Investigating the interaction of Grammostola rosea venom peptides and model lipid bilayers with solid-state NMR and electron microscopy techniques.

Spider venom contains a number of small peptides that can control the gating properties of a wide range of ion channels with high affinity and specificity. These ion channels are responsible for coordination and control of many bodily functions such as transducing signals into sensory functions, smooth muscle contractions as well as serving as sensors in volume regulation. Hence, these peptides have been the topic of many research efforts in hopes that they can be used as biomedical therapeutics. Several peptides are known to control the gating properties of ion channels by involving the lipid membrane. GsMTx4, originally isolated from the Chilean Rose tarantula (Grammostola rosea), is known to selectively inhibit mechanosensitive ion channels by partitioning into the lipid bilayer. To further understand this indirect gating mechanism, we investigated the interactions between native GsAF2, VsTx1 and a synthetic form of GsMTx4 with model DMPC lipid bilayers using 31P solid-state NMR, 13C CP-MAS NMR, NS-TEM and cryo-TEM. The results reveal that these inhibitor cystine knot peptides perforate the DMPC lipid vesicles similarly with some subtle differences and ultimately create small spherical vesicles and anisotropic cylindrical and discoidal vesicles at concentrations near 1.0-1.5 mol% peptide. The anisotropic components align with their long axes along the NMR static B0 magnetic field, a property that should be useful in future NMR structural investigations of these systems. These findings move us forward in our understanding of how these peptides bind and interact with the lipid bilayer.

GsMTx4-D provides protection to the D2.mdx mouse.

Duchenne muscular dystrophy is a life-limiting muscle disease that has no current effective therapy. Despite mounting evidence that dysregulation of mechanosensitive ion channels is a significant contributor to dystrophy pathogenesis, effective pharmacologic strategies targeting these channels are lacking. GsMTx4, and its enantiomer GsMTx4-D, are peptide inhibitors of mechanosensitive channels with identical activity. In previous studies, acute in vitro application of GsMTx4 to dystrophic murine muscle effectively reduced the excess MSC dependent calcium influx linked to contraction-induced muscle damage. Here we sought to determine if in vivo treatment with GsMTx4-D proffered benefit in the D2.mdx mouse. GsMTx4-D showed a 1-week half-life when administered by subcutaneous injection over four weeks. Informed by these results, D2.mdx mice were then treated by a subcutaneous injection regimen of GsMTx4-D for six weeks followed by determination of muscle mass, muscle susceptibility to eccentric contraction injury and multiple histological indicators of disease progression. The mice showed a reduction in the loss of muscle mass and a decrease in susceptibility to contraction induced injury. These protective effects were realized without reduction in fibrosis, supporting a model where GsMTx4-D acts directly on muscle cells. We propose GsMTx4-D represents a promising new therapy to slow disease progression and may complement other therapies such as anti-inflammatory agents and gene-replacement strategies.

Piezo channels and GsMTx4: Two milestones in our understanding of excitatory mechanosensitive channels and their role in pathology.

Discovery of Piezo channels and the reporting of their sensitivity to the inhibitor GsMTx4 were important milestones in the study of non-selective cationic mechanosensitive channels (MSCs) in normal physiology and pathogenesis. GsMTx4 had been used for years to investigate the functional role of cationic MSCs, especially in muscle tissue, but with little understanding of its target or inhibitory mechanism. The sensitivity of Piezo channels to bilayer stress and its robust mechanosensitivity when expressed in heterologous systems were keys to determining GsMTx4's mechanism of action. However, questions remain regarding Piezo's role in muscle function due to the non-selective nature of GsMTx4 inhibition toward membrane mechanoenzymes and the implication of MCS channel types by genetic knockdown. Evidence supporting Piezo like activity, at least in the developmental stages of muscle, is presented. While the MSC targets of GsMTx4 in muscle pathology are unclear, its muscle protective effects are clearly demonstrated in two recent in situ studies on normal cardiomyocytes and dystrophic skeletal muscle. The muscle protective function may be due to the combined effect of GsMTx4's inhibitory action on cationic MSCs like Piezo and TRP, and its potentiation of repolarizing K+ selective MSCs like K2P and SAKCa. Paradoxically, the potent in vitro action of GsMTx4 on many physiological functions seems to conflict with its lack of in situ side-effects on normal animal physiology. Future investigations into cytoskeletal control of sarcolemma mechanics and the suspected inclusion of MSCs in membrane micro/nano sized domains with distinct mechanical properties will aide our understanding of this dichotomy.

GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels.

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.

GsMTx4-D is a cardioprotectant against myocardial infarction during ischemia and reperfusion.

GsMTx4 is a selective inhibitor of cationic mechanosensitive ion channels (MSCs) and has helped establish the role of MSCs in cardiac physiology. Inhomogeneous local mechanical stresses due to hypercontracture and swelling during ischemic reperfusion injury (IRI) likely induce elevated MSC activity that can contribute to cation imbalance. The aim of this study was to determine if the D enantiomer of GsMTx4 can act as a cardioprotectant in a mouse IRI model. Ischemia and reperfusion involved ligating a coronary artery followed by release of the ligature. GsMTx4-D was tested by either acute intravenous injection during the ischemic event or by two day pretreatment by intraperitoneal injection, both methods achieving similar results. Based on pharmacokinetic studies, GsMTx4-D dosage was set to achieve expected plasma concentrations between 50 and 5000nM and heart tissue concentrations between 1 and 200nM by intravenous injection. Relative to vehicle injected animals, GsMTx4-D reduced infarct area by ~40% for acute and pretreated animals for both 20 and 45min ischemic challenges. Many indicators of cardiac output were indistinguishable from sham-treated control hearts after GsMTx4-D treatment showing improvement at both 4 and 48h post ischemia, and premature ventricular beats immediately following reperfusion were also significantly reduced. To determine if GsMTx4-D cardioprotection could act directly at the level of cardiomyocytes, we tested its effects in vitro on indicators of IRI damage like cation influx and activation of inflammatory kinases in isolated myocytes cultured under hypoxic conditions. Hypoxia challenged cardiomyocytes treated with 10µM GsMTx4-D showed improved contractility and near normal contraction-related Ca(2+) influx. GsMTx4-D inhibited indicators of ischemic damage such as the apoptotic signaling system JNK/c-Jun, but also inhibited the energy response signaling system Akt kinase. We conclude that GsMTx4-D is a potent cardioprotectant in vivo that may act directly on cardiomyocytes and potentially be useful in multidrug strategies to treat IRI.

Human PIEZO1 Ion Channel Functions as a Split Protein.

PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the "split protein" is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone.

Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations.

GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5e) due to six Lys residues. The peptide does not have a stereospecific binding site on the channel but acts from the boundary lipids within a Debye length of the pore probably by changing local stress. To gain insight into how these Lys residues interact with membranes, we performed molecular dynamics simulations of Lys to Glu mutants in parallel with our experimental work. In silico, K15E had higher affinity for 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine bilayers than wild-type (WT) peptide or any other mutant tested, and showed deeper penetration than WT, a finding consistent with the experimental data. Experimentally, the inhibitory activities of K15E and K25E were most compromised, whereas K8E and K28E inhibitory activities remained similar to WT peptide. Binding of WT in an interfacial mode did not influence membrane thickness. With interfacial binding, the direction of the dipole moments of K15E and K25E was predicted to differ from WT, whereas those of K8E and K28E oriented similarly to that of WT. These results support a model in which binding of GsMTx4 to the membrane acts like an immersible wedge that serves as a membrane expansion buffer reducing local stress and thus inhibiting channel activity. In simulations, membrane-bound WT attracted other WT peptides to form aggregates. This may account for the positive cooperativity observed in the ion channel experiments. The Lys residues seem to fine-tune the depth of membrane binding, the tilt angle, and the dipole moments.

