Báo cáo khoa học: Modulation of the Arabidopsis KAT1 channel by an activator of protein kinase C in Xenopus laevis oocytes potx

Modulation of the Arabidopsis KAT1 channel by an activator of protein kinase C in Xenopus laevis oocytes Aiko Sato 1 , Franco Gambale 2 , Ingo Dreyer 3 and Nobuyuki Uozumi 1 1 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan 2 Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, Italy 3 Heisenberg Group of Biophysics and Molecular Plant Biology, Institute for Biochemistry and Biology, University of Potsdam, Potsdam- Golm, Germany Introduction Plants possess guard cells in leaves to control gas exchange and water loss. Guard cells control stomatal aperture by osmotic swelling and shrinking in response to, for example, carbon dioxide concentration, humidity and light irradiation. The volume change in guard cells is regulated by fluxes of K + ,Cl ) and organic compounds via diverse transport systems. The hyper- polarization-activated (inward-rectifying) K + channel KAT1 expressed in guard cells is of great interest as it has been suggested to play a key role in controlling Keywords K + channel; KAT1; kinase; phosphorylation; protein kinase C Correspondence N. Uozumi, Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, Japan Fax: +81 22 795 7293 Tel: +81 22 795 7280 E-mail: uozumi@biophy.che.tohoku.ac.jp (Received 19 November 2009, revised 17 February 2010, accepted 10 March 2010) doi:10.1111/j.1742-4658.2010.07647.x The Arabidopsis thaliana K + channel KAT1 has been suggested to play a key role in the regulation of the aperture of stomatal pores on the surface of plant leaves. Calcium-dependent and calcium-independent signaling pathways are involved in abscisic acid-mediated regulation of guard cell turgidity. Although the activity of the KAT1 channel is thought to be regu- lated by calcium-dependent protein kinases, the effect of phosphorylation on KAT1 and the phosphorylated target sites remain elusive. Because it has been proposed that the phosphorylation recognition sequence of plant calcium-dependent protein kinases resembles that of animal protein kinases C, in this study, we used the Xenopus laevis oocyte protein kinase C to identify the target sites of calcium-dependent protein kinases. KAT1 expressed in Xenopus oocytes was inhibited by the protein kinase C activa- tor phorbol 12-myristate 13-acetate. On the basis of an in silico search, we selected S ⁄ T-X-K ⁄ R motifs facing the cytosol, as it has been reported that protein kinase C and calcium-dependent protein kinase share a common consensus sequence. Mutagenesis analyses revealed that six Ser ⁄ Thr residues were responsible for the reduction in activity after phorbol 12-myristate 13-acetate application. Simultaneous mutation of the five residues located in the carboxyl-terminus region of KAT1 led to a K + channel mutant that was insensitive to protein kinase C. These results indicate that, in plant cells, a kinase analogous to protein kinase C might exist that may modulate KAT1 channel activity through calcium-dependent phosphorylation at some of the pinpointed residues in the cytosolic region of KAT1. Abbreviations AAPK ⁄ ABR kinase, ABA-activated protein kinase ⁄ ABA-responsive kinase; ABA, abscisic acid; CDPK, calcium-dependent protein kinase; DAG, diacylglycerol; InsP 3, inositol 1,4,5-trisphosphate; Kv, voltage-activated K + channel; PAs, phosphatidic acids; PI, phosphatidylinositol; PI-PLC, PI-specific phospholipase C; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SnRK, SNF1-related protein kinase; WT, wild-type. 2318 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS the volume of guard cells in Arabidopsis thaliana leaves [1–3]. KAT1 has been proposed to be involved in the mediation of K + uptake during stomatal opening. The plasma membrane H + -ATPase establishes a negative membrane voltage which, in turn, results in the open- ing of inward-rectifying K + channels, allowing the influx of K + ions [4]. For stomatal closure, increased levels of cytosolic Ca 2+ inhibit plasma membrane pro- ton pumps [5], leading to a depolarization of the mem- brane. This activates anion efflux channels and inhibits inward K + uptake channels [6] to reduce the turgor pressure of the cells. The involvement of KAT1 in these regulatory processes has been suggested in sev- eral reports. For example, the co-injection of KAT1 cRNA into oocytes with transcripts extracted from Vicia faba guard cells decreases KAT1 channel activity, unlike that with transcripts from mesophyll cells [7]. In the same heterologous system, KAT1 cur- rent amplitudes decrease in the presence of a soybean calcium-dependent protein kinase (CDPK) [8]. Consis- tent with this, CDPKs in guard cells are involved in Ca 2+ and anion channel activation and stomatal closure [9,10]. In line with these data is the finding that Ca 2+ channels are activated by abscisic acid (ABA) [11] and, as a consequence of cytosolic Ca 2+ elevation, inward K + channel activity is reduced, resulting in stomatal closure [12]. In addition to these Ca 2+ -dependent reactions, a calcium-indepen- dent pathway also contributes to the control of guard cell volume. The calcium-independent, ABA-activated protein kinase ⁄ ABA-responsive kinase (AAPK ⁄ ABR kinase) from Vicia faba has been found to be present in guard cells and to control stomatal response to ABA [13–15]. In an in vitro phosphorylation assay, the Vicia AAPK ⁄ ABR kinase has been shown to phos- phorylate the C-terminal region of KAT1 [16]. One of the 10 members of the SNF1-related protein kinase 2 in