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1.
GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata folded into an inhibitory cysteine knot and it selectively affects gating of some mechanosensitive channels. Here we report the effects of cytoplasmic GsMTx4 on the two bacterial channels, MscS and MscL, in giant Escherichia coli spheroplasts. In excised inside-out patches, GsMTx4 sensitized both channels to tension by increasing the opening rate and decreasing the closing rate. With ascending and descending pressure ramps, GsMTx4 increased the gating hysteresis for MscS, a consequence of slower gating kinetics. Quantitative kinetic analysis of the primary C↔O transition showed that the hysteresis is a result of the decreased closing rate. The gating barrier location relative to the open state energy well was unaffected by GsMTx4. A reconstructed energy profile suggests that the peptide prestresses the resting state of MscS, lowering the net barrier to opening and stabilizes the open conformation by ∼8 kT. In excised patches, both MscL and MscS exhibit reversible adaptation, a process separable from inactivation for MscS. GsMTx4 decreased the rate of reversible adaptation for both channels and the MscS recovery rate from the inactivation. These measurements support a mechanism where GsMTx4 binds to the lipid interface of the channel, increasing the local stress that is sensed by the channels and stabilizing the expanded conformations. 相似文献
2.
Ian Rowe Merina Elahi Anwar Huq Sergei Sukharev 《The Journal of general physiology》2013,142(1):75-85
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 p0.5MscS/p0.5MscL 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. 相似文献
3.
Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the “flying-patch” patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by “shielding” the cytoplasmic domain of the channels. 相似文献
4.
《Channels (Austin, Tex.)》2013,7(4):262-271
Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the “flying-patch” patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by “shielding” the cytoplasmic domain of the channels. 相似文献
5.
Shaista Shaikh Charles D Cox Takeshi Nomura Boris Martinac 《Channels (Austin, Tex.)》2014,8(4):321-326
Mechanosensitive (MS) ion channels are molecular sensors that detect and transduce signals across prokaryotic and eukaryotic cell membranes arising from external mechanical stimuli or osmotic gradients. They play an integral role in mechanosensory responses including touch, hearing, and proprioception by opening or closing in order to facilitate or prevent the flow of ions and organic osmolytes. In this study we use a linear force model of MS channel gating to determine the gating membrane tension (γ) and the gating area change (ΔA) associated with the energetics of MscS channel gating in giant spheroplasts and azolectin liposomes. Analysis of Boltzmann distribution functions describing the dependence of MscS channel gating on membrane tension indicated that the gating area change (ΔA) was the same for MscS channels recorded in both preparations. The comparison of the membrane tension (γ) gating the channel, however, showed a significant difference between the MscS channel activities in these two preparations. 相似文献
6.
From membrane tension to channel gating: A principal energy transfer mechanism for mechanosensitive channels
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Xuejun C. Zhang Zhenfeng Liu Jie Li 《Protein science : a publication of the Protein Society》2016,25(11):1954-1964
Mechanosensitive (MS) channels are evolutionarily conserved membrane proteins that play essential roles in multiple cellular processes, including sensing mechanical forces and regulating osmotic pressure. Bacterial MscL and MscS are two prototypes of MS channels. Numerous structural studies, in combination with biochemical and cellular data, provide valuable insights into the mechanism of energy transfer from membrane tension to gating of the channel. We discuss these data in a unified two‐state model of thermodynamics. In addition, we propose a lipid diffusion‐mediated mechanism to explain the adaptation phenomenon of MscS. 相似文献
7.
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. 相似文献
8.
9.
Lipid bilayers provide a natural anisotropic environment for membrane proteins and can serve as apolar reservoirs for lipid-derived second messengers or lipophilic drugs. Partitioning of lipophilic agents changes the lateral pressure distribution in the bilayer, affecting integral proteins. p-Hydroxybenzoic acid esters (parabens) are amphipathic compounds widely used as food and cosmetics preservatives, but the mechanisms of their broad antibacterial action are unknown. Here we describe effects of ethyl, propyl, and butyl parabens on the gating of the bacterial mechanosensitive channel of small conductance (MscS) and compare them with the surface activity and lateral pressure changes measured in lipid monolayers in the presence of these substances. Near the bilayer-monolayer equivalence pressure of 35 mN/m, ethyl, propyl, or butyl paraben present in the subphase at 1 mM increased the surface pressure of the monolayer by 5, 12.5, or 20%, respectively. No spontaneous activation of MscS channels was observed in patch-clamp experiments with parabens added from either the cytoplasmic or periplasmic side. Increasing concentrations of parabens on the cytoplasmic side of excised patches shifted activation curves of MscS toward higher tensions. A good correlation between the pressure increases in monolayers and shifts in activation midpoints in patch-clamp experiments suggested that the more hydrophobic parabens partition more strongly into the lipid and exert larger effects on channel gating through changes in lateral pressure. We show that cytoplasmically presented ethyl or butyl parabens both hasten the process of desensitization of MscS and influence inactivation differently. The higher rate of desensitization is likely due to increased lateral pressure in the cytoplasmic leaflet surrounding the gate. Neither of the parabens strongly affects the rate of recovery and does not seem to penetrate the TM2-TM3 interhelical clefts in MscS. We conclude that the bacterial mechanosensitive channel MscS provides a sensitive readout of lateral membrane pressure exerted by amphipathic molecules but may not be the primary target for the parabens in their antimicrobial activity. 相似文献
10.
