Strain Transfer in Ventricular Cardiomyocytes to Their Transverse Tubular System Revealed by Scanning Confocal Microscopy

The transverse tubular system (t-system) is a major site for signaling in mammalian ventricular cardiomyocytes including electrical signaling and excitation-contraction coupling. It consists of membrane invaginations, which are decorated with various proteins including mechanosensitive ion channels. Here, we investigated mechanical modulation of the t-system. By applying fluorescent markers, three-dimensional scanning confocal microscopy, and methods of digital image analysis, we studied isolated ventricular cardiomyocytes under different strains. We demonstrate that strain at the cellular level is transmitted to the t-system, reducing the length and volume of tubules and altering their cross-sectional shape. Our data suggest that a cellular strain of as little as 5% affects the shape of transverse tubules, which has important implications for the function of mechanosensitive ion channels found in them. Furthermore, our study supports a prior hypothesis that strain can cause fluid exchange between the t-system and extracellular space.     

Curvature Generation and Pressure Profile Modulation in Membrane by Lysolipids: Insights from Coarse-Grained Simulations

Although many membrane additives are known to modulate the activities of membrane proteins via perturbing the properties of lipid membrane, the underlying mechanism is often not precisely understood. In this study, we investigate the impact of asymmetrically incorporating single-tailed lysophosphatidylcholine (LPC) into a membrane bilayer using coarse-grained molecular dynamics simulations. Using a simple computational protocol designed to approximately mimic a micropipette setting, we show that asymmetric incorporation of LPC can lead to significant curvature in a bilayer. Detailed analysis of geometrical and mechanical properties (pressure profile) of the resulting mound structure indicates that the degree of pressure profile perturbation is determined not by the local curvature per se but by the packing of lipid headgroups (i.e., area-per-lipid). The findings help provide a concrete basis for understanding the activation mechanism of mechanosensitive channels by asymmetric incorporation of LPC into membrane patches in patch-clamp experiments. The calculated local pressure profiles are valuable to the construction of realistic membrane models for the analysis of mechanosensation in a continuum mechanics framework.     

Molecular dynamics study of gating in the mechanosensitive channel of small conductance MscS

Mechanosensitive channels are a class of ubiquitous membrane proteins gated by mechanical strain in the cellular membrane. MscS, the mechanosensitive channel of small conductance, is found in the inner membrane of Escherichia coli and its crystallographic structure in an open form has been recently solved. By means of molecular dynamics simulations we studied the stability of the channel conformation suggested by crystallography in a fully solvated lipid (POPC) bilayer, the combined system encompassing 224,340 atoms. When restraining the backbone of the protein, the channel remained in the open form and the simulation revealed intermittent permeation of water molecules through the channel. Abolishing the restraints under constant pressure conditions led to spontaneous closure of the transmembrane channel, whereas abolishing the restraints when surface tension (20 dyn/cm) was applied led to channel widening. The large balloon-shaped cytoplasmic domain of MscS exhibited spontaneous diffusion of ions through its side openings. Interaction between the transmembrane domain and the cytoplasmic domain of MscS was observed and involved formation of salt bridges between residues Asp62 and Arg128; this interaction may be essential for the gating of MscS. K+ and Cl- ions showed distinctively different distributions in and around the channel.     

Mechanical properties of the red cell membrane skeleton: analysis of axisymmetric deformations

The mechanical properties of erythrocyte membrane composed of a membrane bilayer and membrane skeleton are considered. Two membrane models are described: the model of free boundaries (MFB) and the model of immobilized boundaries (MIB). In MFB, the skeleton is assumed to be attached to the bilayer at a finite number of points, whereas MIB allows the interaction of each spectrin filament with the bilayer along its whole length. For MFB an estimate was made of the mechanical strain generated in the membrane by sucking erythrocytes into a micropipette. The existence of the deformation threshold is demonstrated, below which no mechanical strain, except that of bending, appears in the membrane. Thus only deformations exceeding this threshold result in strain. The relationship between the applied tension and the height of erythrocyte "tongue" sucked into a micropipette was determined. The MIB characteristics correspond to the model of Evans: strains in the membrane are generated at any deformation, however small, i.e. the threshold is equal to zero. A basic feature of this model is quite a different distribution of the skeleton deformations in the membrane. A comparison of the theoretical models and experimental data demonstrated the possibility of either MFB or MIB occurring, depending on the characteristic measurement time.     

