Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch

When skeletal muscle is stretched or injured, satellite cells, resident myogenic stem cells positioned beneath the basal lamina of mature muscle fibers, are activated to enter the cell cycle. This signaling pathway is a cascade of events including calcium-calmodulin formation, nitric oxide (NO) radical production by NO synthase, matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the receptor c-met, as demonstrated by assays of primary cultures and in vivo experiments. Here, we add evidence that two ion channels, the mechanosensitive cation channel (MS channel) and the long-lasting-type voltage-gated calcium-ion channel (L-VGC channel), mediate the influx of extracellular calcium ions in response to cyclic stretch in satellite cell cultures. When applied to 1-h stretch cultures with individual inhibitors for MS and L-VGC channels (GsMTx-4 and nifedipine, respectively) or with a less specific inhibitor (gadolinium chloride, Gd), satellite cell activation and upstream HGF release were abolished, as revealed by bromodeoxyuridine-incorporation assays and Western blotting of conditioned media, respectively. The inhibition was dose dependent with a maximum at 0.1 μM (GsMTx-4), 10 μM (nifedipine), or 100 μM (Gd) and canceled by addition of HGF to the culture media; a potent inhibitor for transient-type VGC channels (NNC55-0396, 100 μM) did not show any significant inhibitory effect. The stretch response was also abolished when calcium-chelator EGTA (1.8 mM) was added to the medium, indicating the significance of extracellular free calcium ions in our present activation model. Finally, cation/calcium channel dependencies were further documented by calcium-imaging analyses on stretched cells; results clearly demonstrated that calcium ion influx was abolished by GsMTx-4, nifedipine, and EGTA. Therefore, these results provide an additional insight that calcium ions may flow in through L-VGC channels by possible coupling with adjacent MS channel gating that promotes the local depolarization of cell membranes to initiate the satellite cell activation cascade.     

The Combined Effect of Hydrophobic Mismatch and Bilayer Local Bending on the Regulation of Mechanosensitive Ion Channels

The hydrophobic mismatch between the lipid bilayer and integral membrane proteins has well-defined effect on mechanosensitive (MS) ion channels. Also, membrane local bending is suggested to modulate MS channel activity. Although a number of studies have already shown the significance of each individual factor, the combined effect of these physical factors on MS channel activity have not been investigated. Here using finite element simulation, we study the combined effect of hydrophobic mismatch and local bending on the archetypal mechanosensitive channel MscL. First we show how the local curvature direction impacts on MS channel modulation. In the case of MscL, we show inward (cytoplasmic) bending can more effectively gate the channel compared to outward bending. Then we indicate that in response to a specific local curvature, MscL inserted in a bilayer with the same hydrophobic length is more expanded in the constriction pore region compared to when there is a protein-lipid hydrophobic mismatch. Interestingly in the presence of a negative mismatch (thicker lipids), MscL constriction pore is more expanded than in the presence of positive mismatch (thinner lipids) in response to an identical membrane curvature. These results were confirmed by a parametric energetic calculation provided for MscL gating. These findings have several biophysical consequences for understanding the function of MS channels in response to two major physical stimuli in mechanobiology, namely hydrophobic mismatch and local membrane curvature.     

Functional design of bacterial mechanosensitive channels. Comparisons and contrasts illuminated by random mutagenesis

MscS and MscL are mechanosensitive channels found in bacterial plasma membranes that open large pores in response to membrane tension. These channels function to alleviate excess cell turgor invoked by rapid osmotic downshock. Although much is known of the structure and molecular mechanisms underlying MscL, genes correlating with MscS activity have only recently been identified. Previously, it was shown that eliminating the expression of Escherichia coli yggB removed a major portion of MscS activity. YggB is distinct from MscL by having no obvious structural similarity. Here we have reconstituted purified YggB in proteoliposomes and have successfully detected MscS channel activity, confirming that purified YggB protein encodes MscS activity. Additionally, to define functional regions of the channel protein, we have randomly mutagenized the structural gene and isolated a mutant that evokes a gain-of-function phenotype. Physiological experiments demonstrate that the mutated channel allows leakage of solutes from the cell, suggesting inappropriate channel opening. Interestingly, this mutation is analogous in position and character to mutations yielding a similar phenotype in MscL. Hence, although MscS and MscL mechanosensitive channels are structurally quite distinct, there may be analogies in their gating mechanisms.     

