scholarly journals The Purified Mechanosensitive Channel TREK-1 Is Directly Sensitive to Membrane Tension

2013 ◽  
Vol 288 (38) ◽  
pp. 27307-27314 ◽  
Author(s):  
Catherine Berrier ◽  
Alexandre Pozza ◽  
Agnes de Lacroix de Lavalette ◽  
Solenne Chardonnet ◽  
Agnes Mesneau ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Mechanosensitive Channel ◽  
Positive Pressure ◽  
Mechanosensitive Channels ◽  
Membrane Tension ◽  
Animal Cells ◽  
Patch Clamp Recording ◽  
Electrophysiological Properties ◽  
Pressure Suction ◽  
Pore Domain

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.

Sensing bilayer tension: bacterial mechanosensitive channels and their gating mechanisms

2011 ◽  
Vol 39 (3) ◽  
pp. 733-740 ◽  
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.

scholarly journals Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

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.

scholarly journals Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

2021 ◽  
Author(s):  
Charles D Cox ◽  
Yixiao Zhang ◽  
Zijing Zhou ◽  
Thomas Walz ◽  
Boris Martinac
Keyword(s):  
Conformational Changes ◽  
Functional Characterization ◽  
Mechanosensitive Channel ◽  
Mechanical Forces ◽  
Mechanosensitive Channels ◽  
Membrane Tension ◽  
E Coli ◽  
Lipid Environment ◽  
Small Conductance

AbstractThe 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 E. 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. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. CDs can also activate the structurally unrelated MscL. 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. CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

scholarly journals Lipid Bilayer Pressure Profiles and Mechanosensitive Channel Gating

2004 ◽  
Vol 86 (6) ◽  
pp. 3496-3509 ◽  
Author(s):  
Justin Gullingsrud ◽  
Klaus Schulten
Keyword(s):  
Lipid Bilayer ◽  
Channel Gating ◽  
Mechanosensitive Channel ◽  
Pressure Profiles

scholarly journals Electrophysiological Properties of Substantia Gelatinosa Neurons in the Preparation of a Slice of Middle-Aged Rat Spinal Cord

2021 ◽  
Vol 13 ◽  
Author(s):  
Yang Li ◽  
Shanchu Su ◽  
Jiaqi Yu ◽  
Minjing Peng ◽  
Shengjun Wan ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Patch Clamp ◽  
Molecular Mechanisms ◽  
Substantia Gelatinosa ◽  
Membrane Properties ◽  
Middle Aged ◽  
Patch Clamp Recording ◽  
Electrophysiological Properties ◽  
Glutamatergic Neurons ◽  
Single Spike

A patch-clamp recording in slices generated from the brain or the spinal cord has facilitated the exploration of neuronal circuits and the molecular mechanisms underlying neurological disorders. However, the rodents that are used to generate the spinal cord slices in previous studies involving a patch-clamp recording have been limited to those in the juvenile or adolescent stage. Here, we applied an N-methyl-D-glucamine HCl (NMDG-HCl) solution that enabled the patch-clamp recordings to be performed on the superficial dorsal horn neurons in the slices derived from middle-aged rats. The success rate of stable recordings from substantia gelatinosa (SG) neurons was 34.6% (90/260). When stimulated with long current pulses, 43.3% (39/90) of the neurons presented a tonic-firing pattern, which was considered to represent γ-aminobutyric acid-ergic (GABAergic) signals. Presumptive glutamatergic neurons presented 38.9% (35/90) delayed and 8.3% (7/90) single-spike patterns. The intrinsic membrane properties of both the neuron types were similar but delayed (glutamatergic) neurons appeared to be more excitable as indicated by the decreased latency and rheobase values of the action potential compared with those of tonic (GABAergic) neurons. Furthermore, the glutamatergic neurons were integrated, which receive more excitatory synaptic transmission. We demonstrated that the NMDG-HCl cutting solution could be used to prepare the spinal cord slices of middle-aged rodents for the patch-clamp recording. In combination with other techniques, this preparation method might permit the further study of the functions of the spinal cord in the pathological processes that occur in aging-associated diseases.

Electrophysiological properties of channel formed by bovine FOF1 ATP synthase in planar lipid bilayer

2018 ◽  
Vol 1859 ◽  
pp. e84
Author(s):  
Andrea Urbani ◽  
Valentina Giorgio ◽  
Chimari Jiko ◽  
Janna F.M. Bogers ◽  
Duncan G.G. McMillan ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Atp Synthase ◽  
Planar Lipid Bilayer ◽  
Electrophysiological Properties ◽  
Fof1 Atp Synthase

Mechanosensitive Channels Gated by Membrane Tension

2008 ◽  
pp. 71-101 ◽  
Author(s):  
Paul Blount ◽  
Li Yuezhou ◽  
Paul C. Moe ◽  
Irene Iscla
Keyword(s):  
Mechanosensitive Channels ◽  
Membrane Tension

scholarly journals Development of a Novel Automated Ion Channel Recording Method Using “Inside-Out” Whole-Cell Membranes

2005 ◽  
Vol 10 (8) ◽  
pp. 806-813 ◽  
Author(s):  
Dmitry V. Vasilyev ◽  
Thomas L. Merrill ◽  
Mark R. Bowlby
Keyword(s):  
Ion Channels ◽  
Parallel Implementation ◽  
Drug Application ◽  
K Channel ◽  
Positive Pressure ◽  
Screening Methods ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Configuration ◽  
Inside Out

Efforts to develop novelmethods for recording from ion channels have been receiving increased attention in recent years. In this study, the authors report a unique “inside-out” whole-cell configuration of patch-clamp recording that has been developed. This method entails adding cells into a standard patch pipette and, with positive pressure, obtaining a gigaseal recording from a cell at the inside tip of the electrode. In this configuration, the cellmay be moved through the air, first rupturing part of the cellularmembrane and enabling bath access to the intracellular side of the membrane, and then into a series of wells containing differing solutions, enabling robotic control of all the steps in an experiment. The robotic system developed here fully automates the electrophysiological experiments, including gigaseal formation, obtaining whole-cell configuration, data acquisition, and drug application. Proof-of-principle experiments consisting of application of intracellularly acting potassium channel blockers to K+ channel cell lines resulted in a very rapid block, aswell as block reversal, of the current. This technique allows compound application directly to the intracellular side of ion channels and enables the dissociation of compound inactivities due to cellular barrier limitations. This technique should allow for parallel implementation of recording pipettes and the future development of larger array-based screening methods.

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

2018 ◽  
Vol 200 (23) ◽  
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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