mechanosensitive channels/MSCs
GsMTx4 inhibits cationic mechanosensitive channels (MSCs), specifically Piezo1 channels. It also potentiates TREK-1 (K+ selective 2P domain) channels. [1]
GsMTx4 specifically blocks cationic stretch-activated ion channels (SACs). The equilibrium dissociation constant (Kd) for blocking SACs in outside-out patches from adult rat astrocytes is approximately 630 nM (calculated from rate constants) or 415 nM (calculated from steady-state current reduction). [2]

GsMTx4 (5 μM) decreased Piezo1-mediated charge transfer to 38% of the starting level in HEK293 cells transfected with Piezo1 cDNA [1]. GsMTx4 (5 μM) inhibits smooth and skeletal muscle cells, cardiomyocytes, and astrocytes' cation-selective stretch-activated channels [2]. In mammary epithelial cells (MCF10A), leptin-induced AMPK and MLC-2 phosphorylation is dramatically reduced by GsMTx4 (2.5 μM, 16 hours) [3]. In organotypic cerebellar slices, GsMTx4 (500 nM, 48 h) attenuates demyelination caused by psychopyrimidine and cytotoxic lipids [4].

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GsMTx4 (5 µM) reduces Piezo1-mediated charge transfer to 38% of initial levels in outside-out patches from HEK293 cells expressing mouse Piezo1. Four lysine-to-glutamate analogs (K15E, K20E, K22E, K25E) show compromised inhibition, reducing charge transfer to 55–60% of initial levels. The wild-type (WT) peptide's effective equilibrium dissociation constant (KD) for Piezo1 inhibition is approximately 2.0 ± 0.6 µM. GsMTx4 (5 µM) potentiates TREK-1 channels in HEK cells. [1]
GsMTx4 (at a dilution corresponding to 8 µg/mL or ~2 µM from whole venom) produces a complete block of stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. The block occurs rapidly upon superfusion. [2]
In outside-out patches from astrocytes, the association rate constant (k_a) for GsMTx4 (5 µM) is 3.4 × 10^5 M^-1 s^-1, and the dissociation rate constant (k_d) is 0.21 s^-1. [2]
GsMTx4 (5 µM) reduces the peak swelling-activated whole-cell current in adult rat astrocytes by approximately 38% at +100 mV and 48% at -100 mV, 30-40 seconds after exposure to hypotonic saline. It does not significantly change the reversal potential of the swelling-activated current. [2]
In ventricular myocytes isolated from rabbits with aortic regurgitation-induced congestive heart failure (CHF), GsMTx4 (0.4 µM) produces a nearly complete block of the constitutively active, inwardly rectifying, cation-selective swelling-activated current (I_Cl,swell), but has no effect on the outwardly rectifying anion current (I_Cl,swell). [2]
GsMTx4 (5 µM) does not significantly affect voltage-sensitive whole-cell currents in astrocytes under isotonic conditions, as determined by perforated-patch clamp. [2]

Stereotaxic injection (3 μM, 1 μL, single dose) reduces cerebral cortical demyelination and lysophosphatidylcholine-induced astrocyte toxicity while also having neuroprotective effects [4]. In the Von Frey test, GsMTx-4 (intraperitoneal injection, single dose 270 μg/kg) lessens mechanical allodynia brought on by inflammation and sciatic nerve damage [6].

GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ∼20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between “deep” and “shallow” binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as “area reservoirs” leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.[1]

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We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance ( approximately 45 pS at -100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of approximately 630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces approximately 40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.[2]

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Isothermal titration calorimetry (ITC) was used to measure peptide-lipid binding. POPG:POPC (3:1) liposomes were prepared in buffer. Titrations were performed with peptide in the cell and liposome suspension in the syringe. Injections of 20 µL were spaced by 300 s intervals. Thermograms were fitted with single-site and two-site binding models. Binding constants for low-affinity sites ranged from 8×10^4 to 8×10^5 M^-1, and for higher-affinity sites from 2×10^6 to 3×10^7 M^-1. Interaction energies (ΔG) ranged from -28 to -32 kJ/mol for low-affinity sites. [1]
Tryptophan fluorescence quenching was used to determine peptide partitioning into lipid bilayers. Peptides (2 µM) were titrated with large unilamellar vesicles (LUVs) in the presence of aqueous quencher KI. Fluorescence intensity changes were used to calculate free energy of partitioning (ΔG), which ranged from 27–34 kJ/mol for both WT and analogs. [1]

Western Blot Analysis[3]
Cell Types: MCF10A Cell
Tested Concentrations: 2.5 μM
Incubation Duration: 16 hrs (hours)
Experimental Results: Leptin-induced phosphorylation of AMPK and MLC-2 was attenuated.