TRPA1 is differentially modulated by the amphipathic molecules trinitrophenol and chlorpromazine

TRPA1, a poorly selective Ca(2+)-permeable cation channel, is expressed in peripheral sensory neurons, where it is considered to contribute to a variety of sensory processes such as the detection of painful stimuli. Furthermore, TRPA1 was also identified in hair cells of the inner ear, but its involvement in sensing mechanical forces is still being controversially discussed. Amphipathic molecules such as trinitrophenol and chlorpromazine have been shown to provide useful tools to study mechanosensitive channels. Depending on their charge, they partition in the inner or outer sheets of the lipid bilayer, causing a curvature of the membrane, which has been demonstrated to activate or inhibit mechanosensitive ion channels. In the present study, we investigated the effect of these molecules on TRPA1 gating. TRPA1 was robustly activated by the anionic amphipathic molecule trinitrophenol. The whole-cell and single channel properties resemble those previously described for TRPA1. Moreover, we could show that the toxin GsMTx-4 acts on TRPA1. In addition to its recently described role as an inhibitor of stretch-activated ion channels, it serves as a potent activator of TRPA1 channels. On the other hand, the positively charged drug chlorpromazine modulates activated TRPA1 currents in a voltage-dependent way. The exposure of activated TRPA1 channels to chlorpromazine led to a block at positive potentials and an increased open probability at negative potentials. The variability in the shape of the I-V curve gives a first indication that native mechanically activated TRPA1 currents must not necessarily exhibit the same biophysical properties as ligand-activated TRPA1 currents.     

Entropic tension in crowded membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function in vivo. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than [Formula: see text] in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.     

Membrane interaction of chrysophsin-1, a histidine-rich antimicrobial peptide from red sea bream

Chrysophsin-1 is an amphipathic alpha-helical antimicrobial peptide produced in the gill cells of red sea bream. The peptide has broad range activity against both Gram-positive and Gram-negative bacteria but is more hemolytic than other antimicrobial peptides such as magainin. Here we explore the membrane interaction of chrysophsin-1 and determine its toxicity, in vitro, for human lung fibroblasts to obtain a mechanism for its antimicrobial activity and to understand the role of the unusual C-terminal RRRH sequence. At intermediate peptide concentrations, solid-state NMR methods reveal that chrysophsin-1 is aligned parallel to the membrane surface and the lipid acyl chains in mixed model membranes are destabilized, thereby being in agreement with models where permeabilization is an effect of transient membrane disruption. The C-terminal RRRH sequence was shown to have a large effect on the insertion of the peptide into membranes with differing lipid compositions and was found to be crucial for pore formation and toxicity of the peptide to fibroblasts. The combination of biophysical data and cell-based assays suggests likely mechanisms involved in both the antibiotic and toxic activity of chrysophsins.     

Inhibiting and stimulating effect of amiloride on potential-dependent cation channels in K562 cells

Non-voltage-gated ion channels play an essential role in cellular signalling and ionic homeostasis in nonexcitable cells. The patch clamp method in cell-attached configuration was used to search for the effects of amiloride and gadolinium (Gd3+) exerted on two types of voltage-insensitive cationic channels in plasma membrane of human leukemia K562 cells: Na-selective channels activated by actin disassembly, and mechanosensitive channels. Here we demonstrate that amiloride in high concentrations (1 mM) caused a full inhibition of mechanosensitive channels in K562 cells similarly to Gd3+ effect in micromolecular concentration range. Na-selective channels controlled by actin dynamics were shown to be unaffected by Gd3+ similarly as by amiloride. We also found that application of amiloride to the extracellular surface of membrane patch resulted in a significant increase in the activity of sodium channels. This unexpected stimulatory effect of amiloride may represent an unknown mechanism of activation of non-voltage-gated sodium channels. The data show an essential difference of the activation and blockage of these types of cation-selective channels.     

