Figure 2
Mechanosensitive channel activities detected from C. glutamicum giant spheroplasts. (A) A diagram showing C. glutamicum giant spheroplasts preparation (top). Images of normal cells (left), round cells after treatment of ethambutol and lysozyme (center) and enlarged cells (right) (bottom). (B) Transmission electron microscopy of C. glutamicum giant spheroplasts. The shape of C. glutamicum normal cell (left). Image of a giant spheroplast (right). Magnified images of the cell wall are shown at the bottom. Scale bars correspond to 200 nm. (C) Image of cell-attached configuration of the C. glutamicum giant spheroplasts. Scale bar corresponds to 5 μm. (D) Three types of mechanosensitive currents in the C. glutamicum native membrane of WT strain at the pipette voltage of +60 mV. Channel current traces (top) and pressure traces applied to the membrane (bottom) in the inside-out excised patch configuration are shown. Magnified traces of CgMscCG (top), and CgMscCG2 and CgMscL (bottom) channels of the traces enclosed by dotted squares are shown. (E) Two types of mechanosensitive currents in the native membrane of CgmscCG-KO strain recorded at the pipette voltage of +30 mV. Magnified trace of CgMscCG2 and CgMscL channels from the traces enclosed by dotted squares is shown. (F) Single channel conductance of CgMscCG, CgMscCG2, and CgMscL. Channel currents were recorded in 200 mM KCl, 90 mM MgCl2, 10 mM CaCl2, 5 mM HEPES-KOH pH 7.0 at the pipette voltage of +30 mV. The channel conductance was calculated as the single channel current divided by the voltage. Bars show standard errors (n = 3). (*Student’s t-test, P < 0.05, **Student’s t-test, P < 0.01). (G) Activation thresholds of CgMscCG and CgMscCG2. The pressure required for the first activation of CgMscCG (PCG), CgMscCG2 (PCG2), and CgMscL (PL) was measured as shown in D and E, and the activation threshold ratio against PL was calculated. Bars show standard errors (n = 5) (*student’s t-test, P < 0.05).