A. thaliana, SnRK2.6, is an ortholog of AAPK and shares 79% amino acid identity [15]. SnRK2.6 has been identified as an essential element of the ABA signaling pathway that mediates stomatal regulation [17–20]. Recently, it has been shown that SnRK2.6, after heterologous expression and purification from Escherichia coli, can phosphorylate the residues T306 and T308 in KAT1. Modification of T306 abolished KAT1 activity in oocyte recordings, whereas modifica- tion of T308 did not cause a loss of function [21]. In animal cells, one type of Ser ⁄ Thr protein kinase, protein kinase C (PKC), is involved in signal transduc- tion pathways that govern a wide range of physiologi- cal processes, such as proliferation, apoptosis, cell survival and migration [22,23]. The animal Shaker superfamily comprises the so-called voltage-activated K + channel (Kv), Kv long QT, small-conductance cal- cium-activated K + channel, large-conductance Ca 2+ - and voltage-regulated K + channel, hyperpolarization- activated cyclic nucleotide gated channel, ether-a-go-go and cyclic nucleotide-gated channel members. It is well known that some of these are modulated by PKC [24– 27]. In addition, G-protein-coupled inward rectifier K + channels are inhibited by PKC phosphorylation [28]. Diacylglycerol (DAG), a natural degradation prod- uct of phosphatidylinositol (PI), allosterically activates PKC and regulates the activity of other proteins involved in carcinogenesis and metastasis, as well as in cell growth, development, survival and apoptosis [29– 33]. DAG, generated from PI in the PI-specific phos- pholipase C (PI-PLC) pathway, and elevated Ca 2+ induce the activation of conventional animal PKCs. Although canonical PKC-encoding genes have not been found in plants, a large family of CDPKs, some of them being activated by phospholipids (e.g. CPK1 in A. thaliana), has been documented [34–36]. PKC can be classified as conventional PKCs (cPKC; a, bI, bII and c), which contain a putative Ca 2+ -binding site, novel PKCs (nPKC; d, h, g and e), which lack Ca 2+ -binding sites, and atypical PKCs (aPKC; f, k ⁄ i and l), which are Ca 2+ -insensitive and are not acti- vated by phorbol esters [37]. Both cPKCs and nPKCs are activated by phorbol esters, such as phorbol 12- myristate 13-acetate (PMA). In Xenopus oocytes, the presence of all 11 PKC isozymes in mammals (a, bI, bII, c, d, f, e, h, g, k ⁄ i and l) has been reported [38]. In Arabidopsis, the existence of a PI-PLC pathway has been reported [39,40]. DAG has been considered to be rapidly converted to phosphatidic acids (PAs) by DAG kinases in plant cells [41]. Therefore, it may be possible that the other downstream events uncovered in animal cells may also have an equivalent in plant cells. Ca 2+ plays an important role as an intracellular signal in both plants and animals, including its involvement in the regulation of CDPK activity [42]. Despite the absence of PKC in plant cells, PKC-like enzymes have been reported to be present in protein extracts from various plant species. For example, an enzyme (ZmcPKC70) has been extensively purified and characterized in leaf protein extracts from the C4 plant maize [43], which belongs to the cPKC family, because it is activated by both PMA, a well-known agonist of animal PKC, and Ca 2+ . In addition, a PKC homolog, which can be detected with the PKC antibody in Brassica juncea, is activated by PMA and inhibited by the general kinase inhibitor H-7 and the PKC-specific inhibitor staurosporine [44]. Moreover, a large family of CDPKs, including some showing cPKC-like charac- A. Sato et al. Phosphorylation of KAT1 channel FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS 2319 teristics, is present in plant genomes [34–36]. Maize CDPK-1 phosphorylates in vitro sequence motifs simi- lar to those recognized by animal PKCs [45]. On the basis of these facts, we examined the effect of PKC activation on KAT1 channel activity by the PKC activator PMA in Xenopus oocytes. We also pin- pointed the phosphorylation target sites which regulate KAT1 channel activity. We uncovered a complex pat- tern of sites that are involved in channel regulation, indicating that phospho-regulation of plant K + chan- nels should not be considered as a ‘single switch’, but rather as the result of a multistage process. Results Reduction of KAT1 currents by PKC activation Earlier studies have reported the phosphorylation of the KAT1 channel expressed in guard cells by CDPK. On co-expression of KAT1 with a CDPK from soy- bean in oocytes, a decrease in the current amplitude was monitored [8,46]. To further evaluate whether KAT1 channel activity is regulated by phosphoryla- tion, we expressed KAT1 in Xenopus laevis oocytes and applied PMA, which is known to activate endoge- nous PKC. The recognition sequence of PKC for phosphorylation resembles that of plant CDPKs [45,47,48]. In KAT1-expressing oocytes, we measured inward-rectifying K + currents as reported previously [2,49]. After the addition of 1 lm PMA to the bath solution [50], the current amplitude apparently began to decrease. At 30 min after PMA application, currents were inhibited by about 45.0 ± 5.6% (Fig. 1A). The normalized current–voltage characteristics were almost identical before and after PMA application (Fig. 1B). Likewise, the normalized cord conductance was not altered as a result of PMA treatment (Fig. 1C). These results suggest that KAT1 is inhibited by PKC in oocytes without affecting its voltage-dependent properties. To confirm the regulation of KAT1 channel activity by PKC, we applied a different voltage pulse protocol to