Effect of Mutation of Potassium-Efflux System,KefA, on Mechanosensitive Channels in the Cytoplasmic Membrane of Escherichia coli

The effect of a kefA mutation on the mechanosensitive channels in the cytoplasmic membrane of Escherichia coli was established by introducing a mutation of the kefA gene into wild-type E. coli by P1 transduction. The mutation of the kefA gene not only made the cells sensitive to K⁺ in the medium but also changed the mechanosensitive channel activity. The kefA mutation did not change the conductances of the two mechanosensitive channels in the cytoplasmic membrane of E. coli, but it prolonged the channel open time. Also, the kefA mutation made the cells more sensitive to pressure in comparison to wild-type cells. The high sensitivity to pressure of the kefA mutant was not modulated by betaine or by the potassium gradient across the membrane. The effect of the kefA mutation on mechanosensitive channels was not due to a membrane fluidity change. KefA might be a regulator for mechanosensitive channels. Received: 6 September 1995/Revised: 13 December 1995     

Mechanical properties of lipid bilayers and regulation of mechanosensitive function

Material properties of lipid bilayers, including thickness, intrinsic curvature and compressibility regulate the function of mechanosensitive (MS) channels. This regulation is dependent on phospholipid composition, lateral packing and organization within the membrane. Therefore, a more complete framework to understand the functioning of MS channels requires insights into bilayer structure, thermodynamics and phospholipid structure, as well as lipid-protein interactions. Phospholipids and MS channels interact with each other mainly through electrostatic forces and hydrophobic matching, which are also crucial for antimicrobial peptides. They are excellent models for studying the formation and stabilization of membrane pores. Importantly, they perform equivalent responses as MS channels: (1) tilting in response to tension and (2) dissipation of osmotic gradients. Lessons learned from pore forming peptides could enrich our knowledge of mechanisms of action and evolution of these channels. Here, the current state of the art is presented and general principles of membrane regulation of mechanosensitive function are discussed.     

Mechanical properties of lipid bilayers and regulation of mechanosensitive function: From biological to biomimetic channels

Material properties of lipid bilayers, including thickness, intrinsic curvature and compressibility regulate the function of mechanosensitive (MS) channels. This regulation is dependent on phospholipid composition, lateral packing and organization within the membrane. Therefore, a more complete framework to understand the functioning of MS channels requires insights into bilayer structure, thermodynamics and phospholipid structure, as well as lipid-protein interactions. Phospholipids and MS channels interact with each other mainly through electrostatic forces and hydrophobic matching, which are also crucial for antimicrobial peptides. They are excellent models for studying the formation and stabilization of membrane pores. Importantly, they perform equivalent responses as MS channels: (1) tilting in response to tension and (2) dissipation of osmotic gradients. Lessons learned from pore forming peptides could enrich our knowledge of mechanisms of action and evolution of these channels. Here, the current state of the art is presented and general principles of membrane regulation of mechanosensitive function are discussed.     

Mechanical deformation of ventricular myocytes modulates both TRPC6 and Kir2.3 channels

Cardiomyocytes respond to mechanical stretch with an increase [Ca2+]i. Here, we analyzed which ion channels could mediate this effect. Murine ventricular myocytes were attached to a glass coverslip and a cell-attached glass stylus sheared the upper cell part versus the attached cell bottom. At negative clamp potentials, stretch induced inward currents that increased with the extent of stretch and reversed within 2 min after relaxation from stretch. Stretch activated a nearly voltage-independent GsMTx-4-sensitive non-selective cation conductance Gns, antibodies against TRPC6 prevented Gns activation. In addition, stretch deactivated a Cs+-sensitive inwardly rectifying potassium conductance GK1, antibodies against Kir2.3 inhibited this effect. Immunolabeling localized TRPC6 and Kir2.3 in T-tubular membranes, and stretch-induced changes in membrane currents were absent in cells whose T-tubules had been removed. In absence of stretch, we could activate Gns and deactivate GK1 by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and other amphipaths. We interpret that the function of TRPC6 and Kir2.3 channels is controlled by both tension and curvature of the surrounding lipid bilayer that are changed by incorporation of amphipaths. Stretch-activation of TRPC6 channels may increase Ca2+ influx directly and indirectly, by membrane depolarization (activation of voltage-gated Ca2+ channels) and by elevated [Na+]i (augmented Na+,Ca2+-exchange).     

