Journal of Experimental Botany

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Journal of Experimental Botany, Vol. 54, No. 383, pp. 663-667, February 1, 2003
© 2003 Oxford University Press

Potassium-selective channel in the red beet vacuolar membrane

Received 13 June 2002; Accepted 3 October 2002

Igor I. Pottosin^1,, Manuel Martínez-Estévez^2,, Oxana R. Dobrovinskaya and Jesús Muñiz

Centro Universitario de Investigaciones Biomedicas, Universidad de Colima, 28047 Colima, Col., México

¹ To whom correspondence should be addressed. Fax: +52 312 31 27581. E-mail: pottosin{at}cgic.ucol.mx
² Present address: Centro de Investigación Científica de Yucatán, 97200 Mérida, Yucatán, México.

	Abstract

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In higher plants the vacuolar K⁺-selective (VK) channel was identified solely in guard cells. This patch-clamp study describes a 40 pS homologue of the VK channel in Beta vulgaris taproot vacuoles. This voltage-independent channel is activated by submicromolar Ca²⁺, and is ideally selective for K⁺ over Cl^– and Na⁺.

Key words: Beta vulgaris, patch-clamp, potassium channel, salt stress, vacuole.

	Introduction

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Two major currents in the tonoplast of higher plants are mediated by non-selective cation channels, the so-called SV channel (slowly-activating) and the FV channel (fast activating) (Hedrich and Neher, 1987; Tikhonova et al., 1997; Allen et al., 1998; Dobrovinskaya et al., 1999). The FV channel conducts various monovalent cations with poor selectivity amongst them (Brüggemann et al., 1999a), whereas the SV channel, besides alkali cation species, also conducts Ca²⁺ and Mg²⁺ (Amodeo et al., 1994; Ward and Schroeder, 1994; Pottosin et al., 2001). Neither of these two channels is able to discriminate well between K⁺ and Na⁺. The only vacuolar K⁺-selective channel, the so-called VK one, has been identified in the vacuolar membrane of guard cells (Ward and Schroeder, 1994; Allen et al., 1998). The VK channel is voltage-independent, is activated by cytosolic Ca²⁺ in the submicromolar range, and is practically impermeable for monovalent cations other than K⁺. To date there have been no reports of tonoplast K⁺-selective channels of other origin. However, vacuolar cation channels, that differ in their biophysical characteristics from SV and FV channels, have also been registered in red beet vacuoles and are the object of the present study (Hedrich and Neher, 1987; Gambale et al., 1996). Cytosolic potassium regulates various metabolic functions, for example, protein synthesis, and can not be substituted by Na⁺ which behaves as a competitive inhibitor. Under salt stress conditions, Na⁺ entering via cation channels in the plasma membrane needs to be extruded from the cytosol to maintain a high K⁺/Na⁺ ratio in this compartment. On the other hand, high external salt implies a hypertonic stress. To cope with this dual problem, halophyte plants accumulate Na⁺ in the vacuole, and Beta vulgaris in particular is able to concentrate Na⁺ against a 10-fold concentration gradient (Blumwald et al., 2000). To avoid the passive leak of Na⁺ from the vacuole, only strictly K⁺-selective cation channels may be allowed to function in the tonoplast under salt-stress conditions. This study presents direct evidence for the existence of such tonoplast K⁺-selective channels in the red beet taproot and provides a link between this VK channel and single channel activities in the vacuolar membrane reported elsewhere.

