All cellular organisms respond to vibration, touch, gravity or changes in osmolarity, although the molecules on which such mechanosensations depend are unknown. Candidates include certain channels that gate in response to membrane stretch. Patch-clamp experiments with Escherichia coli envelope have revealed a mechanosensitive channel with very large conductance (MscL) and one with a smaller conductance (MscS) which may be important in osmoregulation. Here we have solubilized and fractionated the envelope, reconstituted the MscL activity in vitro, and traced it to a small protein, whose gene, mscL, we then cloned. Insertional disruption of mscL removes the channel activity, whereas re-expression of mscL borne on an expression plasmid restores it. MscL-channel activities were observed in material from a cell-free expression system with mscL as the only template. The mscL nucleotide sequence predicts a unique protein of only 136 amino acids, with a highly hydrophobic core and very different from porins or other known proteins.

MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore. To determine functionally important residues within this molecule, we have randomly mutagenized mscL, expressed the genes in living bacteria, and screened for gain-of-function mutants with hampered growth. Expression of these genes caused leakage of cytoplasmic solutes on little or no hypo-osmotic stress. In excised patches, the mutant channels gated at membrane tensions that are less than that required for the gating of the wild-type MscL. Hence, the data suggest that the slowed or no-growth phenotype is caused by solute loss because of inappropriate gating of the channel. Most of the mutations mapped to the first transmembrane domain. When this domain is modeled as an ␣-helix, the most severe mutations are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating.

Mechanosensitive channel large (MscL) encodes the large conductance mechanosensitive channel of the Escherichia coli inner membrane that protects bacteria from lysis upon osmotic shock. To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly(22) with all other common amino acids. Gly(22) was highlighted in random mutagenesis screens of E. coli MscL (, Proc. Nat. Acad. Sci. USA. 95:11471-11475). By analogy to the recently published MscL structure from Mycobacterium tuberculosis (, Science. 282:2220-2226), Gly(22) is buried within the constriction that closes the pore. Substituting Gly(22) with hydrophilic residues decreased the threshold pressure at which channels opened and uncovered an intermediate subconducting state. In contrast, hydrophobic substitutions increased the threshold pressure. Although hydrophobic substitutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions correlated with decreased proliferation. These results suggest a model for gating in which Gly(22) moves from a hydrophobic, and through a hydrophilic, environment upon transition from the closed to open conformation.

MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions ofa lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.

4Corresponding author P.Blount and S.I.Sukharev contributed equally to this studyWe have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.

Although mechanosensory responses are ubiquitous and diverse, the molecular bases of mechanosensation in most cases remain mysterious MscL, a mechanosensitive channel of large conductance of Escherichia coli and its bacterial homologues are the first and currently only channel molecules shown to directly sense mechanical stretch of the membrane. In response to the tension conveyed via the lipid bilayer, MscL increases its open probability by several orders of magnitude. In the present review we describe the identification, cloning, and first sets of biophysical and structural data on this simplest mechanosensory molecule. We discovered a 2.5-ns mechanosensitive conductance in giant E. coli spheroplasts. Using chromatographies to enrich the target and patch clamp to assay the channel activity in liposome-reconstituted fractions, we identified the MscL protein and cloned the mscL gene. MscL comprises 136 amino acid residues (15 kDa), with two highly hydrophobic regions, and resides in the inner membrane of the bacterium. PhoA-fusion experiments indicate that the protein spans the membrane twice with both termini in the cytoplasm. Spectroscopic techniques show that it is highly helical. Expression of MscL tandems and covalent cross-linking suggest that the active channel complex is a homo-hexamer. We have identified several residues, which when deleted or substituted, affect channel kinetics or mechanosensitivity. Although unique when discovered, highly conserved MscL homologues in both gram-negative and gram-positive bacteria have been found, suggesting their ubiquitous importance among bacteria.

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The mechanism(s) by which infectious or malignant material is cleared by the host has long been an area of intensive study. We have used the murine model of infection with lymphocytic choriomeningitis virus (LCMV) to look at immune clearance during persistent infection. LCMV was selected because the mouse is its natural host, it easily induces acute or persistent infection in vivo, and the mechanism by which it is cleared in vivo during acute infection is now well understood. Clearance, although associated with several antiviral immune effector mechanisms, is primarily dependent on the activity of virus-specific cytotoxic T lymphocytes (CTL) restricted by H-2 molecules of the mouse major histocompatibility complex (MHC). If these cells fail to generate or are depleted, progression from acute to persistent infection occurs. Here, using molecular probes, we show that viral nucleic acid sequences, viral proteins and infectious materials can be efficiently and effectively cleared by adoptive transfer of antiviral H-2-restricted lymphocytes bearing the Lyt 2+ phenotype. Viral materials are cleared from a wide variety of tissues and organs where they normally lodge during persistent infection. Unexpectedly, the mode by which viral materials are removed from the central nervous system (CNS) differed markedly from the mechanism of clearance occurring at other sites. These observations indicate the possible use of adoptive lymphocyte therapy for treatment of persistent infections and suggest that immune clearance of products from the CNS probably occurs by a process distinct from those in other organs.