record changes in KAT1 currents over time. For this purpose, we applied a voltage pulse to –150 mV every 30 s. The current amplitude decreased, and the inhibition appeared to be saturated at 30 min after PMA application (Fig. 2A). In addition, we measured the current–voltage characteristics of KAT1 every 5.5 min with or without pre-incubation of oocytes in 2 lm calphostin C, a PKC-specific inhibitor, for 12–24 h (Fig 2B, C). KAT1 currents measured in calphostin-pre-incubated oocytes were less susceptible to PMA than those in nontreated oocytes. Taken together, these results demonstrate that KAT1 is inhibited by PMA-stimulated activation of the oocyte intrinsic PKC. Identification of Ser ⁄ Thr PKC phosphorylation sites influencing KAT1 channel activity Several groups have reported different sequence motifs that are recognized by PKC [51,52]. We used 2 µA 200 ms 0 min 15 min 30 min –0.5 0 –200 –150 –100 –50 0 V (mV) 0 min –2 –1.5 –1 Normalized current 15.5 min 32 min A B C PMA No addition Fig. 1. Effects of PMA on KAT1 WT chan- nel activity. (A) Representative current profile of KAT1 expressed in Xenopus oocytes before (0 min) and 15 and 30 min after the addition of 1 l M PMA (a PKC activator in oocytes). (B) Current–voltage relationship of the current at 0, 15.5 and 32 min after PMA application. Currents were normalized with respect to the current at )150 mV. (C) Normalized KAT1 conduc- tance G Nor before (full line; squares) and after (broken line; circles) PMA application. Phosphorylation of KAT1 channel A. Sato et al. 2320 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS the program prosite (http://ca.expasy.org/prosite/) to predict residues of KAT1 that might be phosphory- lated by PKC. All of the resultant sequences con- tained the classical S ⁄ T-X-K ⁄ R motif, which can be recognized by PKCs in Xenopus laevis oocytes [38,53]. Moreover, ‘S ⁄ T-X-K ⁄ R’ matches with the consensus sequences recognized by plant CDPKs [54,55]. In addition, we employed the prediction program Netph- osK 1.0 (http://www.cbs.dtu.dk/services/NetPhosK/) using the Phospho.ELM database (http://phospho. elm.eu.org/) containing experimentally verified phos- phorylation patterns of Ser ⁄ Thr ⁄ Tyr residues in eukaryotic proteins. The analysis for 11 Ser ⁄ Thr resi- dues in the cytosolic C-terminus and for Thr at posi- tion 45 resulted in a relatively high score (more than 0.7). Consequently, we selected these 12 Ser ⁄ Thr resi- dues as possible phosphorylation sites for PKC within the N- and C-terminal cytosolic regions of KAT1 (Fig. 3A). We systematically screened all of them by replacing the Ser ⁄ Thr residues by an Ala residue which mimics the dephosphorylated form. All vari- ants, except KAT1 T303A, showed detectable inwardly rectifying currents. We also tested the K + transport activities of KAT1 T303D. However, as for the mutant T303A, we could not obtain K + currents in oocytes (data not shown). The remaining mutants could be subdivided into three datasets: (a) the muta- tions T45A, T308A, S312A, S589A, S590A and S641A displayed a decrease in PMA-induced channel inhibition (27.1 ± 2.9%, 20.6 ± 3.2%, 17.0 ± 6.5%, 28.4 ± 6.0%, 23.9 ± 5.0% and 24.9 ± 6.6%, respec- tively; Fig. 3B, top panel); (b) the mutants T22A, S44A, S125A and S529A behaved similarly to the wild-type (WT) (43.2 ± 7.2%, 36.9 ± 3.3%, 34.3 ± 5.6% and 44.5 ± 0.7%, respectively; Fig. 3B, middle panel); (c) T458A showed slightly greater inhibition by PMA-induced PKC activation compared with WT (52.5 ± 2.3%; Fig. 3B, bottom panel). The data sug- gested that six Ser ⁄ Thr residues – one in the cytosolic N terminus (T45) and five in the cytosolic C-terminus (T308, S312, S589, S590 and S641) – were candidates for PKC phosphorylation target sites altering KAT1 channel activity. Quintuple mutation renders KAT1 PKC-insensitive The data in Fig. 3B show that the single mutations do not completely abolish the inhibitory effect of PMA application. This may indicate that PKC stimulates simultaneously multiple phosphorylation events on KAT1. To confirm this, we constructed the quintuple mutant KAT1-T308A-S312A-S589A-S590A-S641A eliminating all putative PKC target sites in the cyto- solic C-terminus. After expression in oocytes, the quin- tuple mutant was no longer sensitive to PMA-induced PKC activation. Even after PMA application, the –3 –4 –2 –1 0 PMA A B C –6 –5 –4.5 0 10 20 30 37.5 Time (min) Current (µA) 0.8 0.6 1 1.2 0 0.2 0.4 0102030 Time (min) No addition PMA PMA + calphostin C 60 37.5 10 20 30 40 50 0 No addition PMA PMA + calphostin C Current inhibition by PMA (%) I/I control Fig. 2. Inhibition of WT KAT1 activity by PKC activation. (A) Time course of a representative WT KAT1 current amplitude at –150 mV after the addition of 1 l M PMA to the bath solution. A black bar indicates the addition and removal of PMA. (B) Changes in the cur- rent at –150 mV in response to 1 l M PMA. PMA was added imme- diately after the recording of currents at t = 0 min. The current amplitude was normalized to the value measured before PMA application (mean ± SEM, n = 3–4). Calphostin C indicates that KAT1-expressing oocytes were pre-incubated for 12–18 h with 2 l M calphostin C, a specific PKC inhibitor. (C) Percentage of cur- rent inhibition 32 min after the addition of PMA. A. Sato et al. Phosphorylation of KAT1 channel FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS 2321 current amplitude behaved similarly to that of WT KAT1 in the absence of PMA (Fig. 4A, B and Table 1). These results suggest that PMA-induced phosphorylation at some or all of the five residues