Characterization of three novel mechanosensitive channel activities in Escherichia coli

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.     

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure

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.     

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.     

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.     

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure

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.     

Managing hypoosmotic stress: aquaporins and mechanosensitive channels in Escherichia coli.

When Escherichia coli cells are subject to hypoosmotic shock they are subject to substantial flows of water that can be equivalent to a 4-5-fold increase in the pressure exerted from the cytoplasm on the membrane and peptidoglycan wall. The recently described aquaporin that facilitates rapid water movement across the cytoplasmic membrane is repressed during growth at high osmolarity. This may enable the cell to reduce the rate of pressure build up during transitions from high to low osmolarity. The presence of multiple mechanosensitive channels in the E. coli cell membrane is well documented. The recent identification of genes that inactivate the MscL and MscS channels has established their role in releasing the pressure built up by hypoosmotic shock. The isolation of specific mutations and the structural studies on MscL now pave the way to a molecular understanding of the mechanism of activation of mechanosensitive channels.     

Ss-bCNGa: a unique member of the bacterial cyclic nucleotide gated (bCNG) channel family that gates in response to mechanical tension

Bacterial cyclic nucleotide gated (bCNG) channels are generally a nonmechanosensitive subset of the mechanosensitive channel of small conductance (MscS) superfamily. bCNG channels are composed of an MscS channel domain, a linking domain, and a cyclic nucleotide binding domain. Among bCNG channels, the channel domain of Ss-bCNGa, a bCNG channel from Synechocystis sp. PCC 6803, is most identical to Escherichia coli (Ec) MscS. This channel also exhibits limited mechanosensation in response to osmotic downshock assays, making it the only known full-length bCNG channel to respond to hypoosmotic stress. Here, we compare and contrast the ability of Ss-bCNGa to gate in response to mechanical tension with Se-bCNG, a nonmechanosensitive bCNG channel, and Ec-MscS, a prototypical mechanosensitive channel. Compared with Ec-MscS, Ss-bCNGa only exhibits limited mechanosensation, which is most likely a result of the inability of Ss-bCNGa to form the strong lipid contacts needed for significant function. Unlike Ec-MscS, Ss-bCNGa displays a mechanical response that increases with protein expression level, which may result from channel clustering driven by interchannel cation?C?? interactions.     

Identification and Analysis of Putative Homologues of Mechanosensitive Channels in Pathogenic Protozoa

Mechanosensitive channels play important roles in the physiology of many organisms, and their dysfunction can affect cell survival. This suggests that they might be therapeutic targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, dysentery, leishmaniasis and trypanosomiasis that are responsible for millions of deaths each year worldwide. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of mechanosensitive channels. Entamoeba histolytica, Leishmania spp., Trypanosoma cruzi and Trichomonas vaginalis have genes encoding homologues of Piezo channels, while most pathogenic protozoa have genes encoding homologues of mechanosensitive small-conductance (MscS) and K⁺-dependent (MscK) channels. In contrast, all parasites examined lack genes encoding mechanosensitive large-conductance (MscL), mini-conductance (MscM) and degenerin/epithelial Na⁺ (DEG/ENaC) channels. Multiple sequence alignments of evolutionarily distant protozoan, amoeban, plant, insect and vertebrate Piezo channel subunits define an absolutely conserved motif that may be involved in channel conductance or gating. MscS channels are not present in humans, and the sequences of protozoan and human homologues of Piezo channels differ substantially. This suggests the possibility for specific targeting of mechanosensitive channels of pathogens by therapeutic drugs.     

Selectivity mechanisms in MscS-like channels: From structure to function

The E. coli mechanosensitive (MS) channel of small conductance (EcMscS) is the prototype of a diverse family of channels present in all domains of life. While EcMscS has been extensively studied, recent developments show that MscS may display some characteristics not widely conserved in this protein subfamily. With numerous members now electrophysiologically characterized, this subfamily of channels displays a breadth of ion selectivity with both anion and cation selective members. The selectivity of these channels may be relatively weak in comparison to voltage-gated channels but their selectivity mechanisms represent great novelty. Recent studies have identified unexpected residues important for selectivity in these homologs revealing different selectivity mechanisms than those employed by voltage gated K⁺, Na⁺, Ca²⁺ and Cl^- channels whose selectivity filters are housed within their transmembrane pores. This commentary looks at what is currently known about MscS subfamily selectivity and begins to unravel the potential physiological relevance of these differences.     