Caveolae regulation of mechanosensitive channel function in myotubes.

Mutations that lead to muscular dystrophy often create deficiencies in cytoskeletal support of the muscle sarcolemma causing hyperactive mechanosensitive cation channel (MSC) activity and elevated intracellular Ca(2+). Caveolae are cholesterol-rich microdomains that form mechanically deformable invaginations of the sarcolemma. Mutations to caveolin-3, the main scaffolding protein of caveolae in muscle, cause Limbe-Girdle muscular dystrophy. Using genetic and acute chemical perturbations of developing myotubes we investigated whether caveolae are functionally linked to MSCs. MSC sensitivity was assayed using suction application to patches and probe-induced indentation during whole-cell recordings. Membrane mechanical stress in patches was monitored using patch capacitance/impedance. Cholesterol depletion disrupted caveolae and caused a large increase in MSC current. It also decreased the membrane mechanical relaxation time, likely reflecting cytoskeleton dissociation from the bilayer. Reduction of Cav3 expression with miRNA also increased MSC current and decreased patch relaxation time. In contrast Cav3 overexpression produced a small decrease in MSC currents. To acutely and specifically inhibit Cav3 interactions, we made a chimeric peptide containing the antennapedia membrane translocation domain and the Cav3 scaffolding domain (A-CSD3). A-CSD3 action was time dependent initially producing a mild Ca(2+) leak and increased MSC current, while longer exposures decreased MSC currents coinciding with increased patch stiffening. Images of GFP labeled Cav3 in patches showed that Cav3 doesn't enter the pipette, showing patch composition differed from the cell surface. However, disruption via cholesterol depletion caused Cav3 to become uniformly distributed over the sarcolemma and Cav3 appearance in the patch dome. The whole-cell indentation currents elicited under the different caveolae modifying conditions mirror the patch response supporting the role of caveolae in MSC function. These studies show that normal expression levels of Cav3 are mechanoprotective to the sarcolemma through multiple mechanisms, and Cav3 upregulation observed in some dystrophies may compensate for other mechanical deficiencies.

Real Time FRET Based Detection of Mechanical Stress in Cytoskeletal and Extracellular Matrix Proteins.

A molecular force sensing cassette (stFRET) was incorporated into actinin, filamin, and spectrin in vascular endothelial cells (BAECs) and into collagen-19 in Caenorhabditis elegans. To estimate the stress sensitivity of stFRET in solution, we used DNA springs. A 60-mer loop of single stranded DNA was covalently linked to the external cysteines of the donor and acceptor. When the complementary DNA was added it formed double stranded DNA with higher persistence length, stretching the linker and substantially reducing FRET efficiency. The probe stFRET detected constitutive stress in all cytoskeletal proteins tested, and in migrating cells the stress was greater at the leading edge than the trailing edge. The stress in actinin, filamin and spectrin could be reduced by releasing focal attachments from the substrate with trypsin. Inhibitors of actin polymerization produced a modest increase in stress on the three proteins suggesting they are mechanically in parallel. Local shear stress applied to the cell with a perfusion pipette showed gradients of stress leading from the site of perfusion. Transgenic C. elegans labeled in collagen-19 produced a behaviorally and anatomically normal animal with constitutive stress in the cuticle. Stretching the worm visibly stretched the probe in collagen showing that we can trace the distribution of mean tissue stress in specific molecules. stFRET is a general purpose dynamic sensor of mechanical stress that can be expressed intracellularly and extracellularly in isolated proteins, cells, tissues, organs and animals.

Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs).

The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4 µM GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca(2+) threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

Biophysics and structure of the patch and the gigaseal.

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1-2 microm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (E(a)), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1-4 mN/m, a significant fraction of the lytic tension of a bilayer ( approximately 10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. E(a) seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased E(a) and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased E(a) and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of approximately 7 gigohm/microm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of E(a) and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.

