• Anderson M, et al. (2013) Opposing effects of podocin on the gating of podocyte TRPC6 channels evoked by membrane stretch or diacylglycerol. Am J Physiol Cell Physiol. PubMed link

Diarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca(2+) signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca(2+) transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1- or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains.

Lee W, et al. (2015) Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage. PNAS. PubMed link

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. ExtracellularGsMTx4 at micromolar concentrations reversibly inhibited ∼80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibition was voltage insensitive, & as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding & unbinding based on Piezo1 current kinetics provided association & dissociation rates of 7.0 × 10(5) M(-1) s(-1) & 0.11 s(-1), respectively, & 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 & 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, & 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 & diagnostic assays.

Bae, C.,  et al. (2011) The Mechanosensitive Ion Channel Piezo1 Is Inhibited by the Peptide GsMTx4. Biochemistry. PMID: 21696149

Our recent molecular dynamics simulation study of hanatoxin 1 (HaTx1), a gating modifier that binds to the voltage sensor of K(+) channels, has shown that HaTx1 has the ability to interact with carbonyl oxygen atoms of both leaflets of the lipid bilayer membrane & to be located at a deep position within the membrane. Here we performed a similar study of GsMTx4, a stretch-activated channels inhibitor, belonging to the same peptide family as HaTx1. Both toxins have an ellipsoidal shape, a belt of positively charged residues around the periphery, & a hydrophobic protrusion. Results show that, like HaTx1, GsMTx4 can interact with the membrane in two different ways. When all the positively charged residues interact with the outer leaflet lipid, GsMTx4 can assume a shallow binding mode. On the other hand, when the electrostatic interaction brings the positively charged groups of K-8 & K-28 into the vicinity of the carbonyl oxygen atoms of the inner leaflet lipids, the system exhibits a deep binding mode. This deep mode is accompanied by local membrane thinning. For both HaTx1 & GsMTx4, our mean force measurement analyses show that the deep binding mode is energetically favored over the shallow mode when a DPPC (dipalmitoyl-phosphatidylcholine) membrane is used at 310 K. In contrast, when a POPC (palmitooleoyl-phosphatidylcholine) membrane is used at 310 K, the two binding modes exhibited similar stability for both toxins. Similar analyses with DPPC membrane at 330 K led to an intermediary result between the above two results. Therefore, the structure of the lipid acyl chains appears to influence the location & the dynamics of the toxins within biological membranes. We also compared the behavior of an arginine & a lysine residue within the membrane. This is of interest because the arginine residue interaction with the lipid carbonyl oxygen atoms mediates the deep binding mode for HaTx1, whereas the lysine residue plays that role for GsMTx4. The arginine residue generally shows smoother dynamics near the lipid carbonyl oxygen atoms than the lysine residue. This difference between arginine & lysine may partly account for the functional diversity of the members of the toxin family.

Nishizawa M, Nishizawa K. (2007) Molecular dynamics simulations of a stretch-activated channel inhibitor GsMTx4 with lipid membranes: two binding modes and effects of lipid structure. Biophys J. PMID: 17384064

A variety of venomous animals produce small protein toxins that impair the function of voltage-dependent cation channels by affecting the motions of the voltage-sensor domains & altering the energetics of the opening of the channel. In this study, we investigate the location of the receptor for tarantula venom voltage-sensor toxins on the voltage-dependent K+ channel from Aeropyrum pernix (KvAP), an archeabacterial channel that is functionally inhibited by members of this toxin family. We show that it is possible to purify the same set of toxins from venom of the tarantula Grammostola spatulata using either the purified KvAP voltage-sensor domain or the full-length KvAP channel. The equivalence of toxin retention profiles for the two channel proteins implies that the tarantula voltage-sensor toxin receptor resides exclusively on the voltage-sensor domain & that the pore is not required for the toxin-channel interaction. We have identified & characterized the functional properties of a subset of the tarantula toxins that bind to the KvAP voltage-sensor domain. Some of these toxins, VSTX1 & GSMTX4, have been previously isolated, while others, VSTX2 & VSTX3, are new members of the tarantula voltage-sensor toxin family. Some but not all toxins that bind to the voltage-sensor domain affect voltage-dependent gating of KvAP channels in lipid membranes.

Ruta, V., and MacKinnon, R. (2004) Localization of the voltage-sensor toxin receptor on KvAP, Biochemistry. PMID: 15287735

The peptide GsMTx4 from the tarantula venom (Grammostola spatulata) inhibits mechanosensitive ion channels. In this work, we report the cDNA sequence encoding GsMTx4. The gene is translated as a precursor protein of 80 amino acids. The first 21 amino acids are a predicted signal sequence & the C-terminal residues are a signal for amidation. An arginine residue adjacent to the N-terminal glycine of GsMTx4 is the cleavage site for release. The resulting peptide is 34 amino acids in length with a C-terminal phenylalanine & not a serine-alanine previously identified [J. Gen. Physiol. 115 (2000) 583]. We chemically synthesized this peptide & folded it in 0.1 M Tris, pH 7.9 with oxidized/reduced glutathione (1/10). Properties of the synthetic peptide were identical to the wild type for high performance liquid chromatography (HPLC), mass spectrometry, CD, & NMR. We also cloned GsMTx4 in a thioredoxin fusion protein system containing six histidines. Nickel affinity columns allowed rapid purification & folding occurred in conditions described above with 0.5 M guanidiniumHCl present. Thrombin cleavage liberated GsMTx4 with three extra amino acids at the N-terminus. The retention time in HPLC analysis & the CD spectrum was similar to wild type. Both the synthetic & cloned peptides were active in the patch clamp assay.

Ostrow, K. L., et al. (2003) cDNA sequence and in vitro folding of GsMTx4, a specific peptide inhibitor of mechanosensitive channels. Toxicon. PMID: 14559077

Mechanosensitive channels (MSCs) play key roles in sensory processing & have been implicated as primary transducers for a variety of cellular responses ranging from osmosensing to gene expression. This paper presents the first structures of any kind known to interact specifically with MSCs. GsMTx-4 & GsMtx-2 are inhibitor cysteine knot peptides isolated from venom of the tarantula, Grammostola spatulata (Suchyna, T. M., Johnson, J. H., Hamer, K., Leykam, J. F., Gage, D. A., Clemo, H. F., Baumgarten, C. M., & Sachs, F. (2000) J. Gen. Physiol. 115, 583-598). Inhibition of cationic MSCs by the higher affinity GsMtx-4 (K(D) approximately 500 nm) reduced cell size in swollen & hypertrophic heart cells, swelling-activated currents in astrocytes, & stretch-induced arrhythmias in the heart. Despite the relatively low affinity, no cross-reactivity has been found with other channels. Using two-dimensional NMR spectroscopy, we determined the solution structure of GsMTx-4 & a lower affinity (GsMTx-2; K(D) approximately 6 microm) peptide from the same venom. The dominant feature of the two structures is a hydrophobic patch, utilizing most of the aromatic residues & surrounded with charged residues. The spatial arrangement of charged residues that are unique to GsMTx-4 & GsMTx-2 may underlie the selectivity of these peptides.

Oswald, R. E., et al. (2002) Solution structure of peptide toxins that block mechanosensitive ion channels. J Biol Chem. PMID: 12082099

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 & has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, & 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 & outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance ( approximately 45 pS at -100 mV) & a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches & 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.

Suchyna, T. M., et al. (2000) Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels. J Gen Physiol. PMID: 10779316