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Human embryonic kidney (HEK)293 cells were transfected with mouse Piezo1 cDNA. Mechanically activated currents were recorded from outside-out patches using an amplifier. Pressure steps (500 ms) were applied via a pressure clamp. Peptides (5 µM) were superfused onto patches, and charge transfer (integrated current) was measured before, during, and after peptide application. Inhibition kinetics were analyzed by fitting decay and recovery phases with exponential equations to derive association (k_a) and dissociation (k_d) rate constants. [1]
Astrocyte Single-Channel Patch Clamp: Outside-out patches were formed from cultured adult rat astrocytes. Pipettes were filled with KCl saline. Bath saline contained NaCl, KCl, MgSO4, CaCl2, glucose, and HEPES. Stretch-activated channels (SACs) were activated by applying pressure or suction to the pipette via a pressure clamp. Currents were recorded using an amplifier, sampled at 10 kHz, and filtered at 2 kHz. For toxin screening, HPLC fractions of GsMTx4 were dissolved in saline and perfused onto the patches. Channel activity and block were analyzed offline. [2]
Astrocyte Whole-Cell Current Clamp (Swelling-activated currents): Whole-cell currents were measured using the nystatin-perforated patch technique. Astrocytes were voltage-clamped. To induce swelling, cells were perfused with hypotonic saline (isotonic saline minus mannitol). Currents were monitored using voltage-step protocols or voltage ramps. GsMTx4 was applied during hypotonic exposure, and its effect on the swelling-activated current was measured. [2]
Cardiac Myocyte Electrophysiology: Ventricular myocytes from rabbits with CHF were studied using the amphotericin perforated-patch technique. Whole-cell currents were recorded with an amplifier. Cells were exposed to isotonic (1.0T) or hypertonic (1.5T) bath solutions. GsMTx4 was perfused into the bath, and its effect on constitutive and swelling-activated currents was assessed. Cell volume was simultaneously monitored via video microscopy. [2]

Animal/Disease Models: Male C57BL/6 mice (toxin induces focal demyelination in cortical brain tissue) [4]
Doses: 3 μM/1 μL, single dose.
Route of Administration: Stereotactic injection in the left and right cerebral hemispheres (sacrifice 4 days after injection)
Experimental Results: Prevented lysophosphatidylcholine (LPC)-induced enhanced increase in microglial reactivity and microglia number. Prevents LPC-mediated astrocyte toxicity by attenuating GFAP+ cells and reducing GFAP fluorescence intensity.

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Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rat sciatic nerve injury model [6]
Doses: 270 μg/kg, single dose
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Reduce inflammation-induced mechanical allodynia.

[1]. GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels. Biophys J. 2017 Jan 10;112(1):31-45.

[2]. Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels. J Gen Physiol. 2000 May;115(5):583-98.

[3]. Adipokine Leptin Co-operates With Mechanosensitive Ca 2 +-Channels and Triggers Actomyosin-Mediated Motility of Breast Epithelial Cells. Front Cell Dev Biol. 2021 Jan 6;8:607038.

[4]. Inhibition of Piezo1 attenuates demyelination in the central nervous system. Glia. 2020 Feb;68(2):356-375.

[5]. Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells. Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):16148-53.

[6]. A tarantula spider toxin, GsMTx4, reduces mechanical and neuropathic pain. Pain. 2008 Jul;137(1):208-217.

