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Plant Physiol. (1999) 120: 331-338 K+-Selective Inward-Rectifying Channels and Apoplastic pH in Barley Roots1
The Plant Laboratory, Department of Biology, University of York, P.O. Box 373, York YO1 5YW, United Kingdom
Recent structure-function analysis of heterologously expressed K+-selective inward-rectifying channels (KIRCs) from plants has revealed that external protons can have opposite effects on different members of the same gene family. An important question is how the diverse response of KIRCs to apoplastic pH is reflected at the tissue level. Activation of KIRCs by acid external pH is well documented for guard cells, but no other tissue has yet been studied. In this paper we present, for the first time to our knowledge, in planta characterization of the effects of apoplastic pH on KIRCs in roots. Patch-clamp experiments on protoplasts derived from barley (Hordeum vulgare) roots showed that a decrease in external pH shifted the half-activation potential to more positive voltages and increased the limit conductance. The resulting enhancement of the KIRC current, together with the characteristic voltage dependence, strongly relates the KIRC of barley root cells to AKT1-type as opposed to AKT3-type channels. Measurements of cell wall pH in barley roots with fluorescent dye revealed a bulk apoplastic pH close to the pK values of KIRC activation and significant acidification of the apoplast after the addition of fusicoccin. These results indicate that channel-mediated K+ uptake may be linked to development, growth, and stress responses of root cells via the activity of H+-translocating systems.
Changes in apoplastic pH are involved in many physiological
processes in plants, such as development, growth, leaf movement, gas
exchange, and pathogen defense (Grignon and Sentenac, 1991 Low-affinity K+ uptake by plants is mediated by
KIRCs that activate at negative membrane potentials when
K+ concentrations are in the high micromolar or
millimolar range, ensuring that K+ movement is
directed inward (Maathuis et al., 1997 In contrast to the findings on H+-induced
activation of IKIRC, work on
suspension-cultured cells from Arabidopsis demonstrated that currents
through the KIRCs of these cells decreased upon acidification of the
external medium (Giromini et al., 1997 We studied the effect of apoplastic pH on the
IKIRC of root cells and show activation of
this channel type by external protons (as was previously described for
guard cells) and a partial dependence of the pH effect on the external
KCl concentration. Our study also included measurements of the
apoplastic pH in root tissue, which enabled us to put channel
activity into a physiological context.
Growth of Plant Material
Preparation of Protoplasts For protoplast preparation 3- to 4-cm distal segments of primary roots (excluding about 1 cm at the root tip) were chopped and incubated with 1.5% (w/v) cellulase ("Onozuka" Yakult Honsha Co., Tokyo, Japan), 0.15% (w/v) pectolyase (Sigma), and 0.1% (w/v) BSA in solution A for 2 h at room temperature. Protoplasts were released from the tissue by gentle squeezing in solution B, washed, and collected. Solutions A and B contained 10 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and 2 mM Mes at pH 5.7 (Tris) and were adjusted to 600 mOsm (solution A) and 400 mOsm (solution B) with sorbitol. Alternatively, cortical tissue was stripped off and discarded before enzyme incubation. Protoplasts obtained from whole-root and stelar preparations were very similar in size, appearance, and features, and we therefore assumed that both preparations produced stelar protoplasts only, probably originating from xylem parenchyma (Wegner and Raschke, 1994 ).
Patch-Clamp Setup and Data Acquisition and Analysis Standard patch-clamp techniques (Hamill et al., 1981) were
applied. Details for pipette preparation, voltage-clamp, and recording equipment were as as described by Amtmann et al. (1997). Protoplasts of
similar size (approximately 30 µm in diameter) were chosen for the
experiments. Whole-cell inward currents were elicited by 3-s bipolar
voltage pulses from holding potentials more positive than
EK (equilibrium potential for
K+) filtered at 300 Hz, and recorded with a sample
frequency of 1.5 kHz. The access resistance was measured with the
amplifier but not corrected for; measurements were discarded when
errors in the clamp voltage larger than 5% arose from high access
resistance. Conductance of the instantaneous current component was
ohmic over the whole voltage range analyzed. Therefore, leak
subtraction was used as the general method for obtaining time-dependent
IKIRC. Steady-state
IKIRC were measured 3 s after onset of
the voltage pulse and averaged over three identical voltage protocols.