In vitro synthesis and oligomerization of the mechanosensitive channel of large conductance, MscL, into a functional ion channel

Elucidation of high-resolution structures of integral membrane proteins is drastically lagging behind that of cytoplasmic proteins. In vitro synthesis and insertion of membrane proteins into synthetic membranes could circumvent bottlenecks associated with the overexpression of membrane proteins, producing sufficient membrane-inserted, correctly folded protein for structural studies. Using the mechanosensitive channel of large conductance, MscL, as a model protein we show that in vitro synthesized MscL inserts into YidC-containing proteoliposomes and oligomerizes to form a homopentamer. Using planar membrane bilayers, we show that MscL forms functional ion channels capable of ion transport. These data demonstrate that membrane insertion of MscL is YidC mediated, whereas subsequent oligomerization into a functional homopentamer is a spontaneous event.     

Estimating the sensitivity of mechanosensitive ion channels to membrane strain and tension

Bone adapts to its environment by a process in which osteoblasts and osteocytes sense applied mechanical strain. One possible pathway for the detection of strain involves mechanosensitive channels and we sought to determine their sensitivity to membrane strain and tension. We used a combination of experimental and computational modeling techniques to gain new insights into cell mechanics and the regulation of mechanosensitive channels. Using patch-clamp electrophysiology combined with video microscopy, we recorded simultaneously the evolution of membrane extensions into the micropipette, applied pressure, and membrane currents. Nonselective mechanosensitive cation channels with a conductance of 15 pS were observed. Bleb aspiration into the micropipette was simulated using finite element models incorporating the cytoplasm, the actin cortex, the plasma membrane, cellular stiffening in response to strain, and adhesion between the membrane and the micropipette. Using this model, we examine the relative importance of the different cellular components in resisting suction into the pipette and estimate the membrane strains and tensions needed to open mechanosensitive channels. Radial membrane strains of 800% and tensions of 5 10(-4) N.m(-1) were needed to open 50% of mechanosensitive channels. We discuss the relevance of these results in the understanding of cellular reactions to mechanical strain and bone physiology.     

Ionic regulation of MscK,a mechanosensitive channel from Escherichia coli

Three gene products that form independent mechanosensitive channel activities have been identified in Escherichia coli. Two of these, MscL and MscS, play a vital role in allowing the cell to survive acute hypotonic stress. Much less is known of the third protein, MscK (KefA). Here, we characterize the MscK channel activity and compare it with the activity of its structural and functional homologue, MscS. While both show a slight anionic preference, MscK appears to be more sensitive to membrane tension. In addition, MscK, but not MscS activity appears to be regulated by external ionic environment, requiring not only membrane tension but also high concentrations of external K(+), NH(4)(+), Rb(+) or Cs(+) to gate; no activity is observed with Na(+), Li(+) or N-methyl-D-glucamine (NMDG). An MscK gain-of-function mutant gates spontaneously in the presence of K(+) or similar ions, and will gate in the presence of Na(+), Li(+) and NMDG, but only when stimulated by membrane tension. Increased sensitivity and the highly regulated nature of MscK suggest a more specialized physiological role than other bacterial mechanosensitive channels.     

基于机械敏感离子通道的超声神经调制研究进展

机械敏感离子通道（mechanosensitive channels,MSCs）是一类分布于各种细胞膜上可将细胞受到的机械刺激转化为电信号或化学信号的特殊膜蛋白。由于机械敏感通道所具有的特性,使其成为超声调控的重要潜在靶点。超声由于具有良好的空间分辨率和聚焦效果,并且理论上可实现无创条件下的全脑范围定位,具有用于进行物理性神经调制和治疗神经系统疾病的潜力。近年来,越来越多的离子通道被鉴定出具有机械敏感特性,但其中有明确报道可以被超声激活的依然数量较少。此外,现阶段超声激励下机械敏感通道的开放过程和机制仍未被阐明。本文着重介绍了大电导机械敏感通道、瞬时受体电位通道、退化蛋白/上皮钠通道、双孔钾通道和Piezo通道等机械敏感离子通道在超声神经调制中的研究进展及其应用,为未来超声神经调制的深入研究和临床应用提供参考。     

