutions in the main hydrophobic gate display altered patterns of subconducting states, providing valuable information about gating intermediates. Here we present thermodynamic analysis of several GOF mutants to clarify the nature and position of low-conducting conformations in the transition pathway. Unlike wild-type (WT) MscL, which predominantly occupies the closed and fully open states with very brief substates, the mild V23T GOF mutant frequently visits a multitude of short-lived subconducting states. Severe mutants V23D and G22N open in sequence: closed (C) → low-conducting substate (S) → open (O), with the first subtransition occurring at lower tensions. Analyses of equilibrium state occupancies as functions of membrane tension show that the C→S subtransition in WT MscL is associated with only a minor conductance increment, but the largest in-plane expansion and free energy change. The GOF substitutions strongly affect the first subtransition by reducing area (ΔA) and energy (ΔE) changes between C and S states commensurably with the severity of mutation. GOF mutants also exhibited a considerably larger ΔE associated with the second (S→O) subtransition, but a ΔA similar to WT. The area changes indicate that closed conformations of GOF mutants are physically preexpanded. The tension dependencies of rate constants for channel closure (koff) predict different positions of rate-limiting barriers on the energy-area profiles for WT and GOF MscL. The data support the two-gate mechanism in which the first subtransition (C→S) can be viewed as opening of the central (M1) gate, resulting in an expanded water-filled “leaky” conformation. Strong facilitation of this step by polar GOF substitutions suggests that separation of M1 helices associated with hydration of the pore in WT MscL is the major energetic barrier for opening. Mutants with a stabilized S1 gate demonstrate impeded transitions from low-conducting substates to the fully open state, whereas extensions of S1–M1 linkers result in a much higher probability of reverse O→S transitions. These data strongly suggest that the bulk of conductance gain in the second subtransition (S→O) occurs through the opening of the NH2-terminal (S1) gate and the linkers are coupling elements between the M1 and S1 gates.

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Lipid–protein forces predict conformational changes in a mechanosensitive channel

European Biophysics Journal ◽

10.1007/s00249-020-01488-z ◽

2020 ◽

Author(s):

Csaba Daday ◽

Bert L. de Groot

Keyword(s):

Potassium Channel ◽

Conformational Change ◽

Conformational Changes ◽

Mechanosensitive Channel ◽

Computational Studies ◽

Interaction Patterns ◽

Membrane Tension ◽

Lipid Interaction

AbstractThe mechanosensitive TREK-2 potassium channel, a member of the K2P family, has essential physiological roles and is, therefore, a pharmaceutical target. A combination of experimental and computational studies have established that of the two known conformations, “up” and “down”, membrane tension directly favors the “up” state, which displays a higher conductance. However, these studies did not reveal the exact mechanism by which the membrane affects the channel conformation. In this work, we show that changes in protein–lipid interaction patterns suffice in predicting this conformational change, and pinpoint potentially important residues involved in this phenomenon.

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Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2104820118 ◽

2021 ◽

Vol 118 (36) ◽

pp. e2104820118

Author(s):

Charles D. Cox ◽

Yixiao Zhang ◽

Zijing Zhou ◽

Thomas Walz ◽

Boris Martinac

Keyword(s):

Escherichia Coli ◽

Conformational Changes ◽

Functional Characterization ◽

Mechanosensitive Channel ◽

Mechanical Forces ◽

Mechanosensitive Channels ◽

Membrane Tension ◽

Lipid Environment ◽

Small Conductance

The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the “force-from-lipids” principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

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