Patch clamp studies on ion pumps of the cytoplasmic membrane of Escherichia coli: Formation, preparation, and utilization of giant vacuole- like structures consisting of everted cytoplasmic membrane

Formation of giant protoplasts from normal Escherichia coli cells resulted in the formation of giant vacuole-type structures (which we designate as provacuoles) in the protoplasts. Electron microscopic observation revealed that these provacuoles were surrounded by a single membrane. We detected inner (cytoplasmic) membrane proteins in the provacuolar membrane but not outer membrane proteins. Biochemical analyses revealed that the provacuoles consist of everted cytoplasmic membranes. We applied the patch clamp method to the giant provacuoles. We have succeeded in measuring current that represents inward movement of H⁺ because of respiration and to ATP hydrolysis by the F(o)F₁-ATPase. Such current was inhibited by inhibitors of the respiratory chain or F(o)F₁-ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.