determines an inhibition of the K + currents. Discussion Phosphorylation and dephosphorylation events are critical for the modulation of the activity of the guard cell-expressed K + uptake channel KAT1. Nevertheless, information on the kinase-mediated phosphorylation of KAT1 and the target sites involved in the regulation of channel activity is scarce. In this study, we investi- gated the effect of the PKC-mediated phosphorylation of KAT1 expressed in Xenopus oocytes. On stimulation of the oocyte endogenous PKC by the application of Putative cyclic A B nucleotide binding domain (CNBD) 22 458 529 45 303 308 312 589 590 641 S S S T T S 44 125 T S T T S S T45A T308A S312A S589A S590A S641A T22A S44A S125A S529A 1 1.2 WT (no addition) 0 0.2 0.4 0.6 0.8 1 I/I control 1 1.2 0 0.2 0.4 0.6 0.8 1 I/I control 1.2 0 0.2 0.4 0.6 0.8 1 I/I control T458A WT (PMA) 10 20 30 37.50 Time (min) 10 20 30 37.50 Time (min) 10 20 30 37.50 Time (min) Fig. 3. Effects of mutations on possible phosphorylation sites on KAT1 currents. (A) Schematic representation of the consensus PKC phosphorylation sites at the cytosolic face of KAT1. (B) Changes in the current amplitudes of the different KAT1 mutants after PMA application. The characteristics of WT KAT1 in the presence and absence of PMA are displayed as broken lines. The mutants are divided into three groups: top panel, smaller degree of inhibition compared with WT; middle panel, WT-like behavior; bottom panel, larger degree of inhibition compared with WT. 0.2 0.4 0.6 0.8 1 1.2 I/I control Quintuple mutant WT (no addition) WT (PMA) 0 0102030 Time (min) 40 50 60 37.5 N.D. 0 10 20 30 T22A S44A T45A S125A T303A T308A S312A T458A S529A S589A S590A S641A WT Current inhibition by PMA (%) B A Quintuple mutant No addition Fig. 4. Quintuple mutations confer insensitivity to PKC activation. (A) Change in the current amplitudes of the quintuple KAT1 mutant KAT1-T308A-S312A-S589A-S590A-S641A after PMA application. The characteristics of WT KAT1 in the presence and absence of PMA are displayed as broken lines. (B) Inhibition of individual mutants by PMA application at 32 min (mean ± SEM, n = 3–5). *Student’s t-test, P < 0.05. N.D., no detectable currents. Phosphorylation of KAT1 channel A. Sato et al. 2322 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS PMA to the bath medium, the KAT1 current ampli- tude decreased more strongly than under control con- ditions. This indicates that phosphorylation by PKC has a downregulatory effect on KAT1. Subsequently, we pinpointed by in silico analyses 12 putative target sites for PKC in KAT1 and evaluated their role on channel regulation. Among the 12 Ser ⁄ Thr sites in KAT1, we identified experimentally six residues that were involved in the regulation by PKC (Figs 3 and 5). The behavior of the different channel mutants on PMA application could be divided into four groups: (a) loss of function; (b) increase in the inhibitory effect; (c) decrease in the inhibitory effect; (d) inhibi- tion comparable with WT. In the first case, the replacements T303A and T303D abolished the KAT1 current. It is possible that the mutation of Thr at position 303 immediately after the S6 segment may interfere with KAT1 channel gating, as illustrated for residues a few positions upstream [56]. In addi- tion, for other K + channels, it has been shown that the region immediately after the last transmembrane segment is strongly involved in channel gating [57– 61]. The inhibition of the KAT1-mediated K + current on PMA application may depend on a decline in the number of active channels or on a lower single-channel conductance, as the voltage-dependent characteristics were not affected by PMA application in oocytes injected with the WT KAT1 channel (as shown in Fig. 1B, C); notably, almost identical I–V characteris- tics were also observed in oocytes injected with mutants before and after the addition of PMA (data not shown). As the A. thaliana genome does not comprise a gene encoding a protein that is homologous to animal PKC, there is no evidence that the reduction in KAT1 by phosphorylation suggested in this study actually occurs in vivo in plant cells. Instead of PKC, in the genome sequence of A. thaliana, 34 different genes encoding CDPKs are present, which is currently recognized as a major group of Ca 2+ -stimulated pro- tein kinases [35]. A calcium-dependent kinase from Vicia faba was found to phosphorylate the KAT1 protein translated in vitro [46], and a CDPK from soybean decreased KAT1-mediated K + current ampli- tudes in Xenopus oocytes [8]. To date, several CDPK phosphorylation target sequences have been reported [62–64]. The two most classical motifs are S-X-R ⁄ K and R ⁄ K-X-X-S ⁄ T [54,55]. The S-X-R ⁄ K sequence is included in the optimal oligopeptide which may be phosphorylated by PKCs [53]. On the other hand, PKC may also recognize Ser ⁄ Thr in the sequence, as PKC recognizes preferentially substrates with a basic residue at position )3 [53]. KAT1 comprises 10 R ⁄ K- X-X-S ⁄ T motifs in the cytosolic N- and C-terminal regions. Among them, T308 and S641 are matching both motifs S ⁄ T-X-R ⁄ K as well as R-X-X-S ⁄ T (Figs 3 and 4). Interestingly, in a recent study, residue T308 was also identified as a target site for the Ca 2+ -indepen- dent ABA-activated SnRK2.6 kinase [21]. In addition, the SnRK2.6 kinase could phosphorylate T306 in an in vitro kinase assay. This evidence suggests that multi- ple protein kinases may participate in the regulation of KAT1 channel activity to respond to various Table 1. Percentage of current inhibition by PKC activation. Current