11.
Mechanosensors are important for many life functions, including the senses of touch, balance, and proprioception; cardiovascular regulation; kidney function; and osmoregulation. Many channels from an assortment of families are now candidates for eukaryotic mechanosensors and proprioception, as well as cardiovascular regulation, kidney function, and osmoregulation. Bacteria also possess two families of mechanosensitive channels, termed MscL and MscS, that function as osmotic emergency release valves. Of the two channels, MscL is the most conserved, most streamlined in structure, and largest in conductance at 3.6 nS with a pore diameter in excess of 30 Å; hence, the structural changes required for gating are exaggerated and perhaps more easily defined. Because of these properties, as well as its tractable nature, MscL represents a excellent model for studying how a channel can sense and respond to biophysical changes of a lipid bilayer. Many of the properties of the MscL channel, such as the sensitivity to amphipaths, a helix that runs along the membrane surface and is connected to the pore via a glycine, a twisting and turning of the transmembrane domains upon gating, and the dynamic changes in membrane interactions, may be common to other candidate mechanosensors. Here we review many of these properties and discuss their structural and functional implications. 相似文献
12.
《Channels (Austin, Tex.)》2013,7(4):321-326
Mechanosensitive (MS) ion channels are molecular sensors that detect and transduce signals across prokaryotic and eukaryotic cell membranes arising from external mechanical stimuli or osmotic gradients. They play an integral role in mechanosensory responses including touch, hearing, and proprioception by opening or closing in order to facilitate or prevent the flow of ions and organic osmolytes. In this study we use a linear force model of MS channel gating to determine the gating membrane tension (γ) and the gating area change (ΔA) associated with the energetics of MscS channel gating in giant spheroplasts and azolectin liposomes. Analysis of Boltzmann distribution functions describing the dependence of MscS channel gating on membrane tension indicated that the gating area change (ΔA) was the same for MscS channels recorded in both preparations. The comparison of the membrane tension (γ) gating the channel, however, showed a significant difference between the MscS channel activities in these two preparations. 相似文献
13.
We have successfully expressed and characterized mechanosensitive channel of small conductance (MscS) from Escherichia coli in oocytes of the African clawed frog, Xenopus laevis. MscS expressed in oocytes has the same single-channel conductance and voltage dependence as the channel in its native environment. Two hallmarks of MscS activity, the presence of conducting substates at high potentials and reversible adaptation to a sustained stimulus, are also exhibited by oocyte-expressed MscS. In addition to its ease of use, the oocyte system allows the user to work with relatively large patches, which could be an advantage for the visualization of membrane deformation. Furthermore, MscS can now be compared directly to its eukaryotic homologues or to other mechanosensitive channels that are not easily studied in E. coli. 相似文献
14.
We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained. 相似文献
15.
We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained. 相似文献
16.
This study aims to explore gating mechanisms of mechanosensitive channels in terms of membrane tension, membrane adaptation, protein conformation, and energetics. The large conductance mechanosensitive channel from Mycobacterium tuberculosis (Tb-MscL) is used as a model system; Tb-MscL acts as a safety valve by releasing small osmolytes through the channel opening under extreme hypoosmotic conditions. Based on the assumption that the channel gating involves tilting of the transmembrane (TM) helices, we have performed free energy simulations of Tb-MscL as a function of TM helix tilt angle in a dimyristoylphosphatidylcholine bilayer. Based on the change in system dimensions, TM helix tilting is shown to be essentially equivalent to applying an excess surface tension to the membrane, causing channel expansion, lipid adaptation, and membrane thinning. Such equivalence is further corroborated by the observation that the free energy cost of Tb-MscL channel expansion is comparable to the work done by the excess surface tension. Tb-MscL TM helix tilting results in an expanded water-conducting channel of an outer dimension similar to the proposed fully open MscL structure. The free energy decomposition indicates a possible expansion mechanism in which tilting and expanding of TM2 facilitates the iris-like motion of TM1, producing an expanded Tb-MscL. 相似文献
17.
MscL is a mechanosensitive channel gated by membrane tension in the lipid bilayer alone. Its structure, known from x-ray crystallography, indicates that it is a homopentamer. Each subunit comprises two transmembrane segments TM1 and TM2 connected by a periplasmic loop. The closed pore is lined by five TM1 helices. We expressed in Escherichia coli and purified two halves of the protein, each containing one of the transmembrane segments. Their electrophysiological activity was studied by the patch-clamp recording upon reconstitution in artificial liposomes. The TM2 moiety had no electrophysiological activity, whereas the TM1 half formed channels, which were not affected by membrane tension and varied in conductance between 50 and 350 pS in 100 mM KCl. Coreconstitution of the two halves of MscL however, yielded mechanosensitive channels having the same conductance as the native MscL (1500 pS), but exhibiting increased sensitivity to pressure. Our results confirm the current view on the functional role of TM1 and TM2 helices in the MscL gating and emphasize the importance of helix-helix interactions for the assembly and functional properties of the channel protein. In addition, the results indicate a crucial role of the periplasmic loop for the channel mechanosensitivity. 相似文献
18.
