INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION LITERATURE CITED

Mg²⁺ is an abundant cytoplasmic cation in higher plants (Epstein, 1965), with concentrations of 2 to 10 mM in leaf cells (Leigh and Wyn Jones, 1986). Mg²⁺ ions exist as free cations and are also sequestered in internal organelles, bound by cytosolic proteins, or complexed with small organic molecules. Many enzymes require or are strongly activated by Mg²⁺, for example, plasma membrane ATPases, protein kinases, type-2C phosphatases, glutathione synthase, and RuBP carboxylase (Marschner, 1995; Leube et al., 1998). The important role of Mg²⁺ as a regulator of various ion channels is well established in animal cells (Agus and Morad, 1991; Flatman, 1991; Murphy et al., 1991; Hille, 1992; Chuang et al., 1997; Kerschbaum and Cahalan, 1999). Matsuda et al. (1987) and Vandenberg (1987) demonstrated the direct blockage of inward rectifier K⁺ channels in animal cells by Mg²⁺; however, little is known about how Mg²⁺ affects ion channel activities in plant cells.

Two types of ion channels have been characterized in most plant vacuolar membranes studied to date. These are the Ca²⁺-permeable, cation-selective slow vacuolar (SV) channels and the cation-selective fast vacuolar (FV) channels (Hedrich and Neher, 1987; Weiser et al., 1991; Bethke and Jones, 1994; Ward and Schroeder, 1994; Allen and Sanders, 1996). SV channels are activated by cytosolic Ca²⁺, whereas FV channels are inhibited by elevations in cytosolic Ca²⁺ (Allen and Sanders, 1996).

FV channels show instantaneous currents in response to voltage pulses (Hedrich and Neher, 1987; Allen and Sanders, 1996; Tikhonova et al., 1997). FV channels are cation-selective (Allen and Sanders, 1996; Tikhonova et al., 1997). The functions of FV channels remain unknown (Allen and Sanders, 1997), although proposals of functions have been made on the basis of their properties, including mediating K⁺ release from guard cell vacuoles during stomatal closing (Allen and Sanders, 1996). However, at physiological resting cytosolic Ca²⁺ concentrations of 0.1 to 0.2 µM, FV current activities can be very high (Hedrich and Neher, 1987; Allen and Sanders, 1996; Tikhonova et al., 1997). This raised the possibility that FV channels need to be further down-regulated by factors other than Ca²⁺ in order to maintain vacuolar membrane ion gradients. Recently, physiological polyamine levels have been shown to partially down-regulate FV channels (Brüggemann et al., 1998; Dobrovinskaya et al., 1999).

Voltage- and time-dependent SV channels, as well as vacuolar K⁺ selective (VK) channels, are activated by cytosolic Ca²⁺ (Hedrich and Neher, 1987; Bethke and Jones, 1994; Ward and Schroeder, 1994; Allen and Sanders, 1996; Pottosin et al., 1997). In addition, SV channels are regulated by ATP, calmodulin, protein kinases, and phosphatases (Weiser et al., 1991; Bethke and Jones, 1994, 1997; Allen and Sanders, 1995).

Although a significant anion permeability of SV channels had been proposed (Coyaud et al., 1987; Hedrich and Kurkdjian, 1988; Schulz-Lessdorf and Hedrich, 1995), detailed studies unequivocally demonstrated the cation selectivity of SV channels with negligible anion permeability (Colombo et al., 1988; Ward and Schroeder, 1994; Ward et al., 1995; Allen and Sanders, 1996; Pottosin et al., 1997). Studies showed substantial Ca²⁺ and Mg²⁺ permeabilities of SV currents (Ward and Schroeder, 1994; Allen and Sanders, 1996). Therefore, SV channels are cation selective with poor selectivity among monovalent cations (K⁺, Na⁺, and Cs⁺) and divalent cations (Ca²⁺, Mg²⁺, and Ba²⁺). The finding that Ca²⁺-activated SV channels are Ca²⁺ permeable has led to the suggestion that these channels may provide an important mechanism not only for K⁺ transport but also for Ca²⁺-induced Ca²⁺ release (Ward and Schroeder, 1994). A recent study showed that conditions favoring Ca²⁺ release from vacuoles decrease the SV channel open probability, leading to a counter-hypothesis in which SV channels cannot mediate Ca²⁺-induced Ca²⁺ release from vacuoles (Pottosin et al., 1997).

At physiological cytosolic Ca²⁺ concentrations, SV channel activities are generally negligible in many plants (Hedrich and Neher, 1987; Ward and Schroeder, 1994; Barkla and Pantoja, 1996; Allen and Sanders, 1997; Allen et al., 1998). Moreover, the activation potentials of SV channels lie positive of physiological vacuolar membrane potentials of 0 to 40 mV (Hedrich and Neher, 1987; Sze et al., 1992; Bethke and Jones, 1994; Ward and Schroeder, 1994; Allen and Sanders, 1996; Pottosin et al., 1997; Allen et al., 1998). A study on fava bean (Vicia faba) guard cell vacuoles led to the suggestion that cytosolic Mg²⁺ activates SV channels in the absence of cytosolic Ca²⁺ (Allen and Sanders, 1996). However, a more recent study on barley mesophyll vacuoles suggested that Mg²⁺ does not activate SV channels (Pottosin et al., 1997).

