Mechanosensitive Channel Of Small Conductance Research Articles

The MscS-like channel YnaI has a gating mechanism based on flexible pore helices

The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. Escherichia coli has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood. The medium-sized channel YnaI was extracted and delivered in native nanodiscs in closed-like and open-like conformations using the copolymer diisobutylene/maleic acid (DIBMA) for structural studies. Here we show by electron cryomicroscopy that YnaI has an extended sensor paddle that during gating relocates relative to the pore concomitant with bending of a GGxGG motif in the pore helices. YnaI is the only one of the six paralogs that has this GGxGG motif allowing the sensor paddle to move outward. Access to the pore is through a vestibule on the cytosolic side that is fenestrated by side portals. In YnaI, these portals are obstructed by aromatic side chains but are still fully hydrated and thus support conductance. For comparison with large-sized channels, we determined the structure of YbiO, which showed larger portals and a wider pore with no GGxGG motif. Further in silico comparison of MscS, YnaI, and YbiO highlighted differences in the hydrophobicity and wettability of their pores and vestibule interiors. Thus, MscS-like channels of different sizes have a common core architecture but show different gating mechanisms and fine-tuned conductive properties.

Structural mechanism for gating of a eukaryotic mechanosensitive channel of small conductance

Mechanosensitive ion channels transduce physical force into electrochemical signaling that underlies an array of fundamental physiological processes, including hearing, touch, proprioception, osmoregulation, and morphogenesis. The mechanosensitive channels of small conductance (MscS) constitute a remarkably diverse superfamily of channels critical for management of osmotic pressure. Here, we present cryo-electron microscopy structures of a MscS homolog from Arabidopsis thaliana, MSL1, presumably in both the closed and open states. The heptameric MSL1 channel contains an unusual bowl-shaped transmembrane region, which is reminiscent of the evolutionarily and architecturally unrelated mechanosensitive Piezo channels. Upon channel opening, the curved transmembrane domain of MSL1 flattens and expands. Our structures, in combination with functional analyses, delineate a structural mechanism by which mechanosensitive channels open under increased membrane tension. Further, the shared structural feature between unrelated channels suggests the possibility of a unified mechanical gating mechanism stemming from membrane deformation induced by a non-planar transmembrane domain.

Symmetry of membrane protein polyhedra with heterogeneous protein size.

In experiments on membrane protein polyhedral nanoparticles (MPPNs) [Basta et al., Proc. Natl. Acad. Sci. USA 111, 670 (2014)PNASA60027-842410.1073/pnas.1321936111], it has been observed that membrane proteins and lipids can self-assemble into closed lipid bilayer vesicles with a polyhedral arrangement of membrane proteins. In particular, MPPNs formed from the mechanosensitive channel of small conductance (MscS) were found to have the symmetry of the snub cube-a chiral, Archimedean solid-with one MscS protein located at each one of the 24 vertices of the snub cube. It is currently unknown whether MPPNs with heterogeneous protein composition maintain a high degree of symmetry. Inspired by previous work on viral capsid symmetry, we employ here computational modeling to study the symmetry of MPPNs with heterogeneous protein size. We focus on MPPNs formed from MscS proteins, which can exist in closed or open conformational states with distinct sizes. We find that, as an increasing number of closed-state MscS proteins transitions to the open conformational state of MscS, the minimum-energy MscS arrangement in MPPNs follows a strikingly regular pattern, with the dominant MPPN symmetry always being provided by the snub cube. Our results suggest that MPPNs with heterogeneous protein size can be highly symmetric, with a well-defined polyhedral ordering of membrane proteins of different sizes.

Influence of the Lipid-Protein Interface on MSCS Mechanosensitive Channel Gating at High Resolutions

Prokaryotic mechanosensitive (MS) channels gate by sensing forces through interactions with the lipid bilayer. This points to a critical role of lipid-protein interface in defining force-from-lipid gating, and defining the specific structural and energetic interplay between channels and the lipids that surround them represents a fundamental challenge in mechanobiology. Recently, we have determined several high resolution cryo-EM structures of MscS (the small-conductance mechanosensitive channel) in nanodisc (ND) environments that varied membrane composition. Surprisingly, the bulk of the ND-containing lipid annulus surrounding the channel was found to be almost a full bilayer leaflet towards the N-terminal where previous expected. Furthermore, several lipids were identified within these close conformation structures which, we hypothesize, play a critical role in channel gating. We have expanded on these new structures by probing the function of the interacting lipids which varied a number of membrane properties, including membrane thickness, overall charge, size, and membrane rigidity. These observations were further evaluated by several functional and biochemical assays, including crosslinking assays, down shock cell survival and high speed pressure clamp electrophysiology. The present observations redefine the mechanism of force-from-lipids gating and suggest that allosteric interactions between TM and coordinating lipids segments play a fundamental role in MscS gating.

