Plant Physiol. (1998) 118: 173-181

Control of Cl Efflux in Chara corallina by Cytosolic pH, Free Ca²⁺, and Phosphorylation Indicates a Role of Plasma Membrane Anion Channels in Cytosolic pH Regulation¹

Eva Johannes^{2, *}, Alan Crofts, and Dale Sanders

The Plant Laboratory, Biology Department, University of York, P.O. Box 373, York YO1 5YW, United Kingdom

	ABSTRACT

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Enhanced Cl efflux during acidosis in plants is thought to play a role in cytosolic pH (pH_c) homeostasis by short-circuiting the current produced by the electrogenic H⁺ pump, thereby facilitating enhanced H⁺ efflux from the cytosol. Using an intracellular perfusion technique, which enables experimental control of medium composition at the cytosolic surface of the plasma membrane of charophyte algae (Chara corallina), we show that lowered pH_c activates Cl efflux via two mechanisms. The first is a direct effect of pH_c on Cl efflux; the second mechanism comprises a pH_c-induced increase in affinity for cytosolic free Ca²⁺ ([Ca²⁺]_c), which also activates Cl efflux. Cl efflux was controlled by phosphorylation/dephosphorylation events, which override the responses to both pH_c and [Ca²⁺]_c. Whereas phosphorylation (perfusion with the catalytic subunit of protein kinase A in the presence of ATP) resulted in a complete inhibition of Cl efflux, dephosphorylation (perfusion with alkaline phosphatase) arrested Cl efflux at 60% of the maximal level in a manner that was both pH_c and [Ca²⁺]_c independent. These findings imply that plasma membrane anion channels play a central role in pH_c regulation in plants, in addition to their established roles in turgor/volume regulation and signal transduction.

	INTRODUCTION

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Plasma membrane anion channels play fundamental roles in several areas of plant cell biology (Tyerman, 1992; Hedrich, 1994; Schroeder, 1995). Salt loss from guard cells during stomatal closure and from euryhaline algae responding to hypoosmotic stress is effected principally by the opening of anion channels (Okazaki and Tazawa, 1990; Okazaki and Iwasaki, 1992; Schroeder et al., 1993; Schwartz et al., 1995). Thus, because the equilibrium potential of permeant anions is positive of 0 mV, the opening of anion channels depolarizes the membrane and brings the membrane potential into a range in which outward-rectifying K⁺ channels open to provide accompanying cation loss.

A second important function of anion channels is likely to relate to the early stages of signal transduction. A wide range of stimuli, including fungal elicitors (Nürnberger et al., 1994), red light (Ermolayeva et al., 1997), blue light (Cho and Spalding, 1996), and Nod factors (Ehrhardt et al., 1992), evoke a rapid depolarization of the plasma membrane, which, based on the measurements of fluxes, ionic currents, and inhibitor sensitivity, indicates the opening of anion channels. Ward et al. (1995) have proposed that one function of these anion-channel-evoked depolarizations might be to open voltage-dependent Ca²⁺ channels, which, by allowing entry of external Ca²⁺, would elevate [Ca²⁺]_c. As befits these important roles in cell biology, the activities of anion channels are tightly regulated by voltage (Keller et al., 1989; Okihara et al., 1991; Schroeder and Keller, 1992), [Ca²⁺]_c (Shiina and Tazawa 1987, 1988; Schroeder and Hagiwara, 1989; Hedrich et al., 1990; Okihara et al., 1991), nucleotides (Hedrich et al., 1990; Schulz-Lessdorf et al., 1996; Thomine et al., 1997), and phosphorylation/dephosphorylation cascades (Zimmermann et al., 1994; Schmidt et al., 1995; Pei et al., 1997).

A further function for anion channels in the control of pH_c is hinted at by the observation that cytosolic acidification with weak acids leads to an enhancement of cellular Cl efflux in charophyte algae (Sanders, 1980a; Smith and Reid, 1991) and higher plants (Beffagna et al., 1995). Smith and Reid (1991) proposed that the activation of anion channels in these conditions would provide a shunt pathway for charge compensation of H⁺ moved electrogenically out of the cell by the activated H⁺-ATPase. The failure to provide such a shunt pathway would severely compromise the ability of the pump to effect control of pH_c because activation of the pump by cytosolic H⁺ would be countered by membrane hyperpolarization (Blatt et al., 1990). The membrane depolarizations and conductance increases reported for fungi and lower plants in conditions of cytosolic acidification might also have their origins in the opening of anion channels, with similar implications for pH_c regulation (Sanders et al., 1981; Frachisse et al., 1988; Roberts et al., 1997).