The findings that physiological cytosolic Ca²⁺ concentrations do not activate SV channels and over-stimulate FV channels have led to difficulties in predicting their functions in vivo. In the present study, we demonstrate that at physiological concentrations, Mg²⁺ down-regulates vacuolar membrane FV channels in fava bean guard cells, which may provide an efficient down-regulation mechanism of FV channels in vivo. Interestingly, cytosolic Mg²⁺ sensitized SV channels to physiological concentrations of cytosolic Ca²⁺, and data presented here clarify the controversy of Mg²⁺ activation of SV channels raised previously.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION LITERATURE CITED

Isolation of Fava Bean Guard Cell Vacuoles

Fava bean (Vicia faba) plants were grown in a controlled environment growth chamber (model E15, Conviron, Asheville, NC) with 16-h light/8-h dark cycle. Guard cell protoplasts were isolated from 3- to 4-week-old plants by enzymatic digestion of leaf epidermal strips, as previously described (Kruse et al., 1989; Ward and Schroeder, 1994). Vacuoles were released from guard cell protoplasts by osmotic shock and purified using a Ficoll density gradient (Ward and Schroeder, 1994).

Patch Clamp and Data Acquisition

Patch-clamp pipettes were prepared from soft glass capillaries (Kimax 51, Kimble, Toledo, OH), and pulled on a multi-stage programmable puller. Giga- seals between electrode and the vacuolar membrane (>15 G) were obtained by gentle suction. The patch-clamp technique was applied to isolated guard cell vacuoles as previously described (Pei et al., 1996). The whole-vacuole configuration, analogous to the whole-cell configuration (Hamill et al., 1981), was attained by applying high-voltage pulses (usually ±500 mV, 25 ms for each) and slight suction to the interior of the pipette (Pei et al., 1996).

Vacuoles were voltage clamped using an amplifier (Axopatch 200, Axon Instruments, Foster City, CA). All membrane potentials are specified as the potential on the cytosolic side relative to the vacuolar side (Bertl et al., 1992). Data were analyzed using AXOGRAPH software (3.5, Axon Instruments). Statistical analyses were performed using EXCEL (5.0, Microsoft, Redmond, WA). Data are the means ± SE. In Figure 1D, the average percentage of inhibition of SV currents at +100 mV by vacuolar Mg²⁺ is fitted to a Hill equation:
where I is the degree of current inhibition, I_max is the maximum current inhibition, [Mg²⁺] is the Mg²⁺ concentration on the vacuolar side, n is the Hill coefficient, and K_i is the inhibition constant.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Fast-activating vacuolar currents inhibited by vacuolar Mg2+ in fava bean guard cells. A through C, Three representative whole-vacuole recordings are shown at Mg2+ concentrations of 0 mM (A), 0.5 mM (B), and 2 mM (C) in the pipette (luminal) solution. Membrane potential was stepped from 100 to +100 mV in 20-mV increments from a holding potential of 0 mV. In all traces, the vacuolar ion currents have been normalized to the whole-vacuolar capacitance (pA/pF). The solutions for FV current measurement contained 100 mM KCl, 4 mM EGTA, 10 mM HEPES-Tris, pH 7.5, in the bathing medium (cytosolic side), and 100 mM KCl, 5 mM CaCl2, 5 mM MES-Tris, pH 5.5, with varied MgCl2 of 0 to 2 mM in the pipette (vacuolar side). D, Average current-voltage relationships from experiments performed as in A through C at Mg2+ concentrations of 0, 0.1, 0.5, 1.0, and 2.0 mM in the pipette solution. FV currents were measured as the instantaneous component of whole-vacuole currents (n = 3-5 vacuoles per Mg2+ concentration; whole-vacuole capacitance = 9.7 ± 3.4 pF). Inset, Average percentage of inhibition of SV currents at +100 mV is plotted as a function of the concentrations of vacuolar Mg2+ and fitted to a Hill equation. E, Voltage dependence of vacuolar Mg2+ block. Average whole-vacuole currents in the presence of vacuolar Mg2+ as in D were normalized to currents in the absence of Mg2+ (Current0 mM). Symbols are as in D. F, Mg2+ inhibition constant (Ki) plotted as a function of the applied membrane potentials. Inhibition constants at +40 to +100 mV were obtained using the Hill equation (see "Materials and Methods").