inhibition of KAT1 and its mutants by PKC activation at 32 min after PMA (mean ± SEM, n = 3–6). Replacement of T303 by Ala or Asp led to a loss of KAT1 activity. Mutant Inhibition (%) n T22A 43.2 ± 7.2 4 S44A 36.9 ± 3.3 4 T45A 27.1 ± 2.9 4 a S125A 34.3 ± 5.6 3 T308A 20.6 ± 3.2 5 a S312A 17.0 ± 6.5 3 a T458A 52.5 ± 2.3 4 S529A 44.5 ± 0.7 3 S589A 28.4 ± 6.0 3 a S590A 23.9 ± 5.0 3 a S641A 24.9 ± 6.6 3 a T308A ⁄ S312A ⁄ S589A ⁄ S590A ⁄ S641A 14.9 ± 5.2 4 a WT 45.0 ± 5.6 3 Control 10.6 ± 4.5 3 WT + calphostin C 19.5 ± 4.8 4 a P < 0.05. K + KAT1 inhibition 45 308 589 590 641 S S S T T S Phosphorylation 306 303 T T 312 Fig. 5. Possible regulation of KAT1 channel activity by phosphoryla- tion via Ca 2+ -dependent ⁄ independent pathways. The target sites of PKC (CDPK) and ABA-activated SnRK2.6 identified in heterologous expression systems are indicated. T306 and T308 were possible target sites for the Ca 2+ -independent, ABA-activated SnRK2.6 kinase [21]. T45, T308, S312, S589, S590 and S641 were possible targets for PKC in Xenopus oocytes performed in this study. Replacement of T303 by Ala or Asp led to a loss of KAT1 activity. A. Sato et al. Phosphorylation of KAT1 channel FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS 2323 physiological signals (Fig. 5). Indeed, the conversion of T306 to Ala or Asp resulted in a loss of KAT1 activity in Xenopus oocyte and yeast expression systems [21]. In addition, after the replacement of T303 by Asp, no K + transport activity could be measured in oocytes. The C-terminal region after the last transmembrane region of S6 in plant K + channels is involved in chan- nel gating [56,65,66]. If the residue at position 303 can be recognized as a phosphorylation target site, the modification of T303 may lead to a loss of K + trans- port activity. Although, in this study, we took advantage of a sig- naling pathway in Xenopus oocytes to stimulate (by PMA treatment), an animal-specific PKC, the results obtained may have implications on signaling in plants. The application of PMA to plant tissues has been shown to alter the expression level of some genes [67–70]. This fact may indicate that, in plants, similar signaling pathways exist which connect the application of phorbol esters to the activation of certain kinases analogous to PKC. This is in line with other observa- tions. Inositol 1,4,5-trisphosphate (InsP 3 ) and Ca 2+ induce stomatal closure [71]. InsP 3 does not affect out- wardly rectifying K + channels [72,73], but inhibits only inwardly rectifying K + channels [72]. DAG is rapidly converted to PAs by DAG kinases in plants [41], and carrot CDPK, DcCPK1, and maize CDPK, ZmCK11, are activated by PAs and Ca 2+ [48,74]. The phosphoinositide-dependent protein kinase-1 spe- cifically binds PA [75]. In guard cell protoplasts, PA inhibits the activity of inwardly rectifying K + channels and also induces stomatal closure and inhib- its stomatal opening [76]. Through the serial trans- duction pathway, the end result is the alteration of the activity of inwardly rectifying K + channels by phosphorylation. It has been reported that exogenously supple- mented animal protein kinase A greatly retards the rundown rate of KAT1 [77,78]. In the same studies, it was also shown that PKC application was not effective in preventing rundown. This result is in line with our study demonstrating that PKC appli- cation has the inverse effect, and may be different from the phosphorylation mechanism involved in rundown. Taken together, our results suggest the existence of several PKC phosphorylation sites in the cytosolic region of KAT1. K + channel modulation, e.g. K + - uptake channel inhibition during stomatal closure, may occur via protein kinases which have PKC- like characteristics, such as CDPKs. Phospholipid signaling may be involved in the preceding signaling cascades. Materials and methods Channel expression in oocytes and electrical recordings The cDNAs encoding full-length KAT1 WT or its variants were amplified by a two-step PCR using HindIII site-con- taining sense primer and BamHI site-containing antisense primer (Table S1, see Supporting information) [49]. The HindIII-BamHI DNA fragments were ligated into the same sites of a modified pYES2 vector (Invitrogen, Carlsbad, CA, USA) for expression in oocytes and yeast [79]. Capped cRNAs were synthesized in vitro from NotI-linearized plas- mids using an in vitro transcription kit (Ambion, Austin, TX, USA). Xenopus oocytes were defolliculated using colla- genase and microinjected with either 1 or 2 ng of cRNAs after a 1–3 day incubation in Barth’s buffer containing 88 mm NaCl, 1 mm KCl, 0.41 mm CaCl 2 , 0.33 mm Ca(NO 3 ) 2 ,1mm MgSO 4 , 2.4 mm NaHCO 3 ,5mm Hepes and 50 lgÆmL )1 gentamicin sulfate (pH 7.3) at 18 °C. The two-electrode voltage clamp experiments were performed using a voltage clamp amplifier (AxoClamp 2B, Axon Instruments, Foster City, CA, USA) at room temperature in Xenopus laevis oocytes [49]. Microelectrodes contained 3 m KCl with a resistance of 0.3–1.0 MX. The bath solu- tion was 120 mm KCl, 1 mm MgCl 2 ,1mm CaCl 2 and 10 mm Hepes (pH 7.3). Time-dependent changes in current were recorded at –150 mV in single-step pulses every 30 s and in step voltage pulses ()30 to )170 mV with a 20 mV decrement) every 5.5 min. Step voltage pulses were applied from a holding potential of –40 mV, the duration of each pulse being 500 ms. Data acquisition and analysis were performed using pclamp 9.2 (Molecular Devices, Sunnyvale, CA, USA) and origin 5.0 software (Axon Instruments). Drug treatment and application PMA (Alexis Biochemicals, Lausen, Switzerland) and calphostin C (Alexis Biochemicals) were dissolved in dim- ethylsulfoxide as stocks and mixed with the recording solu- tion, reaching the final concentrations indicated in the figures [50]. PMA was applied to oocytes after initial measurements in its absence had been carried out. Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research (17078005, 20246044 and 20-08103 to N.U.) from MEXT Japan Ministry of Education, Culture, Sports, Science & Technology and JSPS (Japan Society for the Promotion of Science) as well as by the JSPS-CNR (National Research Council of Italy) Bilateral Program to N.U. and F.G. Phosphorylation of KAT1 channel A. Sato et al. 2324 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS References 1 Anderson JA, Huprikar SS, Kochian LV, Lucas WJ & Gaber RF (1992) Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomy- ces cerevisiae. Proc Natl Acad Sci USA 89, 3736–3740. 2 Schachtman DP, Schroeder JI, Lucas WJ, Anderson JA & Gaber RF (1992) Expression of an inward-rectifying potassium channel by the Arabidopsis KAT1 cDNA. Science, 258(cDNA), 1654–1658. 3 Nakamura RL, McKendree WL Jr, Hirsch RE, Sed- brook JC, Gaber RF & Sussman MR (1995) Expression of an Arabidopsis potassium channel gene in guard cells. Plant Physiol 109, 371–374. 4 Lebaudy A, Vavasseur A, Hosy E, Dreyer I, Leonhardt N, Thibaud JB, Very AA, Simonneau T & Sentenac H (2008) Plant adaptation to fluctuating environment and biomass production are strongly dependent on guard cell potassium channels. Proc Natl Acad Sci USA 105, 5271–5276. 5 Kinoshita T, Nishimura M & Shimazaki K (1995) Cytosolic concentration of Ca 2+ regulates the plasma membrane H + -ATPase in guard cells of fava bean. Plant Cell 7, 1333–1342. 6 Schroeder JI & Hagiwara S (1989) Cytosolic calcium regulates ion channels in the plasma membrane of Vicia faba guard cells. Nature 338, 427–430. 7 Sutton F, Paul SS, Wang XQ & Assmann SM (2000) Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes. Plant Physiol 124, 223–230. 8 Berkowitz G, Zhang X, Mercie R, Leng Q & Lawton M (2000) Co-expression of calcium-dependent protein kinase with the inward rectified guard cell K + channel KAT1 alters current parameters in Xenopus laevis oocytes. Plant Cell Physiol 41, 785–790. 9 Mori IC, Murata Y, Yang Y, Munemasa S, Wang YF, Andreoli S, Tiriac H, Alonso JM, Harper JF, Ecker JR et al. (2006) CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca 2+ -permeable channels and stomatal closure. PLoS Biol 4, e327. 10 Zhu SY, Yu XC, Wang XJ, Zhao R, Li Y, Fan RC, Shang Y, Du SY, Wang XF, Wu FQ et al. (2007) Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidop- sis. Plant Cell 19, 3019–3036. 11 Hamilton DW, Hills A, Kohler B & Blatt MR (2000) Ca 2+ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid. Proc Natl Acad Sci USA 97, 4967–4972. 12 Grabov A & Blatt MR (1999) A steep dependence of inward-rectifying potassium channels on cytosolic free calcium concentration increase evoked by hyperpo- larization in guard cells. Plant Physiol 119, 277– 288. 13 Li J & Assmann SM (1996) An abscisic acid-activated and calcium-independent protein kinase from guard cells of fava bean. Plant Cell 8, 2359–2368. 14 Mori IC & Muto S (1997) Abscisic acid activates a 48- kilodalton protein kinase in guard cell protoplasts. Plant Physiol 113, 833–839. 15 Li J, Wang XQ, Watson MB & Assmann SM (2000) Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase. Science 287, 300–303. 16 Mori IC, Uozumi N & Muto S (2000) Phosphorylation of the inward-rectifying potassium channel KAT1 by ABR kinase in Vicia guard cells. Plant Cell Physiol 41, 850–856. 17 Yoshida R, Hobo T, Ichimura K, Mizoguchi T, Takahashi F, Aronso J, Ecker JR & Shinozaki K (2002) ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis. Plant Cell Physiol 43, 1473–1483. 18 Mustilli AC, Merlot S, Vavasseur A, Fenzi F & Giraudat J (2002) Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14, 3089–3099. 19 Merlot S, Mustilli AC, Genty B, North H, Lefebvre V, Sotta B, Vavasseur A & Giraudat J (2002) Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation. Plant J 30, 601–609. 20 Yoshida R, Umezawa T, Mizoguchi T, Takahashi S, Takahashi F & Shinozaki K (2006) The regulatory domain of SRK2E ⁄ OST1 ⁄ SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis. J Biol Chem 281, 5310–5318. 21 Sato A, Sato Y, Fukao Y, Fujiwara M, Umezawa T, Shinozaki K, Hibi T, Taniguchi M, Miyake H, Goto DB et al. (2009) Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2 ⁄ OST1 ⁄ SnRK2.6 protein kinase. Biochem J 424, 439–448. 22 Nishizuka Y (1989) The Albert Lasker medical awards. the family of protein kinase c for signal transduction. J Am Med Assoc 262, 1826–1833. 23 Nishizuka Y (1995) Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9, 484–496. 24 Busch AE, Varnum MD, North RA & Adelman JP (1992) An amino acid mutation in a potassium channel that prevents inhibition by protein kinase C. Science 255, 1705–1707. 25 Covarrubias M, Wei A, Salkoff L & Vyas TB (1994) Elimination of rapid potassium channel inactivation by A. Sato et al. Phosphorylation of KAT1 channel FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS 2325 phosphorylation of the inactivation gate. Neuron 13, 1403–1412. 26 Barros F, Gomez-Varela D, Viloria CG, Palomero T, Giraldez T & de la Pena P (1998) Modulation of human erg K + channel gating by activation of a G protein-coupled receptor and protein kinase C. J Physiol 511 (Pt 2) 333–346. 