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Mechanosensitive ion channels are responsible for touch sensation and proprioception in higher level organisms such as humans and recovery after osmotic stress in bacteria. Bacterial mechanosensitive channels are homologous to either the mechanosensitive channel of large conductance (MscL) or the mechanosensitive channel of small conductance (MscS). In the E. coli genome there are seven unique mechanosensitive channels, a single MscL homologue, and six MscS homologues. The six MscS homologues are members of the diverse MscS superfamily of ion channels, and these channels show variation on both the N and C termini when compared to E. coli MscS. In bacterial strains with phenotypic analysis of the endogenous mechanosensors, the quantity of MscS superfamily members in the genome range from 2 to 6 and all of the strains contain a copy of MscL. Here, we show an in-depth analysis of over 150 diverse bacterial genomes, encompassing nine phyla, to determine the number of genomes that contain an MscL homologue and the average number of MscS superfamily members per genome. We determined that the average genome contains 4 ± 3 MscS homologues and 67% of bacterial genomes encode for a MscL homologue.

Bacterial mechanosensitive channels gate in response to membrane tension, driven by shifts in environmental osmolarity. The mechanosensitive channels of small conductance (MscS) and large conductance (MscL) from Escherichia coli (Ec) gate in response to mechanical force applied to the membrane. Ec-MscS is the foundational member of the MscS superfamily of ion channels, a diverse family with at least fifteen subfamilies identified by homology to the pore lining helix of Ec-MscS, as well as significant diversity on the N- and C-termini. The MscL family of channels are homologous to Ec-MscL. In a rhizosphere associated bacterium, Paraburkholderia graminis C4D1M, mechanosensitive channels are essential for cell survival during changing osmotic environments such as a rainstorm. Utilizing bioinformatics, we predicted six MscS superfamily members and a single MscL homologue. The MscS superfamily members fall into at least three subfamilies: bacterial cyclic nucleotide gated, multi-TM, and extended N-terminus. Osmotic downshock experiments show that wildtype P. graminis cells contain a survival mechanism that prevents cell lysis in response to hypoosmotic shock. To determine if this rescue is due to mechanosensitive channels, we developed a method to create giant spheroplasts of P. graminis to explore the single channel response to applied mechanical tension. Patch clamp electrophysiology on these spheroplasts shows two unique conductances: MscL-like and MscS-like. These conductances are due to likely three unique proteins. This indicates that channels that gate in response to mechanical tension are present in the membrane. Here, we report the first single channel evidence of mechanosensitive ion channels from P. graminis membranes.

Traditionally, prokaryotic channels are thought to exist as homomultimeric assemblies, while many eukaryotic ion channels form complex heteromultimers. Here we demonstrate that bacterial cyclic nucleotide-gated channels likely form heteromultimers in vivo. Heteromultimer formation is indicated through channel modeling, pull-down assays, and real-time polymerase chain reaction analysis. Our observations demonstrate that prokaryotic ion channels can display complex behavior and regulation akin to that of their eukaryotic counterparts.

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-π interactions.

We have recently identified and characterized the bacterial cyclic nucleotide gated (bCNG) subfamily of the larger mechanosensitive channel of small conductance (MscS) superfamily of ion channels. The channel domain of bCNG channels exhibits significant sequence homology to the mechanosensitive subfamily of MscS in the regions that have previously been used as a hallmark for channels that gate in response to mechanical stress. However, we have previously demonstrated that three of these channels are unable to rescue Escherichiacoli from osmotic downshock. Here, we examine an additional nine bCNG homologues and further demonstrate that the full-length bCNG channels are unable to rescue E. coli from hypoosmotic stress. However, limited mechanosensation is restored upon removal of the cyclic nucleotide binding domain. This indicates that the C-terminal domain of the MscS superfamily can drive channel gating and further highlight the ability of a superfamily of ion channels to be gated by multiple stimuli.

Mutations that alter the phenotypic behavior of the Escherichia coli mechanosensitive channel of small conductance (MscS) have been identified; however, most of these residues play critical roles in the transition between the closed and open states of the channel and are not directly involved in lipid interactions that transduce the tension response. In this study, we use molecular dynamic simulations to predict critical lipid interacting residues in the closed state of MscS. The physiological role of these residues was then investigated by performing osmotic downshock assays on MscS mutants where the lipid interacting residues were mutated to alanine. These experiments identified seven residues in the first and second transmembrane helices as lipid-sensing residues. The majority of these residues are hydrophobic amino acids located near the extracellular interface of the membrane. All of these residues interact strongly with the lipid bilayer in the closed state of MscS, but do not face the bilayer directly in structures associated with the open and desensitized states of the channel. Thus, the position of these residues relative to the lipid membrane appears related to the ability of the channel to sense tension in its different physiological states.

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.

