Mechanosensitive channels of bacteria and archaea share a common ancestral origin

The ubiquity of mechanosensitive (MS) ion channels set off a search for their functional homologues in archaea, the third domain of life. A new MS channel was identified in the archaeon Methanococcus jannaschii by using the TM1 transmembrane domain of the bacterial MS channel of large conductance, MscL, as a genetic probe to search the archaeal genomic database for MS channel homologues. The hypothetical protein MJ0170 (MscMJ) was found to harbor two MscL-like TM1 structural motifs and showed a high degree of se quence and secondary structure conservation with MscS (YggB) homologues. The alignment of sequences of MscL, MscS and MscMJ homologues further revealed that bacterial and archaeal channels form a phylogenetic tree composed of three main branches and share a common ancestral origin. This suggests the evolution of prokaryotic MS channels via gene duplication of a MscL-like progenitor gene followed by divergence, fur ther indicating that the common ancestor of the prokaryotic MS channels most likely resembled MscL. When expressed in E. coli and functionally examined by the patch clamp, the MscMJ protein behaved as a MS channel with a conductance of 270 pS in 200 mM KCl and a cation selectivity (PK/PC]) of approximately 6. The structural and functional homologue of MscMJ, MscMJLR, was identified as a second type of MS channel in M. jannaschii. The channel has a conductance of approximately 2 nS, rectifies with voltage and shares cation selectivity with MscMJ. The stoichiometry of both types of MS channels revealed that the free energy of activation, deltaG0 approximately 7kT, obtained for MscMJ matches the one calculated for MscS, deltaG0 approximately 5kT, whereas the free energy of activation approximately deltaG0 approximately 18kT of MscMJLR resembles more the deltaG0 = 14-19kT reported for MscL. The presence of two types of MS channels discovered in the cell envelope of M. jannaschii indicates that multiplicity of MS channels in prokaryotes is a necessary element for their survival in the habitats frequently challenged by sudden changes in osmolarity. Further functional and phylogenetic study of MS channels from all three domains of the universal phylogenetic tree may help to understand the evolution and common biophysical principles that govern mechanosensory transduction.     

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.     

Functional analysis of conserved motifs in the mechanosensitive channel homolog MscS-Like2 from Arabidopsis thaliana

The Mechanosensitive channel of Small conductance (MscS) of Escherichia coli has become an excellent model system for the structural, biophysical, and functional study of mechanosensitive ion channels. MscS, a complex channel with multiple states, contributes to protection against lysis upon osmotic downshock. MscS homologs are widely and abundantly dispersed among the bacterial and plant lineages, but are not found in animals. Investigation into the eukaryotic branch of the MscS family is in the beginning stages, and it remains unclear how much MscS homologs from eukaryotes resemble E. coli MscS with respect to structure, function, and regulation. Here we test the effect of mutating three conserved motifs on the function of MscS-Like (MSL)2, a MscS homolog localized to the plastids of Arabidopsis thaliana. We show that 1) a motif at the top of the cytoplasmic domain, referred to here as the PN(X)(9)N motif, is essential for MSL2 function and for its proper intraplastidic localization; 2) substituting polar residues for two large hydrophobic residues located in the predicted pore-lining transmembrane helix of MSL2 produces a likely gain-of-function allele, as previously shown for MscS; and 3) mis-expression of this allele causes severe defects in leaf growth, loss of chloroplast integrity, and abnormal starch accumulation. Thus, two of the three conserved motifs we analyzed are critical for MSL2 function, consistent with the conservation of structure and function between MscS family members in bacteria and plants. These results underscore the importance of plastidic mechanosensitive channels in the maintenance of normal plastid and leaf morphology.     

Two Families of Mechanosensitive Channel Proteins

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 α-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.     

Two families of mechanosensitive channel proteins.

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.     

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 dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

Negative and positive temperature dependence of potassium leak in MscS mutants: Implications for understanding thermosensitive channels

Bacterial mechanosensitive channel of small conductance (MscS) is a protein, whose activity is modulated by membrane tension, voltage and cytoplasmic crowding. MscS is a homoheptamer and each monomer consists of three transmembrane helices (TM1-3). Hydrophobic pore of the channel is made of TM3s surrounded by peripheral TM1/2s. MscS gating is a complex process, which involves opening and inactivation in response to the increase of membrane tension. A number of MscS mutants were isolated. Among them mutants affecting gating have been found including gain-of-function (GOF) and loss-of-function (LOF) that open at lower or at higher thresholds, respectively. Previously, using an in vivo screen we isolated multiple MscS mutants that leak potassium and some of them were GOF or LOF. Here we show that for a subset of these mutants K⁺ leak is negatively (NTD) or positively (PTD) temperature dependent. We show that temperature reliance of these mutants does not depend on how MS gating is affected by a particular mutation. Instead, we argue that NTD or PTD leak is due to the opposite allosteric coupling of the structures that determine the temperature dependence to the channel gate. In PTD mutants an increased hydration of the pore vestibule is directly coupled to the increase in the channel conductance. In NTD mutants, at higher temperatures an increased hydration of peripheral structures leads to complete separation of TM3 and a pore collapse.     

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.     

