The Experts below are selected from a list of 1923 Experts worldwide ranked by ideXlab platform
Boris Martinac - One of the best experts on this subject based on the ideXlab platform.
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visualizatIon of the Mechanosensitive Ion Channel mscs under membrane tensIon
Nature, 2021Co-Authors: Yixiao Zhang, Boris Martinac, Csaba Daday, Charles D Cox, Bert L De Groot, Thomas WalzAbstract:Mechanosensitive Channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigatIon4. The bacterial Mechanosensitive Channel of small conductance, MscS, is one of the most extensively studied Mechanosensitive Channels4-8, but how it is regulated by membrane tensIon remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tensIon. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformatIon of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensatIon. Pore lipids are necessary to prevent Ion conductIon in the closed state. Gatekeeper lipids stabilize the closed conformatIon and dissociate with membrane tensIon, allowing the Channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tensIon, allowing the Channel to transitIon to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensatIon4,11.
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Fluorescence microscopy of piezo1 in droplet hydrogel bilayers
2019Co-Authors: Oskar B. Jaggers, Boris Martinac, Pietro Ridone, Matthew A B BakerAbstract:Mechanosensitive Ion Channels are membrane gated pores which are activated by mechanical stimuli. The focus of this study is on Piezo1, a newly discovered, large, mammalian, Mechanosensitive Ion Channel, which has been linked to diseases such as dehydrated hereditary stomatocytosis (Xerocytosis) and lymphatic dysplasia. Here we utilize an established in-vitro artificial bilayer system to interrogate single Piezo1 Channel activity. The droplet-hydrogel bilayer (DHB) system uniquely allows the simultaneous recording of electrical activity and fluorescence imaging of labelled protein. We successfully reconstituted fluorescently labelled Piezo1 Ion Channels in DHBs and verified activity using electrophysiology in the same system. We demonstrate successful insertIon and activatIon of hPiezo1-GFP in bilayers of varying compositIon. Furthermore, we compare the Piezo1 bilayer reconstitutIon with measurements of insertIon and activatIon of KcsA Channels to reproduce the Channel conductances reported in the literature. Together, our results showcase the use of DHBs for future experiments allowing simultaneous measurements of Ion Channel gating while visualising the Channel proteins using fluorescence.
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xenon inhibitIon of the mscl mechano sensitive Channel and the copb copper atpase under different conditIons suggests direct effects on these proteins
PLOS ONE, 2018Co-Authors: Evgeny Petrov, Gopalakrishnan Menon, Paul R Rohde, Andrew R Battle, Boris Martinac, Marc SoliozAbstract:Xenon is frequently used as a general anesthetic in humans, but the mechanism remains an issue of debate. While for some membrane proteins, a direct interactIon of xenon with the protein has been shown to be the inhibitory mechanism, other membrane protein functIons could be affected by changes of membrane properties due to partitIoning of the gas into the lipid bilayer. Here, the effect of xenon on a Mechanosensitive Ion Channel and a copper Ion-translocating ATPase was compared under different conditIons. Xenon inhibited spontaneous gating of the Escherichia coli mechano-sensitive mutant Channel MscL-G22E, as shown by patch-clamp recording techniques. Under high hydrostatic pressure, MscL-inhibitIon was reversed. Similarly, the activity of the Enterococcus hirae CopB copper ATPase, reconstituted into proteoliposomes, was inhibited by xenon. However, the CopB ATPase activity was also inhibited by xenon when CopB was in a solubilized state. These findings suggest that xenon acts by directly interacting with these proteins, rather than via indirect effects by altering membrane properties. Also, inhibitIon of copper transport may be a novel effect of xenon that contributes to anesthesia.