The mechanosensitive channel of small conductance (MscS) from Escherichia coli (E. coli) gates in response to mechanical tension that is generated from hypo-osmotic shock. The gating mechanism of E. coli MscS, in response to mechanical tension, is similar to that of a Jack-in-the-Box, the channel springs into the open state when the applied extrinsic tension overcomes the intrinsic lateral tension of the membrane. E. coli MscS is the founding member of the MscS superfamily, a superfamily of 15 subfamilies that all contain significant homology to the pore lining helix of E. coli MscS. In the early identification of MscS a rapid desensitization of MscS when pressure is applied at low pH is observed, however when patched at physiological pH E. coli MscS does not desensitize. To identify the region of the channel that evokes pH sensitivity we will use asymmetric and symmetric pH patch buffer. A recent study explored how significant alterations to the most conserved residues impacted E. coli MscS function, one residue F127 showed significant alteration to the gating phenotype when mutated to different residues. The results of the previous study show that the rapid desensitization classically observed in low pH are observed in a F127 mutations. This suggests that F127 is involved in a long-range interaction, potentially a cation-π with K60. Based on the location of this interaction in multiple crystal structures, the cation-π is potentially involved in the desensitized state of the channel. Through patch clamp electrophysiology we will understand the role of a pH sensitive region in the gating cycle of E. coli MscS.

Phenotypical analysis of the lipid interacting residues in the closed state of the mechanosensitive channel of small conductance (MscS) from Escherichia coli (E. coli) has previously shown that these residues are critical for channel function. In the closed state, mutation of individual hydrophobic lipid lining residues to alanine, thus reducing the hydrophobicity, resulted in phenotypic changes that were observable using in vivo assays. Here, in an analogous set of experiments, we identify eleven residues in the first transmembrane domain of the open state of MscS that interact with the lipid bilayer. Each of these residues was mutated to alanine and leucine to modulate their hydrophobic interaction with the lipid tail-groups in the open state. The effects of these changes on channel function were analyzed using in vivo bacterial assays and patch clamp electrophysiology. Mutant channels were found to be functionally indistinguishable from wildtype MscS. Thus, mutation of open-state lipid interacting residues does not differentially stabilize or destabilize the open, closed, intermediate, or transition states of MscS. Based on these results and other data from the literature, we propose a new gating paradigm for MscS where MscS acts as a “Jack-In-The-Box” with the intrinsic bilayer lateral pressure holding the channel in the closed state. In this model, upon application of extrinsic tension the channel springs into the open state due to relief of the intrinsic lipid bilayer pressure.

Bacterial mechanosensitive channels are important for cell survival in changing osmotic environments. For the mechanosensitive channel of small conductance from Escherichia coli (Ec-MscS), seven residues have previously been shown to form important lipid contacts in the closed state of the channel. Based on open state crystal structures and closed state models, these residues interact with lipids in the closed state of the channel. Decreasing the hydrophobicity of these residues reduces bacterial survival upon osmotic downshock. Since the closed state appears to be stabilized by lipid interactions, we hypothesized that similar stabilizing lipid interactions could be identified in the open state. Using a computational model of open state Ec-MscS embedded in a lipid bilayer, eleven residues were determined to be lipid exposed with ten of these residues being unique to the open state of the channel. To identify the role of lipid interactions these residues were mutated to alanine and leucine to alter their interaction with the hydrophobic lipids. The effects of these mutations on channel function were assayed using osmotic downshock lysis assays, growth assays, and patch clamp electrophysiology. Mutations of the ten residues that are exposed to the lipid bilayer only in the open state of the channel effected a wildtype phenotype in all of these assays. The lack of phenotypic changes, for residues that interact with the lipid bilayer solely in the open state, suggests that these interactions are not critical for channel gating. This has led us to propose a "Jack-In-The-Box" model of gating for MscS, in which intrinsic lipid bilayer pressure holds the channel in the closed state. Upon relief of the intrinsic bilayer pressure by application of opposing extrinsic tension, MscS springs into the open state.

A family of many talents: The mechanosensitive channel of small conductance (MscS) superfamily of ion channels is composed of 15 unique subfamilies. Many of these subfamilies are predicted to be nonmechanosensitive and to have evolved to play critical roles in bacterial signal transduction.

A family of many talents: The mechanosensitive channel of small conductance (MscS) superfamily of ion channels is composed of 15 unique subfamilies. Many of these subfamilies are predicted to be nonmechanosensitive and to have evolved to play critical roles in bacterial signal transduction.

The bacterial cyclic nucleotide gated (bCNG) channel family is a subfamily of the mechanosensitive channel of small conductance (MscS) superfamily. Members of the bCNG channel family have been shown to gate in response to cyclic adenosine monophosephate alone, but are incapable of rescuing E. coli in osmotic downshock assays. In the bCNG channel family, some bacterial genomes encode for multiple bCNG homologues while others encode for a single homologue. The presence of multiple bCNG homologues in a single bacterial genome has lead us to hypothesize that these channels might exist as heteromultimers, which would represent the first observation of heteromultimeric channels in prokaryotes. In a pull-down assay, bCNG homologues from a single bacterial genome, form heteromultimers, when heterologously expressed in E. coli. Additionally, bCNG channels from different bacteria can form heteromultimers when co-expressed with bCNG channels containing the same number of transmembrane domains. To determine if bCNG heteromultimers are formed in vivo, RT-PCR was used to verify the co-translation of bCNG channels.