Michelle D. Edwards Susan Black Tim Rasmussen Akiko Rasmussen Neil R. Stokes Terri-Leigh Stephen Samantha Miller Ian R. Booth 《Channels (Austin, Tex.)》2012,6(4):272-281
Mechanosensitive channels sense elevated membrane tension that arises from rapid water influx occurring when cells move from high to low osmolarity environments (hypoosmotic shock). These non-specific channels in the cytoplasmic membrane release osmotically-active solutes and ions. The two major mechanosensitive channels in Escherichia coli are MscL and MscS. Deletion of both proteins severely compromises survival of hypoosmotic shock. However, like many bacteria, E. coli cells possess other MscS-type genes (kefA, ybdG, ybiO, yjeP and ynaI). Two homologs, MscK (kefA) and YbdG, have been characterized as mechanosensitive channels that play minor roles in maintaining cell integrity. Additional channel openings are occasionally observed in patches derived from mutants lacking MscS, MscK and MscL. Due to their rare occurrence, little is known about these extra pressure-induced currents or their genetic origins. Here we complete the identification of the remaining E. coli mechanosensitive channels YnaI, YbiO and YjeP. The latter is the major component of the previously described MscM activity (~300 pS), while YnaI (~100 pS) and YbiO (~1000 pS) were previously unknown. Expression of native YbiO is NaCl-specific and RpoS-dependent. A Δ7 strain was created with all seven E. coli mechanosensitive channel genes deleted. High level expression of YnaI, YbiO or YjeP proteins from a multicopy plasmid in the Δ7 strain (MJFGH) leads to substantial protection against hypoosmotic shock. Purified homologs exhibit high molecular masses that are consistent with heptameric assemblies. This work reveals novel mechanosensitive channels and discusses the regulation of their expression in the context of possible additional functions. 相似文献
19.
Single-channel recordings from central neurons of the helix snail, Cepaea nemoralis, revealed two types of channels that could be activated by stretch (i.e., by the membrane deformation produced when suction is applied to the patch pipette). One, a K+ channel (58 pS in physiological solution), was evident in excised and cell-attached patches. Its conductance in symmetrical [K+] solutions indicated a channel of high K+ permeability (PK = 3.4 x 10(-13) cm/s). Though osmoregulation has been suggested as a function for such channels, comparisons among molluscs indicate osmotic milieu does not govern their expression; Cepaea is terrestrial, and stretch-activated K+ channels similar to those described here occur in aquatic and marine molluscs. The second type of channel, observed only in excised patches, was Cl- permeant; it had a large conductance (130 pS) and was inactive prior to patch excision. Membrane tension may not be the physiological activator of either the K+ or Cl- channel; the channels are designated as stretch-activated channels on the basis of their experimental behaviour during single-channel recording. 相似文献
20.
The crystal structure of the small conductance mechanosensitive channel (MscS) has been an invaluable tool in the search for the gating mechanism, however many functional aspects of the channel remain unsettled. Here we characterized the gating of MscS in Escherichia coli spheroplasts in a triple mutant (mscL-, mscS-, mscK-) background. We used a pressure clamp apparatus along with software developed in-lab to generate dose-response curves directly from two-channel recordings of current and pressure. In contrast to previous publications, we found that MscS exhibits essentially voltage-independent activation by tension, but at the same time strong voltage-dependent inactivation under depolarizing conditions. The MscS activation curves obtained under saturating ramps of pressure, at different voltages, gave estimates for the energy, area, and gating charge for the closed-to-open transition as 24 kT, 18 nm2, and +0.8, respectively. The character of activation and inactivation was similar in both K+ and Na+ buffers. Perhaps the most salient and intriguing property of MscS gating was a strong dependence on the rate of pressure application. Patches subjected to various pressure ramps from 2.7 to 240 mmHg/s revealed a midpoint of activation almost independent of rate. However, the resultant channel activity was dramatically lower when pressure was applied slowly, especially at depolarizing pipette voltages. It appears that MscS prefers to respond in full to abrupt stimuli but manages to ignore those applied slowly, as if the gate were connected to the tension-transmitting element via a velocity-sensitive "dashpot." With slower ramps, channels inactivate during the passage through a narrow region of pressures below the activation midpoint. This property of "dumping" a slowly applied force may be important in environmental situations where rehydration of cells occurs gradually and release of osmolytes is not desirable. MscS often enters the inactivated state through subconducting states favored by depolarizing voltage. The inactivation rate increases exponentially with depolarization. Based on these results we propose a kinetic scheme and gating mechanism to account for the observed phenomenology in the framework of available structural information. 相似文献