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

Abstract

Inner and outer membranes of Escherichiu 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, pressureinsensitive 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.

YbdG in Escherichia coli is a threshold-setting mechanosensitive channel with MscM activity

“…The screen resulting in isolation of the V229A mutant was conducted at low osmolarity and thus the selection was specifically for channels that could gate under these conditions. In the earliest experiments measuring E. coli MS channels using cytoplasmic membranes fused with phosphatidylcholine liposomes (5)(6)(7)43), multiple activities were observed that could be differentiated by their kinetics. Individual patches contained single types of activities rather than the mixtures of channels routinely measured in cells, suggesting that the channels are clustered in the mixed membranes of the reconstituted system (6).…”

“…Martinac and colleagues discovered that hypoosmotic shock could be mimicked by application of slight pressure (0.1-0.3 atm) across an isolated bacterial membrane patch, leading to increased transmembrane electrical current (5). Subsequently, multiple MS conductances, activated at different pressure thresholds, were identified after solubilization of membrane fractions and reconstitution of protein into artificial lipid bilayers (6,7).…”

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

“…The liposol~es were then fused into giant liposomes using a cycle of dchydration-rehydration, as described [15], A 2-5 #1 drop of the 8taut proteoliposome suspension was deposited on a nunclon plastic tissue dish and diluted with 1,5 ml of tlae bath solution (as defined in the figure legends). Single.channel activity was measured using the method of Hamill et at, [20], as previously described [15].…”

“…Single.channel activity was measured using the method of Hamill et at, [20], as previously described [15]. Records were filtered at 1 or 2 kHz (-3 dB point) through an 8-pole Bessel low pass filter and digitiz~l at a rate of 2 or 10 kHz, The membrane potential refers to the potential in the bath minus the potential in the pipette,…”

Gating the bacterial mechanosensitive channel MscL in vivo

“…But the insights gained in these studies cannot be easily translated into insights in bacterial physiology. We have the following difficulties: (i) There are no electrophysiological methods to study intact bacteria during downshock; (ii) MSCs are mired in a complex tripartite envelope whose mechanical properties are not well understood; (iii) Although there is likely no osmotic pressure difference between the cytoplasm and the periplasm during steady-state growth (14,15), the course of the rise and fall of turgor pressure during downshock, especially at the plasma membrane where MSCs reside (16), is unknown. A 1,000 mmol͞kg downshock could theoretically create a turgor pressure of Ϸ24 atm (1 atm ϭ 101.3 kPa) at 25°C.…”