Abstract
Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca2+ and glutamate−. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.
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We thank Dr. A. Kubolski for his manuscript “Generation of giant protoplasts of E. coli and an inner-membrane selective conductance.” We thank H.C. Berg, I.R. Booth, W. Epstein, and C. Kung for gifts of strains, and B. Martinac and A. Kubalski for the initial help in technique. This work was supported by a grant from the National Institutes of Health (AI08746).
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Cui, C., Smith, D.O. & Adler, J. Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording. J. Membarin Biol. 144, 31–42 (1995). https://doi.org/10.1007/BF00238414
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DOI: https://doi.org/10.1007/BF00238414