Patch Clamp Studies on Ion Pumps of the Cytoplasmic Membrane ofEscherichia coli

Abstract

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 F 1 -ATPase. Such current was inhibited by inhibitors of the respiratory chain or F o F 1 -ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.The patch clamp technique is an excellent method to measure ion movement across cell membranes as current (1). An extremely small glass pipette (about 1 m in diameter) is attached to the membranes, and activity of ion translocating proteins (ion channels, ion pumps, or ion transporters) is directly measured. So far, however, this important method has been mainly utilized for studies on animal or plant cells but scarcely for bacterial cells (2). Bacterial cells are usually too small to be measured by this method.Escherichia coli, a Gram-negative bacterium, is the best characterized organism from both biochemical and genetical points of view. Ion pumps and ion transporters in E. coli are biochemically well characterized. Many mutant E. coli cells are available. Thus, genetical manipulations are very easy with this microorganism. Therefore, development of a patch clamp method applicable to E. coli membranes must be extremely valuable. Cells of E. coli are surrounded by an outer membrane and an inner membrane (cytoplasmic membrane) separated by a peptidoglycan layer and a periplasmic space. All of the major ion pumps and ion transporters such as the respiratory chain, F o F 1 -ATPase, various ion transporters, and ion-coupled solute transporters are located in the cytoplasmic membrane. To measure ion translocation via such ion pumps or transporters of the cytoplasmic membrane, we have to overcome the following three hurdles: 1) we have to prepare giant vesicles, the diameter of which must be at least 10 m (this is important to get high success rate and accuracy of measurement), 2) the pipette must be directly accessible to the cytoplasmic membrane, and 3) the substrates or effectors of the ion pumps or transporters must be easily accessible to the active site of the proteins and easily removable from the system.It would be essential to prepare giant protoplasts to overcome the first two hurdles. Many attempts have been made by many research groups to prepare giant bacterial cells or giant protoplasts. So far, however, no giant protoplasts surrounded by cytoplasmic membranes and suitable for patch clamp analysis have been prepared....

Patch Clamp Studies on V-type ATPase of Vacuolar Membrane of Haploid Saccharomyces cerevisiae

“…Five minutes were usually long enough to exchange the medium inside the vacuole with another medium through the capillary by diffusion. The external medium was changed by a tandem 6-way valve system as described previously (15). Membrane currents were amplified by a patch/whole cell clamp amplifier (CEZ-2300, Nihon Kohden Co., Tokyo), and recorded on a digital audio tape recorder (DTC 55ES, Sony Corp., Tokyo).…”

“…DISCUSSION Kusaka in 1967 (16) used penicillin G as the cell wall synthesis inhibitor in his original SI method for preparing giant protoplasts of B. megaterium. Penicillin G was also effective for E. coli (15). However, no hydrophilic reagent like penicillin G is known for yeast that inhibits cell wall synthesis (42) but does not affect other cellular functions, particularly, transport activities in the membrane.…”

“…Recently, we succeeded in converting Escherichia coli cells into giant protoplasts that are suitable for patch clamp experiments (15). This spheroplast incubation method (SI method) 1 was a modification of the giant cell preparation method originally developed for Bacillus megaterium by Kusaka (16) in * This work was in part supported by Japanese Ministry of Education, Science, Sports and Culture Grant 10219206 (to Y.…”

Retraction: Characterization of giant spheroplasts generated from the aerobic anoxygenic photosynthetic marine bacterium <i>Roseobacter litoralis</i>

“…Incubation of protoplasts formed by lysozymes in the presence of penicillin (an inhibitor of peptidoglycan synthesis) was shown to generate giant protoplasts (Kuroda et al, 1998;Kusaka, 1967;Nakamura et al, 2011). However, protoplasts do not divide under incubation, but DNA replication has been shown to occur.…”

Round Shape Enlargement of the Yeast Spheroplast of Saccharomyces cerevisiae by HM-1 Toxin.

“…[33][34][35][36] Lipophilic antibiotics, aculeacin A and papula- The incubation conditions and rhodamine phalloidin staining of spheroplasts are described in Materials and Methods. The incubation temperature was 30°C for the first 3 h and 25°C for the following 20 h. A and B. Spheroplasts were incubated in the control medium.…”