Experimental Solutions

The standard solutions used in patch-clamp experiments were composed of 200 mM KCl and 20 mM HEPES-Tris, pH 8.0, in the bathing medium (cytosolic side), and 20 mM KCl, 2 mM EGTA, and 5 mM HEPES-Tris, pH 7.0, in the pipette (vacuolar side) unless otherwise noted. Free cytosolic Ca²⁺ concentrations ranging from 10 nM to 1 µM were buffered with EGTA. Total CaCl₂ concentrations in bath solutions (Fig. 5) were changed to give the indicated cytosolic free Ca²⁺ of 10 nM (0.8 mM total CaCl₂ concentration), 50 nM (2 mM), 150 nM (3 mM), and 1 µM (3.8 mM), pH 7.8, with 4 mM EGTA in all solutions. Free Ca²⁺ concentrations were calculated after accounting for 10 mM MgCl₂, ionic strength, and temperature (24°C) with CALCV22 software (Foehr et al., 1993). For 10 and 50 µM Ca²⁺ in Figure 5, 10 and 50 µM CaCl₂ were added to the bath solution without the addition of the Ca²⁺ buffer EGTA, as these concentrations lie outside the range of effective EGTA-buffering capacity. The bath solution was exchanged either by bath perfusion using a peristaltic pump (Rainin, Woburn, MA) or by a local perfusion pipette. Osmolalities of all solutions were adjusted to 600 mmol kg¹ by the addition of D-sorbitol.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION LITERATURE CITED

Inhibition of Vacuolar FV Channels by Luminal Mg²⁺

At zero cytosolic Ca²⁺, vacuolar currents were almost entirely instantaneous and were larger at positive potentials (on the cytoplasmic side of the membrane) compared with negative potentials (Fig. 1A). The activation of previously described Ca²⁺-activated VK channels (Ward and Schroeder, 1994) was avoided by buffering cytosolic Ca²⁺ to nominally zero. The steady-state current-voltage characteristics were similar to FV channel-mediated currents previously described in beet root vacuoles (Hedrich and Neher, 1987), barley mesophyll vacuoles (Tikhonova et al., 1997), and fava bean guard cell vacuoles (Allen and Sanders, 1996; Allen et al., 1998). The instantaneous FV currents were carried by monovalent cations including K⁺ and Cs⁺ (data not shown) as shown for FV currents (Allen and Sanders, 1996; Tikhonova et al., 1997).

Whole-vacuolar currents were analyzed at 0 to 2 mM vacuolar Mg²⁺ concentrations (Fig. 1). FV current amplitudes were reduced by increasing the vacuolar Mg²⁺ concentration from 0 (Fig. 1A) to 0.5 mM (Fig. 1B) or 2 mM (Fig. 1C). FV currents measured at five different vacuolar Mg²⁺ concentrations confirmed the strong down-regulation of FV currents by vacuolar Mg²⁺ (Fig. 1D). The average effect of vacuolar Mg²⁺ shows a 14.3- ± 2.1-fold decrease of FV currents at +100 mV by increasing vacuolar Mg²⁺ from 0 to 2 mM (Fig. 1D). FV currents at negative potentials were also reduced (2.4- ± 0.4-fold). A Hill curve could be fitted to the currents at +100 mV showing a K_i of approximately 0.23 mM and a Hill coefficient of 0.67 (Fig. 1D, inset), indicating that FV current amplitudes are inhibited by vacuolar Mg²⁺ within the physiological range (Yazaki et al., 1988). The Hill coefficient of 0.67 is consistent with one Mg²⁺ binding site per FV channel.

Whole-vacuole currents measured at different vacuolar Mg²⁺ concentrations were normalized to the control currents measured in the absence of Mg²⁺, and plotted as a function of applied voltage (Fig. 1E). Voltage-dependent block was observed at positive membrane potentials, with a continuous decrease in current by decreasing the voltage from +100 to +40 mV. Furthermore, the apparent K_i at different membrane potentials also shows the voltage dependence of Mg²⁺ block (Fig. 1F).

Inhibition of Vacuolar FV Channels by Cytosolic Mg²⁺

Experiments were designed to analyze whether, in addition to vacuolar Mg²⁺ (Fig. 1), cytosolic Mg²⁺ affects FV currents. A local perfusion system was used that allowed multiple changes of cytosolic solutions bathing single vacuoles. In the whole-vacuole configuration with zero Mg²⁺ on the cytosolic side, large instantaneous currents were recorded (Fig. 2A). When Mg²⁺ (1 mM) was applied to the cytosolic side, FV currents were decreased dramatically at both positive and negative vacuolar potentials (Fig. 2B). Activation of time-dependent SV currents in Figure 2B will be described later. Quantitative analysis showed a 3-fold inhibition of instantaneous currents by varying cytosolic Mg²⁺ from 0 to 1 mM at +100 mV (Fig. 2C). These results indicate that both luminal (Fig. 1) and cytosolic Mg²⁺ (Fig. 2) down-regulate FV currents at both positive and negative vacuolar potentials. As predicted, elimination of Mg²⁺ and Ca²⁺ from both the luminal and cytosolic sides gave rise to large FV currents (Fig. 2D), further illustrating the inhibitory effects of Mg²⁺ and Ca²⁺.

View larger version (25K): [in this window] [in a new window]   Figure 2.   Cytosolic Mg2+ inhibits FV current. A and B, Whole-vacuole currents recorded in the absence (0 Mgcyt; A) or presence of Mg2+ (1 mM Mgcyt; B) in bath (cytosolic) solutions of the same vacuole. The holding potential was 40 mV. The pipette solution contained 20 mM KCl, 2 mM EGTA, and 5 mM HEPES-Tris, and the bath solution contained 100 mM KCl, 20 mM HEPES-Tris, pH 8.0, with the addition of 10 µM CaCl2, in the absence or presence of 1 mM MgCl2. Whole-vacuole capacitance = 8.4 pF. C, Average FV currents at +100 mV as recorded in A and B. Currents recorded in the absence of cytosolic Mg2+ were normalized as 1 (64.2 ± 4.5 pA/pF; n = 11 vacuoles for each condition; whole-vacuole capacitance = 8.3 ± 1.2 pF). r.u., Relative unit. D, Representative whole-vacuole FV currents recorded in the absence of Mg2+ and Ca2+ on both cytosolic and vacuolar membrane sides (n > 15 vacuoles). The solution contained 200 mM KCl, 5 mM HEPES-Tris, pH 7.0, in the pipette and 50 mM KCl, 2 mM EGTA, and 20 mM HEPES-Tris, pH 8.0, in the bath. No Mg2+ or Ca2+ was added to solutions. E, Current-voltage relationships as recorded in D. Currents from five representative recordings are averaged and plotted as a function of the applied membrane potentials.

Does Cytosolic Mg²⁺ Activate SV Currents?

In fava bean guard cells, at high cytosolic Ca²⁺ concentrations SV currents are the major vacuolar conductance. However, the cytosolic Ca²⁺ concentration required for SV current activation is larger than known resting cytosolic Ca²⁺ levels and the upper limit of free cytosolic Ca²⁺ concentrations measured during Ca²⁺-dependent signal transduction (Ward and Schroeder, 1994; Bush, 1995; Allen and Sanders, 1996; McAinsh et al., 1997). This has contributed to difficulties in predicting the physiological roles of SV channels. We therefore designed experiments to determine whether the Ca²⁺ sensitivity of SV activation could be modified. At 10 µM cytosolic Ca²⁺, instantaneous currents were recorded in the absence of cytosolic Mg²⁺ (Fig. 3A).

View larger version (28K): [in this window] [in a new window]   Figure 3.   Possible up-regulation of slowly activating vacuolar currents by cytosolic Mg2+ in fava bean guard cells. Whole-vacuole currents measured from one vacuole at different Mg2+ concentrations in the bath solution. The holding potential was 40 mV with an interval time between pulses of 1 s. Standard bath and pipette solutions (see "Materials and Methods") were used with varying Mg2+ concentrations (0, 1, and 5 mM) in the bath. Note that 10 µM CaCl was added to the bath solution. A, Current recordings started in a bath solution containing no added Mg2+. B and C, Bath solutions containing 1 mM (B) and 5 mM Mg2+ (C) were subsequently added by local perfusion (see "Materials and Methods"). D and E, The vacuole was then perfused with a bath solution containing no added Mg2+. Similar experiments were repeated on eight vacuoles.

Cytosolic Mg²⁺ of 1 mM was applied by local perfusion in the continued presence of 10 µM Ca²⁺. Interestingly, time-dependent SV currents were increased dramatically at positive vacuolar potentials (Fig. 3B). The Mg²⁺ concentration was then further increased to 5 mM. SV currents were similar in magnitude to those at 1 mM cytosolic Mg²⁺ (Fig. 3C). Finally cytosolic Mg²⁺ was removed by slow bath perfusion, during which the time-dependent SV currents vanished (Fig. 3, D and E), while instantaneous currents increased (Fig. 3, D and E), also confirming the inhibitory effect of cytosolic Mg²⁺ on FV currents described in Figure 2. These data suggest that Mg²⁺ might up-regulate SV current as previously proposed (Allen and Sanders, 1996). However, in a recent study, no Mg²⁺ activation of SV currents was found in barley mesophyll vacuoles, and Mg²⁺ activation of SV channels described previously (Allen and Sanders, 1996) were concluded to be an artifact (Pottosin et al., 1997). To further examine these differences among previous reports, we investigated whether Mg²⁺ activation of SV currents depends on the presence of physiological levels of cytosolic Ca²⁺.

Mg²⁺ Sensitizes SV Currents to Cytosolic Ca²⁺

To test whether cytosolic Ca²⁺ is necessary for activation of SV currents by Mg²⁺ in fava bean guard cells, whole-vacuolar currents were measured at 10 mM cytosolic Mg²⁺ in the absence or presence of the Ca²⁺ buffer EGTA (4 mM) in the bath solution (Fig. 4). Small time-dependent SV currents were observed in the absence of EGTA (Fig. 4A). However, in the presence of EGTA, SV currents were reduced (Fig. 4B). Figure 4C () shows the dramatic reduction in time-dependent SV currents at positive potentials, when EGTA was added to the cytosolic side. These data (Figs. 3 and 4) indicate the possibility that cytosolic Mg²⁺ might modify the sensitivity of SV channels to cytosolic Ca²⁺.

View larger version (31K): [in this window] [in a new window]   Figure 4.   Cytosolic EGTA inhibits Mg2+ activation of SV currents. A and B, Whole-vacuole currents recorded in the absence (A) or presence (B) of 4 mM EGTA in the bath solutions in one vacuole. Standard pipette and bath solutions were used without or with the addition of 4 mM EGTA. C, Current-voltage relationships from experiments performed in the absence or presence of 4 mM EGTA as in A and B. SV currents were measured as time-dependent components of whole-vacuole currents. Symbols are as given in A and B (n = 8; whole-vacuole capacitance = 7.1 ± 2.6 pF).