Domain organization of the MscS mechanosensitive channel of Escherichia coli

The major structural features of the Escherichia coli MscS mechanosensitive channel protein have been explored using alkaline phosphatase (PhoA) fusions, precise deletions and site-directed mutations. PhoA protein fusion data, combined with the positive-inside rule, strongly support a model in which MscS crosses the membrane three times, adopting an N(out)-C(in) configuration. Deletion data suggest that the C-terminal domain of the protein is essential for the stability of the MscS channel, whereas the protein will tolerate small deletions at the N-terminus. Four mutants that exhibit either gain-of-function (GOF) or loss-of-function have been identified: a double mutation I48D/S49P inactivates MscS, whereas the MscS mutants T93R, A102P and L109S cause a strong GOF phenotype. The similarity of MscS to the last two domains of MscK (formerly KefA) is reinforced by the demonstration that expression of a truncated MscK protein can substitute for MscL and MscS in downshock survival assays. The data derived from studies of the organization, conservation and the influence of mutations provide significant insights into the structure of the MscS channel.     

Bacterial mechanosensitive channels as a paradigm for mechanosensory transduction

Research on bacterial mechanosensitive (MS) channels has since their discovery been at the forefront of the MS channel field due to extensive studies of the structure and function of MscL and MscS, two of the several different types of MS channels found in bacteria. Just a few years after these two MS channels were cloned their 3D structure was solved by X-ray crystallography. Today, the repertoire of multidisciplinary approaches used in experimental and theoretical studies following the cloning and crystallographic determination of the MscL and MscS structure has expanded by including electronparamagnetic resonance (EPR) and F?rster resonance energy transfer (FRET) spectroscopy aided by computational modelling employing molecular dynamics as well as Brownian dynamics simulations, which significantly advanced the understanding of structural determinants of the gating and conduction properties of these two MS channels. These extensive multidisciplinary studies of MscL and MscS have greatly contributed to elucidation of the basic physical principles of MS channel gating by mechanical force. This review summarizes briefly the major experimental and conceptual advancements, which helped in establishing MscL and MscS as a major paradigm of mechanosensory transduction in living cells.     

Toward Understanding Protocell Mechanosensation

Mechanosensitive (MS) channels can prevent bacterial bursting during hypo-osmotic shocks by responding to increases in lateral tension at the membrane level through an integrated and coordinated opening mechanism. Mechanical regulation in protocells could have been one of the first mechanisms to evolve in order to preserve their integrity against changing environmental conditions. How has the rich functional diversity found in present cells been created throughout evolution, and what did the primordial MS channels look like? This review has been written with the aim of identifying which factors may have been important for the appearance of the first osmotic valve in a prebiotic context, and what this valve may have been like. It highlights the mechanical properties of lipid bilayers, the association of peptides as aggregates in membranes, and the conservation of sequence motifs as central aspects to understand the evolution of proteins that gate below the tension required for spontaneous pore formation and membrane rupture. The arguments developed here apply to both MscL and MscS homologs, but could be valid to mechano-susceptible proteins in general.     

From membrane tension to channel gating: A principal energy transfer mechanism for mechanosensitive channels

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.     

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.     

Sensing and Responding to Membrane Tension: The Bacterial MscL Channel as a Model System

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.     

Rapid and improved reconstitution of bacterial mechanosensitive ion channel proteins MscS and MscL into liposomes using a modified sucrose method

The bacterial mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance have functionally been reconstituted into giant unilamellar liposomes (GUVs) using an improved reconstitution method in the presence of sucrose. This method gives significant time savings (preparation times as little as 6 h) compared to the classical method of protein reconstitution which uses a dehydration/rehydration (D/R) procedure (minimum 2 days preparation time). Moreover, it represents the first highly reproducible method for functional reconstitution of MscS as well as MscS/MscL co-reconstitution. This novel procedure has the potential to be used for studies of other ion channels by liposome reconstitution.     

Engineering covalent oligomers of the mechanosensitive channel of large conductance from Escherichia coli with native conductance and gating characteristics

To obtain a gene construct for making single substitutions per channel and to determine the quaternary structure of the mechanosensitive channel MscL from Escherichia coli, covalent oligomers (monomer to hexamer) were engineered by gene fusion; up to six copies of the mscL gene were fused in tandem. All the multimeric tandem constructs yielded functional channels with wild-type conductance and dwell times. Importantly, only the covalent pentamer opened at the same relative pressure (compared to the pressure required to open MscS) as the wild-type MscL channel. The in vivo data strongly suggest that pentameric MscL represents the functional state of the channel.     

Structural and functional differences between two homologous mechanosensitive channels of Methanococcus jannaschii

We report the molecular cloning and characterization of MscMJLR, a second type of mechanosensitive (MS) channel found in the archaeon Methanococcus jannaschii. MscMJLR is structurally very similar to MscMJ, the MS channel of M.jannaschii that was identified and cloned first by using the TM1 domain of Escherichia coli MscL as a genetic probe. Although it shares 44% amino acid sequence identity and similar cation selectivity with MscMJ, MscMJLR exhibits other major functional differences. The conductance of MscMJLR of approximately 2 nS is approximately 7-fold larger than the conductance of MscMJ and rectifies with voltage. The channel requires approximately 18 kT for activation, which is three times the amount of energy required to activate MscMJ, but is comparable to the activation energy of Eco-MSCL: Our study indicates that a multiplicity of conductance-wise and energetically well-tuned MS channels in microbial cell membranes may provide for cell survival by the sequential opening of the channels upon challenge with different osmotic cues.     

Mechanosensitive channel of Thermoplasma, the cell wall-less archaea

By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less archaeon Thermoplasma volcanium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation by the anionic amphipath trinitrophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of T. acidophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL ⁻ knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was charaterized by very brief openings of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL ⁺ strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or modulate each other activity. Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels.