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

Abstract

Ca(2+), Mg(2+), and K(+) activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca(2+), by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca(2+) activity was approximately 0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca(2+) 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 Ca(2+)) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca(2+) within a physiological range of concentrations (0.1-1.0 mM). Aggregation of the physiological vacuolar Na(+) (60 mM) and Mg(2+) (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca(2+) variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca(2+) activates the SV channel in a voltage-independent manner with K(d)=0.7-1.5 microM. Comparison of the vacuolar Ca(2+) 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 microM) cytosolic Ca(2+), only 0.5% of SV channels are open. This mediates a Ca(2+) release of only a few pA per vacuole (approximately 0.1 pA per single SV channel). Overall, our data suggest that the release of Ca(2+) through SV channels makes little contribution to a global cytosolic Ca(2+) signal.

Fig. 1.

Effects of cytosolic Ca²⁺ on trans-tonoplast Ca²⁺ fluxes and the membrane potential difference in isolated red beet vacuoles. (A) Ca²⁺ fluxes from individual vacuoles measured with the MIFE technique (n=6–19 vacuoles for each Ca²⁺ concentration in the bath). Negative flux implies a net Ca²⁺ transport from the vacuole to the extravacuolar (cytosolic) side. (B) Vacuolar Ca²⁺ flux stimulation by cytosolic Mg²⁺ and inhibition by Zn²⁺ (n=8–10 vacuoles). (C) Electric potential difference across the tonoplast as a function of bath free Ca²⁺ (n=8–11 vacuoles). All data are presented as mean ±SEM. Bath: 100 mM KCl, pH 7.5.

Fig. 2.

Vacuolar pH modifies the dependence of the SV channel voltage activation on the vacuolar Ca²⁺. (A) Voltage dependence of the SV channel activation at different vacuolar free Ca²⁺ concentrations (indicated in mM at the right of the corresponding symbols) and pH 5.5. SV channel activity was analysed in large isolated tonoplast patches (n=4–8 patches for each Ca²⁺ concentration, means ±SEM). Dashed horizontal line indicates the voltage at which 1% of maximum number of SV channels are open. (B) Dependence of the threshold for SV channel voltage activation (a membrane potential at which 1% of maximal number of SV channels are open) on the vacuolar free Ca²⁺ at different vacuolar pH. Data for pH 7.5 were calculated from Pottosin et al. (2004). Solution composition was: symmetric 100 mM KCl; 2 mM free Ca²⁺ at cytosolic side; cytosolic and vacuolar pH values were 7.5 and 5.5, respectively.

Fig. 3.

Vacuolar Na⁺ has opposite effects on the SV channel voltage dependence at zero and submillimolar vacuolar Ca²⁺. Voltage dependence of the SV channel activity in large isolated tonoplast patches at two vacuolar free Ca²⁺ concentrations, c. 10 nM (A) and 0.5 mM (B). Vacuolar K⁺ and Na⁺ concentrations (in mM) for each condition are indicated, 100 mM KCl, 2 mM free Ca²⁺ (pH 7.5) at cytosolic side. Data are means ±SEM, n=3 patches for each condition.

Fig. 4.

The presence of various cations at the luminal side makes the SV channel almost insensitive to changes in vacuolar Ca²⁺ from zero up to the millimolar range. (A) Voltage dependence of the SV channel activity in large isolated tonoplast patches at different vacuolar free Ca²⁺ concentrations. The solution at the vacuolar side contained (in mM): 125 KCl, 62 NaCl, 8 MgCl₂ (pH 5.5), and at the cytosolic side: 100 mM KCl, 2 mM free Ca²⁺ (pH 7.5). (B) Dependence of the threshold for SV channel voltage activation (membrane potential at which 1% of maximum number of SV channels are open) on the vacuolar free Ca²⁺. Other components of the vacuolar and cytosolic solutions are as above. For comparison, a dependence obtained in 100 mM KCl (pH 5.5) was redrawn from Fig. 2B (dashed line). The range of values obtained in the attached patches is indicated (grey zone). Data are means ±SEM, n=3–5 patches for each condition.

Fig. 5.

Sensitivity of the SV channel to cytosolic Ca²⁺ and Mg²⁺ at physiological concentrations of vacuolar cations. (A) An example of a SV current record from a small (c ∼1 pF) outside (cytosolic side)-out vesicle at different concentrations of cytosolic Ca²⁺. External (cytosolic) solution contained 100 mM KCl (pH 7.4). The composition of a pipette (vacuolar) solution is as in Fig. 4. (B) The Ca²⁺-dependence of the SV current (nA/pF) at +180 and +100 mV. Cytosolic free Mg²⁺ was 0.5 mM, and other ionic conditions are as in (A). Data are presented as mean ±SEM (n=6–10 vacuoles). Solid lines are the best fits to the Hill equation, with K_d for cytosolic Ca²⁺ at 1.4±0.7 μM and 0.7±0.1 μM, and Hill coefficients of 1.4±0.2 and 1.3±0.2 for +100 and +180 mV, respectively. (C) Voltage dependence of the SV current at different cytosolic free Ca²⁺ and Mg²⁺ levels. A macroscopic SV current was divided by the single channel current at each potential and by membrane capacitance, yielding the mean number of the open channels per pF. Data are means ±SEM (n=4–8 vacuoles). Solid lines are the best fits to the Boltzmann equation with parameter values given in Table 3.

Fig. 6.

Slow-vacuolar channel activity in vacuole-attached patches. Bath and pipette solutions are identical, 100 mM KCl (pH 7.4) and free Ca²⁺ and Mg²⁺ concentrations are as indicated at the top of the curves. A macroscopic SV current was divided by the single channel current at each potential, yielding the mean number of open channels per patch (NPo). For comparison, assuming the same channel density as in outside-out patches (Fig. 5), the mean open SV channel number per typical vacuole of 50 μm diameter is given at the second y-axis on the right. Data are means ±SEM (n=4). Solid lines are the best fits to the Boltzmann equation with parameter values given in Table 3. Voltages were command potentials for this case; to convert them to the actual tonoplast electric potential difference, the resting potential value (about +10 mV at these conditions, as verified by 3 M KCl-filled microelectrode impalements) should be totalled.