The effects of parabens on the mechanosensitive channels of E. coli

Parabens are alkyl esters of p-hydroxybenzoic acid used as preservatives in a wide range of food, pharmaceutical, and cosmetic products (Soni et al. Food Chem. Toxicol. 39:513–532, 2001). Despite their common use for over 50 years, their mechanism of action is still unclear. In this study we examined the effects of ethyl and propyl paraben, on gating of the E. coli mechanosensitive channel of large conductance (MscL) reconstituted into azolectin liposomes. We found that propyl and ethyl paraben spontaneously activate MscL. Moreover, the addition of propyl paraben caused an increase in MscL activity and the lowering of p_1/2, the pressure at which the MscL was opened 50% of the time, the G_o, the free energy required to open the MscL, and the parameter , which describes the channel sensitivity to pressure. In addition, in silico studies showed that propyl paraben binds to the channel gate of the MscL. The mechanosensitive channel of small conductance was also found to be spontaneously activated by parabens. In summary, our study indicates that one of the previously unidentified mechanisms of action of parabens as antimicrobial agents is via an interaction with the mechanosensitive channels to upset the osmotic gradients in bacteria.This revised version was published online in March 2005 with corrections to Figure 6.     

Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy

The mechanosensitive channel of large conductance (MscL) plays an important role in the survival of bacterial cells to hypo-osmotic shock. This channel has been extensively studied and its sequence, structure and electrophysiological characteristics are well known. Here we present a method to visualise MscL in living bacteria using confocal microscopy. By creating a gene fusion between mscl and the gene encoding the green fluorescent protein (GFP) we were able to express the fusion protein MscL-GFP in bacteria. We show that MscL-GFP is present in the cytoplasmic membrane and forms functional channels. These channels have the same characteristics as wild-type MscL, except that they require more pressure to open. This method could prove an interesting, non-invasive, tool to study the localisation and the regulation of expression of MscL in bacteria.     

Theoretical evaluation of cell membrane ion channel activation by applied magnetic fields

This letter re-examines a recently published calculation of the forces exerted on a membrane ion channel by a cation passing through in the presence of an externally applied magnetic field. We show here, in contradiction to the originally published calculation, that the forces generated due to the Lorentz force of the magnetic field on the cation are negligible compared with the forces required to activate an ion channel protein conformation change associated with the gating of the channel. Received: 11 August 1998 / Revised version: 25 October 1998 / Accepted: 11 November 1998     

Lipid membrane interaction and antimicrobial activity of GsMTx-4, an inhibitor of mechanosensitive channel

GsMTx-4, a polypeptide from the spider Grammostola spatulata, is an inhibitor of mechanosensitive channels. It is known to interact with lipid membranes, suggesting it partitions into the membrane to alter the channel gating, but the effect of the membrane charge on GsMTx-4 activity remains unknown. In this study, we found that GsMTx-4 more effectively interacts with anionic lipids than zwitterionic ones. The effect of GsMTx-4 on negatively charged membranes was similar to that of the antimicrobial peptide melittin, which led us to assess GsMTx-4's antimicrobial activity. Interestingly, we found that, in contrast to other neurotoxins, GsMTx-4 exhibited antimicrobial properties and was more active against Gram-positive than Gram-negative bacteria. These results suggest that GsMTx-4 exerts its antimicrobial effect by altering the packing of the membrane and/or inhibiting mechanosensitive channels. These findings could point the way towards a new class of antimicrobial peptides.     

机械敏感性离子通道蛋白Piezo2的研究进展

2010年科学家在小鼠神经瘤母细胞中筛选鉴定出Piezo1和Piezo2蛋白.Piezo2是一个由机械刺激直接激活且可将机械刺激转换为电信号进而形成机械敏感性电流通道的蛋白质.Piezo2自发现以来一直受到广泛的关注,在触觉、本体感觉、痛觉、肿瘤癌症等多种生理病理过程中发挥重要作用.本文在前期研究的基础上阐述了机械敏感...     