	Materials and methods

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Red beet (Beta vulgaris L.) vacuoles were isolated mechanically as described previously (Pottosin et al., 2001). Standard bath and pipette solutions contained 92.5 mM KCl and 15 mM HEPES-KOH (pH 7.5). In selectivity experiments K⁺ was replaced for Na⁺, or the K⁺ concentration in the bath was decreased to 10 mM (HEPES reduced to 10 mM in this case). Free cytosolic Ca²⁺ was either decreased to a nM level by the addition of 2 mM EGTA (the estimated Ca²⁺ contamination in the reagents is about 10 µM, free Ca²⁺ <2 nM), buffered in either a 0.1–25 µM or a 27–100 µM range using a triple chelator mixture, 2 mM each of EGTA, HEDTA and nitrilotriacetic acid (NTA), or just 2 mM NTA, respectively, with variable CaCl₂ additions, or adjusted to a 1–2 mM level without the use of chelating agents. In order to inhibit FV channels, dominating at low cytosolic Ca²⁺, Mg²⁺ at a free concentration of 1 mM (Brüggemann et al., 1999b) was introduced into the bath solution. Free Ca²⁺ and Mg²⁺ concentrations were calculated using Winmaxc v1.78 software by Chris Patton (Stanford University). Osmolalities of solutions were adjusted to 670–700 mOsm with sorbitol. The resistance of patch-pipettes filled with 100 mM K⁺ solution was 2–3 M. All reagents were of analytical grade (Sigma Chemical Co, St Louis, MO). Current measurements were performed using an Axopatch 200A Integrating Patch-Clamp amplifier and analyses were carried out using the pClamp 6.0 software package (Axon Instruments, Foster City, CA), as described in detail previously (Pottosin et al., 2001). Ion currents were recorded across large (C=0.2–1 pF) cytosolic-side–out patches. The sign of the voltage referred to the cytosolic side, and positive currents represent the flux of cations from the cytosol into the vacuole. Continuous current–voltage (I/V) relations were obtained by application of 15 ms ramps from –150 to 150 mV. Each trial consisted of 10 ramps, separated one from another by variable (20 ms to 1 s) intervals. To make separate recordings of currents through VK and SV channels present in the same patch, the holding voltage preceding a ramp was set to –60 to –100 mV or to 0–100 mV, respectively. At the first holding range SV channels were closed, and, due to their slow and delayed activation there were no SV channel openings during the ramp, even the patch might contain several tens of active SV channel copies. On the other hand, the voltage independent VK channel (only few copies in the patch) displayed bursts of openings separated by long-lasting shut periods (Figs 1B, 2). Thus, by chance, the VK channel could be trapped either in the closed or open configuration during the ramp (Fig. 3A, inset). At the second holding voltage range, SV channel activity was dominant; in every case the precise holding voltage level and the interval between ramps were adjusted to ensure the opening of few (0–3) SV channels during the ramp. Currents elicited by ramps with no open channels (leak currents) were subtracted from those with open channels, either VK or SV, plotted against voltage, adjusted for the occasional channel closures and averaged, so that resulting unitary I/V relations were presented as mean ±SD

View larger version (36K): [in this window] [in a new window]   Fig. 1. The 40 pS Beta vulgaris vacuolar channel requires sub micromolar cytosolic Ca2+ for its activation. (A) In the virtual absence (<2 nM) of Ca2+ only flickering currents through low-conductance FV channels could be detected, these currents were abolished by the application of 1 mM Mg2+ in the bath (cytosolic) solution. (B) In the presence of 1 mM Mg2+ free Ca2+ was varied at the cytosolic face of an isolated tonoplast patch. Symbols c and o indicate closed and open channel states, respectively. Inset shows a fragment of the open channel current–voltage relationship at 1 µM cytosolic Ca2+, used to evaluate single channel conductance. Symmetrical 100 mM K+, pH=7.5. Holding potential was –50 mV; traces were filtered at 0.7 kHz.

 

View larger version (32K): [in this window] [in a new window]   Fig. 2. Membrane voltage has little effect on the open probability of the 40 pS channel. An isolated tonoplast patch containing two channel copies and lacking the SV channel activity (free cytosolic Ca2+ was 27 µM) was analysed. Symmetrical 100 mM K+, pH=7.5. Amplitude histograms were constructed for 5 s stretches of single channel activity at –50 and +50 mV and presented at the right hand side. Original traces (approximately one-third of the total record at corresponding potential) were filtered at 0.7 kHz; c, o1 and o2 indicate current levels with no, 1 or 2 channels open. The amplitude histograms were fitted by a sum of three Gaussians, and the mean number of open channels was evaluated from the relative area under peaks corresponding to 1 and 2 open channels taken with respective weights; open probability was calculated as the mean number of open channels divided by the total number (2) of channels present in the patch. Similar analysis was performed at different holding voltages, and the resulting data points were plotted against membrane voltage (below); the solid line has no theoretical meaning.

 

View larger version (25K): [in this window] [in a new window]   Fig. 3. A small conductance VK channel is ideally selective for K+ over Cl– and Na+. (A) Two distinct channel types coexist in the same patch. Mean unitary I/V relations (mean ±SD) of the open slow activating (SV, n=3 ramps) and small conductance (VK, n=11 ramps) channels recorded on the same tonoplast patch. Symmetrical 100 mM KCl, free vacuolar and cytosolic Ca2+ were <2 nM and 27 µM, respectively. The inset shows the original current traces evoked by voltage ramp (–150 to +150 mV), the black and the grey ones correspond to those containing single open VK channel and the leak current only, respectively; the difference between the two was calculated (brief channel closure indicated by the arrow was omitted), plotted against voltage and averaged with similarly obtained curves. (B) K+/Cl– selectivity of the VK channel. Recorded first in symmetrical 100 mM KCl (n=11 ramps, grey trace) conditions, the open VK channel I/V curves were then registered after the decrease of cytosolic K+ to 10 mM (n=9 ramps, black trace), and Nernst potentials for K+ (EK+) and Cl– (ECl–) are indicated. (C) Whilst the SV channel displays a weak (Na+>K+) selectivity, the VK channel does not conduct Na+ at all. Unitary I/V curves for the two channels were recorded on the same tonoplast patch, the data are mean ±SD, n=18 and 28 ramps for SV and VK, respectively. Ionic concentrations (in mM) in the bath (cytosolic side) and in the patch-pipette are indicated.