A fluorescence energy transfer-based mechanical stress sensor for specific proteins in situ.

To measure mechanical stress in real time, we designed a fluorescence resonance energy transfer (FRET) cassette, denoted stFRET, which could be inserted into structural protein hosts. The probe was composed of a green fluorescence protein pair, Cerulean and Venus, linked with a stable alpha-helix. We measured the FRET efficiency of the free cassette protein as a function of the length of the linker, the angles of the fluorophores, temperature and urea denaturation, and protease treatment. The linking helix was stable to 80 degrees C, unfolded in 8 m urea, and rapidly digested by proteases, but in all cases the fluorophores were unaffected. We modified the alpha-helix linker by adding and subtracting residues to vary the angles and distance between the donor and acceptor, and assuming that the cassette was a rigid body, we calculated its geometry. We tested the strain sensitivity of stFRET by linking both ends to a rubber sheet subjected to equibiaxial stretch. FRET decreased proportionally to the substrate strain. The naked cassette expressed well in human embryonic kidney-293 cells and, surprisingly, was concentrated in the nucleus. However, when the cassette was located into host proteins such alpha-actinin, nonerythrocyte spectrin and filamin A, the labeled hosts expressed well and distributed normally in cell lines such as 3T3, where they were stressed at the leading edge of migrating cells and relaxed at the trailing edge. When collagen-19 was labeled near its middle with stFRET, it expressed well in Caenorhabditis elegans, distributing similarly to hosts labeled with a terminal green fluorescent protein, and the worms behaved normally.

Mechanosensitive channel properties and membrane mechanics in mouse dystrophic myotubes.

Muscular dystrophy is associated with increased activity of mechanosensitive channels (MSCs) and increased cell calcium levels. MSCs in patches from mdx mouse myotubes have higher levels of resting activity, compared to patches from wild-type mice, and a pronounced latency of activation and deactivation. Measurements of patch capacitance and geometry reveal that the differences are linked to cortical membrane mechanics rather than to differences in channel gating. We found unexpectedly that patches from mdx mice are strongly curved towards the pipette tip by actin pulling normal to the membrane. This force produces a substantial tension (approximately 5 mN m(-1)) that can activate MSCs in the absence of overt stimulation. The inward curvature of patches from mdx mice is eliminated by actin inhibitors. Applying moderate suction to the pipette flattens the membrane, reducing tension, and making the response appear to be stretch inactivated. The pronounced latency to activation in patches from mdx mice is caused by the mechanical relaxation time required to reorganize the cortex from inward to outward curvature. The increased latency is equivalent to a three-fold increase in cortical viscosity. Disruption of the cytoskeleton by chemical or mechanical means eliminates the differences in kinetics and curvature between patches from wild-type and mdx mice. The stretch-induced increase in specific capacitance of the patch, approximately 80 fF microm(-2), far exceeds the specific capacitance of bilayers, suggesting the presence of stress-sensitive access to large pools of membrane, possibly caveoli, T-tubules or portions of the gigaseal. In mdx mouse cells the intrinsic gating property of fast voltage-sensitive inactivation is lost. It is robust in wild-type mouse cells (observed in 50% of outside-out patches), but never observed in mdx cells. This link between dystrophin and inactivation may lead to increased background cation currents and Ca2+ influx. Spontaneous Ca2+ transients in mdx mouse cells are sensitive to depolarization and are inhibited by the specific MSC inhibitor GsMTx4, in both the D and L forms.

Mechanosensitive ion channels and the peptide inhibitor GsMTx-4: history, properties, mechanisms and pharmacology.

