Catherine Berrier

Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure.

Abstract. 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.

Release of Thioredoxin via the Mechanosensitive Channel MscL during Osmotic Downshock of Escherichia coli Cells

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− cells. Release was restored by expression of the gene coding for MscL. Thus MscL is not necessary for the excretion of osmoprotectants, but it does open in vivoduring shock and catalyzes the efflux of thioredoxin and possibly other small cytosolic proteins. It follows that the other mechanosensitive channels, which are known to be activated at lower tension, must also open during osmotic shock. Their opening and that of MscL could account for the rapid release of osmolytes.

The Destabilization of Lipid Membranes Induced by the C-terminal Fragment of Caspase 8-cleaved Bid Is Inhibited by the N-terminal Fragment

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 (tcBid) and a 6.5 kDa N-terminal fragment (tnBid). Following the cleavage, tcBid translocates to the mitochondria and promotes the release of cytochromec into the cytosol by a mechanism that is not understood. Here we report that recombinant tcBid can act as a membrane destabilizing agent. tcBid 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, tcBid, but not full-length Bid, permeabilizes liposomes at physiological pH. The destabilizing effect of tcBid on liposomes and planar bilayers is independent of the BH3 domain. In contrast, mutations in the BH3 domain impair tcBid ability to induce cytochrome c release from mitochondria. The permeabilizing effect of tcBid on planar bilayers, liposomes, and mitochondria can be inhibited by tnBid. 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.

A patch-clamp study of ion channels of inner and outer membranes and of contact zones of E. coli, fused into giant liposomes. Pressure-activated channels are localized in the inner membrane.

Inner and outer membranes of Escherichia coli and contact zones were isolated and fused separately with giant liposomes amenable to patch‐clamp recording. Different types of large pressure‐activated channels were localized in the inner membrane fraction which also contained smaller, pressure‐insensitive channels. The outer membrane contained pressure‐insensitive channels with large conductances and long opening and closing times which are likely to be porins. Large channels were also observed in contact zones.

Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis

Hemifluorinated and fluorinated surfactants are lipophobic and, as such, non-detergent. Although they do not solubilize biological membranes, they can, after conventional solubilization, substitute for detergents to keep membrane proteins soluble, which generally improves their stability [Breyton, Chabaud, Chaudier, Pucci and Popot (2004) FEBS Lett. 564, 312-318]. In the present study, we show that (hemi)fluorinated surfactants can be used for in vitro synthesis of membrane proteins: they do not interfere with protein synthesis, and they provide a suitable environment for MscL, a pentameric mechanosensitive channel, to fold and oligomerize to its native functional state. Following synthesis, both types of surfactants can be used to deliver MscL directly to pre-formed lipid vesicles. The electrophysiological activity of MscL synthesized in vitro in the presence of either hemi- or per-fluorinated surfactant is similar to that of the protein expressed in vivo.

Fast and slow kinetics of porin channels from Escherichia coli reconstituted into giant liposomes and studied by patch‐clamp

E. coli porins (OmpF and OmpC) were purified and reconstituted into liposomes which were enlarged to giant proteoliposomes by dehydration—rehydration and studied by patch‐clamp. The porins could be closed by voltage pulses under −100mV. The kinetics of closure was slow, with closure events of about 200 pS in 0.1 M KCl. Rapid fluctuations (in the millisecond range) of about one third (60–70 pS) of the large closure steps were also observed. The data are interpreted as follows: an increase in membrane potential favours the cooperative transition of multimers towards an inactivated state, while monomers which have not been inactivated can flicker rapidly between an open and a short‐lived closed state.

The Purified Mechanosensitive Channel TREK-1 Is Directly Sensitive to Membrane Tension

Background: For eukaryotic cells, a direct gating of mechanosensitive channels by membrane tension has not been demonstrated. Results: The mouse TREK-1 was purified and reconstituted in liposomes amenable to patch clamp recording. Conclusion: The channel displayed expected electrophysiological properties, and positive pressure could reversibly close it. Significance: TREK-1, a eukaryotic mechanosensitive channel, is directly sensitive to a modification of membrane tension. Mechanosensitive channels are detected in all cells and are speculated to play a key role in many functions including osmoregulation, growth, hearing, balance, and touch. In prokaryotic cells, a direct gating of mechanosensitive channels by membrane tension was clearly demonstrated because the purified channels could be functionally reconstituted in a lipid bilayer. No such evidence has been presented yet in the case of mechanosensitive channels from animal cells. TREK-1, a two-pore domain K+ channel, was the first animal mechanosensitive channel identified at the molecular level. It is the target of a large variety of agents such as volatile anesthetics, neuroprotective agents, and antidepressants. We have produced the mouse TREK-1 in yeast, purified it, and reconstituted the protein in giant liposomes amenable to patch clamp recording. The protein exhibited the expected electrophysiological properties in terms of kinetics, selectivity, and pharmacology. Negative pressure (suction) applied through the pipette had no effect on the channel, but positive pressure could completely and reversibly close the channel. Our interpretation of these data is that the intrinsic tension in the lipid bilayer is sufficient to maximally activate the channel, which can be closed upon modification of the tension. These results indicate that TREK-1 is directly sensitive to membrane tension.

MECHANOSENSITIVE ION CHANNELS AND THEIR MODE OF ACTIVATION

Mechanosensitive channels are ion channels whose activity is dependent on a mechanical stress on the membrane. They are believed to play a central role in mechanotransduction, the process by which mechanical energy is converted into electrical or chemical signals in biological cells. Recent progress, which has been made at the molecular level, is presented, and the two current models of activation of these channels are discussed.

Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL: MscL does not need the insertase YidC for insertion in vitro

The mechanosensitive channel MscL of the plasma membrane of bacteria is a homopentamer involved in the protection of cells during osmotic downshock. The MscL protein, a polypeptide of 136 residues, was recently shown to require YidC to be inserted in the inner membrane of E. coli. The insertase YidC is a component of an insertion pathway conserved in bacteria, mitochondria and chloroplasts. MscL insertion was independent of the Sec translocon. Here, we report sucrose gradient centrifugation and freeze-etching microscopy experiments showing that MscL produced in a cell-free system complemented with preformed liposomes is able to insert directly in a pure lipid bilayer. Patch-clamp experiments performed with the resulting proteoliposomes showed that the protein was highly active. In vitro cell-free synthesis targeting to liposomes is a new promising expression system for membrane proteins, including those that might require an insertion machinery in vivo. Our results also question the real role of insertases such as YidC for membrane protein insertion in vivo.

Structural study of the membrane protein MscL using cell-free expression and solid-state NMR.

High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75kDa pentameric alpha-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.