 

	Results

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Figure 1 shows single channel records made on isolated tonoplast patches. At zero (<2 nM) cytosolic Ca²⁺, only flickering currents through single fast-activating FV channels, having a conductance of approximately 20 pS under symmetrical 100 mM KCl, could be detected (n=17 patches). Because these currents are suppressed only at high (tens of µM, Dobrovinskaya et al., 1999) cytosolic Ca²⁺, 1 mM Mg²⁺ was introduced to the cytosolic side, completely abolishing the FV currents (Fig. 1A). Under these conditions (nanomolar cytosolic Ca²⁺, 1 mM Mg²⁺) no channel activity could be seen, however, when cytosolic Ca²⁺ was increased to a submicromolar range, the activity of a novel channel could be detected (n=5 patches). The channel under study had a conductance of around 40 pS in symmetrical 100 mM KCl, and displayed a characteristic bursting behaviour (groups of openings separated by long pauses, Fig. 1B). Apparently, Mg²⁺ alone was not the cause of the channel activation, as evident by the disappearance of the channel activity on decreasing Ca²⁺ to 100 nM. Moreover, the activity of this channel could be stably recorded in the virtual absence of Mg²⁺ with cytosolic Ca²⁺ in the range of 10–100 µM; no significant effect of open probability was observed when Ca²⁺ was increased to 2 mM (result not shown). When activated by cytosolic Ca²⁺, the new channel did not show any notable voltage dependence, i.e. the open probability was between 0.5 and 0.75 in the potential range –125 to 100 mV (Fig. 2). Therefore, this channel could easily be discerned from the large conductance depolarization-activated slow vacuolar (SV) channels, almost always (but not in the patch shown in Fig. 2) present in the same tonoplast patches at elevated cytosolic Ca²⁺. In fact, SV channels dominate in these conditions, yielding several tens to hundreds of copies per patch. The mean number of the SV channel copies present in a macropatch was estimated by dividing macroscopic SV currents at saturation Ca²⁺ (1–2 mM) and voltage (120 mV) conditions through single channel currents. The density of the new channel was 0–4 copies per patch as estimated from the maximal number of simultaneous openings at –100 mV. The analysis yielded 216±12 large conductance SV channel copies for each smaller channel copy (n=24 separate patches).

Therefore, a strategy that allows the separate recording of these two channel types coexisting in the same patch has been developed (see Materials and methods for details). An example of such separate recording in symmetrical 100 mM K⁺ is given in Fig. 3A. Whereas the SV channel I/V relationship displays a moderate inward rectification, that of the smaller channel was fairly linear between –150 and +150 mV under symmetrical ionic conditions (Fig. 3A, B). Averaging the data obtained on n=5 separate patches, with a comparative number of successful voltage ramps applied to each one, yielded a mean slope conductance of 39.8±0.8 pS for this channel. The I/V relationship of the smaller conductance channel was then measured after lowering the cytosolic K⁺ level to 10 mM (Fig. 2B). The reversal potential of a single channel current in this case was +57.5±1.5 mV (n=3 separate patches), which fairly fits the equilibrium potential for K⁺, +55 mV, as calculated after correction for the activity coefficients. In these conditions the equilibrium potential for Cl^– was –73 mV. Therefore, the smaller conductance channel was ideally selective for K⁺ over Cl^–. In the next experimental series K⁺ (100 mM) was substituted for Na⁺ at the cytosolic side. The I/V curve of the smaller conductance channel under these bi-ionic conditions asymptotically approached the voltage axis without any measurable outward current component, corresponding to the efflux of Na⁺ from the cytosol (Fig. 3C). This result was reproduced on n=5 separate membrane patches. For the comparison, the unitary I/V relationship of the SV channel has been measured in the same experiment (Fig. 3C). In contrast to the smaller conductance channel, the SV channel readily conducts Na⁺, as evident by the large outward current. Moreover, in accord with the reversal potential shift (–7 mV), the SV channel in red beet vacuoles has a somewhat higher Na⁺ over K⁺ relative permeability.