Sensing the energy from mechanical inputs is ubiquitous--and perhaps the oldest form of biological energy transduction. However, the tools available to probe the mechanisms of transduction are far fewer than for the chemical and electric field sensitive transducers. The one pharmacological tool available for mechansensitive ion channels (MSCs) is a peptide (GsMTx-4) isolated from venom of the tarantula, Grammostola spatulata, that blocks cationic MSCs found in non-specialized eukaryotic tissues. In this review, we summarize the current knowledge of GsMTx-4, and discuss the inevitable crosstalk between the MSC behavior and the mechanical properties of the cell cortex.

Properties and Mechanism of the Mechanosensitive Ion Channel Inhibitor GsMTx4, a Therapeutic Peptide Derived from Tarantula Venom.

Mechanosensitive ion channels (MSCs) are found in all types of cells ranging from Escherichia coli to morning glories to humans. They seem to fall into two families: those in specialized receptors, such as the hair cells of the cochlea, and those in cells not clearly differentiated for sensory duty. The physiological function of the channels in nonspecialized cells has not been demonstrated, although their activity has been demonstrated innumerable times in vitro. The only specific reagent to block MSCs isGsMTx4, a 4-kDa peptide isolated from tarantula venom. Despite being isolated from venom, it is nontoxic to mice. GsMTx4 is specific for an MSC subtype, the nonselective cation channels that may be members of the transient receptor potential (TRP) family. GsMTx4 acts as a gating modifier, increasing the energy of the open state relative to the closed state. The mirror image D enantiomer of GsMTx4 is equally active, so mode of action is not via the traditional lock and key model. GsMTx4 probably acts in the boundary lipid of the channel by changing local curvature and mechanically stressing the channel toward the closed state. Despite the lack of definitive physiological data on the function of the cationic MSCs, GsMTx4 may prove useful as a drug or lead compound that can affect physiological processes. These processes would be those driven by mechanical stress, such as blood vessel autoregulation, stress-induced contraction of smooth muscle, and Ca(2+) loading in muscular dystrophy.

Effects of stretch-activated channel blockers on [Ca2+]i and muscle damage in the mdx mouse.

The mdx mouse lacks dystrophin and is a model of human Duchenne muscular dystrophy. Single mdx muscle fibres were isolated and subjected to a series of stretched (eccentric) contractions while measuring intracellular calcium concentration ([Ca(2+)](i)) with fluo-3 and confocal microscopy. Following the stretched contractions there was a slow rise in resting [Ca(2+)](i) and after 30 min both the [Ca(2+)](i) during a tetanus (tetanic [Ca(2+)](i)) and the tetanic force were reduced. Two blockers of stretch-activated channels, streptomycin and the spider venom toxin GsMTx4, prevented the rise of resting [Ca(2+)](i) and partially prevented the decline of tetanic [Ca(2+)](i) and force. Reducing extracellular calcium to zero also prevented the rise in resting [Ca(2+)](i) and prevented some of the decline in tetanic [Ca(2+)](i) and force. Patch-clamping experiments identified a stretch-activated channel in both wild-type and mdx myotubes which was blocked by GsMTx4. These data suggest that blockers of stretch-activated channels can ameliorate the force reduction following stretched contractions by reducing the influx of Ca(2+) into the muscle. We therefore tested whether in intact mdx mice streptomycin, added to the drinking water, was capable of reducing muscle damage. mdx mice show a period of muscle damage from 20 to 40 days of life and fibres which regenerate from this damage display central nuclei. We measured the frequency of central nuclei in control mdx mice compared to streptomycin-treated mdx mice and showed that the incidence of central nuclei was significantly reduced by streptomycin treatment. This result suggests that blockers of stretch-activated channels may protect against muscle damage in the intact mdx mouse.

Mechanosensitive ion channels as drug targets.