Characterizing the mechanosensitive response of Paraburkholderia graminis membranes

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.

Molecular basis of force-from-lipids gating in the mechanosensitive channel MscS.

Prokaryotic mechanosensitive (MS) channels open by sensing the physical state of the membrane. As such, lipid-protein interactions represent the defining molecular process underlying mechanotransduction. Here, we describe cryo-electron microscopy (cryo-EM) structures of the E. coli small-conductance mechanosensitive channel (MscS) in nanodiscs (ND). They reveal a novel membrane-anchoring fold that plays a significant role in channel activation and establish a new location for the lipid bilayer, shifted ~14 Å from previous consensus placements. Two types of lipid densities are explicitly observed. A phospholipid that 'hooks' the top of each TM2-TM3 hairpin and likely plays a role in force sensing, and a bundle of acyl chains occluding the permeation path above the L105 cuff. These observations reshape our understanding of force-from-lipids gating in MscS and highlight the key role of allosteric interactions between TM segments and phospholipids bound to key dynamic components of the channel.

Interaction between mechanosensitive channels embedded in lipid membrane

The study of the gating mechanism of mechanosensitive channels opens a window to the exploration of how different mechanical stimuli induce adaptive cellular behaviors of both the protein and the lipid, across different time and length scales. In this work, through a molecular dynamics-decorated finite element method (MDeFEM), the gating behavior of mechanosensitive channels of small conductance (MscS) in Escherichia coli (E. coli) is studied upon membrane stretch or global bending. The local membrane curvature around MscS is incorporated, as well as multiple MscL (mechanosensitive channels of large conductance) molecules in proximity to MscS. The local membrane curvature is found to delay MscS opening and diminishes moderately upon membrane stretching. Mimicking the insertion of lysophosphatidylcholine (LPC) molecules into the lipid, both downward and upward bending can active MscS, as long as the global membrane curvature radius reaches 34 nm. Based on the different MscS pore evolutions observed with the presence of one or more MscLs nearby, we propose that when coreconstituted, multiple MscL molecules tend to be located at the local membrane curvature zone around MscS. In another word, as MscL “swims around” in the lipid bilayer, it can be trapped by the membrane's local curvature. Collectively, the current study provides valuable insights into the interplay between mechanosensitive channels and lipid membrane at structural and physical levels, and specific predictions are proposed for further experimental investigations.

Structure of the Mechanosensitive Channel MscS Embedded in the Membrane Bilayer

Since life has emerged, gradients of osmolytes over the cell membrane cause pressure changes in the cell and require tight regulation to prevent cell rupture. The mechanosensitive channel of small conductance (MscS) releases solutes and water when a hypo-osmotic shock raises the pressure in the cell. It is a member of a large family of MscS-like channels found in bacteria, archaea, fungi and plants and model for mechanosensation. MscS senses the increase of tension in the membrane directly by the force from the lipids, but the molecular mechanism is still elusive. We determined the lipid interactions of MscS by resolving the structure of Escherichia coli MscS embedded in membrane discs to 2.9-Å resolution using cryo-electron microscopy. The membrane is attached only to parts of the sensor paddles of MscS, but phospholipid molecules move through grooves into remote pockets on the cytosolic side. On the periplasmic side, a lipid bound by R88 at the pore entrance is separated from the membrane by TM1 helices. The N-terminus interacts with the periplasmic membrane surface. We demonstrate that the unique membrane domain of MscS promotes deep penetration of lipid molecules and shows multimodal interaction with the membrane to fine-tune tension sensing.

Genetic and physical interactions between the organellar mechanosensitive ion channel homologs MSL1, MSL2, and MSL3 reveal a role for inter-organellar communication in plant development.