Partners in Protection: Interdependence of Cytoskeleton and Plasma Membrane in Adaptations to Applied Forces

In mechanically active environments mammalian cells must cope with potentially injurious forces to survive, but the most proximal mechanosensors are largely unknown. How mechanoprotective responses to applied forces are generated and regulated is still a mystery. We consider recent evidence that suggests cellular mechanoprotective adaptations involve a coordinated remodeling of the cell membrane and the associated cytoskeleton. The plasma membrane ``protects' the cytoskeleton by maintenance of intracellular ionic balance and can modulate force-induced cytoskeletal rearrangements by stretch-activated (e.g., Ca²⁺) ion channels and mechanosensitive enzymes (e.g., Phospholipase A₂ and Phospholipase C). Conversely, the cytoskeleton protects the plasma membrane by providing structural support, reinforcement of the cortical framework at sites of force application, modulation of mechanosensitive ion channels and by potentially contributing to the membrane resealing process after mechanical rupture. We suggest that the plasma membrane and the cytoskeleton are partners in the cytoprotective response to physical forces. Received: 8 September 1999/Revised: 15 December 1999     

Mechanosensitive channels: what can they do and how do they do it?

While mechanobiological processes employ diverse mechanisms, at their heart are force-induced perturbations in the structure and dynamics of molecules capable of triggering subsequent events. Among the best characterized force-sensing systems are bacterial mechanosensitive channels. These channels reflect an intimate coupling of protein conformation with the mechanics of the surrounding membrane; the membrane serves as an adaptable sensor that responds to an input of applied force and converts it into an output signal, interpreted for the cell by mechanosensitive channels. The cell can exploit this information in a number of ways: ensuring cellular viability in the presence of osmotic stress and perhaps also serving as a signal transducer for membrane tension or other functions. This review focuses on the bacterial mechanosensitive channels of large (MscL) and small (MscS) conductance and their eukaryotic homologs, with an emphasis on the outstanding issues surrounding the function and mechanism of this fascinating class of molecules.     

Genetic screen for potassium leaky small mechanosensitive channels (MscS) in Escherichia coli: recognition of cytoplasmic β domain as a new gating element

Mechanosensitive membrane channels in bacteria respond to the mechanical stretching of the membrane. They will open when bacteria are subjected to rapid osmotic down shock. MscS is a bacterial mechanosensitive channel of small conductance. It is a heptameric membrane protein whose transmembrane part, including the gate and its kinetics, has been well characterized. MscS has a large cytoplasmic domain of a cage-like shape that changes its conformation upon gating, but its involvement in gating is not understood. We screened MscS for mutations that cause potassium leak in Escherichia coli strains deficient in potassium transport systems. We did a phenotypic analysis of single and multiple mutants and recorded the single channel activities of some of them. After these analyses, we attributed the effects of a number of mutations to particular functional states of the channel. Our screen revealed that MscS leaks potassium in a desensitized and in an inactivated state. It also appeared that the lower part of TM3 (transmembrane, pore-forming helix) and the cytoplasmic β domain are tightly packed in the inactivated state but are dissociated in the open state. We attribute the TM3-β interaction to stabilization of the inactivated state in MscS and to the control of tight closure of its membrane pore.     

On the Role of the Difference in Surface Tensions Involved in the Allosteric Regulation of NHE-1 Induced by Low to Mild Osmotic Pressure,Membrane Tension and Lipid Asymmetry