Mechanically sensitive ion channels (MSCs) are ubiquitous. They exist as two major types: those in specialized receptors that require fibrous proteins to transmit forces to the channel, and those in non-specialized tissues that respond to stress in the lipid bilayer. While few MSCs have been cloned, the existing structures show no sequence or structural homology--an example of convergent evolution. The physiological function of MSCs in many tissues is not known, but they probably arose from the need for cell volume regulation. Recently, a peptide called GsMTx4 was isolated from tarantula venom and is the first specific reagent for mechanosensitive channels. GsMTx4 is a approximately 4 kD peptide with a hydrophobic face opposite a positively charged face. It is active in the D and L forms, and appears non-toxic to mice. GsMTx4 has shown physiological effects on cationic MSCs in heart, smooth muscle, astrocytes, and skeletal muscle. By itself, GsMTx4 can serve as a lead compound or as a potential drug. Its availability opens clinical horizons in the diagnosis and treatment of pathologies including cardiac arrhythmia, muscular dystrophy and glioma.

Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers.

The peptide GsMTx4, isolated from the venom of the tarantula Grammostola spatulata, is a selective inhibitor of stretch-activated cation channels (SACs). The mechanism of inhibition remains unknown; but both GsMTx4 and its enantiomer, enGsMTx4, modify the gating of SACs, thus violating a trademark of the traditional lock-and-key model of ligand-protein interactions. Suspecting a bilayer-dependent mechanism, we examined the effect of GsMTx4 and enGsMTx4 on gramicidin A (gA) channel gating. Both peptides are active, and the effect increases with the degree of hydrophobic mismatch between bilayer thickness and channel length, meaning that GsMTx4 decreases the energy required to deform the boundary lipids adjacent to the channel. GsMTx4 decreases inward SAC single-channel currents but has no effect on outward currents, suggesting it is located within a Debye length of the outer vestibule of the SACs, but significantly farther from the inner vestibule. Likewise, GsMTx4 decreases gA single-channel currents. Our results suggest that modulation of membrane proteins by amphipathic peptides--mechanopharmacology--involves not only the protein itself but also the surrounding lipids. The surprising efficacy of the d form of GsMTx4 peptide has important therapeutic implications, because d peptides are not hydrolysed by endogenous proteases and may be administered orally.

Dynamic regulation of mechanosensitive channels: capacitance used to monitor patch tension in real time.

All cells, from bacteria to human, are mechanically sensitive. The most rapid of these membrane protein transducers are mechanosensitive ion channels, ionic pores in the membrane that open and close in response to membrane tension. In specific sensory organs, these channels serve the senses of touch and hearing, and inform the central nervous system about the filling of hollow organs such as the bladder. Non-specialized cells use these channels to report on changes in cell volume and local strain. To preserve dynamic sensitivity, sensory receptors adapt to steady-state stimuli. Here we show that in rat astrocytes, the most abundant cells in the brain, this apparent adaptation to the stimulus is actually an inactivation. We have been able to track the time course of local strain by measuring attofarad changes in membrane capacitance and show that it is not correlated with loss of channel activity. The reduction in current with time is caused by an increased occupancy of low conductance states, and a reduction in the probability of opening, not a relaxation of local stress. The occupancy of these substates depends on the integrity of the cell's cytoplasm. However, while disruption of the cytoskeleton leads to a loss of inactivation, it leaves activation unaffected. The activation process is voltage-insensitive, closely correlated with changes in capacitance, and seems to arise solely from stress in the bilayer. The inactivation rate decreases with depolarization, and kinetic analysis suggests that the process involves multiple cytoplasmic ligands. Surprisingly, multivalent ions such as Gd(+3) and Ca(+2) that bind to the lipids and affect channel gating, do not affect the strain-induced increase in membrane capacitance; contrary to expectations, membrane elasticity is unchanged.

Solution structure of peptide toxins that block mechanosensitive ion channels.

Mechanosensitive channels (MSCs) play key roles in sensory processing and 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 and 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., and 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 and hypertrophic heart cells, swelling-activated currents in astrocytes, and 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 and 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 and surrounded with charged residues. The spatial arrangement of charged residues that are unique to GsMTx-4 and GsMTx-2 may underlie the selectivity of these peptides.