A pivotal question relating to the role of anion channels in responding to pH_c concerns the mechanism of action of cytosolic H⁺. Does H⁺ directly activate anion efflux, or are the effects mediated by one of the other well-established regulators of anion channels? In the present study we have addressed this question using internodal cells of charophyte algae. These large and robust cells can be internally perfused, thereby permitting experimental control of medium composition at the inner surface of the plasma membrane in a manner analogous to the whole-cell patch-clamp technique. The results reveal not only that cytosolic H⁺ activates anion efflux directly, but also that indirect effects can prevail, which are mediated through a pH-induced decrease in K_Ca. Furthermore, a profound influence of phosphorylation state on anion efflux has been discovered that might underlie the refractory period that follows electrical excitation of these cells.

	MATERIALS AND METHODS

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Plant Material

Media

1

[H⁺][EGTA]/[H·EGTA] = 3.39 × 10¹⁰ M; [H⁺]²[EGTA]/[H₂·EGTA] = 4.57 × 10¹⁹ M²; [H⁺]³[EGTA]/[H₃·EGTA] = 1.05 × 10²¹ M³; [H⁺]⁴[EGTA]/[H₄·EGTA] = 1.05 × 10²³ M⁴; [Mg²⁺][EGTA]/[Mg·EGTA] = 6.18 × 10⁶ M; [Mg²⁺][H⁺] [EGTA]/[Mg·H·EGTA] = 1.20 × 10¹³ M²; [Ca²⁺][EGTA]/[Ca·EGTA] = 1.07 × 10¹¹ M; [Ca²⁺][H⁺][EGTA]/[Ca·H·EGTA] = 1.74 × 10¹⁵ M²; [H⁺][HEDTA]/[H·HEDTA] = 3.39 × 10¹⁰ M; [H⁺]²[HEDTA]/[H₂·HEDTA] = 4.57 × 10¹⁹ M²; [H⁺]³[HEDTA]/[H₃·HEDTA] = 1.05 × 10²¹ M³; [H⁺]⁴[HEDTA]/[H₄·HEDTA] = 1.05 × 10²³ M⁴; [Mg²⁺][HEDTA]/[Mg·HEDTA] = 6.18 × 10⁶ M; [Mg²⁺][H⁺][HEDTA]/[Mg·H·HEDTA] = 1.20 × 10¹³ M²; [Ca²⁺][HEDTA]/[Ca·HEDTA] = 1.07 × 10¹¹ M; [Ca²⁺][H⁺][HEDTA]/[Ca·H·HEDTA] = 1.74 × 10¹⁵ M²; [H⁺][NTA]/[H·NTA] = 2.24 × 10¹⁰ M; [H⁺]²[NTA]/[H₂·NTA] = 7.41 × 10¹³ M²; [H⁺]³[NTA]/[H₃·NTA] = 1.175 × 10¹⁴ M³; [H⁺]⁴[NTA]/[H₄·NTA] = 1.86 × 10¹⁵ M⁴; [Mg²⁺][NTA]/[Mg·NTA] = 3.39 × 10⁶ M; [Ca²⁺][NTA]/[Ca·NTA] = 3.63 × 10⁷ M; [Ca²⁺][NTA]²/[Ca·NTA₂] = 1.55 × 10⁹ M².

For phosphorylation experiments 1 µM PKA catalytic subunit (isolated from bovine heart) and 3 mM BTP-ATP were added to the perfusion medium. PKA was reconstituted in deionized water containing 6 mg/mL DTT and was used the same day or the day after. BTP-ATP was prepared by adding 1 g of Dowex 50-X8 cation-exchange resin to 50 mL of a 30 mM solution of Na₂ATP. After stirring, filtration, and addition of BTP, the resulting solution was stored at 20°C. Alkaline phosphatase (from bovine intestinal mucosa, prepared in deionized water), ATP--S, and anion channel antagonists were added to the perfusion medium as indicated.

When tested externally, anion-channel antagonists were added to APW and introduced into the central chamber of the perfusion apparatus (see below) after the 2-min recovery period. The cell was then left for another 10 min before addition of fresh medium for monitoring efflux. The inhibitors IAA-94/95, ISA-94/95, NPPB, and NPPB-S were made up in 0.5% (v/v) DMSO, which on its own had no discernible effect. NPPB, NPPB-S, IAA-94/95, and ISA-94/95 were a kind gift of Dr. A. Pope (SmithKline Beecham Pharmaceuticals, Welwyn, Hertfordshire, UK). Na³⁶Cl was obtained from Amersham, and all other chemicals were obtained from Sigma.

