Gsmtx4

Manufactured by Alomone

Sourced in Israel

GsMTx4 is a potent and selective inhibitor of mechanosensitive ion channels. It functions by blocking the opening of these channels in response to mechanical stimuli.

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Lab products found in correlation

17 protocols using gsmtx4

Pharmacological Modulation of Ion Channels

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All salts were acquired from Sigma and were of analytical grade or better. Dooku1: 2-[(2,6-Dichlorobenzyl)thio)-5-(1H-pyrrol-2-yl)-1,3,4-oxadiazole (Sigma-Aldrich, France). Yoda1: 2- [5-[[(2,6-Dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]pyrazine (Tocris, France). Charybdotoxin (Alomone labs, Israel). CCCP: carbonyl cyanide 3-chlorophenylhydrazone (Sigma-Aldrich, France). GsMTx4 (Alomone labs, Israel). Fluo-4, AM (Invitrogen, France). All drugs are used at 1000X stock solution in DMSO, except Charybdotoxin and GsMTx4 in water.

Hatem A., Poussereau G., Gachenot M., Pérès L., Bouyer G, & Egée S. (2023). Dual action of Dooku1 on PIEZO1 channel in human red blood cells. Frontiers in Physiology, 14, 1222983.

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Probing RBC Mechanics and Signaling

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Human RBCs were obtained from voluntary healthy donors who were fully informed regarding the purposes of the study and gave their consent. All blood samples were deidentified prior to use in the study. RBCs were used within 6 h, except for spherocytic and sickle RBCs which were delivered 1 d after harvesting from patients. RBCs were stored in DPBS-G [Dulbecco’s Phosphate Buffered Saline solution (Gibco) + glucose] at a volume fraction (hematocrit, Hct) of 50%, and diluted at 1% Hct right before experiment. When calcium was present in the external medium, DPBS-G was supplemented with 10% plasma. Membrane rigidity was increased with 1 mM diamide (Sigma-Aldrich). PIEZO1 and Gardos ion channels were inhibited with 5

μ

M GsMTx4 (Alomone Labs) and 10

μ

M TRAM34 (Sigma-Aldrich), respectively. RBC membrane and intracellular calcium were labeled using CellTrace Yellow and Fluo-4 AM (ThermoFisher), respectively.

Moreau A., Yaya F., Lu H., Surendranath A., Charrier A., Dehapiot B., Helfer E., Viallat A, & Peng Z. (2023). Physical mechanisms of red blood cell splenic filtration. Proceedings of the National Academy of Sciences of the United States of America, 120(44), e2300095120.

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Mechanosensitive calcium signaling in VSMC

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Mice primary VSMC seeded on micropillar array were washed twice using normal physiological saline solution (NPSS) with 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 1 mM calcium chloride, 10 mM HEPES, and 10 mM glucose. Cells were then loaded for 20 mins with calcium probe Fluo-4 (F14201, Thermo Fisher Scientific) followed by two washes with NPSS. Imaging was performed using a Zeiss Axio observer with a ×20 objective excited at 480 nm. Before recording data, Ca²⁺ signal was stabilized by imaging VSMC continuously every 1 s for 6 min. To image Ca²⁺ in VSMC upon mechanical stimulation, VSMC were imaged every 200 ms and mechanical stimulation was applied 3 s from the start of the recording. To image Ca²⁺ in VSMC with drug stimulation, Yoda1 (#21904, Cayman Chemical) or recombinant mouse Netrin-1 (#1109-N1, R&D Systems) at a final concentration of 30 µM and 2.5 µg/ml were respectively added to VSMC media. For Piezo1 inhibition assays, cells were preincubated with 2.5 µM GsMTx4 (#STG-100, Alomone labs) for 10 min or 10 µM Dooku1 (#6568, R&D Systems) for 30 min prior to stimulation.

Qian W., Hadi T., Silvestro M., Ma X., Rivera C.F., Bajpai A., Li R., Zhang Z., Qu H., Tellaoui R.S., Corsica A., Zias A.L., Garg K., Maldonado T., Ramkhelawon B, & Chen W. (2022). Microskeletal stiffness promotes aortic aneurysm by sustaining pathological vascular smooth muscle cell mechanosensation via Piezo1. Nature Communications, 13, 512.

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Mechanosensitivity of PIEZO1 Channel

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Inhibitors were used to evaluate the effect of several factors on the mechanosensitivity of the PIEZO1 channel. We used GsMTx-4 to show that the responses to mechanical loading and different rates of loading are due to the activation of the PIEZO1 channel. We used EGTA and Thapsigargin to determine the role of extracellular and intracellular Ca²⁺ sources, respectively, in the activation of the PIEZO1 channel. The concentration of each inhibitor and the duration of treatment are as follows: GsMTx-4 (20 μM, 15 min prior to loading, Alomone Lab), EGTA (10 mM, Sigma Aldrich), and Thapsigargin (3 μM, 30 min prior to loading, Sigma Aldrich).

Savadipour A., Nims R.J., Rashidi N., Garcia-Castorena J.M., Tang R., Marushack G.K., Oswald S.J., Liedtke W.B, & Guilak F. (2023). Membrane stretch as the mechanism of activation of PIEZO1 ion channels in chondrocytes. Proceedings of the National Academy of Sciences of the United States of America, 120(30), e2221958120.

