Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock. Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 M KCl) activated by stretch. The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances. In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch. These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E. coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes. These conductances could be grouped into three subfamilies of poorly selective channels. In both preparations, the higher the conductance, the higher was the negative pressure needed for activation. We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system.

Bacteria subjected to a hypotonic osmotic shock lose internal ions and also metabolites, without lysis of the cells. We show that the presence in the shock medium, at submillimolar concentrations, of the ion gadolinium, recently shown to block stretch-activated channels in Xenopus oocytes [Yang, X.-C. & Sachs, F. (1989) Science 243,1068 -10711, was sufficient to inhibit shock-induced release of metabolites such as lactose and ATP in Escherichia coli and ATP in Streptococcus faecalis. Moreover, gadolinium was observed, in patch-clamp experiments, to inhibit the giant stretch-activated channels of E. coli , S. faecalis. and Bacillus subtilis. Taken together, these data suggest that stretch-activated channels are localized in the cytoplasmic membrane of Gram-negative and Gram-positive bacteria, where they control the efflux of osmotic solutes, thus probably playing a major role in the response to hypotonic osmotic shock.

Escherichia coli cells possess several mechanosensitive ion channels but only MscL, the channel with the highest conductance, which is activated at the highest membrane tension, has been cloned. We investigated the putative involvement of MscL in the effluxes caused by osmotic downshock. Osmotic shock caused the release of potassium glutamate, trehalose, and glycine betaine from wild type cells and cells lacking MscL. There was no difference between the two strains, but the extreme rapidity of the efflux process, as shown herein for glycine betaine, suggests that it is channel-mediated. Osmotic downshock also induces the release of some cytosolic proteins from EDTA-treated cells. We investigated the release of thioredoxin. This protein was totally released from wild type cells but was retained by MscL

Bid is a proapoptotic, BH3-domain-only member of the Bcl-2 family. In Fas-induced apoptosis, Bid is activated through cleavage by caspase 8 into a 15.5-kDa C-terminal fragment (t c Bid) and a 6.5 kDa N-terminal fragment (t n Bid). Following the cleavage, t c Bid translocates to the mitochondria and promotes the release of cytochrome c into the cytosol by a mechanism that is not understood. Here we report that recombinant t c Bid can act as a membrane destabilizing agent. t c Bid induces destabilization and breaking of planar lipid bilayers without appearance of ionic channels; its destabilizing activity is comparable with that of Bax and at least 30-fold higher than that of full-length Bid. Consistently, t c Bid, but not full-length Bid, permeabilizes liposomes at physiological pH. The destabilizing effect of t c Bid on liposomes and planar bilayers is independent of the BH3 domain. In contrast, mutations in the BH3 domain impair t c Bid ability to induce cytochrome c release from mitochondria. The permeabilizing effect of t c Bid on planar bilayers, liposomes, and mitochondria can be inhibited by t n Bid. In conclusion, our results suggest a dual role for Bid: BH3-independent membrane destabilization and BH3-dependent interaction with other proteins. Moreover, the dissociation of Bid after cleavage by caspase 8 represents an additional step at which apoptosis may be regulated.

We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent. We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein. A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experiments showed that the protein was able to oligomerize in detergents. When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult.

MscL is a mechanosensitive channel that is gated by tension in the membrane bilayer alone. It is a homooligomer of a protein comprising two transmembrane segments connected by an external loop, with the NH 2 and COOH termini located in the cytoplasm. The contributions of the extramembranous domains of the channel to its activity were investigated by specific proteolysis during patch-clamp experiments. Limited proteolysis of the COOH terminus or the NH 2 terminus increased the mechanosensitivity of the channel without changing its conductance. Strikingly, after cleavage of the external loop of each monomer, the channel was still functional, and its mechanosensitivity was increased dramatically, indicating that the loop acts as a spring that resists the opening of the channel and promotes its closure when it is open. These results indicate that the integrity of most of the extramembranous domains is not essential for mechanosensitivity. They suggest that these domains counteract the movement of the transmembrane helices to which they are connected, thus setting the level of sensitivity of the channel to tension.Mechanosensation and mechanotransduction, the processes by which mechanical force is detected and transduced into electrical and chemical signals by living cells, are at the basis of the physiology of osmoregulation, touch, hearing, proprioception, as well as detection of wind and gravity by plants. Since their discovery by patch-clamp experiments (1, 2), mechanosensitive ion channels (Msc)

Upon osmotic downshock, a few cytoplasmic proteins, including thioredoxin, elongation factor Tu (EF-Tu), and DnaK, are released from Tris-EDTA-treated Escherichia coli cells by an unknown mechanism. We have shown previously that deletion of mscL, the gene coding for the mechanosensitive channel of the plasma membrane with the highest conductance, prevents the release of thioredoxin. We confirm and extend the implication of MscL in this process by showing that the release of EF-Tu and DnaK is severely impaired in MscL-deficient strains. Release of these proteins is not observed in the absence of a Tris-EDTA treatment which disrupts the outer membrane, indicating that, in intact cells, they are transferred to the periplasm upon shock, presumably through the MscL channel.

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