Plant development requires communication on many levels, including between cells and between organelles within a cell. For example, mitochondria and plastids have been proposed to be sensors of environmental stress and to coordinate their responses. Here we present evidence for communication between mitochondria and chloroplasts during leaf and root development, based on genetic and physical interactions between three Mechanosensitive channel of Small conductance‐Like (MSL) proteins from Arabidopsis thaliana. MSL proteins are Arabidopsis homologs of the bacterial Mechanosensitive channel of Small conductance (MscS), which relieves cellular osmotic pressure to protect against lysis during hypoosmotic shock. MSL1 localizes to the inner mitochondrial membrane, while MSL2 and MSL3 localize to the inner plastid membrane and are required to maintain plastid osmotic homeostasis during normal growth and development. In this study, we characterized the phenotypic effect of a genetic lesion in MSL1, both in wild type and in msl2 msl3 mutant backgrounds. msl1 single mutants appear wild type for all phenotypes examined. The characteristic leaf rumpling in msl2 msl3 double mutants was exacerbated in the msl1 msl2 msl3 triple mutant. However, the introduction of the msl1 lesion into the msl2 msl3 mutant background suppressed other msl2 msl3 mutant phenotypes, including ectopic callus formation, accumulation of superoxide and hydrogen peroxide in the shoot apical meristem, decreased root length, and reduced number of lateral roots. All these phenotypes could be recovered by molecular complementation with a transgene containing a wild type version of MSL1. In yeast‐based interaction studies, MSL1 interacted with itself, but not with MSL2 or MSL3. These results establish that the abnormalities observed in msl2 msl3 double mutants is partially dependent on the presence of functional MSL1 and suggest a possible role for communication between plastid and mitochondria in seedling development.

Gating and inactivation of mechanosensitive channels of small conductance: A continuum mechanics study

Mechanosensitive channels of small conductance (MscS) in Escherichia coli (E. coli) serve as a paradigm for understanding the gating behaviors of the MscS family of ion channels. In this work, we develop a continuum mechanics framework to explore the conformational states of MscS during the gating transition. A complete gating transition trajectory from the closed to the open state along with partially open intermediates is obtained, and the open structure is close to the available structural model from crystallographic studies. The computational efficiency of the modeling framework makes it possible to explore the roles of various structural elements (e.g., loops that connect transmembrane helices) and specific interactions in the gating transition. It is observed that removing either the Asp62-Arg131 salt bridge or the Phe68-Leu111 non-polar interaction leads to essentially non-conducting structures even with a membrane tension close to the lysis limit. The loop connecting TM2 (the second transmembrane helix) and TM3 is found to be essential for force transmission during gating, while the loop connecting TM1 and TM2 does not make any major contribution. Based on the different structural evolutions observed when the TM3 kink is treated as a loop or a helical segment, we propose that the helical propensity of the kink plays a central role in inactivation; i.e., under prolonged sub-threshold membrane tension, transition of the initially flexible loop to a helical segment in TM3 may lead to MscS inactivation. Finally, the gating transition of MscS under different transmembrane voltages is explored and found to be essentially voltage independent. Collectively, results from the current continuum mechanics analysis provide further insights into the gating transition of MscS at structural and physical levels, and specific predictions are proposed for further experimental investigations.

A Novel Corynebacterium glutamicum l-Glutamate Exporter.

Besides metabolic pathways and regulatory networks, transport systems are also pivotal for cellular metabolism and hyperproduction of biochemicals using microbial cell factories. The identification and characterization of transporters are therefore of great significance for the understanding and engineering of transport reactions. Herein, a novel l-glutamate exporter, MscCG2, which exists extensively in Corynebacterium glutamicum strains but is distinct from the only known l-glutamate exporter, MscCG, was discovered in an industrial l-glutamate-producing C. glutamicum strain. MscCG2 was predicted to possess three transmembrane helices in the N-terminal region and located in the cytoplasmic membrane, which are typical structural characteristics of the mechanosensitive channel of small conductance. MscCG2 has a low amino acid sequence identity (23%) to MscCG and evolved separately from MscCG with four transmembrane helices. Despite the considerable differences between MscCG2 and MscCG in sequence and structure, gene deletion and complementation confirmed that MscCG2 also functioned as an l-glutamate exporter and an osmotic safety valve in C. glutamicum Besides, transcriptional analysis showed that MscCG2 and MscCG genes were transcribed in similar patterns and not induced by l-glutamate-producing conditions. It was also demonstrated that MscCG2-mediated l-glutamate excretion was activated by biotin limitation or penicillin treatment and that constitutive l-glutamate excretion was triggered by a gain-of-function mutation of MscCG2 (A151V). Discovery of MscCG2 will enrich the understanding of bacterial amino acid transport and provide additional targets for exporter engineering.IMPORTANCE The exchange of matter, energy, and information with surroundings is fundamental for cellular metabolism. Therefore, studying transport systems that are essential for these processes is of great significance. Besides, transport systems of bacterial cells are usually related to product excretion as well as product reuptake, making transporter engineering a useful strategy for strain improvement. The significance of our research is in identifying and characterizing a novel l-glutamate exporter from the industrial workhorse Corynebacterium glutamicum, which will enrich the understanding of l-glutamate excretion and provide a new target for studying bacterial amino acid transport and engineering transport reactions.