GsMTx4 is a peptide that acts as a blocker of mechanosensitive ion channels. GsMTx-4 is a peptide found in tarantula venom that inhibits the activity of mechanosensitive ion channels (MSCs). GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs), such as the Piezo1 and TRP channels. It is an amphiphilic peptide with an inhibitory cysteine knot (ICK) backbone, carrying a net positive charge of +5 due to its six lysine residues. Its inhibitory effect is not stereospecific (both L- and D-type are active). The proposed mechanism is a "tension-clamping" model: in a relaxed membrane, GsMTx4 binds superficially to the lipid-water interface; as membrane tension increases (lateral pressure decreases), the peptide penetrates deeper, acting as an "area reservoir," reducing the effective tension transmitted to the MSC gate, thereby inhibiting channel activation. This model is supported by Langmuir monolayer experiments and molecular dynamics simulations. [1]
GsMTx4 is a 35-amino acid peptide toxin isolated from the venom of the spider Grammostola spatulata. It belongs to the neuroactive peptide family of inhibitory cysteine knots (ICK). Its sequence shows less than 50% homology with other known peptide toxins. The peptide has a molecular weight of 4,093.90 Da and a net positive charge of +5. [2]
This is the first reported peptide toxin that specifically blocks stretch-activated ion channels. GsMTx4 is considered a novel pharmacological tool for studying the role of cationic SACs in physiological processes, such as astrocyte volume regulation and the development of cardiomyocyte arrhythmias and hypertrophy. [2]
Preliminary results from Langendorff perfusion rabbit heart experiments (cited but not elaborated in this paper) indicate that GsMTx4 (0.17 µM) can inhibit dilation-related atrial fibrillation without blocking normal electrical activity, suggesting its potential clinical significance. [2]

C185H273N49O45S6

4095.83845305443

4093.893

1209500-46-8

GsMTx4 TFA;D-GsMTx4 TFA;D-GsMTx4

90488987

H-Gly-DL-Cys(1)-DL-Leu-DL-Glu-DL-Phe-DL-Trp-DL-Trp-DL-Lys-DL-Cys(2)-DL-Asn-DL-Pro-DL-Asn-DL-Asp-DL-Asp-DL-Lys-DL-Cys(3)-DL-Cys(1)-DL-Arg-DL-Pro-DL-Lys-DL-Leu-DL-Lys-DL-Cys(2)-DL-Ser-DL-Lys-DL-Leu-DL-Phe-DL-Lys-DL-Leu-DL-Cys(3)-DL-Asn-DL-Phe-DL-Ser-DL-Phe-NH2

GCLEFWWKCNPNDDKCCRPKLKCSKLFKLCNFSF-NH2 (Disulfide bridge:Cys2-Cys17, Cys9-Cys17, Cys16-Cys30); GCLEFWWKCNPNDDKCCRPKLKCSKLFKLCNFSF

White to off-white solid powder

-12.7

52

59

75

285

9620

0

S1CC2C(NC(CCCNC(=N)N)C(N3CCCC3C(NC(C(NC(C(NC(C(NC3C(NC(C(NC(C(NC(C(NC(CC4C=CC=CC=4)C(NC(CCCCN)C(NC(CC(C)C)C(NC(C(NC(C(NC(C(NC(C(NC(C(N)=O)CC4C=CC=CC=4)=O)CO)=O)CC4C=CC=CC=4)=O)CC(N)=O)=O)CSSCC(C(N2)=O)NC(C(CCCCN)NC(C(CC(=O)O)NC(C(CC(=O)O)NC(C(CC(N)=O)NC(C2CCCN2C(C(CC(N)=O)NC(C(CSSC3)NC(C(CCCCN)NC(C(CC2=CNC3C=CC=CC2=3)NC(C(CC2=CNC3C=CC=CC2=3)NC(C(CC2C=CC=CC=2)NC(C(CCC(=O)O)NC(C(CC(C)C)NC(C(CS1)NC(CN)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)CC(C)C)=O)CCCCN)=O)CO)=O)=O)CCCCN)=O)CC(C)C)=O)CCCCN)=O)=O)=O