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Reagents for Tyrode and Ion Channel Studies

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Reagents used to make Tyrode solutions, and 2-aminoethoxydiphenyl borate (2-APB), apyrase, carbenoxolone, CdCl 2 , GdCl 3 , diphenyleneiodonium (DPI), NiCl 2 , nifedipine, probenecid, suramin and tetrodotoxin (TTX) were purchased from Sigma-Aldrich. 9-phenanthrol, KB-R7943, MRS-2179 and pyridoxalphosphate-6-azophenyl-2',5'-disulfonic acid (iso-PPADS) were supplied by Tocris Bioscience (Avonmouth, Bristol, BS11 9QD, UK). Fluo-4 AM and fluo-3 K 5 were from Thermo Fisher Scientific (Waltham, MA, USA). Ryanodine and U73122 were purchased from Calbiochem (Merck Millipore Corporation, Darmstadt, Germany). GsMTx-4 was obtained from Alomone Labs (Jerusalem, Israel).

Kim J.C., Son M.J, & Woo S.H. (2018). Ca2+ Signaling Triggered by Shear-Autocrine P2X Receptor Pathway in Rat Atrial Myocytes. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 50(6).

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Fluorescent Labeling of Red Blood Cells

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During the staining procedure and the experiments, RBCs were kept in Tyrode solution and incubated with Fluo-4, AM or Calcein Red-Orange, AM (both ThermoFisher Scientific, Waltham, MA, USA) at a concentration of 5 μM (from a 1 mM stock solution in dimethyl sulfoxide (DMSO) containing 20% Pluronic F-127) for 1 h. After the staining, cells were washed three times by 3 min centrifugation with 1,000 × g. Then RBCs were plated on coverslips and 20 min were allowed for sedimentation and deesterification of the Fluo-4. Mechano-sensitive channels and the Gardos-channel were inhibited by GsMTx-4 and TRAM-34, respectively. GsMTx-4 was purchased from Alomone Labs (Jerusalem, Israel) and TRAM-34 from Sigma-Aldrich (St. Louis, MO, USA). For both substances, stock solutions were prepared at 1 mM in aqua dest. Further applications were performed at concentrations indicated in the experimental description.

Danielczok J.G., Terriac E., Hertz L., Petkova-Kirova P., Lautenschläger F., Laschke M.W, & Kaestner L. (2017). Red Blood Cell Passage of Small Capillaries Is Associated with Transient Ca2+-mediated Adaptations. Frontiers in Physiology, 8, 979.

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Piezo1 Inhibitor GsMTx4 Protocol

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The Piezo1 inhibitor GsMTx4 (Alomone Labs, Jerusalem, Israel) was purchased and dissolved in PBS solution. According to a previous study, 5 µM GsMTx4 was used for all our experiments^{27 (link)}.

He J., Fang B., Shan S., Xie Y., Wang C., Zhang Y., Zhang X, & Li Q. (2021). Mechanical stretch promotes hypertrophic scar formation through mechanically activated cation channel Piezo1. Cell Death & Disease, 12(3), 226.

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Pharmacological Inhibition of Piezo

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For pharmacological inhibition of Piezo 5µM GsMTx4 (Alomone Labs) was added to HBSS. The genetically encoded GsMTx4 strains are described above; these were crossed to either Tin-Gal4 or Piezo-Gal4 and experiments were carried out on 3-day old adults.

Zechini L., Camilleri-Brennan J., Walsh J., Beaven R., Moran O., Hartley P.S., Diaz M, & Denholm B. (2022). Piezo buffers mechanical stress via modulation of intracellular Ca2+ handling in the Drosophila heart. Frontiers in Physiology, 13, 1003999.

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Piezo1 Inhibition by GsMTx4

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The Piezo1 inhibitor GsMTx4 (Alomone Labs, Jerusalem, Israel) was purchased and dissolved in PBS solution. 5 µM GsMTx4 was used for all experiments according to the manufacturer’s protocol. The incubation time with GsMTx4 is 24 h, accompanying by stretch process.

He J., Shan S., Li Q., Fang B, & Xie Y. (2022). Mechanical Stretch Triggers Epithelial-Mesenchymal Transition in Keratinocytes Through Piezo1 Channel. Frontiers in Physiology, 13, 745572.

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Peptide Stock Preparation Protocol

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The peptide GsMTx4 was purchased from Alomone Labs. All short peptides were commercially synthesized with a purity >98% (Sangon Biotech). The aqueous stock solutions were prepared at 5 mM or 10 mM in distilled water. Pept 02-0 was dissolved in N,N-dimethylformamide with a concentration of 50 to 100 μg/μl (∼23–46 mM) and further diluted to 10 mM in distilled water for the stock solution. The stock solutions (5 mM or 10 mM) were stored at −80 °C, and an appropriate amount of the stock aliquot was diluted in normal pipette solution freshly to the concentrations used on the day when patch-clamp experiments were performed. Other chemicals were purchased from Sigma–Aldrich unless otherwise noted.

Zhou N., Li H., Xu J., Shen Z.S., Tang M., Wang X.H., Su W.X., Sokabe M., Zhang Z, & Tang Q.Y. (2022). Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate. The Journal of Biological Chemistry, 298(9), 102326.

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