Genome-wide identification of MscS like gene family in Cicer arietinum using bioinformatics approach

In plants, the mechanosensitive channels have important roles in proprioception, gravity sensing, growth of pollen tube and controlling the shape and size of plastid. In the present study, we performed computational analysis and identified the mechanosensitive channel of small conductance like (MscS) gene family in Cicer arietinum (chickpea). Six Cicer arietinum MscS-like (CaMSL) genes were identified. These six genes were distributed on four different chromosomes. A detailed overview of CaMSL genes in Cicer arietinum is explored by using a total of 33107 proteins from chickpea. On the basis of BLAST analysis, presence of complete ORF and evolutionary relationship we identified 6 genes (named as CaMSL1, CaMSL2, CaMSL3, CaMSL4, CaMSL5 and CaMSL6). Further, the transmembrane regions, subcellular localization, physico-chemical properties, gene structure analysis and genome structure annotation of the identified genes confirmed that the identified genes might act for transmembrane proteins as well as their subcellular localization prediction demonstrated three genes to be located in the plasma membrane, two in the chloroplast membrane, one in the mitochondrion. Thus, further analysis of the data obtained from this study will help to make a baseline to increase the understanding of involvement of MSL gene families in plant growth, development and other functions in C. arietinum.

Identification of Intracellular β-Barrel Residues Involved in Ion Selectivity in the Mechanosensitive Channel of Thermoanaerobacter tengcongensis.

The mechanosensitive channel of small conductance (MscS) is a bacterial membrane pore that senses membrane tension and protects cells from lysis by releasing osmolytes. MscS is a homoheptameric channel with a cytoplasmic domain with seven portals and a β-barrel opening to the cytoplasm. TtMscS, an MscS channel from Thermoanaerobacter tengcongensis, is an anion-selective channel. A previous study from our laboratory has defined the crucial role of β-barrel in the anion selectivity of TtMscS (Zhang et al., 2012). However, the mechanistic details by which the β-barrel determines anion selectivity remain unclear. Here, using mutagenesis and patch-clamp recordings, we investigated the function and structural correlations between β-barrels and the anion selectivity of TtMscS at the atomic level. Our results indicated that mutation of V274, a residue at the center of the inner wall of the β-barrel in TtMscS, caused the anion selectivity of TtMscS reverse to cation selectivity. Moreover, the electrostatic potential (T272) and physical size (L276) of residues in the inner wall of β-barrel also determine the anion selectivity of TtMscS. In summary, the present study confirmed that the β-barrel region of TtMscS acts as a “selective filter” that renders TtMscS anion selectivity.

Exploring the pH Sensitivity of E. Coli MscS

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.

MscS-like Mechanosensitive Ion Channels: Modular Sensors and Reporters of Membrane Tension

A long-standing question is how biological systems sense and perceive mechanical signals such as osmotic pressure, gravity, and touch. One well-established molecular mechanism for force sensing is the activation of mechanosensitive (MS) ion channels. The Mechanosensitive channel of Small conductance (MscS) from E. coli functions as a hypo-osmotic safety valve, opening in response to increased membrane tension and preventing cellular rupture. Genes predicted to encode MscS homologs are found in genomes from all three kingdoms of life. We have been characterizing the structure, function, and regulation of ten MscS-Like (MSL) proteins in the model plant Arabidopsis thaliana. Based on their modest homology to MscS and high topological diversity, we have proposed that MSLs might (1) sense and respond to sources of membrane tension other than environmental hypo-osmotic shock; (2) be regulated by mechanisms in addition to membrane tension; and (3) signal in ways that are separable from ion flux. Evidence in support of all three of these hypotheses will be presented. I will also discuss our current effort to engineer novel biosensors based on MSL proteins.

How do mechanosensitive channels sense membrane tension?