27 Thomas D, Zhang W, Wu K, Wimmer AB, Gut B, Wendt-Nordahl G, Kathofer S, Kreye VA, Katus HA, Schoels W et al. (2003) Regulation of HERG potassium channel activation by protein kinase C independent of direct phosphorylation of the channel protein. Cardio- vasc Res 59, 14–26. 28 Mao J, Wang X, Chen F, Wang R, Rojas A, Shi Y, Piao H & Jiang C (2004) Molecular basis for the inhibi- tion of G protein-coupled inward rectifier K + channels by protein kinase C. Proc Natl Acad Sci USA 101, 1087–1092. 29 Asaoka Y, Nakamura S, Yoshida K & Nishizuka Y (1992) Protein kinase C, calcium and phospholipid deg- radation. Trends Biochem Sci 17, 414–417. 30 Ebinu JO, Bottorff DA, Chan EY, Stang SL, Dunn RJ & Stone JC (1998) RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol- binding motifs. Science 280, 1082–1086. 31 Ron D & Kazanietz MG (1999) New insights into the regulation of protein kinase C and novel phorbol ester receptors. FASEB J 13, 1658–1676. 32 Sakane F, Imai S, Yamada K, Murakami T, Tsushima S & Kanoh H (2002) Alternative splicing of the human diacylglycerol kinase delta gene generates two isoforms differing in their expression patterns and in regulatory functions. J Biol Chem 277, 43519–43526. 33 Tognon CE, Kirk HE, Passmore LA, Whitehead IP, Der CJ & Kay RJ (1998) Regulation of RasGRP via a phorbol ester-responsive C1 domain. Mol Cell Biol 18, 6995–7008. 34 Harper JF, Binder BM & Sussman MR (1993) Calcium and lipid regulation of an Arabidopsis protein kinase expressed in Escherichia coli. Biochemistry 32, 3282– 3290. 35 Cheng SH, Willmann MR, Chen HC & Sheen J (2002) Calcium signaling through protein kinases. The Arabid- opsis calcium-dependent protein kinase gene family. Plant Physiol 129, 469–485. 36 Hrabak EM, Chan CW, Gribskov M, Harper JF, Choi JH, Halford N, Kudla J, Luan S, Nimmo HG, Sussman MR et al. (2003) The Arabidopsis CDPK- SnRK superfamily of protein kinases. Plant Physiol 132, 666–680. 37 Spitaler M & Cantrell DA (2004) Protein kinase C and beyond. Nat Immunol 5 , 785–790. 38 Rajagopal S, Fang H, Patanavanich S, Sando JJ & Kamatchi GL (2008) Protein kinase C isozyme-specific potentiation of expressed Cav2.3 currents by acetyl- beta-methylcholine and phorbol-12-myristate, 13-ace- tate. Brain Res 1210, 1–10. 39 Tasma IM, Brendel V, Whitham SA & Bhattacharyya MK (2008) Expression and evolution of the phosphoino- sitide-specific phospholipase C gene family in Arabidopsis thaliana. Plant Physiol Biochem 46, 627–637. 40 Gomez-Merino FC, Brearley CA, Ornatowska M, Abdel-Haliem ME, Zanor MI & Mueller-Roeber B (2004) AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2-arachi- donoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression. J Biol Chem 279, 8230–8241. 41 Laxalt AM & Munnik T (2002) Phospholipid signalling in plant defence. Curr Opin Plant Biol 5, 332–338. 42 Harper JF, Breton G & Harmon A (2004) Decoding Ca 2+ signals through plant protein kinases. Annu Rev Plant Biol 55, 263–288. 43 Chandok MR & Sopory SK (1998) ZmcPKC70, a pro- tein kinase C-type enzyme from maize. Biochemical characterization, regulation by phorbol 12-myristate 13-acetate and its possible involvement in nitrate reductase gene expression. J Biol Chem 273, 19235– 19242. 44 Deswal R, Chowdhary GK & Sopory SK (2004) Purifi- cation and characterization of a PMA-stimulated kinase and identification of PMA-induced phosphorylation of a polypeptide that is dephosphorylated by low tempera- ture in Brassica juncea. Biochem Biophys Res Commun 322, 420–427. 45 Loog M, Toomik R, Sak K, Muszynska G, Jarv J & Ek P (2000) Peptide phosphorylation by calcium-depen- dent protein kinase from maize seedlings. Eur J Bio- chem 267, 337–343. 46 Li J, Lee YR & Assmann SM (1998) Guard cells pos- sess a calcium-dependent protein kinase that phospho- rylates the KAT1 potassium channel. Plant Physiol 116 , 785–795. 47 Pearson RB & Kemp BE (1991) Protein kinase phos- phorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol 200, 62–81. 48 Farmer PK & Choi JH (1999) Calcium and phospho- lipid activation of a recombinant calcium-dependent protein kinase (DcCPK1) from carrot (Daucus carota L.). Biochim Biophys Acta 1434, 6–17. 49 Uozumi N, Gassmann W, Cao Y & Schroeder JI (1995) Identification of strong modifications in cation selectiv- ity in an Arabidopsis inward rectifying potassium chan- nel by mutant selection in yeast. J Biol Chem 270, 24276–24281. 50 Zhang L, Lee JK, John SA, Uozumi N & Kodama I (2004) Mechanosensitivity of GIRK channels is medi- ated by protein kinase C-dependent channel–phosphati- dylinositol 4,5-bisphosphate interaction. J Biol Chem 279, 7037–7047. Phosphorylation of KAT1 channel A. Sato et al. 2326 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS 51 Kishimoto A, Nishiyama K, Nakanishi H, Uratsuji Y, Nomura H, Takeyama Y & Nishizuka Y (1985) Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3¢:5¢-monophosphate- dependent protein kinase. J Biol Chem 260, 12492– 12499. 52 Woodgett JR, Gould KL & Hunter T (1986) Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for sub- strate recognition requirements. Eur J Biochem 161, 177–184. 53 Nishikawa K, Toker A, Johannes FJ, Songyang Z & Cantley LC (1997) Determination of the specific sub- strate sequence motifs of protein kinase C isozymes. J Biol Chem 272, 952–960. 54 Roberts DM & Harmon AC (1992) Calcium-modulated proteins: targets of intracellular calcium signals in higher plants. Annu Rev Plant Physiol Plant Mol Biol 43, 375–414. 55 Neumann GM, Thomas I & Polya GM (1996) Identifi- cation of the site on potato carboxypeptidase inhibitor that is phosphorylated by plant calcium-dependent protein kinase. Plant Sci 114, 45–51. 