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 ] 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.

No abstract

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 (∼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 ∼630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces ∼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.

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.

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Mechanical stress is one of the most influential physical factors in biology and one of the least characterized. Whereas it is obvious from molecular dynamics [1][2][3][4] and force spectroscopy [5][6][7][8][9][10][11][12] that forces deform molecules, the mechanics of cells are much more complicated, involving the interaction of heterogeneous polymers and membranes and their interaction with both two-dimensional heterogeneous liquid membranes [13,14] and three-dimensional cytoplasmic solutions, where signaling factors can vary in time and space [15][16][17] 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 a-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°C, unfolded in 8 m urea, and rapidly digested by proteases, but in all cases the fluorophores were unaffected. We modified the a-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 a-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.Abbreviations CFP, cyan fluorescent protein; COL-19, collagen-19; D ⁄ A ratio, donor emission to acceptor emission ratio; DIC, differential interference contrast; E, fluorescence resonance energy transfer energy transfer efficiency; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; HEK, human embryonic kidney; YPF, yellow fluorescent protein.

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.

The neuronal mechano-gated K2P channels TREK-1 and TRAAK show pronounced desensitization within 100 ms of membrane stretch. Desensitization persists in the presence of cytoskeleton disrupting agents, upon patch excision, and when channels are expressed in membrane blebs. Mechanosensitive currents evoked with a variety of complex stimulus protocols were globally fit to a four-state cyclic kinetic model in detailed balance, without the need to introduce adaptation of the stimulus. However, we show that patch stress can be a complex function of time and stimulation history. The kinetic model couples desensitization to activation, so that gentle conditioning stimuli do not cause desensitization. Prestressing the channels with pressure, amphipaths, intracellular acidosis, or the E306A mutation reduces the peak-to-steady-state ratio by changing the preexponential terms of the rate constants, increasing the steady-state current amplitude. The mechanical responsivity can be accounted for by a change of in-plane area of Ϸ2 nm 2 between the closed and open conformations. Desensitization and its regulation by chemical messengers is predicted to condition the physiological role of K2P channels.M ost receptors, including ligand-gated ion channels, desensitize with continuous stimulation. Desensitization is a reversible, use-dependent, form of signal plasticity critical for maintaining the dynamic sensitivity over a wide dynamic range. Desensitization also protects cells from inappropriate persistent activation. Many mechano-gated ion channels desensitize (1-5). There are two basic mechanisms that account for desensitization: adaptation and inactivation. Adaptation refers to the uncoupling of the stimulus from the channel, whereas inactivation refers to a block of the permeation path. In hair cells of the inner ear, deflection opens a mechano-gated channel at the tip of the stereocilium leading to depolarization (6). A fast adaptation mechanism (0.3-5 ms) is attributed to the binding of calcium near the channel after permeation through the open ionic pore (6), i.e., Ca 2ϩ block. A slower adaptation (10-100 ms) occurs when the calcium-dependent molecular motor myosin-1c, which pulls on the channels, slips down the actin cytoskeleton reducing the force applied to the channel (6).Rapid desensitization also occurs in mechanosensitive channels expressed in nonspecialized cells. For instance, the stretchactivated cation channel of Xenopus oocyte opens transiently in response to a step change in patch pressure (5). The decrease in current has been attributed to relaxation of the mechanical stimulus, although as shown in this work, that interpretation may not be a good discriminator. Desensitization of the oocyte channel is fragile and may disappear when patches are repetitively stimulated (5). This fragility was attributed to decoupling of the bilayer from the cytoskeleton (5).Stretch-activated K ϩ channels are present in neurons of both the central and the peripheral nervous systems (7-9), and they also have been described in nonne...