Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca²⁺ fluxes in situ

Ca²⁺, Mg²⁺, and K⁺ activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca²⁺, by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca²⁺ activity was ∼0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca²⁺ in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca²⁺) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca²⁺ within a physiological range of concentrations (0.1-1.0 mM). Aggregation of the physiological vacuolar Na ⁺ (60 mM) and Mg²⁺ (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca²⁺ variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca²⁺ activates the SV channel in a voltage-independent manner with K_d=0.7-1.5 μM. Comparison of the vacuolar Ca²⁺ fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 μM) cytosolic Ca ²⁺, only 0.5% of SV channels are open. This mediates a Ca ²⁺ release of only a few pA per vacuole (∼0.1 pA per single SV channel). Overall, our data suggest that the release of Ca²⁺ through SV channels makes little contribution to a global cytosolic Ca²⁺ signal.