Mechanosensitive (MS) channels provide protection against hypo-osmotic shock in bacteria whereas eukaryotic MS channels fulfil a multitude of important functions beside osmoregulation. Interactions with the membrane lipids are responsible for the sensing of mechanical force for most known MS channels. It emerged recently that not only prokaryotic, but also eukaryotic, MS channels are able to directly sense the tension in the membrane bilayer without any additional cofactor. If the membrane is solely viewed as a continuous medium with specific anisotropic physical properties, the sensitivity towards tension changes can be explained as result of the hydrophobic coupling between membrane and transmembrane (TM) regions of the channel. The increased cross-sectional area of the MS channel in the active conformation and elastic deformations of the membrane close to the channel have been described as important factors. However, recent studies suggest that molecular interactions of lipids with the channels could play an important role in mechanosensation. Pockets in between TM helices were identified in the MS channel of small conductance (MscS) and YnaI that are filled with lipids. Less lipids are present in the open state of MscS than the closed according to MD simulations. Thus it was suggested that exclusion of lipid fatty acyl chains from these pockets, as a consequence of increased tension, would trigger gating. Similarly, in the eukaryotic MS channel TRAAK it was found that a lipid chain blocks the conducting path in the closed state. The role of these specific lipid interactions in mechanosensation are highlighted in this review.

English

Mechanosensitive (MS) ion channels are transmembrane proteins that open and close in response to mechanical forces produced by osmotic pressure, sound, touch and gravity. In plants, MS have an important role in different biological processes like gravity detection, maintenance of plastid shape and size, lateral root emergence, growth of pollen tube, and plant-pathogen interactions. In this study, homologous mechanosensitive channel of small conductance (MscS)-like gene family in common bean was identified. Nine Phaseolus vulgaris MscS-like (PvMSL) genes were found to be distributed on five chromosomes. A complete overview of PvMSL genes in common bean is presented, including gene structures, chromosome locations, phylogeny, protein motifs and expression pattern. Subcellular localization predictions of PvMSL family revealed their location to plasma and chloroplast membrane. Phylogenetic analysis of nine PvMSL proteins resulted in two main classes. The predicted gene structure, conserved motif, domain and presence of transmembrane regions in each PvMSL strongly supported their identity as members of MscS-like gene family. Four duplicate events of PvMSL genes were discovered in P. vulgaris chromosomes, and tandem and segmental duplication may cause the expansion of PvMSL genes. Furthermore, PvMSL genes displayed differential expression patterns in tissues and organs. This is the first step towards genome-wide analyses of MSL genes in common bean. Thus, the data obtained in this study provide resources to select candidate genes for future functional analyses that will help understand plant growth, development, and function of MSL gene family in P. vulgaris . Key words : Mechanosensitive, phylogenetic analysis, gene duplication, plant, in silico.

Voltage-Dependent Inactivation of MscS Occurs Independently of the Positively Charged Residues in the Transmembrane Domain.

MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and plays a major role in avoiding cell lysis upon rapid osmotic downshock. The gating of MscS is modulated by voltage, but little is known about how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-54, and Arg-74) in the transmembrane (TM) domain are possible to respond to voltage judging from the MscS structure. To examine whether these residues are involved in the voltage dependence of MscS, we neutralized the charge of each residue by substituting with asparagine (R46N, R54N, and R74N). Mechanical threshold for the opening of the expressed wild-type MscS and asparagine mutants did not change with voltage in the range from −40 to +100 mV. By contrast, inactivation process of wild-type MscS was strongly affected by voltage. The wild-type MscS inactivated at +60 to +80 mV but not at −60 to +40 mV. The voltage dependence of the inactivation rate of all mutants tested, that is, R46N, R54N, R74N, and R46N/R74N MscS, was almost indistinguishable from that of the wild-type MscS. These findings indicate that the voltage dependence of the inactivation occurs independently of the positive charges of R46, R54, and R74.

The role of lipids in mechanosensation.

The ability of proteins to sense membrane tension is pervasive in biology. A higher resolution structure of E. coli MscS, the channel of small conductance, identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E. coli lipids and fluorescence quenching demonstrates that phospholipid acyl chains exchange between bilayer and TM pockets. Molecular dynamics and biophysical analyses show that the volume of the pockets and thus the number of lipid acyl chain within them decreases upon channel opening. Phospholipids with one acyl chain per head group (lysolipids) displace normal phospholipids (two acyl chains) from MscS pockets and trigger channel opening. We propose the extent of acyl chain interdigitation in these pockets determines the conformation of MscS. Where interdigitation is perturbed by increased membrane tension or by lysolipids, the closed state becomes unstable and the channel gates.

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.