WVDNTWXIIKNMHY-UHFFFAOYSA-N

InChI=1S/C185H273N49O45S6/c1-98(2)72-121-160(255)203-114(54-27-33-65-188)156(251)229-141-96-284-283-95-140-178(273)225-134(84-147(195)239)184(279)234-71-39-60-144(234)182(277)224-131(83-146(194)238)170(265)222-133(86-151(245)246)172(267)223-132(85-150(243)244)171(266)206-116(56-29-35-67-190)158(253)230-142(97-285-282-94-139(177(272)221-130(82-145(193)237)169(264)218-127(79-105-46-19-12-20-47-105)166(261)226-136(91-236)174(269)211-120(152(196)247)76-102-40-13-9-14-41-102)231-163(258)124(75-101(7)8)214-153(248)112(52-25-31-63-186)204-164(259)125(77-103-42-15-10-16-43-103)217-162(257)123(74-100(5)6)213-154(249)113(53-26-32-64-187)207-173(268)135(90-235)227-179(141)274)180(275)232-138(176(271)210-119(58-37-69-199-185(197)198)183(278)233-70-38-59-143(233)181(276)209-117(155(250)212-121)57-30-36-68-191)93-281-280-92-137(202-148(240)87-192)175(270)215-122(73-99(3)4)161(256)208-118(61-62-149(241)242)159(254)216-126(78-104-44-17-11-18-45-104)165(260)219-129(81-107-89-201-111-51-24-22-49-109(107)111)168(263)220-128(80-106-88-200-110-50-23-21-48-108(106)110)167(262)205-115(157(252)228-140)55-28-34-66-189/h9-24,40-51,88-89,98-101,112-144,200-201,235-236H,25-39,52-87,90-97,186-192H2,1-8H3,(H2,193,237)(H2,194,238)(H2,195,239)(H2,196,247)(H,202,240)(H,203,255)(H,204,259)(H,205,262)(H,206,266)(H,207,268)(H,208,256)(H,209,276)(H,210,271)(H,211,269)(H,212,250)(H,213,249)(H,214,248)(H,215,270)(H,216,254)(H,217,257)(H,218,264)(H,219,260)(H,220,263)(H,221,272)(H,222,265)(H,223,267)(H,224,277)(H,225,273)(H,226,261)(H,227,274)(H,228,252)(H,229,251)(H,230,253)(H,231,258)(H,232,275)(H,241,242)(H,243,244)(H,245,246)(H4,197,198,199)

3-[77-[(2-aminoacetyl)amino]-30-[[4-amino-1-[[1-[[1-[(1-amino-1-oxo-3-phenylpropan-2-yl)amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-1,4-dioxobutan-2-yl]carbamoyl]-22,36,45,54,60,95-hexakis(4-aminobutyl)-4,13-bis(2-amino-2-oxoethyl)-39,86-dibenzyl-69-(3-carbamimidamidopropyl)-16,19-bis(carboxymethyl)-48-(hydroxymethyl)-89,92-bis(1H-indol-3-ylmethyl)-33,42,57,80-tetrakis(2-methylpropyl)-2,3a,5,11,14,17,20,23,32,35,38,41,44,47,50,53,56,59,62,68,71,78,81,84,87,90,93,96-octacosaoxo-a,27,28,74,75,99-hexathia-2a,3,6,12,15,18,21,24,31,34,37,40,43,46,49,52,55,58,61,67,70,79,82,85,88,91,94,97-octacosazapentacyclo[49.46.4.225,72.06,10.063,67]trihectan-83-yl]propanoic acid TFA salt

GsMTX 4; 1209500-46-8; GsMTx-4; H-Gly-DL-Cys(1)-DL-Leu-DL-Glu-DL-Phe-DL-Trp-DL-Trp-DL-Lys-DL-Cys(2)-DL-Asn-DL-Pro-DL-Asn-DL-Asp-DL-Asp-DL-Lys-DL-Cys(3)-DL-Cys(1)-DL-Arg-DL-Pro-DL-Lys-DL-Leu-DL-Lys-DL-Cys(2)-DL-Ser-DL-Lys-DL-Leu-DL-Phe-DL-Lys-DL-Leu-DL-Cys(3)-DL-Asn-DL-Phe-DL-Ser-DL-Phe-NH2; CHEBI:194078; GsMTX-4

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~12.21 mM)
DMSO : ~50 mg/mL (~12.21 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (0.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (0.31 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.25 mg/mL (0.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)