Intracellular Perfusion and Removal of Vacuolar Membrane and Streaming Cytoplasm

1

Membrane Potential Measurements

	RESULTS

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To determine whether reverse action of the previously described Cl-H⁺ symporter (Sanders, 1980c) contributes to efflux of radiolabeled Cl, the effect of external Cl in the range 0 to 5 mM was tested. For [Ca²⁺]_c = 5 µM and pH_c = 7.8, negligible variation in Cl efflux was found (mean value of approximately 40 pmol cm² s¹) at all Cl concentrations tested (data not shown). The absence of self-exchange of Cl supports the conclusion that the Cl-H⁺ symporter is not effectively reversible (Sanders and Hansen, 1981). It can be argued, therefore, that the observed Cl efflux through the plasma membrane of C. corallina cells is mediated by anion channels.

To test the possibility that variation in the membrane potential might contribute to modulation of Cl efflux in the experimental conditions of the present study, membrane potential measurements were performed on perfused cells. At the extremes of both pH_c and [Ca²⁺]_c (i.e. at pH_c values of 6.8 and 7.8 and [Ca²⁺]_c values of 0.01 and 100 µM), variations in the membrane potential were not very pronounced. For example, at low [Ca²⁺]_c (0.01 µM), the membrane potential was not significantly different over the pH range tested (83 ± 5 mV at pH 6.8; 85 ± 10 mV at pH 7.8). At high [Ca²⁺]_c (100 µM), the membrane potential was generally about 20 mV more negative (e.g. 103 ± 8 mV at pH 7.8) but still fell within a range in which channel activity would be expected to be mainly unchanged, since Ca²⁺-dependent anion channels in C. corallina show maximum activity in the range between 80 and 100 mV (Okihara et al., 1991).

Direct Effect of pH_c

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View larger version (13K): [in this window] [in a new window]   Figure 1. Direct effect of pHc on Cl efflux. Cl efflux was measured in the absence of Ca2+ in the perfusion medium. Each point is the mean of three replicates. The fitted curve describes the relationship JCl = Jmax/(1 + 10(pH-pK)), where JCl is the Cl efflux, Jmax is the Cl efflux at the optimum pH (fitted as 132 pmol cm2 s1), and pK = 6.1 ± 0.5.

In intact cells, Cl efflux increases from <1 to 16 pmol cm² s¹ when pH is decreased from 7.5 to 6.8 by the uptake of butyric acid (Smith and Reid, 1991), compared with 4 to 24 pmol cm² s¹ in perfused cells over the same pH range. These values are in reasonable agreement given the known variability of different C. corallina cultures.

pH_c Modulates Activation by [Ca²⁺]_c

Figure 2A shows that at the resting pH of 7.8, Cl efflux increased as a function of [Ca²⁺]_c, as shown previously by Shiina and Tazawa (1988). It is noteworthy that a large portion (70%) of the Cl efflux was activated by an increase in [Ca²⁺]_c over the physiological range of 0.2 to 6.0 µM. As pH was progressively decreased from 7.8 to 7.0, two important things occurred: First, Cl efflux saturated to a common pH-independent value of about 53 pmol cm² s¹ at maximum permissive levels of free Ca²⁺ despite the fact that the flux in the absence of Ca²⁺ was pH sensitive (Fig. 1). Second, K_Ca declined as pH was decreased (Fig. 2B).

View larger version (21K): [in this window] [in a new window]   Figure 2. Modulation of Ca2+-dependent Cl efflux by pHc. A, Cl efflux as a function of [Ca2+]c over the physiological pH range. Each point is the mean of three replicates: SE values, omitted for clarity, were between 5% and 10%. Data were fitted by Equation 3 with S = 0.95,  = 55, KH = 7.94 × 107, and variable Kca values. B, Affinity of Ca2+ binding (KCa) as a function of pHc. Values were derived from the fits shown in A.

