We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherchia coli and have discovered pressure-activated ion channels. The channels have the following properties: (t) activation by slight positive or negative pressure; (it) voltage dependence; (ifi) large conductance; (iv) selectivity for anions over cations; (v) dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.Ion channels are gated protein pores found in biological membranes; these channels regulate many cellular interactions with the environment, including responses to hormonal, neuronal, and sensory stimuli (1). Ion channels have been studied in animals, plants, and microorganisms (2-4). In bacteria, in vivo channel activity has not been demonstrated, although the activity of isolated channel proteins has been measured in artificial membranes (5, 6).The patch-clamp technique allows recording of current through individual ion channels in the native membrane by sucking the membrane onto a recording pipette to form a tight (gigaohm) electrical seal (7). This method has been used to study single channels in vivo in many eukaryotic cells, and it has demonstrated that the large currents measured across the membranes of a whole cell are really composed ofmany small currents passing through individual channels.The lower limit to the diameter ofthe patch-pipette opening is about 1 ,um (1); this precludes measurement ofion channels in bacteria directly. Cells of Escherichia coli, however, can become giant spheroplasts when grown in the presence of chemicals such as mecillinam to prevent cell wall (peptidoglycan) synthesis, and membrane potential has been measured in such spheroplasts by conventional electrophysiology (8). Giant spheroplasts can also be formed by growth of cells in the presence of cephalexin to prevent cell division and form filamentous "snakes"; these snakes can then be treated with lysozyme and EDTA to dissolve the cell wall (the spheroplasts can revert to normal form when returned to growth medium in the absence of these chemicals) (9). We used this latter method to make spheroplasts with a diameter of -6 ,.m. We demonstrate here the application of in vivo patch-clamp recording to such giant spheroplasts. This method should be generally applicable to any bacterial species.We discovered that a low positive or negative pressure (tens of millimeters of mercury; 1 mm Hg = 133 Pa) applied to the spheroplast membrane activates ion channels. This pressure could be caused by an osmotic difference of as little as a few milliosmolar across the membrane. We believe that these channels may allow E. coli to detect and to respond to small osmotic changes in the surrounding medium. The preliminary work has been reported in abstract form (10). MATERIALS AND METHODSMaterials. Organic components of the growth medium were purchased from Difco. Tris was purchased from Boehringer Mannheim; other salts and chemicals for preparation of...

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 multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (P o) has a steep sigmoidal dependence on T, with a midpoint (T 1/2) of 11.8 dyn/cm. The maximal slope sensitivity of P o/P c was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was ΔE = 18.6 k B T. If the mechanosensitivity arises from tension acting on a change of in-plane area (ΔA), the free energy, TΔA, would correspond to ΔA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest ΔA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to ΔA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.