Values are means ± SE. Curve fitting was
carried out using the program FigP (Biosoft, Cambridge, UK).
Experimental Solutions Patch pipettes contained 100 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1.4 mM EGTA (4 mM KOH), 1.5 mM MgATP, and 2 mM Hepes or Tes buffered to pH 7.4 with Tris. The free Ca2+ concentration was 176 nM, as calculated with the CALCIUM program (Föhr et al., 1993). Bath solutions contained 10 or 1 mM KCl, 1 mM MgCl2,
and 1 mM CaCl2, at various pH values
(2-5 mM Mes, Tes, or Hepes and Tris or NaOH). Liquid
junction potentials were measured and corrected when larger than 3 mV
according to the method of Amtmann and Sanders (1997). All solutions
were adjusted to 500 mOsm with sorbitol and sterile-filtered before
use.
Determination of Apoplastic pH Calibration and Dye Loading Stock solutions (10 mM) of the pH-sensitive dye NERF-DM (Molecular Probes, Leiden, Netherlands) were prepared in 20 mM Mes buffer, pH 6.3, and stored at 4°C. For calibration measurements the dye was diluted to 10 µM in 100 mM phosphate buffer at an appropriate pH. Calibration points were fitted with Equation 3 (see ``Results''). pH sensitivity (ratiomax ratiomin = 0.54) and affinity (pK = 4.9) of the in vitro calibration were
satisfactory (compare with Whitaker et al., 1992). For in situ
calibration, 1-cm-long fragments of barley roots (2 cm above the tip)
were incubated for 2 h in 10 µM NERF-DM.
Imaging of root fragments was performed immediately after they were
rinsed in dye-free buffer. We found that root fragments had to be
exposed to buffer concentrations greater than 200 mM to influence apoplastic pH. However, when 1 mM KCN, 1 mM
salicylhydroxamic acid, and 1 µM carbonyl
cyanide p-trifluoromethoxyphenylhydrazone (all from Sigma)
were added during dye incubation, in situ calibration was achieved in
100 mM phosphate buffers. Compared with the in
vitro calibration, ratio values were shifted to higher values in situ
(by 0.24 unit on average) and the sensitivity of the dye was slightly
reduced (ratiomax ratiomin = 0.49), whereas the affinity for
H+ was only slightly affected (pK 5.1). The in
situ calibration curve is shown in Figure 6. For in vivo measurements
root fragments were incubated for 1 h in 100 µM Mes-Tris, pH 5.9, and 10 µM NERF-DM with and without 5 µM fusicoccin. The ethanol concentration in both solutions was 0.5% (v/v).
Imaging Confocal fluorescence imaging (model MRC 1000, Bio-Rad) was controlled by CoMOS and TCSM software (Bio-Rad). The confocal system was interfaced with an upright microscope (Optiphot 300, Nikon). Imaging was performed using a ×60 (1.4 numerical aperture) oil-immersion objective (PlanApo, Nikon) and a ×3.5 electronic zoom. Fluorescence images were acquired using the 514- and 488-nm excitation lines of an argon laser at intensities between 1% and 10%, depending on dye loading into the cell walls. Emission was recorded using the integral emission filter (540 DF 30, Nikon) of the confocal microscope. The pinhole size (4) and gain setting (900) were chosen to give optimal fluorescence signals at all pH values in the calibration curve and were identical for all experiments. Fluorescence ratio values (514/488 nm) were calculated pixel by pixel using TCSM software. Average ratio values for NERF-DM-loaded cell walls were extracted using the Histogram command in CoMOS. Care was taken not to include areas close to the edge of the cell wall, since low fluorescence intensity caused artifacts in this region.
Typical IKIRC in the Plasma Membrane of Barley Root Cells Hyperpolarization of the plasma membrane to voltages more negative than80 mV evoked large inward currents with the typical features of
KIRCs, such as time-dependent double-exponential activation, single-exponential deactivation, high selectivity for
K+ over anions and other cations, and blockage by
tetraethylammonium ions. The IKIRC has been
described for many plants and in particular for barley root xylem
parenchyma cells (Wegner and Raschke, 1994; Wegner et al., 1994).
Response of IKIRC to Varying External pH Figure 1 shows a typical response of IKIRC to changes in external pH. A shift of pH from 7.5 to 5.5 increased IKIRC considerably. Furthermore, the voltage needed for half-activation of IKIRC was more positive at pH 5.5 than at pH 7.5. The effects of external pH on IKIRC were fully reversible. The reversal potentials (Erev) of the time-dependent currents as determined in tail-current experiments (resolution ± 5 mV) were not significantly different at different pH values: for 10 mM external KCl (n = 5 protoplasts), Erev =54 ± 2 mV at pH 5.5 and 58 ± 3 mV at pH 7.5; for 1 mM external KCl (n = 4 protoplasts), Erev = 101 ± 4 mV at
pH 5.5 and 98 ± 2 mV at pH 7.5.
Fitting the IKIRC-Voltage Relations Figure 2 depicts typical current-voltage (I-V) relationships of the KIRC at pH 5.5 and 7.5 (same protoplast as in Fig. 1) recorded with 10 or 1 mM KCl in the bathing medium. All I-V relationships were well fitted by a simple function in which the Goldman-Hodgkin-Katz equation for the open-channel current is multiplied by the Boltzman distribution of the open probability, the maximal open probability, and the total number of channels in the protoplast. Thus,
V50 and Glim at pH 5.5 and 7.5 Figure 3 displays the results of a statistical evaluation of V50 and Glim measured at external pH values of 5.5 and 7.5 and external KCl concentrations of 1 and 10 mM. For both external KCl concentrations, mean absolute magnitudes of V50 (Fig. 3A) were significantly more positive at pH 5.5 than at pH 7.5. The difference in absolute magnitudes of Glim determined at pH 5.5 and 7.5 was not significant because variation of Glim among different protoplasts was high, probably because of varying channel densities in the membrane (for means, see the legend of Fig. 3A). This problem was overcome by analyzing changes of V50 and Glim in response to individual medium-exchange events in each protoplast, where the total number of channels could be assumed to be constant (Fig. 3B). When the external pH changed from 5.5 to 7.5, V50 shifted to a more negative value by an average of 28 ± 3 mV in 1 mM external KCl (n = 5) and 16 ± 2 mV in 10 mM external KCl (n = 6). The Glim at pH 7.5 was, on average, 63% ± 7% (1 mM KCl, n = 5) or 61% ± 5% (10 mM KCl, n = 6) of the Glim at pH 5.5.
Apoplastic pH in Barley Roots
Effects of Apoplastic pH on KIRCs in Different Cell Types
Mechanisms of KIRC Activation by External Protons The effect of external pH on V50 reflects proton-dependent modulation of channel gating. Ilan et al. (1996) suggested that external protons affect
V50 by decreasing the negative
surface-charge density in the vicinity of the channel gates. Our
observation that the effect of external protons on
V50 was attenuated by increasing external
KCl concentrations points to the same conclusion. One possibility is
that protons act on V50 by screening the
surface potential of the entire membrane, which would result in a
different proportion of clamp voltage being sensed by the channel. The
second possibility is that the pH effect on
V50 involves protonation and charge
screening of intrinsic channel sites that are exposed to the
extracellular side of the membrane. Analysis of mutations performed on
KST1 suggested that a His residue situated in the outer-pore region of
the channel is crucial for the proton action on
V50 (Hoth et al., 1997) and the pK of 6.5, which in our study was derived from the pH titration of
V50 and is approximately the pK of His
protonation (Creighton, 1993).
