Figures

Figure 1 14-3-3B protein inhibits the SV channel and activates the FV channel

(A) Modulation of SV current by 14-3-3. Protoplasts were prepared from barley mesophyll cells as described [33] and a small aliquot was released in the measuring chamber filled with SV bath solution (in mM): 95 KCl, 10 Hepes/KOH (pH 7.5), 2 CaCl₂ (total [K⁺], 101 mM). The pipette solution contained (in mM): 95 KCl, 5 EGTA, 10 Hepes/KOH (pH 7.5) (total [K⁺], 113 mM). The latter solution was identical to the FV bath solution. Osmolarities of all solutions were adjusted to 525 mOsm with sorbitol. Test voltages ranged from +100 to -60 mV in steps of 20 mV. Before and after test steps vacuoles were held at 0 mV. Recording and analysis conditions were essentially as described in [33]. When a stable recording was obtained, bath perfusion (0.25 ml/min) was stopped, several recordings were made to test whether this did not affect the SV or FV current and then at t = 0 recombinant 14-3-3B protein was added (final concentration, 100 nM). As observed before [33] the inhibitory effect of 14-3-3 was very rapid, but a partial recovery of the current was observed within 5 min. At t = 6.5 the bath was washed again with SV solution and the recording at 9 min shows that the magnitude of the current did not increase but the kinetics of channel activation were restored (see the -1 and +9 min recordings). (B) Modulation of the FV current by 14-3-3. Recording of the whole-vacuole FV current. After the addition of 100 nM 14-3-3 to the bath solution (t = 0) a steady increase in current density was observed. The 14-3-3 effect was reversible after washing (started at t = 9.5 min) the bath with 14-3-3 free FV solution. The numbers refer to the time (in min) before and after the moment of 14-3-3B addition (t = 0 min) to the bath.