The sodium-proton exchanger 1 (NHE-1) is a membrane transporter that exchanges Na⁺ for H⁺ ion across the membrane of eukaryotic cells. It is cooperatively activated by intracellular protons, and this allosteric regulation is modulated by the biophysical properties of the plasma membrane and related lipid environment. Consequently, NHE-1 is a mechanosensitive transporter that responds to osmotic pressure, and changes in membrane composition. The purpose of this study was to develop the relationship between membrane surface tension, and the allosteric balance of a mechanosensitive transporter such as NHE-1. In eukaryotes, the asymmetric composition of membrane leaflets results in a difference in surface tensions that is involved in the creation of a reservoir of intracellular vesicles and membrane buds contributing to buffer mechanical constraints. Therefore, we took this phenomenon into account in this study and developed a set of relations between the mean surface tension, membrane asymmetry, fluid phase endocytosis and the allosteric equilibrium constant of the transporter. We then used the experimental data published on the effects of osmotic pressure and membrane modification on the NHE-1 allosteric constant to fit these equations. We show here that NHE-1 mechanosensitivity is more based on its high sensitivity towards the asymmetry between the bilayer leaflets compared to mean global membrane tension. This compliance to membrane asymmetry is physiologically relevant as with their slower transport rates than ion channels, transporters cannot respond as high pressure-high conductance fast-gating emergency valves.     

Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation

The skeleton adapts to its mechanical usage, although at the cellular level, the distribution and magnitude of strains generated and their detection are ill-understood. The magnitude and nature of the strains to which cells respond were investigated using an atomic force microscope (AFM) as a microindentor. A confocal microscope linked to the setup enabled analysis of cellular responses. Two different cell response pathways were identified: one, consequent upon contact, depended on activation of stretch-activated ion channels; the second, following stress relaxation, required an intact microtubular cytoskeleton. The cellular responses could be modulated by selectively disrupting cytoskeletal components thought to be involved in the transduction of mechanical stimuli. The F-actin cytoskeleton was not required for responses to mechanical strain, whereas the microtubular and vimentin networks were. Treatments that reduced membrane tension, or its transmission, selectively reduced contact reactions. Immunostaining of the cell cytoskeleton was used to interpret the results of the cytoskeletal disruption studies. We provide an estimate of the cellular strain magnitude needed to elicit intracellular calcium responses and propose a model that links single cell responses to whole bone adaptation. This technique may help to understand adaptation to mechanical usage in other organs.     

Intramembrane electrostatic interactions destabilize lipid vesicles

Membrane stability is of central concern in many biology and biotechnology processes. It has been suggested that intramembrane electrostatic interactions play a key role in membrane stability. However, due primarily to a lack of supporting experimental evidence, they are not commonly considered in mechanical analyses of lipid membranes. In this paper, we use the micropipette aspiration technique to characterize the elastic moduli and critical tensions of lipid vesicles with varying surface charge. Charge was induced by doping neutral phosphatidylcholine vesicles with anionic lipids phosphatidylglycerol and phosphatidic acid. Measurements were taken in potassium chloride (moderate ion-lipid binding) and tetramethylammonium chloride (low ion-lipid binding) solutions. We show that inclusion of anionic lipid does not appreciably alter the areal dilation elasticity of lipid vesicles. However, the tension required for vesicle rupture decreases with increasing anionic lipid fraction and is a function of electrolyte composition. Using vesicles with 30% charged (i.e., unbound) anionic lipid, we measured critical tension reductions of 75%, demonstrating the important role of electrostatic interactions in membrane stability.     

A three-dimensional random network model of the cytoskeleton and its role in mechanotransduction and nucleus deformation

We have developed a three-dimensional random network model of the intracellular actin cytoskeleton and have used it to study the role of the cytoskeleton in mechanotransduction and nucleus deformation. We use the model to predict the deformation of the nucleus when mechanical stresses applied on the plasma membrane are propagated through the random cytoskeletal network to the nucleus membrane. We found that our results agree with previous experiments utilizing micropipette pulling. Therefore, we propose that stress propagation through the random cytoskeletal network can be a mechanism to effect nucleus deformation, without invoking any biochemical signaling activity. Using our model, we also predict how nucleus strain and its relative displacement within the cytosol vary with varying concentrations of actin filaments and actin-binding proteins. We find that nucleus strain varies in a sigmoidal manner with actin filament concentration, while there exists an optimal concentration of actin-binding proteins that maximize nucleus displacement. We provide a theoretical analysis for these nonlinearities in terms of the connectivity of the random cytoskeletal network. Finally, we discuss laser ablation experiments that can be performed to validate these results in order to advance our understanding of the role of the cytoskeleton in mechanotransduction.     