Contrasting Responses of Plant KIRCs to External Protons Recent studies established that the Arabidopsis K+-channel AKT3 displays unique properties with respect to pH sensitivity (Marten et al., 1998), even though its cDNA
sequence is highly similar to that of the other Arabidopsis KIRCs, KAT1
and AKT1 (Ketchum and Slayman, 1996). When expressed in X. laevis oocytes, AKT3 shows weak inward rectification and blockage
by external protons. Acidification of the external medium does not
affect V50 but decreases Glim because of a decrease in
single-channel conductance (Marten et al., 1998). From these findings
one may speculate that the KIRC of cultured Arabidopsis cells, which is
inhibited by external protons (Giromini et al., 1997), is closely
related to or identical to AKT3. Similarities between other
characteristics of both channel currents have been highlighted
previously (Colombo and Cerana, 1991; compare with Ketchum and Slayman,
1996). The expression pattern of AKT3 in the plant has not yet been
established. pH dependence and gating properties clearly relate the
barley root KIRC to AKT1 and SKT1 but not to AKT3-type channels.
Implications of the Effect of External pH on IKIRC for Low-Affinity K+ Uptake KIRCs have been shown to be the major pathway for low-affinity K+ uptake by plant cells (Maathuis et al., 1997).
Any change in KIRC activity caused by changes in the external pH can
therefore be expected to change K+-uptake rates.
However, it has to be taken into account that a change in the amount of
K+ inward current may influence the membrane
potential and, since the KIRC is voltage dependent, this will also
affect its activity. Roelfsema and Prins (1998) calculated that
apoplastic acidification would ultimately reduce
K+ influx into guard cell protoplasts from
Arabidopsis since activation of the KIRC caused a strong
depolarization. However, experimental modification of apoplastic pH as
applied by Roelfsema and Prins (1998) does not reflect the
physiological conditions in which acidification is generated. In many
cases apoplastic acidification is achieved via enhanced proton-pumping
activity (Palmgren, 1998), which will not only acidify the apoplast but
also hyperpolarize the membrane. To assess pH-linked effects on
low-affinity K+ uptake, both parameters,
apoplastic pH and membrane potential, would have to be measured
simultaneously.
Apoplastic pH in Barley Root Tissue To determine whether the pH effects on the KIRC in root cells could have a physiological role we measured the steady-state pH in cell walls of barley roots and found that the pH-dependent fluorescent dye DM-NERF was suitable for measurements of apoplastic pH in root tissue insofar as it failed to permeate the plasma membrane. The values of apoplastic pH determined here are in the same range as those derived for other tissues (Grignon and Sentenac, 1991), e.g. in the vicinity of
guard cells (pH 6.0-7.0, Edwards et al., 1988), leaf epidermis
(5.2-5.9, Mühling et al., 1995), various leaf tissues (5.5-6.5,
Hoffmann and Kosegarten, 1995), and root epidermis (4.5-4.9,
Taylor et al., 1996). Furthermore, we found that the apoplastic pH of
barley root cells changes when fusicoccin is added. This result agrees
well with recent data from maize roots, in which the addition of 2 µM fusicoccin caused apoplast acidification from pH 5.6 to 4.8, as measured with pH-sensitive microelectrodes (Felle, 1998).
The conclusion that the plasma membrane H+-ATPase
strongly regulates apoplastic pH is further supported by our
observation that tissue calibration could be achieved only in the
presence of respiratory blockers (see ``Materials and Methods'').
Accordingly, Felle (1998) reported increased apoplastic pH after the
addition of KCN and salicylhydroxamic acid. Taking into account that
the pH effect on the KIRC has a pk of 5.9, we can predict a tight linkage between activities of KIRC and H+-ATPase
via apoplastic pH.
Putative Physiological Roles of pH Modulation of KIRC Activity One of the few external factors of physiological relevance that has been specifically shown to influence the plasma membrane H+-ATPase is salt stress (Palmgren, 1998).