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activatIon of the Mechanosensitive Ion Channel mscl by mechanical stimulatIon of supported droplet hydrogel bilayers
Scientific Reports, 2017Co-Authors: Kadla R Rosholm, Paul R Rohde, Matthew A B Baker, Pietro Ridone, Yoshitaka Nakayama, Luis G Cuello, Lawrence K Lee, Boris MartinacAbstract:The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the functIon of Ion Channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigatIon of Mechanosensitive (MS) Ion Channels, since they are highly sensitive to their lipid environment. However, the activatIon of MS Ion Channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activatIon of the large conductance MS Channel of E. coli, (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injectIons of buffer, we induced quantifiable DHB tensIon, which could be related to Channel activity. The MscL activity response revealed that the droplet monolayer tensIon equilibrated over time, likely by insertIon of lipid from solutIon. Our study thus establishes a method to controllably activate MS Channels in DHBs and thereby advances studies of MS Channels in this novel platform.
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Hidden Markov analysis of improved bandwidth Mechanosensitive Ion Channel data
European Biophysics Journal, 2015Co-Authors: Ibrahim M. Almanjahie, Robin K Milne, R. Nazim Khan, Takeshi Nomura, Boris MartinacAbstract:The gating behaviour of a single Ion Channel can be described by hidden Markov models (HMMs), forming the basis for statistical analysis of patch clamp data. Extensive improved bandwidth (25 kHz, 50 kHz) data from the Mechanosensitive Channel of large conductance in Escherichia coli were analysed using HMMs, and HMMs with a moving average adjustment for filtering. The aim was to determine the number of levels, and mean current, mean dwell time and proportIon of time at each level. Parameter estimates for HMMs with a moving average adjustment for low-pass filtering were obtained using an expectatIon-maximisatIon algorithm that depends on a generalisatIon of Baum’s forward–backward algorithm. This results in a simpler algorithm than those based on meta-states and a much smaller parameter space; hence, the computatIonal load is substantially reduced. In additIon, this algorithm maximises the actual log-likelihood rather than that for a related meta-state process. Comprehensive data analyses and comparisons across all our data sets have consistently shown five subconducting levels in additIon to the fully open and closed levels for this Channel.
Philip A Gottlieb - One of the best experts on this subject based on the ideXlab platform.
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Mechanosensitive Ion Channel piezo2 is inhibited by d gsmtx4
Channels, 2017Co-Authors: Constanza Alcaino, Kaitlyn R. Knutson, Philip A Gottlieb, Gianrico Farrugia, Arthur BeyderAbstract:Enterochromaffin (EC) cells are the primary mechanosensors of the gastrointestinal (GI) epithelium. In response to mechanical stimuliEC cells release serotonin (5-hydroxytryptamine; 5-HT). The molecular details ofEC cell mechanosensitivity are poorly understood. Recently, our group found that human and mouseEC cells express the Mechanosensitive Ion Channel Piezo2. The Mechanosensitive currents in a humanEC cell model QGP-1 were blocked by the Mechanosensitive Channel blocker D-GsMTx4. In the present study we aimed to characterize the effects of the Mechanosensitive Ion Channel inhibitor spider peptide D-GsMTx4 on the mechanically stimulated currents from both QGP-1 and human Piezo2 transfected HEK-293 cells. We found co-localizatIon of 5-HT and Piezo2 in QGP-1 cells by immunohistochemistry. QGP-1 Mechanosensitive currents had biophysical properties similar to dose-dependently Piezo2 and were inhibited by D-GsMTx4. In response to direct displacement of cell membranes, human Piezo2 transiently expressed in HEK-293 cells produced robust rapidly activating and inactivating inward currents. D-GsMTx4 reversibly and dose-dependently inhibited both the potency and efficacy of Piezo2 currents in response to mechanical force. Our data demonstrate an effective inhibitIon of Piezo2 Mechanosensitive currents by the spider peptide D-GsMTx4.