The data can be described by a function based on a model that assumes competition between H⁺ and Ca²⁺ for a regulatory binding site according to the scheme: where C and O represent the closed and open states of the channel, respectively, P_o denotes the open probability, and K_Ca and K_H are the affinities of the binding site for Ca²⁺ and H⁺, respectively. The Cl flux J_Cl can be described as:

(1)

with

(2)

Let  =  and S be a scaling factor (which includes P_o^H). Then

(3)

The data are well described by Equation 3 when pH dependence is incorporated into the K_Ca component, as shown in Figure 2B. At low [Ca²⁺]_c, channel opening is pH dependent, but this component becomes insignificant at high [Ca²⁺]_c, where Cl efflux saturates at a common value independent of pH_c. The affinity for Ca²⁺ binding is dramatically enhanced when cytosolic [H⁺] increases. The pH-dependent increase in Ca²⁺ affinity was strongest for small decreases in pH_c in the vicinity of the resting pH of 7.8 and became progressively less pronounced for stronger cytosolic acidification.

Effect of Phosphorylation/Dephosphorylation and Nucleotides

View larger version (12K): [in this window] [in a new window]   Figure 3. Inhibition of Cl efflux in phosphorylating conditions. A, pH dependence of Cl efflux in Ca2+-free medium. B, Ca2+ dependence of Cl efflux at pHc = 7.8. , Control; , plus 1 µM PKA catalytic subunit and 3 mM ATP. Each point is the mean ± SE of three independent replicates.

The effect of dephosphorylation was tested by inclusion of 30 nM alkaline phosphatase in the perfusion medium. When tested in the absence of Ca²⁺ in the pH range of 7.8 to 6.8, the phosphatase caused a pH-independent increase in Cl efflux to levels of 30 to 35 pmol cm² s¹ (Fig. 4A). Figure 4B shows that in dephosphorylating conditions Cl efflux is insensitive to [Ca²⁺]_c. In the presence of defined [Ca²⁺]_c (0.1-100 µM) tested at pH_c = 7.8, addition of the phosphatase to the perfusion medium reduced Cl efflux to the same level as in the absence of internal Ca²⁺, which was about 60% of the maximal level obtained in the control with optimal [Ca²⁺]_c. Thus, depending on the [Ca²⁺]_c level, dephosphorylating conditions can either stimulate ([Ca²⁺]_c < 2 µM) or inhibit ([Ca²⁺]_c > 2 µM) Cl efflux.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effect of dephosphorylating conditions on Cl efflux. A, pH dependence of Cl efflux in Ca2+-free medium. B, Ca2+ dependence of Cl efflux at pHc = 7.8. , Control; , plus 30 nM nonspecific alkaline phosphatase. Each point is the mean ± SE of three independent replicates.

Plant anion channels have repeatedly been shown to be subject to regulation by nucleotides (Hedrich et al., 1990; Schulz-Lessdorf et al., 1996; Thomine et al., 1997). This possibility was tested for the Cl efflux pathway in C. corallina using the nonhydrolyzable analog ATP--S, which was included in the perfusion medium in preference to ATP to eliminate possible inhibition by endogenous kinases. Figure 5A illustrates that in the absence of Ca²⁺, addition of ATP--S elevated Cl efflux by a constant value of 11.0 ± 0.6 pmol cm² s¹ over the whole pH range tested (7.8-6.8). The effect of ATP--S on Cl efflux in the presence of [Ca²⁺]_c over the range of 0.1 to 100 µM (at a constant pH_c of 7.8) is shown in Figure 5B. The presence of ATP--S enhanced the sensitivity of Cl efflux to [Ca²⁺]_c such that saturation of the flux, which occurred at a 1.5-fold higher level than in the control, was achieved at a [Ca²⁺]_c of 10 µM compared with 50 µM in the control.

View larger version (15K): [in this window] [in a new window]   Figure 5. Stimulation of Cl efflux by ATP--S. A, pH dependence of Cl efflux in Ca2+-free medium. B, Ca2+ dependence of Cl efflux at pHc = 7.8. , Control; , plus 3 mM ATP--S. Each point is the mean ± SE of three independent replicates.

Pharmacology

View this table: [in this window] [in a new window]   Table I. Effect of anion-channel blockers on Cl efflux Anion-channel antagonists were either added to the perfusion medium (Internal Inhibition) or to APW (External Inhibition). When applied externally, the inhibitors were present for 10 min. Three replicates were performed for each condition. Data are means ± SE.