Salt-induced gene expression and increased activity of the
H+-ATPase have been reported for many plant
species (Braun et al., 1986; Niu et al., 1993; Binzel, 1995; Ayala et
al., 1996; Wu and Seliskar, 1998) and seem to be positively correlated
with salt tolerance (Niu et al., 1993). Our results are in accord with
the notion that activation of the H+-ATPase would
acidify the apoplast and increase IKIRC.
This might reverse the inhibitory effect of salt-induced membrane
depolarization (Cakirlar and Bowling, 1981; Katsuhara and Tazawa, 1990;
Kourie and Findlay, 1990) on the KIRCs and allow the root cells to
maintain a relatively high
K+/Na+-influx ratio under
saline conditions (Amtmann and Sanders, 1999).
* Corresponding author; e-mail aa15{at}york.ac.uk; fax 44-1904-434-317. Received September 10, 1998;
accepted February 4, 1999.
Abbreviations: Glim, limit conductance. IKIRC, KIRC current(s). KIRC, K+-selective inward-rectifying channel. V50, half-activation potential.
We thank Alison Karley for assistance with plant maintenance and Frans Maathuis, Steve Roberts, and Richard Parton for useful discussions.
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  J. E. Hayes and R. J. Reid Boron Tolerance in Barley Is Mediated by Efflux of Boron from the Roots Plant Physiology, October 1, 2004; 136(2): 3376 - 3382. [Abstract] [Full Text] [PDF]
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L. Fan and P. M. Neumann The Spatially Variable Inhibition by Water Deficit of Maize Root Growth Correlates with Altered Profiles of Proton Flux and Cell Wall pH Plant Physiology, August 1, 2004; 135(4): 2291 - 2300. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
2 s
, means ± SE; n = 9) was
obtained by ratio-imaging NERF-DM fluorescence from barley root cells
incubated in 100 mM phosphate buffers and respiratory
inhibitors (see ``
) and with (
) 5 µM fusicoccin.
1, 
![<FR><NU>NP(F<SUP>2</SUP>/RT)<UP>V([K</UP><SUP><UP>+</UP></SUP><UP>]</UP><SUB><UP>c</UP></SUB><UP> − [K</UP><SUP><UP>+</UP></SUP><UP>]</UP><SUB><UP>o</UP></SUB><UP> exp(</UP>−FV/RT))</NU><DE>(1−<UP>exp(</UP>−FV/RT))p<FENCE>1−<FR><NU>1</NU><DE>1+<UP>exp(</UP>−z<SUB>g</SUB>(F/RT) (<UP>V − </UP>V<SUB>50</SUB>))</DE></FR></FENCE></DE></FR>](/content/vol120/issue1/fulltext/331/img002.gif)
![G<SUB>lim</SUB> = NPp(F<SUP>2</SUP>/RT)<UP>[K</UP><SUP><UP>+</UP></SUP><UP>]</UP><SUB><UP>o</UP></SUB>](/content/vol120/issue1/fulltext/331/img003.gif)
) external K+
at pH 5.5 and 7.5 (same protoplast as in Fig. 
V50 and the
Glim ratio: in the same protoplast pH could
have a relatively strong effect on V50 and
a relatively weak effect on Glim or
vice versa: the correlation coefficient was 0.7 (10 mM external KCl, n = 6) and 0.6 (1 mM KCl, n = 5), respectively,
and in both P > 0.1. Therefore, external pH affects KIRC activity
via two independent modulation mechanisms: the first one shifts the
Boltzman distribution of the open probability along the voltage axis
(effect on V50), and the second changes the
asymptote of the Boltzman distribution by affecting the maximal open
probability or the conductance of the open channel (effect on
Glim). Both effects of external pH were
independent of the type of pH buffer used (Mes or HCl for pH 5.5, Hepes
or Tes for pH 7.5, Tris or NaOH) and the buffer concentration (between
2 and 5 mM) used.