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Mechanosensitive Ion Channel piezo2 is important for enterochromaffin cell response to mechanical forces
The Journal of Physiology, 2017Co-Authors: David R. Linden, Kaitlyn R. Knutson, Fan Wang, Constanza Alcaino, Simon J Gibbons, Purna C Kashyap, Madhusudan Grover, Richard A Oeckler, Philip A GottliebAbstract:Key points The gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransductIon is unknown. In the present study, we show, for the first time, that the Mechanosensitive Ion Channel Piezo2 is specifically expressed by the human and mouse EC cells. ActivatIon of Piezo2 by mechanical forces results in a characteristic Ionic current, the release of serotonin and stimulatIon of gastrointestinal secretIon. Piezo2 inhibitIon by drugs or molecular knockdown decreases Mechanosensitive currents, serotonin release and downstream physiological effects. The results of the present study suggest that the Mechanosensitive Ion Channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransductIon. Abstract The enterochromaffin (EC) cell in the gastrointestinal (GI) epithelium is the source of nearly all systemic serotonin (5-hydroxytryptamine; 5-HT), which is an important neurotransmitter and endocrine, autocrine and paracrine hormone. The EC cell is a specialized mechanosensor, and it is well known that it releases 5-HT in response to mechanical forces. However, the EC cell mechanotransductIon mechanism is unknown. The present study aimed to determine whether Piezo2 is involved in EC cell mechanosensatIon. Piezo2 mRNA was expressed in human jejunum and mouse mucosa from all segments of the small bowel. Piezo2 immunoreactivity localized specifically within EC cells of human and mouse small bowel epithelium. The EC cell model released 5-HT in response to stretch, and had Piezo2 mRNA and protein, as well as a mechanically-sensitive inward non-selective catIon current characteristic of Piezo2. Both inward currents and 5-HT release were inhibited by Piezo2 small interfering RNA and antagonists (Gd3+ and D-GsMTx4). Jejunum mucosal pressure increased 5-HT release and short-circuit current via submucosal 5-HT3 and 5-HT4 receptors. Pressure-induced secretIon was inhibited by the Mechanosensitive Ion Channel antagonists gadolinium, ruthenium red and D-GsMTx4. We conclude that the EC cells in the human and mouse small bowel GI epithelium selectively express the Mechanosensitive Ion Channel Piezo2, and also that activatIon of Piezo2 by force leads to inward currents, 5-HT release and an increase in mucosal secretIon. Therefore, Piezo2 is critical to EC cell mechanosensitivity and downstream physiological effects.
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Mechanosensitive Ion Channel piezo2 is important for enterochromaffin cell response to mechanical forces
The Journal of Physiology, 2017Co-Authors: David R. Linden, Kaitlyn R. Knutson, Fan Wang, Constanza Alcaino, Simon J Gibbons, Purna C Kashyap, Madhusudan Grover, Richard A Oeckler, Philip A GottliebAbstract:Key points The gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransductIon is unknown. In the present study, we show, for the first time, that the Mechanosensitive Ion Channel Piezo2 is specifically expressed by the human and mouse EC cells. ActivatIon of Piezo2 by mechanical forces results in a characteristic Ionic current, the release of serotonin and stimulatIon of gastrointestinal secretIon. Piezo2 inhibitIon by drugs or molecular knockdown decreases Mechanosensitive currents, serotonin release and downstream physiological effects. The results of the present study suggest that the Mechanosensitive Ion Channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransductIon. Abstract The enterochromaffin (EC) cell in the gastrointestinal (GI) epithelium is the source of nearly all systemic serotonin (5-hydroxytryptamine; 5-HT), which is an important neurotransmitter and endocrine, autocrine and paracrine hormone. The EC cell is a specialized mechanosensor, and it is well known that it releases 5-HT in response to mechanical forces. However, the EC cell mechanotransductIon mechanism is unknown. The present study aimed to determine whether Piezo2 is involved in EC cell mechanosensatIon. Piezo2 mRNA was expressed in human jejunum and mouse mucosa from all segments of the small bowel. Piezo2 immunoreactivity localized specifically within EC cells of human and mouse small bowel epithelium. The EC cell model released 5-HT in response to stretch, and had Piezo2 mRNA and protein, as well as a mechanically-sensitive inward non-selective catIon current characteristic of Piezo2. Both inward currents and 5-HT release were inhibited by Piezo2 small interfering RNA and antagonists (Gd3+ and D-GsMTx4). Jejunum mucosal pressure increased 5-HT release and short-circuit current via submucosal 5-HT3 and 5-HT4 receptors. Pressure-induced secretIon was inhibited by the Mechanosensitive Ion Channel antagonists gadolinium, ruthenium red and D-GsMTx4. We conclude that the EC cells in the human and mouse small bowel GI epithelium selectively express the Mechanosensitive Ion Channel Piezo2, and also that activatIon of Piezo2 by force leads to inward currents, 5-HT release and an increase in mucosal secretIon. Therefore, Piezo2 is critical to EC cell mechanosensitivity and downstream physiological effects.