2,2,2-Trifluoroethanol changes the transition kinetics and subunit interactions in the small bacterial mechanosensitive channel MscS

2,2,2-Trifluoroethanol (TFE), a low-dielectric solvent, has recently been used as a promising tool to probe the strength of intersubunit interactions in membrane proteins. An analysis of inner membrane proteins of Escherichia coli has identified several SDS-resistant protein complexes that separate into subunits upon exposure to TFE. One of these was the homo-heptameric stretch-activated mechanosensitive channel of small conductance (MscS), a ubiquitous component of the bacterial turgor-regulation system. Here we show that a substantial fraction of MscS retains its oligomeric state in cold lithium-dodecyl-sulfate gel electrophoresis. Exposure of MscS complexes to 10-15 vol % TFE in native membranes or nonionic detergent micelles before lithium-dodecyl-sulfate electrophoresis results in a complete dissociation into monomers, suggesting that at these concentrations TFE by itself disrupts or critically compromises intersubunit interactions. Patch-clamp analysis of giant E. coli spheroplasts expressing MscS shows that exposure to TFE in lower concentrations (0.5-5.0 vol %) causes leftward shifts of the dose-response curves when applied extracellularly, and rightward shifts when added from the cytoplasmic side. In the latter case, TFE increases the rate of tension-dependent inactivation and lengthens the process of recovery to the resting state. MscS responses to pressure ramps of different speeds indicate that in the presence of TFE most channels reside in the resting state and only at tensions near the activation threshold does TFE dramatically speed up inactivation. The effect of TFE is reversible as normal channel activity returns 15-30 min after a TFE washout. We interpret the observed midpoint shifts in terms of asymmetric partitioning of TFE into the membrane and distortion of the bilayer lateral pressure profile. We also relate the increased rate of inactivation and subunit separation with the capacity of TFE to perturb buried interhelical contacts in proteins and discuss these effects in the framework of the proposed gating mechanism of MscS.     

Adaptive MscS gating in the osmotic permeability response in E. coli: the question of time

Microorganisms adapt to osmotic downshifts by releasing small osmolytes through mechanosensitive (MS) channels. We want to understand how the small mechanosensitive channel's (MscS) activation and inactivation, both driven by membrane tension, optimize survival in varying hypoosmotic shock situations. By measuring light scattering with a stopped-flow device, we estimate bacterial swelling time as 30-50 ms. A partial solute equilibration follows within 150-200 ms, during which optical responses from cells with WT MscS deviate from those lacking MS channels. MscS opening rates estimated in patch clamp show the channels readily respond to tensions below the lytic limit with a time course faster than 20 ms and close promptly upon tension release. To address the role of the tension-insensitive inactivated state in vivo, we applied short, long, and two-step osmotic shock protocols to WT, noninactivating G113A, and fast-inactivating D62N mutants. WT and G113A showed a comparable survival in short 1 min 800 mOsm downshock experiments, but G113A was at a disadvantage under a long 60 min shock. Preshocking cells carrying WT MscS for 15 s to 15 min with a 200 mOsm downshift did not sensitize them to the final 500 mOsm drop in osmolarity of the second step. However, these two-step shocks induced death in D62N more than just a one-step 700 mOsm downshift. We conclude MscS is able to activate and exude osmolytes faster than lytic pressure builds inside the cell under abrupt shock. During prolonged shocks, gradual inactivation prevents continuous channel activity and assists recovery. Slow kinetics of inactivation in WT MscS ensures that mild shocks do not inactivate the entire population, leaving some protection should conditions worsen.     

Investigating the structural dynamics of the PIEZO1 channel activation and inactivation by coarse‐grained modeling

The PIEZO channels, a family of mechanosensitive channels in vertebrates, feature a fast activation by mechanical stimuli (eg, membrane tension) followed by a slower inactivation. Although a medium‐resolution structure of the trimeric form of PIEZO1 was solved by cryo‐electron microscopy (cryo‐EM), key structural changes responsible for the channel activation and inactivation are still unknown. Toward decrypting the structural mechanism of the PIEZO1 activation and inactivation, we performed systematic coarse‐grained modeling using an elastic network model and related modeling/analysis tools (ie, normal mode analysis, flexibility and hotspot analysis, correlation analysis, and cryo‐EM‐based hybrid modeling and flexible fitting). We identified four key motional modes that may drive the tension‐induced activation and inactivation, with fast and slow relaxation time, respectively. These modes allosterically couple the lateral and vertical motions of the peripheral domains to the opening and closing of the intra‐cellular vestibule, enabling external mechanical forces to trigger, and regulate the activation/inactivation transitions. We also calculated domain‐specific flexibility profiles, and predicted hotspot residues at key domain‐domain interfaces and hinges. Our results offer unprecedented structural and dynamic information, which is consistent with the literature on mutational and functional studies of the PIEZO channels, and will guide future studies of this important family of mechanosensitive channels.     

