The Arabidopsis AtPP2CA Protein Phosphatase Inhibits the GORK K+ Efflux Channel and Exerts a Dominant Suppressive Effect on Phosphomimetic-activating Mutations

Abstract

The regulation of the GORK (Guard Cell Outward Rectifying) Shaker channel mediating a massive K(+) efflux in Arabidopsis guard cells by the phosphatase AtPP2CA was investigated. Unlike the gork mutant, the atpp2ca mutants displayed a phenotype of reduced transpiration. We found that AtPP2CA interacts physically with GORK and inhibits GORK activity in Xenopus oocytes. Several amino acid substitutions in the AtPP2CA active site, including the dominant interfering G145D mutation, disrupted the GORK-AtPP2CA interaction, meaning that the native conformation of the AtPP2CA active site is required for the GORK-AtPP2CA interaction. Furthermore, two serines in the GORK ankyrin domain that mimic phosphorylation (Ser to Glu) or dephosphorylation (Ser to Ala) were mutated. Mutations mimicking phosphorylation led to a significant increase in GORK activity, whereas mutations mimicking dephosphorylation had no effect on GORK. In Xenopus oocytes, the interaction of AtPP2CA with "phosphorylated" or "dephosphorylated" GORK systematically led to inhibition of the channel to the same baseline level. Single-channel recordings indicated that the GORK S722E mutation increases the open probability of the channel in the absence, but not in the presence, of AtPP2CA. The dephosphorylation-independent inactivation mechanism of GORK by AtPP2CA is discussed in relation with well known conformational changes in animal Shaker-like channels that lead to channel opening and closing. In plants, PP2C activity would control the stomatal aperture by regulating both GORK and SLAC1, the two main channels required for stomatal closure.

Keywords: Arabidopsis thaliana; Xenopus; abscisic acid (ABA); mutagenesis; phosphatase; plant molecular biology; plant physiology; potassium channel; potassium transport; protein-protein interaction.

FIGURE 1.

Decreased transpiration in the atpp2ca mutants. A, transpiration rate of wild-type (black, n = 14), atpp2ca-1 (light gray, n = 10), and atpp2ca-2 (dark gray, n = 14) living plants grown in pots for different time periods before and after light switch-off (0 in the time scale). Student's t tests for comparisons between the mutants and the wild type were used. *, p < 0.05 (for atpp2ca-2, p = 0.031 between 0 and 15 min and p = 0.015 between 15 and 30 min); **, p < 0.01; ***, p < 0.001. Two sets of measurements were performed, and results were reproducible for the two mutants. B, cumulated water loss in excised rosettes of wild-type and atpp2ca mutant plants (n = 10–15, a pool of two experiments was performed under the same conditions).

FIGURE 2.

Physical interaction between AtPP2CA and the C-terminal cytosolic region of GORK. A, quantitative two-hybrid β-galactosidase activity assays with AtPP2CA as the bait. The reciprocal interactions with GORK as the bait could not be assayed because of the low sensitivity of the test. β-Galactosidase activities (n = 7, mean ± S.D.) are expressed as A₄₂₀ per min × 1000/A₆₀₀ of the culture medium. B, two-hybrid tests using HIS3 as the reporter gene in strain AH109. Drop tests (dilution series, from A 0,066 at 600 nm (1/3)) showing growth of co-transformed yeast cells on medium without tryptophan and leucine (selection of plasmids, control medium) or also without histidine (interaction tests). GORK was used as a positive control (strong GORK-GORK interaction because of subunit assembly via the C-terminal intracytoplasmic domains (25)). In this experiment and all subsequent drop test experiments, each series of dilutions was made in duplicate with two different yeast colonies, and tests were repeated with colonies from a different yeast transformation. DBD, DNA-binding domain of GAL4; ACT, activator domain. ACT alone indicates transformations with empty pGAD10 vector. C, co-purification on a His trap column. An E. coli extract containing either the His₆ tag alone, or the AtPP2CA- His₆ tag fusion protein was loaded onto nickel-coated beads (control column and AtPP2CA column, respectively). After washing, Saccharomyces cerevisiae extract containing lexA::CT-GORK protein (CT) was loaded on both columns. Unbound proteins were washed again, and aliquots (15 μl) of the last wash fraction (W), the first elution (E1), and the second elution (E2) fractions were loaded on SDS-polyacrylamide gel. Right panel, the Western blot obtained from the gel, revealed with anti-LexA antibody. Left panel, Western blot analysis performed with an extract obtained from yeast expressing the LexA tag alone (yeast transformed with empty vector, EV).