To analyze quantitatively whether Mg²⁺ could shift the sensitivity of SV activation to physiological cytosolic Ca²⁺ concentrations and to determine cytosolic Ca²⁺ concentrations required for Mg²⁺ activation of SV currents, SV currents were measured over a range of cytosolic Ca²⁺ concentrations from 10 nM to 50 µM with a constant cytosolic Mg²⁺ concentration of 10 mM (Fig. 5). At 10 nM Ca²⁺, SV currents were not activated (Fig. 5A). Strikingly, when the Ca²⁺ concentration was subsequently increased to 50 nM, 150 nM, and up to 1 µM, SV currents measured in the same vacuole were gradually activated (Fig. 5). At 10 and 50 µM Ca²⁺, SV currents were close to saturation (Fig. 5). In contrast, in the absence of Mg²⁺ in the bath solution, physiological concentrations of Ca²⁺ could not activate SV currents (Fig. 5B, ). A Hill curve could be fitted to the data for cytosolic Ca²⁺ concentrations from 10 nM to 1 µM, showing a K_d of approximately 227 nM for a Hill coefficient of 0.95 (Fig. 5B, inset). The Hill coefficient of approximately 1 indicates binding of one Ca²⁺ ion per SV channel. These data demonstrate that physiological concentrations of Ca²⁺ can activate SV currents, if Mg²⁺ is also present on the cytosolic side, showing a sensitization of the SV channel to Ca²⁺ by cytosolic Mg²⁺.

View larger version (23K): [in this window] [in a new window]   Figure 5.   Cytosolic Mg2+ sensitizes SV channels to cytosolic Ca2+. A, Representative whole-vacuole currents recorded at different cytosolic Ca2+ concentrations in two separate vacuoles. In one vacuole, cytosolic Ca2+ concentrations were changed from 10 nM to 1 µM by either local or bath perfusion (vacuolar capacitance = 3.5 pF). In another vacuole, 10 µM Ca2+ in the bath solution was replaced by 50 µM Ca2+ (vacuolar capacitance = 4.2 pF). Only current traces at +100 mV are shown. Dashed lines show zero current levels. Pipette solution contained 20 mM KCl, 2 mM EGTA, and 5 mM HEPES-Tris, pH 7. Bath solution contained 200 mM KCl, 10 mM MgCl2, and 20 mM HEPES-Tris, pH 8.0, with varying free Ca2+ concentrations of 0, 10 nM, 50 nM, 150 nM, 1 µM, 10 µM, and 50 µM (see "Materials and Methods" for details). B, Effect of cytosolic Mg2+ on cytosolic Ca2+ activation of SV currents at +100 mV as performed in A. In control experiments, SV currents were recorded at 0 mM Mg2+ in bath solutions (). Values are from three to eight vacuoles (capacitance = 4.7 ± 1.2 pF). A Hill curve is fitted to the data for the SV currents activated by Ca2+ at 10 mM cytosolic Mg2+. Data obtained at 10 nM to 1 µM cytosolic Ca2+ are shown in the inset (Kd approximately 227 nM).

Differential Activation Time Course of SV Currents by Saturating Cytosolic Ca²⁺ or Mg²⁺

The data presented above suggested two ways to activate SV currents: first by high concentrations of cytosolic Ca²⁺ alone and second by combining cytosolic Mg²⁺ with low physiological concentrations of Ca²⁺. To test whether these two putative mechanisms of SV channel activation were kinetically distinguishable, experiments were designed using saturating Ca²⁺ (10 mM) in the absence of Mg²⁺; or using saturating Mg²⁺ (10 mM) in the presence of 10 µM cytosolic Ca²⁺. Under these two conditions, activation time courses for SV currents were different (Fig. 6, A and B). The time constants for SV current activation by Mg²⁺ in the presence of 10 µM Ca²⁺ were approximately three times more rapid than by Ca²⁺ alone (Fig. 6C), further supporting the hypothesis that there are two distinct mechanisms for the activation of SV channels (see "Discussion").

View larger version (34K): [in this window] [in a new window]   Figure 6.   Effect of cytosolic Mg2+ on the activation time course of SV currents. A and B, SV currents recorded at saturated cytosolic 10 mM Ca2+ (A) are compared with SV currents at saturated cytosolic 10 mM Mg2+ (B). For Mg2+ activation of SV currents, 10 µM CaCl2 was added to the bath solution to saturate the proposed high-affinity Ca2+ binding site (see "Discussion"). Standard pipette and bath solutions (see "Materials and Methods") were used with the addition of 10 mM CaCl2 in A and with the addition of 10 mM MgCl2 in B. C, Fitted time constants of the activation of SV currents plotted against the applied vacuolar membrane potentials (n = 3 vacuoles for each condition). Symbols are as in A and B.