Colitis decreases mechanosensitive K2P channel expression and function in mouse colon sensory neurons

TREK-1, TREK-2 and TRAAK are mechanosensitive two-pore domain K(+) (K(2P)) channels thought to be involved in the attenuation of mechanotransduction. Because colon inflammation is associated with colon mechanohypersensitivity, we hypothesized that the role of these channels in colon sensory (dorsal root ganglion, DRG) neurons would be reduced by colon inflammation. Accordingly, we studied the functional expression of mechanosensitive K(2P) channels in colon sensory neurons in both thoracolumbar (TL) and lumbosacral (LS) DRG that represent the splanchnic and pelvic nerve innervations of the colon, respectively. In colon DRG neurons identified by retrograde tracer previously injected into the colon wall, 62% of TL neurons and 83% of LS neurons expressed at least one of three K(2P) channel mRNAs; the proportion of neurons expressing the TREK-1 gene was greater in LS than in TL DRG. In electrophysiological studies, single-channel activities of TREK-1a, TREK-1b, TREK-2, and TRAAK-like channels were detected in cultured colon DRG neuronal membranes. After trinitrobenzene sulfonic acid-induced colon inflammation, we observed significant decreases in the amount of TREK-1 mRNA, in the response of TREK-2-like channels to membrane stretch, and in the whole cell outward current during osmotic stretch in LS colon DRG neurons. These findings document that the majority of DRG neurons innervating the mouse colon express mechanosensitive K(2P) channels and suggest that a decrease in their expression and activities contributes to the increased colon mechanosensitivity that develops in inflammatory bowel conditions.     

Effect of proline position on the antimicrobial mechanism of buforin II

Buforin II (BF2) is a histone-derived antimicrobial peptide that causes cell death by translocating across membranes and interacting with nucleic acids. It contains one proline residue critical for its function. Previous research found that mutations replacing proline lead to decreased membrane translocation and antimicrobial activity as well as increased membrane permeabilization. This study further investigates the role of proline in BF2's antimicrobial mechanism by considering the effect of changing proline position on membrane translocation, membrane permeabilization, and antimicrobial activity. For this purpose, four mutants were made with proline substitution (P11A) or relocation (P11A/G7P, P11A/V12P, P11A/V15P). These mutations altered the amount of helical content. Although antimicrobial activity correlated with the α-helical content for the peptides containing proline, membrane translocation did not. This observation suggests that factors in BF2's bactericidal mechanism other than translocation must be altered by these mutations. To better explain these trends we also measured the nucleic acid binding and membrane permeabilization of the mutant peptides. A comparison of mutant and wild type BF2 activity revealed that BF2 relies principally on membrane translocation and nucleic acid binding for antimicrobial activity, although membrane permeabilization may play a secondary role for some BF2 variants. A better understanding of the role of proline in the BF2 antimicrobial mechanism will contribute to the further design and development of BF2 analogs. Moreover, since proline residues are prevalent among other antimicrobial peptides, this systematic characterization of BF2 provides general insights that can promote our understanding of other systems.     

Two MscS homologs provide mechanosensitive channel activities in the Arabidopsis root

In bacterial and animal systems, mechanosensitive (MS) ion channels are thought to mediate the perception of pressure, touch, and sound [1-3]. Although plants respond to a wide variety of mechanical stimuli, and although many mechanosensitive channel activities have been characterized in plant membranes by the patch-clamp method, the molecular nature of mechanoperception in plant systems has remained elusive [4]. Likely candidates are relatives of MscS (Mechanosensitive channel of small conductance), a well-characterized MS channel that serves to protect E. coli from osmotic shock [5]. Ten MscS-Like (MSL) proteins are found in the genome of the model flowering plant Arabidopsis thaliana[4, 6, 7]. MSL2 and MSL3, along with MSC1, a MscS family member from green algae, are implicated in the control of organelle morphology [8, 9]. Here, we characterize MSL9 and MSL10, two MSL proteins found in the plasma membrane of root cells. We use a combined genetic and electrophysiological approach to show that MSL9 and MSL10, along with three other members of the MSL family, are required for MS channel activities detected in protoplasts derived from root cells. This is the first molecular identification and characterization of MS channels in plant membranes.     

Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels

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.     

Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.