FIGURE 3.

AtPP2CA inhibits GORK activity in Xenopus oocytes. A, macroscopic current traces recorded in Xenopus oocytes. Shown are representative traces obtained with GORK alone and GORK co-expressed with AtPP2CA. B, current/voltage curves deduced from a pool of recordings. Oocytes were maintained at a holding potential of −60 mV between two voltage pulses. Oocytes were injected with 10 ng of GORK cRNA either alone or with 20 ng of AtPP2CA cRNA (n ≥ 10). C, inhibition of GORK (currents at +50 mV) by AtPP2CA in eight independent experiments. Currents were normalized to the level of GORK in each experiment (n ≥ 10/experiment). The dotted line indicates the mean level of inhibition. D, current/voltage curves obtained from patch clamp recordings in COS cells expressing GORK or co-expressing GORK and AtPP2CA (n ≥ 10). Pulses of 1.6 s were applied from −100 to +100 mV, with a holding potential of −40 mV (steps of 20 mV). E and F, current/voltage curves obtained after expression of GORK alone, GORK with ABI2 (E), or GORK with OST1 (F) in Xenopus oocytes (n ≥ 13). The same electrophysiological protocol was used as in B. All data are mean ± S.E.

FIGURE 4.

Effect of mutations in AtPP2CA. A, GORK inhibition by AtPP2CA or AtPP2CA G139D. The protocol is described in Fig. 3B. Data are mean ± S.E. (n ≥ 10). B, two-hybrid interaction tests between the GORK C-terminal region and AtPP2CA (or AtPP2CA mutant forms) using HIS3 as a reporter gene. Left, control growth medium. Right, selective medium for detection of interactions. DBD, DNA-binding domain; ACT, activator domain. See Fig. 2B for details.

FIGURE 5.

Positions of substituted residues in the AtPPCA sequence. Shown is a sequence comparison of four Arabidopsis PP2C amino acid sequences: ABI1,ABI2, HAB1, and AtPP2CA. Multiple alignment was performed using ClustalW-multialign with Boxshade. Regions of identity are shaded. Mutated amino acids are indicated in red. The locations of invariant metal-coordinating residues (NCBI CDD accession no. cd00143 (73)) are indicated by blue arrows. Amino acids designated by an asterisk are involved in interaction with PYR/PYL/RCAR receptors. The blue frame indicates the conserved tryptophan (Trp-385 in HAB1) of the PYL interaction loop, which inserts directly into the ligand-binding pocket of the receptor and establishes an ABA contact with the ketone group of ABA (74). This Trp-385 also inserts into the catalytic cleft of kinase, mimicking receptor/PP2C interactions (16).

FIGURE 6.