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two segments of the human piezo1 Mechanosensitive Ion Channel can reassemble into a functIonal unit
Biophysical Journal, 2014Co-Authors: Philip A Gottlieb, Chilman Bae, Thomas M Suchyna, Frederick SachsAbstract:We created clones that covalently linked a fluorescent protein to PIEZO1 in order to image PIEZO1 on the cell surface. Inserting mCherry1 in internal positIons of the PIEZO1 protein, we found a construct at positIon 1591 that yielded a protein whose Channel kinetics were nearly identical to wild type. Through cotransfectIon, we imaged PIEZO1 (mCherry) and TREK-1 (GFP) on HEK cells. These two Mechanosensitive Channels were at the cell surface in completely different physical domains. PIEZO1 appeared as random clusters but TREK tended to follow cytoskeleton tracks.Given the stability of the PEIZO1 protein with a fluorescent group at 1591, we asked if the PIEZO1 protein could be split at positIon 1591 and still functIon. Fragment 1 (1-1591) was fused to mCherry, and fragment 2 (1592-2520) to GFP. Co-expressIon of the two segments showed currents in whole cell recording, cell-attached patches, and outside-out patches. The biophysical characteristics were nearly identical to the wild type Channel, having a reversal potential around 0 mV and voltage dependent inactivatIon in whole cell and cell attached mode.We next asked if either fragment alone was functIonal. The C-terminal fragment produced Mechanosensitive currents in outside-out patches but the N terminal segment (1-1591) did not in any recording mode. We then shortened the C terminal fragment from its C- and N- termini using the outside-out patches to screen for current. We obtained Mechanosensitive currents from a fragment of ∼ 450 amino acids, so the fundamental properties of the giant PIEZO1 Channel are contained in this small fragment. This protein segment appears to contain the mechanosensor and the gate, and establishes that eight putative transmembrane domains are required for pore formatIon. None of the shortened fragments displayed whole cell currents.
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the Mechanosensitive Ion Channel piezo1 is inhibited by the peptide gsmtx4
Biochemistry, 2011Co-Authors: Chilman Bae, Frederick Sachs, Philip A GottliebAbstract:Cells can respond to mechanical stress by gating Mechanosensitive Ion Channels (MSCs). The cloning of Piezo1, a eukaryotic catIon selective MSC, defines a new system for studying mechanical transductIon at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous catIonic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentratIons reversibly inhibited ∼80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibitIon was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided associatIon and dissociatIon rates of 7.0 × 105 M–1 s–1 and 0.11 s–1, respectively, and a KD of ∼155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier beha...
Frederick Sachs - One of the best experts on this subject based on the ideXlab platform.
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two segments of the human piezo1 Mechanosensitive Ion Channel can reassemble into a functIonal unit
Biophysical Journal, 2014Co-Authors: Philip A Gottlieb, Chilman Bae, Thomas M Suchyna, Frederick SachsAbstract:We created clones that covalently linked a fluorescent protein to PIEZO1 in order to image PIEZO1 on the cell surface. Inserting mCherry1 in internal positIons of the PIEZO1 protein, we found a construct at positIon 1591 that yielded a protein whose Channel kinetics were nearly identical to wild type. Through cotransfectIon, we imaged PIEZO1 (mCherry) and TREK-1 (GFP) on HEK cells. These two Mechanosensitive Channels were at the cell surface in completely different physical domains. PIEZO1 appeared as random clusters but TREK tended to follow cytoskeleton tracks.Given the stability of the PEIZO1 protein with a fluorescent group at 1591, we asked if the PIEZO1 protein could be split at positIon 1591 and still functIon. Fragment 1 (1-1591) was fused to mCherry, and fragment 2 (1592-2520) to GFP. Co-expressIon of the two segments showed currents in whole cell recording, cell-attached patches, and outside-out patches. The biophysical characteristics were nearly identical to the wild type Channel, having a reversal potential around 0 mV and voltage dependent inactivatIon in whole cell and cell attached mode.We next asked if either fragment alone was functIonal. The C-terminal fragment produced Mechanosensitive currents in outside-out patches but the N terminal segment (1-1591) did not in any recording mode. We then shortened the C terminal fragment from its C- and N- termini using the outside-out patches to screen for current. We obtained Mechanosensitive currents from a fragment of ∼ 450 amino acids, so the fundamental properties of the giant PIEZO1 Channel are contained in this small fragment. This protein segment appears to contain the mechanosensor and the gate, and establishes that eight putative transmembrane domains are required for pore formatIon. None of the shortened fragments displayed whole cell currents.