	Discussion

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The 40 pS vacuolar cation channel described in the present work has a genuine K⁺ over Na⁺ selectivity (Fig. 3C). In contrast, other vacuolar cation channels are highly permeable for Na⁺, the SV channel 60–30% better (Amodeo et al., 1994; this work) and the FV one by 20% worse (Brüggemann et al., 1999 a) than for K⁺. The only vacuolar channel, identified to date, which could be compared with the 40 pS channel is the guard cell VK channel. Both channels are activated by submicromolar Ca²⁺ (the effect almost saturated at 1 µM) and lacking voltage dependence (Ward and Schroeder, 1994; Allen et al., 1998; this study). However, the guard cell VK channel displayed a moderate inward rectification. Linear regression yielded the slope conductance value of 70 pS (Ward and Schroeder, 1994), which was higher than that of the beet channel at equivalent ionic conditions.

Despite the difference in the unitary conductance, the guard cell VK channel and the 40 pS beet K⁺ channel share a high degree of functional similarity, including characteristic bursting kinetics. One may conclude, therefore, that the vacuolar K⁺ channel described in the present study is a homologue of the guard cell VK channel.

Due to a relatively low abundance, the beet taproot VK channel escaped detection in the whole vacuole mode. There have been, however, at least two studies where single channels with quite similar properties have been reported (Hedrich and Neher, 1987; Gambale et al., 1996). These reports describe a 40 pS voltage-independent cation channel, displaying long-lasting bursts of activity. The channel required cytosolic Ca²⁺ for its activation, with the open probability at 6 µM being merely 2-fold lower than at 1 mM (Gambale et al., 1996). Although in original publications this channel was confused with the FV one, based on the published characteristics its identity with the beet VK channel characterized here is likely.

What may be the physiological role of the beet VK channel? Beta vulgaris is a plant with an inducible salt-tolerant phenotype, which is related to the increased expression of the tonoplast Na⁺/H⁺ antiporter, mediating the accumulation of large amounts of Na⁺ in the vacuole. The antiporter, in turn, is energized by the H⁺ gradient generated by vacuolar H⁺ ATPase and stimulated by salt stress (see Blumwald et al., 2000, for a review). The activity of the H⁺ pump is electrogenic, i.e. besides the H⁺ concentration gradient it also generates the electric potential difference across the tonoplast. For long-term Na⁺ accumulation in the vacuole, the whole ion transport machinery needs to operate electroneutrally, thus, the generated difference of electric potential needs to be efficiently dissipated via a suitable ionic shunt conductance. Previously, some voltage-independent current, only moderately selective for K⁺ and Na⁺ over Cl^–, was assigned to serve as a shunt conductance for vacuolar H⁺ pumps (Davies and Sanders, 1995). However, under salt stress, a plausible shunt conductance needs to be selective for K⁺ over Na⁺, otherwise futile Na⁺ cycling between vacuole and cytosol will arise. The potassium ion is also a good candidate because the efflux of K⁺ from the vacuole will improve the cytosolic K⁺ to Na⁺ ratio. The VK channel is the only highly K⁺-selective vacuolar ion channel characterized to date. Although in this study perfect K⁺ to Na⁺ selectivity was revealed with Na⁺ on the cytosolic side and K⁺ in the vacuole, it would hold also for the opposite configuration, with Na⁺ in the vacuole, because ion channels, when open, generally mediate ion transport in either direction (except, of course, when the ion does not permeate as in the present example) and the absence of Na⁺ conductance from the cytosolic side will also rule out the possibility of a Na⁺ leak from the vacuole. Based on the approximate channel density of one VK copy per 1 pF, the whole vacuole population of VK channels in red beet will approach several tens of copies. In the physiological voltage range this population is capable of mediating K⁺ currents in the range of a few tens of pA, which is comparable to currents generated by H⁺-pumps under optimal conditions (Gambale et al., 1994). VK channels require cytosolic Ca²⁺ for their activity, so does the product of the SOS3 gene encoding the calcineurin regulatory subunit, whose interaction with downstream elements of the SOS-cascade is crucial for salt tolerance (Halfter et al., 2000), and salt stress is also known to induce cytosolic Ca²⁺ transients (Knight et al., 1997). Therefore, the VK channel is potentially capable of providing an adequate shunt conductance for the vacuolar H⁺-ATPase under salt stress conditions.

	Acknowledgements

 
This work was supported by CONACyT grant 29473N to IIP. The authors are indebted to Dr Gerald Schönknecht for helpful comments on the manuscript.

	References

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