Mutations in a Conserved Domain of E. coli MscS to the Most Conserved Superfamily Residue Leads to Kinetic Changes

In Escherichia coli (E. coli) the mechanosensitive channel of small conductance, MscS, gates in response to membrane tension created from acute external hypoosmotic shock, thus rescuing the bacterium from cell lysis. E. coli MscS is the most well studied member of the MscS superfamily of channels, whose members are found throughout the bacterial and plant kingdoms. Homology to the pore lining helix and upper vestibule domain of E. coli MscS is required for inclusion into the superfamily. Although highly conserved, in the second half of the pore lining helix (TM3B), E. coli MscS has five residues significantly different from other members of the superfamily. In superfamilies such as this, it remains unclear why variations within such a homologous region occur: is it tolerance of alternate residues, or does it define functional variance within the superfamily? Point mutations (S114I/T, L118F, A120S, L123F, F127E/K/T) and patch clamp electrophysiology were used to study the effect of changing these residues in E. coli MscS on sensitivity and gating. The data indicate that variation at these locations do not consistently lead to wildtype channel phenotypes, nor do they define large changes in mechanosensation, but often appear to effect changes in the E. coli MscS channel gating kinetics.     

Effects of GsMTx4 on bacterial mechanosensitive channels in inside-out patches from giant spheroplasts

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.     

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.     

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     

Bacterial mechanosensitive channels: Experiment and theory

Since their discovery in Escherichia coli some 20 years ago, studies of bacterial mechanosensitive (MS) ion channels have been at the forefront of the MS channel research field. Two major events greatly advanced the research on bacterial MS channels: (i) cloning of MscL and MscS, the MS channels of Large and Small conductance, and (ii) solving their 3D crystal structure. These events enabled further experimental studies employing EPR and FRET spectroscopy in addition to patch clamp and molecular biological techniques that have successfully been used in characterization of the structure and function of bacterial MS channels. In parallel with the experimental studies computational modelling has been applied to elucidate the molecular dynamics of MscL and MscS, which has significantly contributed to our understanding of basic physical principles of the mechanosensory transduction in living organisms.     

The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an Escherichiacoli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress.     

Adaptive behavior of bacterial mechanosensitive channels is coupled to membrane mechanics

Mechanosensitive channel of small conductance (MscS), a tension-driven osmolyte release valve residing in the inner membrane of Escherichia coli, exhibits a complex adaptive behavior, whereas its functional counterpart, mechanosensitive channel of large conductance (MscL), was generally considered nonadaptive. In this study, we show that both channels exhibit similar adaptation in excised patches, a process that is completely separable from inactivation prominent only in MscS. When a membrane patch is held under constant pressure, adaptation of both channels is manifested as a reversible current decline. Their dose–response curves recorded with 1–10-s ramps of pressure are shifted toward higher tension relative to the curves measured with series of pulses, indicating decreased tension sensitivity. Prolonged exposure of excised patches to subthreshold tensions further shifts activation curves for both MscS and MscL toward higher tension with similar magnitude and time course. Whole spheroplast MscS recordings performed with simultaneous imaging reveal activation curves with a midpoint tension of 7.8 mN/m and the slope corresponding to ∼15-nm² in-plane expansion. Inactivation was retained in whole spheroplast mode, but no adaptation was observed. Similarly, whole spheroplast recordings of MscL (V23T mutant) indicated no adaptation, which was present in excised patches. MscS activities tried in spheroplast-attached mode showed no adaptation when the spheroplasts were intact, but permeabilized spheroplasts showed delayed adaptation, suggesting that the presence of membrane breaks or edges causes adaptation. We interpret this in the framework of the mechanics of the bilayer couple linking adaptation of channels in excised patches to the relaxation of the inner leaflet that is not in contact with the glass pipette. Relaxation of one leaflet results in asymmetric redistribution of tension in the bilayer that is less favorable for channel opening.     

Recent characterizations of MscS and its homologs provide insight into the basis of ion selectivity in mechanosensitive channels

The bacterial mechanosensitive channel MscS provides an excellent model system for the study of mechanosensitivity and for investigations into the cellular response to hypoosmotic shock. Numerous studies have elucidated the structure, function and gating mechanism of Escherichia coli MscS, providing a wealth of information for the comparative analysis of MscS family members in bacteria, archaea, fungi and plants. We recently reported the electrophysiological characterization of MscS-Like (MSL)10, a MscS homolog from the model flowering plant Arabidopsis thaliana. Here we summarize our results and briefly compare MSL10 to previously described members of the MscS family. Finally, we comment on how this and other recently published studies illuminate the possible mechanisms by which ion selectivity is accomplished in this fascinating family of channels.     