	DISCUSSION

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Activation of Cl Efflux by Cytosolic Ca²⁺ and H⁺

In the present work the perfusion solutions were selected to render the membrane potential relatively insensitive to changes in pH_c and [Ca²⁺]_c by clamping it in the region of the K⁺ equilibrium potential (85 to 100 mV in these experiments; see Sanders, 1980c). This ensured that the effects of changes in ligand composition could be interpreted without the complication of associated changes in membrane potential, to which anion channels are known to be markedly sensitive (Keller et al., 1989; Okihara et al., 1991; Schroeder and Keller, 1992).

The present results demonstrate that Cl efflux from C. corallina is activated directly by cytosolic H⁺. Additionally, elevation in cytosolic H⁺ markedly decreased the Ca²⁺ activation constant for Cl release. In resting conditions at pH_c = 7.8 and [Ca²⁺]_c = 200 nM, Cl efflux is low. However, the Cl efflux pathway is poised such that as either [Ca²⁺]_c or [H⁺]_c is elevated, a marked increase in Cl efflux results. Physiologically, the H⁺-induced decrease in the Ca²⁺ activation constant is primarily responsible for this marked sensitivity to H⁺. Thus, the net effect is that the increase in cytosolic acidity sensitizes the Cl release channels to activation by Ca²⁺.

Physiological Role of Activation by H⁺

Physiological Role of Activation by Ca²⁺

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Ca²⁺-sensitive anion channels that open upon depolarization have been characterized in excised plasma membrane patches of C. corallina (Okihara et al., 1991). Whether these channels have a role in the Ca²⁺- and pH-dependent Cl efflux observed in the present report is difficult to judge, because the pH dependence and pharmacology have not been tested in the patch-clamp study. Simultaneous measurements of action potentials and patch currents (cell-attached mode) in the C. corallina plasma membrane recently revealed two additional types of Cl conductances that are closely associated with excitability (Homann and Thiel, 1994). These channels were active only during the action potential and could not be directly gated open either by depolarizing voltage changes or by elevation of [Ca²⁺]_c, nor could they be observed in excised patches (Thiel et al., 1993; Homann and Thiel, 1994). It has been argued, therefore, that these channels are mainly responsible for the rapid, massive Cl fluxes during the charophyte action potential. Whether the pH- and Ca²⁺-sensitive Cl efflux pathway under investigation in this study has a direct role in excitability is not clear, but the [Ca²⁺]_c changes that occur during the action potential are sufficient to activate Cl efflux through these channels.

Phosphorylation and the Refractory Period of the Charophyte Action Potential

Pharmacology of Cl Efflux

Anion channel antagonists such as A-9-C and ethacrynic acid, which are known to block Ca²⁺-activated Cl efflux and action potentials in charophytes (Shiina and Tazawa, 1987, 1988), inhibited Cl efflux in the present study by 70% and 40% when applied to the cytoplasmic side. The most potent inhibitors of the pH and Ca²⁺-sensitive Cl efflux in C. corallina, however, were the high-affinity ligands IAA-94/95 (90% inhibition, K_i = 53 nM) and NPPB-S (80% inhibition at 10 µM). IAA-94 and NPPB have also been reported to inhibit fast and slow anion channels in guard cells at micromolar concentrations (Marten et al., 1992; Schroeder et al., 1993), although in these cases an extracellular site of action seemed likely. So far, the effect of these high-affinity ligands on charophyte responses involving Cl channel activation, such as excitability and turgor regulation, as well as their effect on single-channel currents, remains to be elucidated.

	FOOTNOTES

   Received February 20, 1998; accepted May 25, 1998.

	ABBREVIATIONS

Abbreviations: A-9-C, anthracene-9-carboxylic acid. APW, artificial pond water. BTP, bis-tris propane. [Ca²⁺]_c, cytosolic free Ca²⁺ concentration. HEDTA, N-hydroxyethylethylenediaminetriacetic acid. IAA-94/95, [(2-cyclopentyl-6,7-dichloro-2-methyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxyl]acetic acid. ISA-94/95, [(2-cyclopentyl-6,7-dichloro-2-methyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]methanesulfonic acid . K_Ca, half-activation constant for Ca²⁺. K_H, half-activation constant for H⁺. NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid. NPPB-S, 5-nitro-2-(3-phenylpropylamino)benzene sulfonic acid. NTA, nitrilotriacetic acid. pH_c, cytosolic pH. PKA, protein kinase A.

	ACKNOWLEDGMENT

We are very grateful to Dr. A. Pope (SmithKline Beecham Pharmaceuticals) for the kind gift of NPPB, NPPB-S, IAA-94/95, and ISA-94/95.

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