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positively charged residues on gsmtx4 are crucial for inhibitIon of the Mechanosensitive Ion Channel piezo1
Biophysical Journal, 2013Co-Authors: Radhakrishnan Gnanasambandam, Frederick Sachs, Kazuhisa Nishizawa, Thomas M SuchynaAbstract:GsMTx4 is the only known peptide inhibitor of Mechanosensitive Channels. It is a gating modifier and presumably acts by modifying the arrangement of lipid molecules in the Channel-lipid interface. Like other ICK peptide toxins GsMTx4 has a hydrophobic face which presumably facilitates partitIoning of the molecule to the water/membrane interface of the bilayer. Prior studies also show that charged lysine residues may further influence the positIoning of the peptide in the bilayer through interactIons with the headgroups of anIonic lipids. Coarse-grained simulatIons of GsMTx4 in the lipid bilayer suggest the existence of two binding modes: a shallow-binding mode and a deep-binding mode. The deep-binding mode in particular involves interactIons between positively charged residues on the peptide and carbonyl atoms of the headgroups of inner-leaflet lipids. To provide insight into these interactIons for GsMTx4 functIon, we investigated if reversing the charge by mutating lysine residues to glutamate affected the activity of GsMTx4. Each lysine residue in GsMTx4 was individually changed to glutamate to generate six mutants: K8E, K15E, K20E, K22E, K25E and K28E. When tested on outside-out patches from HEK cells transfected with mPiezo1 cDNA, all KtoE mutants showed reduced ability to inhibit the Channel. In additIon to reduced activity, K15E peptide altered the inactivatIon rate of the mPiezo1 currents. Even at saturating concentratIons, K28E mutatIon reduced the activity of the peptide to 23% of that of the wild-type peptide. This study shows that charge reversing mutatIons to lysine residues diminish the efficacy of GsMTx4, and this phenomenon may involve disruptIon of peptide partitIoning into the membrane and/or peptide-induced bending of the membrane bilayer.
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the Mechanosensitive Ion Channel piezo1 is inhibited by the peptide gsmtx4
Biochemistry, 2011Co-Authors: Chilman Bae, Frederick Sachs, Philip A GottliebAbstract:Cells can respond to mechanical stress by gating Mechanosensitive Ion Channels (MSCs). The cloning of Piezo1, a eukaryotic catIon selective MSC, defines a new system for studying mechanical transductIon at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous catIonic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentratIons reversibly inhibited ∼80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibitIon was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided associatIon and dissociatIon rates of 7.0 × 105 M–1 s–1 and 0.11 s–1, respectively, and a KD of ∼155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier beha...