Varying the luminal Mg²⁺ concentration had no effect on SV currents in fava bean guard cells (n = 6; data not shown), which was also demonstrated in barley mesophyll vacuoles (Pottosin et al., 1997), suggesting that SV channels are not regulated by luminal Mg²⁺.

Shifting SV Activation to Physiological Potentials and Modification of Outward-Rectifying Properties

In previous studies, steady-state SV currents have only been activated at positive vacuolar potentials (Ward and Schroeder, 1994; Allen and Sanders, 1996; Barkla and Pantoja, 1996; Bethke and Jones, 1997; Pottosin et al., 1997), whereas at physiological vacuolar potentials (from 0 to 40 mV; Sze et al., 1992) SV currents are vanishingly small. Experiments were designed to test whether the activation potential of SV currents could be significantly shifted to negative vacuolar potentials within the physiological range. To maximize SV channel activation, we designed a pipette solution containing 20 mM KCl and 4 mM EGTA (to eliminate the inhibitory effect of Ca²⁺ from the luminal side on SV channels (Allen and Sanders, 1996; Pottosin et al., 1997), and a bath solution containing 200 mM KCl, 10 mM CaCl₂, and 2 mM MgCl₂. Under these conditions the activation potential was shifted to potentials of about 60 to 40 mV (Fig. 7, A and B). In some vacuoles (two out of nine), the activation potential was shifted dramatically to 90 mV. Both inward and outward currents were recorded, and the time-dependent activation of SV channels was not altered (Fig. 7, C and D). A similar modification of the rectification property of the SV current has also been found in barley mesophyll cells (Pottosin et al., 1997). These results suggest that SV currents can activate at physiological vacuolar potentials under specific ionic conditions, and that SV channels may carry both inward and outward currents in vivo depending on conditions. We used extreme experimental conditions to show that the activation potential of SV channels could be strongly shifted (Fig. 7). The variation in activation potential (Fig. 7) suggests that additional unknown factors exist that can greatly shift the activation potential of SV channels.

View larger version (40K): [in this window] [in a new window]   Figure 7.   Shifting SV activation to physiological vacuolar potentials and changing rectification property. A and B, Whole-vacuolar SV currents (A) and current-voltage relationship (B). The pipette solution contained 20 mM KCl, 2 mM EGTA, and 5 mM HEPES-Tris, pH 7.0. The bath contained 200 mM KCl, 10 mM CaCl2, 2 mM MgCl2, and 20 HEPES-Tris, pH 8.0. Similar currents were recorded in seven of nine vacuoles. C, Under the same conditions as in A, time-dependent inward SV currents were recorded occasionally (n = 2 of 9 vacuoles) at negative membrane potentials (C). D, Current-voltage relationship of vacuoles showing bi-directional SV currents. This behavior was observed in two of nine vacuoles recorded under these conditions. As symbolized in C,  and  show the time-dependent peak and steady-state amplitudes of SV currents, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION LITERATURE CITED

In animal cells, Mg²⁺ blocks many cation channels, which includes Ca²⁺ channels, various inward-rectifier K⁺ channels, N-methyl-D-Asp receptor channels, ryanodine receptor-Ca²⁺ release channels, and Ca²⁺ release-activated Ca²⁺ channels (Nowak et al., 1984; Matsuda et al., 1987; Vandenberg, 1987; Agus and Morad, 1991; Hille, 1992; Laver et al., 1997; Kerschbaum and Cahalan, 1999). However, in plant cells, regulation of ion channels by Mg²⁺ has not yet been studied in detail. In guard cells, inward-rectifying K⁺ currents are not blocked by cytosolic Mg²⁺ (Schroeder, 1995), and the only ionic current shown to be activated by Mg²⁺ is a cation current in beet vacuoles, which has been proposed to be a shunt conductance for the vacuolar H⁺-ATPase (Davies and Sanders, 1995). In this study, we show that in fava bean guard cells, Mg²⁺ strongly regulates two major vacuolar currents: down-regulating the vacuolar FV currents from both the cytosolic and luminal sides (Figs. 1 and 2) and up-regulating vacuolar SV currents from the cytosolic side (Figs. 4 and 6). The regulation of vacuolar ion channels by Mg²⁺ may play an important role in guard cells, as ion transport processes across the vacuolar membrane are essential for stomatal movements (MacRobbie, 1981, 1998; Assmann, 1993; Ward et al., 1995; Allen and Sanders, 1997).

Inhibition of Guard Cell Vacuolar FV Currents by Both Cytosolic and Luminal Mg²⁺

Systematic studies of the effect of Ca²⁺ on FV channels in fava bean guard cell and barley mesophyll vacuoles have shown that cytosolic and vacuolar Ca²⁺ inhibits FV channels (Allen and Sanders, 1996; Tikhonova et al., 1997). Higher concentrations of Mg²⁺ are required to inhibit FV currents (Figs. 1 and 2). For half inhibition from the luminal side, Mg²⁺ concentrations of approximately 230 µM were required (Fig. 1). In a previous study of VK currents, 2 mM Mg²⁺ was used to exclude FV currents (Ward and Schroeder, 1994), while Mg²⁺-free conditions result in large FV currents (Allen and Sanders, 1996). These results suggest a divalent ion-binding/block site on the luminal side of FV channels.