Studies of bacterial ion channels have provided significant insights into the structure–function relationships of mechanosensitive and voltage-gated ion channels. However, to date, very few bacterial channels that respond to small molecules have been identified, cloned, and characterized. Here, we use bioinformatics to identify a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels containing a channel domain related by sequence homology to the mechanosensitive channel of small conductance (MscS). In this initial report, we clone selected members of this channel family, use electrophysiological measurements to verify their ability to directly gate in response to cyclic nucleotides, and use osmotic downshock to demonstrate their lack of mechanosensitivity. In addition to providing insight into bacterial physiology, these channels will provide researchers with a useful model system to investigate the role of ligand-gated ion channels (LGICs) in the signaling processes of higher organisms. The identification of these channels provides a foundation for structural and functional studies of LGICs that would be difficult to perform on mammalian channels. Moreover, the discovery of bCNG channels implies that bacteria have cyclic nucleotide-gated and cyclic nucleotide-modulated ion channels, which are analogous to the ion channels involved in eukaryotic secondary messenger signaling pathways.

We have previously used a combination of random mutagenesis and molecular dynamics simulations to identify and understand critical residues for tension sensation in MscL (mechanosensitive channel of large conductance) [Maurer et al JBC 2003, 278, 21076-21082; Elmore et. al Biophysical J., 2003, 85, 1512-1524]. In MscL, we were able to demonstrate a strong correlation between lipid-protein interaction energy, determined using a molecular dynamics simulation of the closed state structure, and mutant channel function. Upon mutation of amino acid residues in transmembrane domains that exhibited significant lipid interaction over the course of the simulation displayed were more likely than other transmembrane residues to display either a loss of function or a gain of function phenotype in bacterial assays. Here, we have employed a similar computational analysis to identify potentially critical lipid-binding residues for tension sensation in MscS. Molecular dynamics simulations of a closed state model of MscS were carried out in the presence of an explicit lipid membrane. Energetic analysis of lipid-protein interactions in the first transmembrane domain (TM1) and the second transmembrane domain (TM2) indicated ten residues (TM1: L35, I39, L42, I43, R46, N50, R54; and TM2: R74, L78, I82) with increased lipid interactions. Using site directed mutagenesis these residues were mutated to alanine and their ability to rescue MscL/MscS/MscK null E. coli from osmotic downshock was determined. Generally, mutating lipid interacting residues to alanine resulted in MscS channels that exhibited decreased ability to rescue bacteria from osmotic downshock, following a similar trend to our previous studies of MscL.

The bacterial cyclic nucleotide gated (bCNG) ion channel family contains a N-terminal channel domain, which is homologous to the Mechanosensitive Channel of Small Conductance (MscS) and a ligand binding domain with sequence similarity to known cyclic nucleotide binding domains. Previously, we have demonstrated that some of these channels gate in response to cyclic adenosine monophosphate alone [Caldwell, et al, BBA 2010, 1798, 1750-1756]. Here we explore the ability of bCNG channels to gate in response to mechanical stress, which one might predict based on their significant sequence homology to MscS. To this end, we measured the ability of fourteen distinct bCNG channels from several different bacterial strains to rescue E. coli lacking mechanosensitive channels (MscS/MscL/MscK null) from osmotic downshock. In our studies, only two bCNG channels exhibit limited ability to rescue bacteria from osmotic downshock. These two channels were found in bacterial strains with genomes encoding for multiple variants of the bCNG gene. Only one bCNG channel variant in each strain was capable of rescuing E.coli from osmotic downshock, implying that each gene product may have a unique role or that the two gene products may work in concert. Additionally, truncation of the cyclic nucleotide binding domain in some non-mechanosensitive variants of bCNG increases their response to mechanical stress.

We have identified, cloned, and characterized a new family of bacterial cyclic nucleotide gated (bCNG) ion channels. While we have demonstrated that these channels gate in response to cyclic adenosine monophosphate (cAMP), all bCNG channels exhibit significant homology to the pore lining helix (TM3) of the mechanosensitive channel of small conductance (MscS). This homology suggests that these channels might gate in response to mechanical stress in addition to ligand binding. To test this hypothesis, we have explored the ability of bCNG channels to rescue MscL/MscS/MscK null E. coli from osmotic downshock. While some homologues, such as bCNG from Synechococcus sp. PCC 6803, show rescue similar to that observed for wild-type E. coli MscS, other homologues, such as bCNG from M. loti, do not exhibit osmotic rescue. In the bCNG channel family, the numbers of transmembrane domains varies from two to six, with high homology between pore lining helices. Our current data implies that bCNG channels with greater homology to MscS are more likely to be mechanosensitive.