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the Mechanosensitive Ion Channel piezo1 is inhibited by the peptide gsmtx4
Biochemistry, 2011Co-Authors: Chilman Bae, Frederick Sachs, Philip A GottliebAbstract:Cells can respond to mechanical stress by gating Mechanosensitive Ion Channels (MSCs). The cloning of Piezo1, a eukaryotic catIon selective MSC, defines a new system for studying mechanical transductIon at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous catIonic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentratIons reversibly inhibited ∼80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibitIon was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided associatIon and dissociatIon rates of 7.0 × 10(5) M(-1) s(-1) and 0.11 s(-1), respectively, and a K(D) of ∼155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier behavior, GsMTx4 produced an ∼30 mmHg rightward shift in the pressure-gating curve and was active on closed Channels. In contrast, streptomycin, a nonspecific inhibitor of catIonic MSCs, showed the use-dependent inhibitIon characteristic of open Channel block. The peptide did not block currents of the mechanical Channel TREK-1 on outside-out patches. Whole-cell Piezo1 currents were also reversibly inhibited by GsMTx4, and although the off rate was nearly identical to that of outside-out patches, differences were observed for the on rate. The ability of GsMTx4 to target the mechanosensitivity of Piezo1 supports the use of this Channel in high-throughput screens for pharmacological agents and diagnostic assays.
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a Mechanosensitive Ion Channel regulating cell volume
American Journal of Physiology-cell Physiology, 2010Co-Authors: Susan Z Hua, Philip A Gottlieb, Jinseok Heo, Frederick SachsAbstract:Cells respond to a hyposmotic challenge by swelling and then returning toward the resting volume, a process known as the regulatory volume decrease or RVD. The sensors for this process have been proposed to include catIonic Mechanosensitive Ion Channels that are opened by membrane tensIon. We tested this hypothesis using a microfluidic device to measure cell volume and the peptide GsMTx4, a specific inhibitor of catIonic Mechanosensitive Channels. GsMTx4 had no effect on RVD in primary rat astrocytes or Madin-Darby canine kidney (MDCK) cells but was able to completely inhibit RVD and the associated Ca(2+) uptake in normal rat kidney (NRK-49F) cells in a dose-dependent manner. Gadolinium (Gd(3+)), a nonspecific blocker of many Mechanosensitive Channels, inhibited RVD and Ca(2+) uptake in all three cell types, demonstrating the existence of at least two types of volume sensors. Single-Channel stretch-activated currents are present in outside-out patches from NRK-49F, MDCK, and astrocytes, and they are reversibly inhibited by GsMTx4. While Mechanosensitive Channels are involved in volume regulatIon, their role for volume sensing is specialized. The NRK cells form a stable platform from which to screen drugs that affect volume regulatIon via mechanosensory Channels and as a sensitive system to clone the Channel.
Valeria Vasquez - One of the best experts on this subject based on the ideXlab platform.
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c elegans pezo 1 is a Mechanosensitive Ion Channel involved in food sensatIon
The Journal of General Physiology, 2022Co-Authors: Jonathan R M Millet, Luis O Romero, Jungsoo Lee, Briar Bell, Valeria VasquezAbstract:PIEZO Channels are force sensors essential for physiological processes, including baroreceptIon and proprioceptIon. The Caenorhabditis elegans genome encodes an orthologue gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contractIon and relaxatIon are modulated by mechanical stimulatIon elicited by food content. Whether pezo-1 encodes a Mechanosensitive Ion Channel and contributes to pharyngeal functIon remains unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal functIon. Knockout (KO) and gain-of-functIon (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, as well as sensing osmolarity and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display Mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a Mechanosensitive Channel. Our findings reveal that pezo-1 is a Mechanosensitive Ion Channel that regulates food sensatIon in worms.
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c elegans pezo 1 is a Mechanosensitive Ion Channel involved in food sensatIon
bioRxiv, 2021Co-Authors: Jonathan R M Millet, Luis O Romero, Jungsoo Lee, Briar Bell, Valeria VasquezAbstract:PIEZO Channels are force sensors essential for physiological processes, including baroreceptIon and proprioceptIon. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contractIon and relaxatIon are modulated by mechanical stimulatIon elicited by food content. Whether pezo-1 encodes a Mechanosensitive Ion Channel and contributes to pharyngeal functIon remain unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal functIon. Knockout (KO) and gain-of-functIon (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity, and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display Mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a Mechanosensitive Channel. Our findings reveal that pezo-1 is a Mechanosensitive Ion Channel that regulates food sensatIon in worms.
Ching Kung - One of the best experts on this subject based on the ideXlab platform.