Osmotic shock: a mechanosensitive channel blocker can prevent release of cytoplasmic but not periplasmic proteins

When over-expressed in the cytoplasm of Escherichia coli, carboxylesterase Est55 of Geobacillus stearothermophilus was found to be released from cells upon osmotic shock. Comparing two osmotic shock protocols showed that release of Est55 was abolished in the absence of mechanosensitive channel MscL by one method but not the other. The discrepancy extended to several previously reported cytoplasmic proteins released by osmotic shock, including: EF-Tu, thioredoxin, and DnaK in E. coli. Stepwise analyses of parameters between these two protocols revealed that the use of mechanical pipetting instead of gentle dilution of cells prior to exposure to hypotonic solution abolished the effect of MscL. Furthermore, while this phenomenon of release of certain cytoplasmic proteins was sustained in all three wild type strains of E. coli, presence of gadolinium was able to serve as an MscL channel blocker and prevented release of Est55 and EF-Tu in the process. An optimized protocol of osmotic shock was developed from this study to provide a more reliable assessment of location of proteins in E. coli. This method allowed release of authentic periplasmic MalE and beta-lactamase proteins comparable to that by EDTA-lysozyme treatment.     

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 μM-1 mM and was induced at different values of intracellular pH and Ca²⁺. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg²⁺ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca²⁺ and their sensitivity to intracellular cAMP are consistent with a role in the Ca²⁺ oscillations associated with cell growth.     

Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum

Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.     

The trajectories of particles suspended in electrolytes under the influence of crossed electric and magnetic fields

We observed that particles, suspended in an electrolyte and brought into crossed magnetic and electric fields of low intensities, will deviate in the central part of the electrophoresis chamber of a standard Zeiss Cytopherometer with a component vertical to both fields. The direction and magnitude, however, were sharply at variance with what would be expected by the action of the Lorentz force (EMF) on the surface of the particles. The magnitude of the deviation depends upon the magnetic and electric field strength, the ion concentration of the suspension medium and the geometry of the chamber. The movement of the particles is due to streaming of the electrolyte which is mainly caused by inhomogeneities of the electric field in the electrophoresis chamber. The magnitude of the effect is high enough to occur under physiological conditions. Magneto-electrophoretic streaming might eventually act as a transducer mechanism which could explain the ability of some animals to orientate themselves in the geomagnetic field.     

Conversion of a mechanosensitive channel protein from a membrane-embedded to a water-soluble form by covalent modification with amphiphiles

Covalent modification of integral membrane proteins with amphiphiles may provide a general approach to the conversion of membrane proteins into water-soluble forms for biophysical and high-resolution structural studies. To test this approach, we mutated four surface residues of the pentameric Mycobacterium tuberculosis mechanosensitive channel of large conductance (MscL) to cysteine residues as anchors for amphiphile attachment. A series of modified ion channels with four amphiphile groups attached per channel subunit was prepared. One construct showed the highest water solubility to a concentration of up to 4mg/ml in the absence of detergent. This analog also formed native-like, alpha-helical homo-pentamers in the absence of detergent as judged by circular dichroism spectroscopy, size-exclusion chromatography and various light-scattering techniques. Proteins with longer, or shorter polymers attached, or proteins modified exclusively with polar cysteine-reactive small molecules, exhibited reduced to no solubility and higher-order aggregation. Electron microscopy revealed a homogeneous population of particles consistent with a pentameric channel. Solubilization of membrane proteins by covalent attachment of amphiphiles results in homogeneous particles that may prove useful for crystallization, solution NMR spectroscopy, and electron microscopy.     

CFTR型氯离子通道研究进展

囊性纤维化跨膜传导调节因子（CFTR）是一种重要的氯离子通道,突变易引起囊性纤维化病变,故得名。一系列研究表明,CFTR由5个结构域组成：两个跨膜结构域形成氯离子通道;两个核苷酸结合结构域调节通道的开闭;一个调节结构域主要影响氯通道的活动。这些结构域通过协同作用共同控制了氯离子的跨膜流动,而一些突变可以影响细胞功能而导致囊性纤维化的发生。本文通过介绍CFTR基本结构、调节机制、与囊性纤维化病变的关系及针对CFTR的治疗而对CFTR型氯离子通道有一个的全面的理解。     