Effect of mutations on Ser-649 on GORK activity and inhibition by AtPP2CA. A, two-hybrid interaction assay between GORK (or GORK mutated on serine 649) and AtPP2CA. Yeast cells were grown on medium without tryptophan, leucine, and histidine. Details of the experimental procedures are in the legend for Fig. 2B. DBD, DNA-binding domain; ACT, activator domain. B, current/voltage curves with oocytes expressing GORK alone, GORK with AtPP2CA, GORK S649A alone, or GORK S649A with AtPP2CA (n ≥ 10). The holding potential is −60 mV. Applied voltages increase from −90 to +50 mV with an increment of +10 mV. C, the same experiment as in B with GORK S649E instead of 649A (n ≥ 12). B and C, oocytes were injected with 10 ng of GORK cRNA and 20 ng of AtPP2CA cRNA. D, quantitative aspects of the experiments in B and C (1 and 2, respectively). Data were normalized with the level of GORK current in each experiment. a, the basic level of inhibition of GORK by AtPP2CA; b, the increase of activity because of the mutation S649E. The white and gray arrows point, respectively, to the expected levels of GORK S649E + AtPP2CA if the effect of AtPP2CA on GORK were completely independent on the activation of the Ser-649 site or if it were proportional to the activity of the channel. The difference between the level of the gray arrow (0.87 ± 0.05) and that of GORK S649E + AtPP2CA (0.62 ± 0.05) is statistically significant (Student's t test, p = 0.002). In reality, the increase of inhibition (b') is roughly equivalent to b, highlighting a complete suppression of the positive effect of the mutation. Data are mean ± S.E.

FIGURE 7.

Effect of mutations on Ser-722 on GORK activity and inhibition by AtPP2CA. A, two-hybrid interaction tests between GORK (or GORK mutated on serine 722) and AtPP2CA. Interacting partners are listed as bait/prey combinations: fusions with the DNA-binding domain and the activator domain of GAL4 (ACT), respectively. See Fig. 2B for details. Yeast cells were grown on medium without tryptophan, leucine, and histidine. B, steady-state currents recorded at +50 mV (holding potential, −60 mV) from oocytes expressing GORK alone (black columns), GORK + AtPP2CA (light gray columns), GORK S722A (dark gray columns), and GORK S722A + AtPP2CA (white columns) in 10 and 100 mm KCl (two independent experiments, n ≥ 10 for each column). C, steady-state currents recorded at + 50 mV (holding potential, −60 mV) from oocytes expressing GORK alone (black columns), GORK + AtPP2CA (light gray columns), GORK S722E (dark gray columns), and GORK S722E + AtPP2CA (white columns) in the experiments, including the four oocyte batches in 10 mm and 100 mm KCl (n ≥ 10 for each column) (three independent experiments, the former and the latter group of plots were obtained with the same batch of oocytes). Note the similarity with Fig. 6D (GORK + AtPP2CA close to GORK S722E + AtPP2CA). B and C, oocytes were injected with 10 ng of GORK (or GORK S722E) cRNA and 20 ng of AtPP2CA cRNA. Data are mean ± S.E. D, Western blotting with anti-GORK antibody after electrophoresis of oocyte plasma membrane proteins (left panel), molecular weight markers (center panel), and staining of the same protein samples with Coomassie Blue R (right panel). S, native GORK; A, GORK S722A; E, GORK S722E. The band is at the expected size (94 kDa for full-length GORK).

FIGURE 8.

Single-channel analyses in Xenopus oocytes. A, traces of currents obtained for GORK, GORK S722E, and GORK S722E + AtPP2CA (depolarization of +80 mV). B, current/voltages traces obtained from the current amplitudes of single opening events at different applied depolarizations and calculations of the corresponding conductances (means of the slopes of the linear regression lines; three, six, and four repeats). C, open probabilities calculated from integrated times of channel openings in two experiments (n ≥ 7). Comparisons of GORK and GORK + AtPP2CA could not be performed because of low signals, moderate inhibition, and variability inherent to single channel measurements. Data are mean ± S.E.

FIGURE 9.

Proposed model representing the effect of PP2C phosphatases on GORK. Two subunits are represented for GORK (ovoid transmembrane domain and spherical C-terminal intracytoplasmic region). There is an equilibrium between PP2C phosphatases bound to GORK and bound to PYR/PYL/RCAR receptors. In the atpp2ca mutant and during stomatal closure, the equilibrium is shifted to the left (less PP2Cs available for GORK inhibition). When conditions become favorable for stomatal opening, PYL/PYL/RCAR receptors release PP2Cs that inhibit GORK. Phosphorylation sites become inactive.