Conserved motifs in mechanosensitive channels MscL and MscS

Mechanosensitive (MS) channels play a major role in protecting bacterial cells against hypo-osmotic shock. To understand their function, it is important to identify the conserved motifs using sequence analysis methods. In this study, the sequence conservation was investigated by an in silico analysis to generate sequence logos. We have identified new conserved motifs in the domains TM1, TM2 and the cytoplasmic helix from 231 homologs of MS channel of large conductance (MscL). In addition, we have identified new motifs for the TM3 and the cytoplasmic carboxy-terminal domain from 309 homologs of MS channel of small conductance (MscS). We found that the conservation in MscL homologs is high for TM1 and TM2 in the three domains of life. The conservation in MscS homologs is high only for TM3 in Bacteria and Archaea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

State-stabilizing Interactions in Bacterial Mechanosensitive Channel Gating and Adaptation

We outline several principles that we believe define the gating of two bacterial mechanosensitive channels, MscL and MscS. Serving as turgor regulators in bacteria and other walled cells, these molecules are tangible models for studying conformational transitions in membrane proteins driven directly by membrane tension. MscL, a compact pentamer, reversibly opens a gigantic 30-Å pore at near-lytic tensions. MscS, a heptameric complex, exhibits transient activation of a smaller pore at moderate tensions, thereby entering a tension-insensitive inactivated state. By comparing the structures and predicted transitions in these channels, we concluded that opening is commonly achieved through tilting and outward motion of the pore-lining helices, which is kinetically limited by hydration of the pore. The intricate adaptive behavior in MscS appears to depend on specific interhelical associations and the flexibility of the pore-lining helices. We discuss physical factors that may direct the transitions and stabilize main functional states in these channels.Osmotic forces are strong, which necessitated development of osmoregulation along with the first semipermeable membrane delineating the early cell. A simple estimation shows that a 1-μm cell behaving as an ideal osmometer would sustain a downshock no stronger than 20 mm, after which membrane tension would exceed the lytic limit of 10–12 dynes/cm. Thus, a cell without a reinforcing envelope or protective valves is very vulnerable. Free-living and enteric microorganisms cycling through the soil and experiencing drastic environmental changes developed robust mechanisms to maintain volume and integrity (1). The mechanosensitive channels MscS and MscL (mechanosensitive channels of small and large conductance, respectively) have been identified as primary osmolyte release valves limiting the turgor pressure under acute osmotic shock (2–4).Without mscS and mscL genes, Escherichia coli survives a 300 mosm osmotic downshock (2), its resistance attributed to the peptidoglycan layer partially restraining swelling. However, expression of either MscS or MscL allows cells to withstand a 700–800 mosm downshock through release of small osmolytes (2). Purification and reconstitution proved that MscL and MscS respond directly to tension in the lipid bilayer (5–7). Both channels reside in the inner (cytoplasmic) membrane (8), with MscL localized at the cell poles, bearing high curvature (9).As primary components of the turgor regulation system, E. coli MscS and MscL became convenient models for studies of tension-driven conformational transitions in membrane proteins (10). The crystal structures of closed-state Mycobacterium tuberculosis MscL (11) and E. coli MscS in two distinct conformations (12, 13) provided invaluable initial points to explore their gating mechanisms, in which computational methods play increasingly important roles.     

Molecular identification of a mechanosensitive channel in archaea

The TM1 domain of the large conductance mechanosensitive (MS) channel of Escherichia coli was used as a genetic probe to search the genomic database of the archaeon Methanoccoccus jannashii for MscL homologs. We report that the hypothetical protein MJ0170 of M. jannashii exhibited 38.5% sequence identity with the TM1 domain of Eco-MscL. Moreover, MJ0170 was found to be a conserved homolog of MscS, the second type of E. coli MS channel encoded by the yggB gene. Furthermore, we identified a cluster of charged residues KIKEE in the C-terminus of MJ0170 that strikingly resembled the charged C-terminal amino acid cluster present in Eco-MscL (RKKEE). We cloned and expressed MJ0170 in E. coli, which when reconstituted into liposomes or expressed in the cell membrane of giant E. coli spheroplasts, exhibited similar activity to the bacterial MS channels. Our study suggests that the M. jannashii MS channel and its homologs evolved as a result of gene duplication of the ancestral MscL-like molecule with the TM1 domain remaining the most conserved structural motif among prokaryotic MS channels.