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helicity membrane incorporatIon orientatIon and thermal stability of the large conductance Mechanosensitive Ion Channel from e coli
Biochimica et Biophysica Acta, 1998Co-Authors: Isaiah T Arkin, Ching Kung, Sergei Sukharev, Paul Blount, Axel T BrungerAbstract:In this report, we present structural studies on the large conductance Mechanosensitive Ion Channel (MscL) from E. coli in detergent micelles and lipid vesicles. Both transmissIon Fourier transform infrared spectroscopy and circular dichroism (CD) spectra indicate that the protein is highly helical in detergents as well as liposomes. The secondary structure of the proteins was shown to be highly resistant towards denaturatIon (25-95 degrees C) based on an ellipticity thermal profile. Amide H+/D+ exchange was shown to be extensive (ca. 66%), implying that two thirds of the protein are water accessible. MscL, reconstituted in oriented lipid bilayers, was shown to possess a net bilayer orientatIon using dichroic ratios measured by attenuated total-reflectIon Fourier transform infrared spectroscopy. Here, we present and discuss this initial set of structural data on this new family of Ion-Channel proteins.
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activities of a Mechanosensitive Ion Channel in an e coli mutant lacking the major lipoprotein
The Journal of Membrane Biology, 1993Co-Authors: Andrzej Kubalski, Boris Martinac, Kityin Ling, Julius Adler, Ching KungAbstract:The activity of the Mechanosensitive (MS) Ion Channels in membrane patches, excised fromE. coli spheroplasts, was analyzed using the patch-clamp technique. Outer membranes from a mutant lacking the major lipoprotein (Lpp) and its wildtype parent were examined. The MS-Channel activities in the wild-type membrane rarely revealed substates at the time resolutIon used. These Channels showed a stretch sensitivity indicated by the IISP (the suctIon for ane-fold increase in Channel open probability) of 4.9 mm Hg suctIon. The MS-Channel activities oflpp included a prominent substate and showed a weaker mechano-sensitivity with an 1/S p of 10.0 mm Hg. Whereas small amphipaths (chlorpromazine, trinitrophenol) or a larger amphipath (lysolecithin) all activated the MS Channel in the wild-type membrane under minimal suctIon, only the larger lysolecithin could activate the MS Channel in thelpp membranes. After lysolecithin additIon, thelpp membrane became more effective in transmitting the stretch force to the MS Channel, as indicated by a steepening of the Boltzmann curve. We discuss one interpretatIon of these results, in which the major lipoprotein serves as a natural amphipath inserted in the inner monolayer and the loss of this natural amphipath makes the bilayer less able to transmit the gating force.
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a Mechanosensitive Ion Channel in schizosaccharomyces pombe
The EMBO Journal, 1992Co-Authors: Xinliang Zhou, Ching KungAbstract:Protoplast protuberances (blebs) of Schizosaccharomyces pombe were examined using the patch-clamp technique. In additIon to several voltage-gated Ion Channels, we encountered the activities of a Mechanosensitive Ion Channel with a conductance of 180 pS. Microscopic currents of one or two units were observed in some excised patches and ensemble currents of several tens of units were observed in all blebs examined in whole-bleb configuratIon. This Channel opens at pressures of cm Hg applied to whole blebs and it passes catIons, including Ca2+. It is inactivated by membrane depolarizatIons and blocked by Gd3+. We discuss the possible functIons of such a Channel, including its activatIon upon cell cycle dependent cytoskeletal reorganizatIons.
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a Mechanosensitive Channel in whole cells and in membrane patches of the fungus uromyces
Science, 1991Co-Authors: Xinliang Zhou, Harvey C. Hoch, Mary Ann Stumpf, Ching KungAbstract:Bean leaf stomata provide a topographical signal that induces germlings of the phytopathogen Uromyces appendiculatus to develop specialized infectIon structures. Protoplasts from germ tubes of this fungus, when examined with patch-clamp electrodes, displayed the activities of a 600-picosiemen Mechanosensitive Ion Channel. This Channel passes a variety of catIons, including Ca2+, and is blocked by Gd3+ at 50 micromolar. This Channel could transduce the membrane stress induced by the leaf topography into an influx of Ions, including Ca2+, that may trigger differentiatIon.