Demonstrating the intrinsic ion channel activity of virally encoded proteins

This review summarizes the types of evidence that can be invoked in order to demonstrate that a virally encoded protein possesses ion channel activity that is intrinsic to the life cycle of the virus. Ion channel activity has been proposed to be a key step in the life cycle of influenza virus, and the protein responsible for this activity has been proposed to be the M2 protein encoded by the virus. This review contrasts the evidence supporting the conclusion that the A/M2 protein of influenza A virus has intrinsic ion channel activity with the evidence that the 3AB protein encoded by the human rhinovirus possesses intrinsic ion channel activity.     

Ca2+ ion transport through patch-clamped cells exposed to magnetic fields

The total current of Ca²⁺ ions through patch-clamped cell membranes was measured while exposing clonal insulin-producing β-cells (RINm5F) to a combination of DC and AC magnetic fields at so-called cyclotron resonance conditions. Previous experimental evidence supports the theory that a resonant interaction between magnetic fields and organisms can exist. This experiment was designed to test one possible site of interaction: channels in the cell membrane. The transport of Ca²⁺ ions through the protein channels of the plasma membrane did not show any resonant behavior in the frequency range studied. © 1995 Wiley-Liss, Inc.     

The effects of static magnetic field on action potential propagation and excitation recovery in nerve

Calculations using the Hodgkin–Huxley and one-dimensional cable equations have been performed to determine the expected sensitivity of conduction and refractoriness to changes in the time constant of sodium channel deactivation at negative potentials, as reported experimentally by Rosen (Bioelectromagnetics 24 (2003) 517) when voltage-gated sodium channels are exposed to a 125 mT static magnetic field. The predicted changes in speed of conduction and refractory period are very small.     

Recent advances in ion channel research

The field of ion channels has entered into a rapid phase of development in the last few years, partly due to the breakthroughs in determination of the crystal structures of membrane proteins and advances in computer simulations of biomolecules. These advances have finally enabled the long-dreamed goal of relating function of a channel to its underlying molecular structure. Here we present simplified accounts of the competing permeation theories and then discuss their application to the potassium, gramicidin A and calcium channels.     

Permeability changes of connexin32 hemi channels reconstituted in liposomes induced by extremely low frequency, low amplitude magnetic fields

The effect of extremely low frequency and low amplitude magnetic fields on gap junctional permeability was investigated by using reconstituted connexin32 hemi channel in liposomes. Cytochrome c was loaded inside these proteoliposomes and its reduction upon addition of ascorbate in the bulk aqueous phase was adopted as the index of hemi channel permeability. The permeability rate of the hemi channels, expressed as ΔA/min, was dependent on the incubation temperature of proteoliposomes. The effect of exposures to magnetic fields at different frequencies (7, 13 and 18 Hz) and amplitudes (50, 50 and 70 μT, respectively), and at different temperatures (16, 18 and 24 °C) was studied. Only the exposure of proteoliposomes to 18-Hz (B_acpeak and B_dc=70 μT) magnetic field for 60 min at 16±0.4 °C resulted in a significant enhancement of the hemi channel permeability from ΔA/min=0.0007±0.0002 to ΔA/min=0.0010±0.0001 (P=0.030). This enhancement was not found for magnetic field exposures of liposomes kept at the higher temperatures tested. Temperature appears to influence lipid bilayer arrangement in such a way as being capable to mask possible effects induced by the magnetic field. Although the observed effect was very low, it seems to confirm the applicability of our model previously proposed for the interaction of low frequency electromagnetic fields with lipid membrane.     