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.     

Bilayer-mediated clustering and functional interaction of MscL channels

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.     

The mechanoelectrical response of the cytoplasmic membrane of Vibrio cholerae

Persistence of Vibrio cholerae in waters of fluctuating salinity relies on the capacity of this facultative enteric pathogen to adapt to varying osmotic conditions. In an event of osmotic downshift, osmolytes accumulated inside the bacterium can be quickly released through tension-activated channels. With the newly established procedure of giant spheroplast preparation from V. cholerae, we performed the first patch-clamp characterization of its cytoplasmic membrane and compared tension-activated currents with those in Esherichia coli. Saturating pressure ramps revealed two waves of activation belonging to the ∼1-nS mechanosensitive channel of small conductance (MscS)-like channels and ∼3-nS mechanosensitive channel of large conductance (MscL)-like channels, with a pressure midpoint ratio p_0.5MscS/p_0.5MscL of 0.48. We found that MscL-like channels in V. cholerae present at a density three times higher than in E. coli, and yet, these vibrios were less tolerant to large osmotic downshocks. The Vibrio MscS-like channels exhibit characteristic inward rectification and subconductive states at depolarizing voltages; they also adapt and inactivate at subsaturating tensions and recover within 2 s upon tension release, just like E. coli MscS. Trehalose, a compatible internal osmolyte accumulated under hypertonic conditions, significantly shifts activation curves of both MscL- and MscS-like channels toward higher tensions, yet does not freely partition into the channel pore. Direct electrophysiology of V. cholerae offers new avenues for the in situ analysis of membrane components critical for osmotic survival and electrogenic transport in this pathogen.     

Brefeldin A block of integrin-dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction

Many animal cells release ATP into the extracellular medium, and often this release is mechanosensitive. However, the mechanisms underlying this release are not well understood. Using the luciferin-luciferase bioluminescent assay we demonstrate that a Xenopus oocyte releases ATP at a basal rate approximately 0.01 fmol/s, and gentle mechanical stimulation can increase this to 50 fmol/s. Brefeldin A, nocodazole, and progesterone-induced- maturation block basal and mechanosensitive ATP release. These treatments share the common feature of disrupting the Golgi complex and vesicle trafficking to the cell surface and thereby block protein secretion and membrane protein insertion. We propose that ATP release occurs when protein transport vesicles enriched in ATP fuse with the plasma membrane. Collagenase, integrin-binding peptides, and cytochalasin D also block ATP release, indicating that extracellular, membrane and cytoskeletal elements are involved in the release process. Elevation of intracellular Ca(2+) does not evoke ATP release but potentiates mechanosensitive ATP release. Our study indicates a novel mechanism of mechanotransduction that would allow cells to regulate membrane trafficking and protein transport/secretion in response to mechanical loading.     

Calcium-permeable channels in plant cells

Calcium signal transduction is a central mechanism by which plants sense and respond to endogenous and environmental stimuli. Cytosolic Ca(2+) elevation is achieved via two cellular pathways, Ca(2+) influx through Ca(2+) channels in the plasma membrane and Ca(2+) release from intracellular Ca(2+) stores. Because of the significance of Ca(2+) channels in cellular signaling, interaction with the environment and developmental processes in plants, a great deal of effort has been invested in recent years with regard to these important membrane proteins. Because of limited space, in this review we focus on recent findings giving insight into both the molecular identity and physiological function of channels that have been suggested to be responsible for the elevation in cytosolic Ca(2+) level, including cyclic nucleotide gated channels, glutamate receptor homologs, two-pore channels and mechanosensitive Ca(2+) -permeable channels. We provide an overview of the regulation of these Ca(2+) channels and their physiological roles and discuss remaining questions.     

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.