In animal cells, both Mg²⁺ and spermine block inward-rectifier K⁺ channels and cause voltage-dependent inward rectification (Matsuda et al., 1987; Vandenberg, 1987; Hille, 1992; Fakler et al., 1995). In the case of NMDA receptors in neurons, Mg²⁺ and spermine share a regulatory site (Paoletti et al., 1995). Similarly, FV channels are inhibited by both Mg²⁺, as shown here, and spermine in barley vacuoles (Figs. 1 and 2; Brüggemann et al., 1998; Dobrovinskaya, et al., 1999). The inhibition of FV channels by vacuolar Mg²⁺ is voltage dependent (Fig. 1), whereas spermine inhibition is voltage independent (Brüggemann et al., 1998; Dobrovinskaya, et al., 1999), suggesting that Mg²⁺ and spermine may not share the same binding site or that the inhibitory mechanisms are different. Whether the inhibitory effects of Mg²⁺ and spermine are additive in FV channel regulation or if Mg²⁺ and spermine share an inhibitory site will require further investigation.

Mg²⁺ Sensitizes SV Channels to Physiological Cytosolic Ca²⁺ Levels and a Model for SV Activation with Two Binding Sites

SV currents in many plant cell types are activated at cytosolic Ca²⁺ concentrations (for example 100 µM), which are >100-fold higher than known resting levels (Ward and Schroeder, 1994; Barkla and Pantoja, 1996; Allen and Sanders, 1996, 1997). The high cytosolic Ca²⁺ levels required for SV channel activation have contributed to the difficulty in assigning a physiological function to the channels. Information on mechanisms that modify the Ca²⁺ sensitivity of SV channel activation could further our understanding of SV function in guard cells. Mg²⁺ activation of SV channels has been proposed in fava bean guard cells (Allen and Sanders, 1996). However, Pottosin et al. (1997) reported that the SV channel activation in barley mesophyll vacuoles is due to Ca²⁺ contamination of the cytosolic bath solution, and that Mg²⁺ does not activate SV channels.

To clarify these controversial conclusions, our results show that in the presence of EGTA, Mg²⁺ does not activate SV currents in fava bean guard cells (Fig. 3, B and C), indicating that Mg²⁺ activation in the previous study can be explained by residual free Ca²⁺, because no Ca²⁺ chelators were added to the cytosolic membrane side (Allen and Sanders, 1996). The conclusion that cytosolic Mg²⁺ does not modulate SV channels was based on experiments with 1 to 2 mM EGTA and no Ca²⁺ added to the cytosolic solutions (Pottosin et al., 1997). Interestingly, however, in our experiments within the range of cytosolic Ca²⁺ concentrations at which SV currents were not normally activated, the addition of Mg²⁺ led to SV current activation (Fig. 4), indicating a synergistic effect between Mg²⁺ and Ca²⁺. Our data show that Mg²⁺ sensitizes SV channels to physiological levels of cytosolic Ca²⁺. Ba²⁺ did not activate SV channels in fava bean guard cell vacuoles (Schulz-Lessdorf and Hedrich, 1995), but did activate SV channels in beet vacuoles (Pantoja et al., 1992).

Based on our results, a simplified model for SV channel regulation in fava bean guard cells is proposed (Fig. 8), which includes two activating cytosolic sites and one inhibitory luminal site. First, low concentrations of cytosolic Ca²⁺ cannot activate SV channels in the absence of cytosolic Mg²⁺, whereas in the presence of cytosolic Mg²⁺, these low concentrations of Ca²⁺ (A₁; Fig. 8) are necessary and sufficient to activate SV channels, implying a synergistic Mg²⁺-binding site (A₂; Fig. 8). Second, Mg²⁺ alone cannot activate SV channels, indicating that a high-affinity (K_d of approximately 227 nM) Ca²⁺-binding site (A₁) is required and is different from the Mg²⁺-binding site (A₂). Mg²⁺ cannot compete with Ca²⁺ for A₁ binding. Both A₁ and A₂ need to be occupied for SV channel activation. Third, a high concentration of cytosolic Ca²⁺ alone can activate SV channels (Ward and Schroeder, 1994; Allen and Sanders, 1996), suggesting (for a simple model) that cytosolic Ca²⁺ can bind to both sites A₁ and A₂. In addition, our results showed that the time course of SV current activation differed when using saturating Ca²⁺ (10 mM) to bind both sites (A₁ and A₂) in the absence of Mg²⁺ as opposed to using saturating Mg²⁺ (10 mM) binding to the low-affinity site (A₂) in the presence of 10 µM Ca²⁺ to bind to the high-affinity site (A₁) (Fig. 6), indicating additional ion-specific effects.

View larger version (24K): [in this window] [in a new window]   Figure 8.   Simplified model for the regulation of SV channels by cytosolic and luminal Ca2+ and Mg2+ in fava bean guard cells. A1, High-affinity Ca2+-binding site on the cytosolic side, which is not activated by Mg2+. A2, Low-affinity binding site on the cytosolic side, which can be occupied by either Mg2+ or Ca2+. B, Vacuolar Ca2+-binding site, which is not affected by vacuolar Mg2+. For the activation of SV channels, both activation sites A1 and A2 need to be occupied (see "Discussion"). The cytosolic and vacuolar membrane sides are labeled.