56 Gajdanowicz P, Garcia-Mata C, Gonzalez W, Morales- Navarro SE, Sharma T, Gonzalez-Nilo FD, Gutowicz J, Mueller-Roeber B, Blatt MR & Dreyer I (2009) Dis- tinct roles of the last transmembrane domain in control- ling Arabidopsis K + channel activity. New Phytol 182, 380–391. 57 Jiang Y, Lee A, Chen J, Cadene M, Chait BT & MacKinnon R (2002) The open pore conformation of potassium channels. Nature 417, 523–526. 58 Sansom MS, Shrivastava IH, Bright JN, Tate J, Capener CE & Biggin PC (2002) Potassium channels: structures, models, simulations. Biochim Biophys Acta 1565, 294–307. 59 Kuo A, Gulbis JM, Antcliff JF, Rahman T, Lowe ED, Zimmer J, Cuthbertson J, Ashcroft FM, Ezaki T & Doyle DA (2003) Crystal structure of the potassium channel KirBac1.1 in the closed state. Science 300, 1922–1926. 60 Long SB, Campbell EB & Mackinnon R (2005) Crystal structure of a mammalian voltage-dependent Shaker family K + channel. Science 309, 897–903. 61 Riedelsberger J, Sharma T, Gonzalez W, Gajdanowicz P, Morales-Navarro SE, Garcia-Mata C, Mueller-Roe- ber B, Gonza ´ lez-Nilo FD, Blatt MR & Dreyer I (2010) Distributed structures underlie gating differences between the K in channel KAT1 and the K out channel SKOR. Mol Plant 3, 236–245. 62 Sebastia ` CH, Hardin SC, Clouse SD, Kieber JJ & Huber SC (2004) Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate. Arch Biochem Biophys 428, 81–91. 63 Klimecka M & Muszynska G (2007) Structure and functions of plant calcium-dependent protein kinases. Acta Biochim Pol 54, 219–233. 64 Vlad F, Turk BE, Peynot P, Leung J & Merlot S (2008) A versatile strategy to define the phosphorylation pref- erences of plant protein kinases and screen for putative substrates. Plant J 55, 104–117. 65 Cherel I, Michard E, Platet N, Mouline K, Alcon C, Sentenac H & Thibaud JB (2002) Physical and func- tional interaction of the Arabidopsis K + channel AKT2 and phosphatase AtPP2CA. Plant Cell 14, 1133–1146. 66 Michard E, Dreyer I, Lacombe B, Sentenac H & Thibaud JB (2005) Inward rectification of the AKT2 channel abolished by voltage-dependent phosphoryla- tion. Plant J 44 , 783–797. 67 Baudouin E, Charpenteau M, Ranjeva R & Ranty B (1999) Involvement of active oxygen species in the regu- lation of a tobacco defence gene by phorbol ester. Plant Sci 142, 67–72. 68 Chandok MR, Sopory SK & Oelmuller R (2001) Cytoplasmic kinase and phosphatase activities can induce PsaF gene expression in the absence of func- tional plastids: evidence that phosphorylation ⁄ dephos- phorylation events are involved in interorganellar crosstalk. Mol Gen Genet 264, 819–826. 69 Wostemeyer A & Oelmuller R (2003) The promoter of the spinach PsaF gene for the subunit III of the pho- tosystem I reaction center directs beta-glucuronidase gene expression in transgenic tobacco roots. Implica- tion of the involvement of phospholipases and protein kinase C in PsaF gene expression. J Plant Physiol 160, 503–508. 70 Chandok MR & Sopory SK (1996) Phosphoryla- tion ⁄ dephosphorylation steps are key events in the phy- tochrome-mediated enhancement of nitrate reductase mRNA levels and enzyme activity in maize. Mol Gen Genet 251, 599–608. 71 Blatt MR, Thiel G & Trentham DR (1990) Reversible inactivation of K + channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-tris- phosphate. Nature 346, 766–769. 72 Gilroy S, Read ND & Trewavas AJ (1990) Elevation of cytoplasmic calcium by caged calcium or caged inositol triphosphate initiates stomatal closure. Nature 346, 769– 771. 73 Schroeder JI (1988) K + transport properties of K + channels in the plasma membrane of Vicia faba guard cells. J Gen Physiol 92, 667–683. 74 Szczegielniak J, Klimecka M, Liwosz A, Ciesielski A, Kaczanowski S, Dobrowolska G, Harmon AC & Muszynska G (2005) A wound-responsive and phospho- lipid-regulated maize calcium-dependent protein kinase. Plant Physiol 139, 1970–1983. 75 Deak M, Casamayor A, Currie RA, Downes CP & Alessi DR (1999) Characterisation of a plant 3-phos- A. Sato et al. Phosphorylation of KAT1 channel FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS 2327 [...]...Phosphorylation of KAT1 channel 76 77 78 79 A Sato et al phoinositide-dependent protein kinase- 1 homologue which contains a pleckstrin homology domain FEBS Lett 451, 220–226 Jacob T, Ritchie S, Assmann SM & Gilroy S (1999) Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity Proc Natl Acad Sci USA 96, 12192–12197 Hoshi T (1995) Regulation of voltage dependence of the KAT1 channel. .. for the Arabidopsis 2328 thaliana Na+ ⁄ K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters Proc Natl Acad Sci USA 98, 6488–6493 Supporting information The following supplementary material is available: Table S1 Primer sequences for plasmid construction This supplementary material can be found in the online version of this article Please note: As a service to our authors and... channel by intracellular factors J Gen Physiol 105, 309–328 Tang XD & Hoshi T (1999) Rundown of the hyperpolarization-activated KAT1 channel involves slowing of the opening transitions regulated by phosphorylation Biophys J 76, 3089–3098 Kato Y, Sakaguchi M, Mori Y, Saito K, Nakamura T, Bakker EP, Sato Y, Goshima S & Uozumi N (2001) Evidence in support of a four transmembrane-poretransmembrane topology... readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS . Modulation of the Arabidopsis KAT1 channel by an activator of protein kinase C in Xenopus laevis oocytes Aiko Sato 1 , Franco Gambale 2 , Ingo Dreyer 3 and. recognized by animal PKCs [45]. On the basis of these facts, we examined the effect of PKC activation on KAT1 channel activity by the PKC activator PMA in