Mechanosensitive ion channels in chara: influence of water channel inhibitors, HgCl2 and ZnCl2, on generation of receptor potential

Characean internodal cells generate receptor potential (ΔE _m) in response to mechanical stimuli. Upon a long-lasting stimulus, the cells generated ΔE _m at the moment of both compression and decompression, and the amplitude of ΔE _m at the moment of decompression, (ΔE _m)_E, was larger than that at compression. The long-lasting stimulus caused a membrane deformation (ΔD _m) having two components, a rapid one, (ΔD _m)_rapid, at the moment of compression and a slower one, (ΔD _m)_slow, during the long-lasting compression. We assumed that (ΔD _m)_slow might have some causal relation with the larger ΔE _m at (ΔE _m)_E. We treated internodal cells with either HgCl₂ or ZnCl₂, water channel inhibitors, to decrease (ΔD _m)_slow. Both inhibitors attenuated (ΔD _m)_slow during compression. Cells treated with HgCl₂ generated smaller (ΔE _m)_E compared to nontreated cells. On the other hand, cells treated with ZnCl₂ never attenuated (ΔE _m)_E but, rather, amplified it. Thus, the amplitude of (ΔD _m)_slow did not always show tight correlation with the amplitude of (ΔE _m)_E. Furthermore, when a constant deformation was applied to an internodal cell in a medium with higher or lower osmotic value, a cell having higher turgor always showed a larger (ΔE _m)_E. Thus, we concluded that changes in tension at the membrane may be the most important factor to induce activation of mechanosensitive Ca²⁺ channel.     

Effects of static magnetic fields on diffusion in solutions

Static magnetic fields affect the diffusion of biological particles in solutions through the Lorentz force and Maxwell stress. These effects were analyzed theoretically to estimate the threshold field strength for these effects. Our results show that the Lorentz force suppresses the diffusion of charged particles such as Na+, K+, Ca2+, Cl-, and plasma proteins. However, the threshold is so high, i.e., more than 10(4) T, that the Lorentz force does not affect the ion diffusion at typical field strengths (a few Tesla at most). Since the threshold of gradient fields for producing a change in ion diffusion through the Maxwell stress is more than 10(5) T2/m for paramagnetic molecules (FeCl3, O2) and plasma proteins, their diffusion would be unaffected by typical gradient fields (100 T2/m at most) and even by high gradient fields (less than 10(5) T2/m) used in magnetic separation techniques. In contrast, movement of deoxygenated erythrocytes and FeCl3 colloids (more than 10(3) molecules) is influenced by the usual gradient fields due to a volume effect.     

The superdiamagnetic effect of magnetic fields on one and two component multilamellar liposomes

For multilamella vesicles of DMPC, DPPC, DSPC, binary mixtures of DMPC-DPPC, DMPC-DSPC, DMPC-DPPE, DOPC and egg lecithin, the optical turbidity decreases significantly on the application of a magnetic field in excess of about 0.2 T, provided that the temperature is above the pretransition value. The turbidity reaches a limiting value for magnetic fields of about 2 T. The effect is attributed to augmentation of the diamagnetic anisotropy of the lipid molecules by clustering within the bilayer, with consequent orientation of either the individual ‘superdiamagnetic’ clusters or the whole liposome. It is suggested that, since most animal cell membranes are largely in the liquid crystalline phase, it is possible that homogeneous magnetic fields as low as 0.2 T may cause biologically significant changes within the membrane.     

Modulation of channel activity and gadolinium block of MscL by static magnetic fields

The magnetic field of the Earth has for long been known to influence the behaviour and orientation of a variety of living organisms. Experimental studies of the magnetic sense have, however, been impaired by the lack of a plausible cellular and/or molecular mechanism providing meaningful explanation for detection of magnetic fields by these organisms. Recently, mechanosensitive (MS) ion channels have been implied to play a role in magnetoreception. In this study we have investigated the effect of static magnetic fields (SMFs) of moderate intensity on the activity and gadolinium block of MscL, the bacterial MS channel of large conductance, which has served as a model channel to study the basic physical principles of mechanosensory transduction in living cells. In addition to showing that direct application of the magnetic field decreased the activity of the MscL channel, our study demonstrates for the first time that SMFs can reverse the effect of gadolinium, a well-known blocker of MS channels. The results of our study are consistent with a notion that (1) the effects of SMFs on the MscL channels may result from changes in physical properties of the lipid bilayer due to diamagnetic anisotropy of phospholipid molecules and consequently (2) cooperative superdiamagnetism of phospholipid molecules under influence of SMFs could cause displacement of Gd³⁺ ions from the membrane bilayer and thus remove the MscL channel block.