Finally, luminal Ca²⁺ inhibits SV currents by shifting the activation potential (Figs. 7B versus 4C; also see Allen and Sanders, 1996; Pottosin et al., 1997), indicating that Ca²⁺ ion binding on the luminal side (B; Fig. 8) is inhibitory and thus limits large Ca²⁺ release currents that could be toxic. Vacuolar Mg²⁺ did not affect SV (data not shown), indicating that Mg²⁺ does not compete for this inhibitory Ca²⁺-binding site (B).

Proposed Physiological Roles of Mg²⁺ Regulation of Vacuolar Currents

The vacuole constitutes 90% of the guard cell volume and functions as a storage organelle for solutes that are important for osmoregulation during stomatal movements (Boller and Wiemken, 1986; Assmann, 1993). More than 90% of the K⁺ and anions released from guard cells during stomatal closing must first be released from vacuoles into the cytosol (MacRobbie, 1981). Studies show that FV channels can mediate both inward and outward currents with large amplitudes (Fig. 2D), which would lead to vacuolar K⁺ release when V-type ATPases are active. Cytosolic Ca²⁺ and polyamines down-regulate FV channels (Hedrich and Neher, 1987; Allen and Sanders, 1996; Tikhonova et al., 1997). The half-inhibitory concentration of cytosolic Ca²⁺ for FV currents is about 6 µM (Tikhonova et al., 1997), which is higher than known physiological levels of free Ca²⁺ (Bush, 1995; McAinsh and Hetherington, 1998). In contrast to Ca²⁺, the half-inhibitory concentration of cytosolic Mg²⁺ was about 230 µM (Fig. 1), which lies within the physiological range of free Mg²⁺ concentrations (400 µM; Yazaki et al., 1988), suggesting that Mg²⁺ might play a major role in the down-regulation of FV channels in vivo during stomatal opening or cell expansion.

A recent study concluded that due to vacuolar Ca²⁺ block (Fig. 8B), SV channels cannot mediate Ca²⁺-induced Ca²⁺ release (Pottosin et al., 1997). However, this model represents a negative hypothesis based on a lack of observation, which, given the complexity of biological systems, may be oversimplified (for review, see Alberts, 1998). This hypothesis (Pottosin et al., 1997) did not consider shifts in the Ca²⁺ sensitivity of SV channels to cytosolic Mg²⁺ (Fig. 7), the effects of the K⁺ gradient across the vacuolar membrane (Fig. 7), nor effects of malate gradients proposed to shift SV activation (Hedrich et al., 1986). Cellular regulation mechanisms of the SV channel, such as calmodulin (Bethke and Jones, 1994), redox agents (Carpaneto et al., 1999), and protein phosphorylation (Allen and Sanders, 1995; Bethke and Jones, 1997) might also shift the voltage dependence of SV channels, and were not considered (Pottosin et al., 1997).

In addition, deactivating time-dependent (tail) SV currents have been shown to unequivocally mediate Ca²⁺ efflux from vacuoles (Ward and Schroeder, 1994; Ward et al., 1995). Therefore, the conclusion that SV channels cannot mediate Ca²⁺ release (Pottosin et al., 1997) is not consistent with these direct recordings. Transient stimulation of second-messenger (cADPR and InP₃)-activated Ca²⁺ selective channels in the vacuolar membrane (Allen et al., 1995; Leckie et al., 1998; Cancela et al., 1999) will polarize the vacuolar potential to positive voltages, which in turn activates SV channels. Subsequently, deactivation of second-messenger-activated Ca²⁺-selective channels (Allen et al., 1995; Leckie et al., 1998) could produce Ca²⁺-induced Ca²⁺ release via tail currents. Rapid vacuolar membrane repolarization could also be mediated by the combination of activated VK channels and proton pumps and/or anion efflux from vacuoles (Ward et al., 1995). In addition, data in Figure 7 show yet to be identified conditions that shift the voltage dependence of SV channels. Further research on mechanisms that could shift the voltage dependence of SV channels, such as cytosolic Mg²⁺ (Fig. 7), calmodulin, redox agents (Bethke and Jones, 1994; Carpaneto et al., 1999), and other regulators, may lead to the identification of additional mechanisms that allow Ca²⁺-induced Ca²⁺ release. Taken together, our data show that Mg²⁺ can play an important role in the regulation of vacuolar ion channels. These findings raise an additional question of whether cytosolic Mg²⁺ activities change during stomatal movements.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION LITERATURE CITED

The effects of both cytosolic and vacuolar Mg²⁺ on SV and FV channels have been systematically investigated. The present study shows that even if cytosolic Mg²⁺ concentrations do not change, physiological levels of Mg²⁺ ions provide a major mechanism for sensitizing SV channels to stimulus-induced elevations in cytosolic Ca²⁺ during signal transduction. Furthermore, both vacuolar and cytosolic Mg²⁺ ensure that FV channels do not function as a continuous leak for K⁺ ions, which would prevent stomatal opening.