Pressure-sensitive ion channel in Escherichia coli.

Abstract

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...

A patch‐clamp study of ion channels of inner and outer membranes and of contact zones of E. coli, fused into giant liposomes

“…We show here that E. coli strain K12 possesses several different pressure-activated channels. The pressure-activated channel described by Martinac et al [4] in giant spheroplasts is similar (in conductances and kinetics) to the 330 pS or the 490 pS (in 0.1 M KCl) channel observed here. Multiple conductances of pressure-sensitive channels have also been recently observed in E. coli giant spheroplasts (Zoratti, M., personal communication).…”

“…Martinac et al applied the patch-clamp technique to E. coli cells [4]. Since normal bacteria are too small to be studied directly by patch-clamp, they used giant spheroplasts obtained by growth of the cells in the presence of an inhibitor of septation followed by a classical lyzozyme-EDTA treatment.…”

“…Nevertheless it was not possible to ascertain whether the record was made from the outer or the inner membrane of the bacteria. Although it is not known from planar lipid bilayer experiments whether some porins are activated by pressure, the very large conductance of this channel made the authors suggest that it could be located in the outer membrane rather than in the inner membrane [4,5]. Nevertheless, Zoratti and Petronilli recently observed by the patch-clamp technique, pressure-activated channels in giant spheroplasts of Gram-positive (devoid of outer membrane) bacteria […”

Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation

“…Early studies detected MS activities when patches of Escherichia coli giant spheroplasts were subjected to negative pressure in the pipette (Martinac et al, 1987). Similar observations have been made in Bacillus subtilis and Streptococcus faecalis protoplasts (Szabo et al, 1993).…”

Organellar mechanosensitive channels in fission yeast regulate the hypo-osmotic shock response

“…Giant spheroplasts were obtained from E. coli cells harbouring the expression vector pB10b as described previously 44 . Gene expression was induced at 37 °C for 3 h. Single-channel currents were recorded from inside-out excise patches from the giant spheroplasts.…”