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Originally published In Press as doi:10.1074/jbc.M708213200 on November 20, 2007

J. Biol. Chem., Vol. 283, Issue 4, 1911-1920, January 25, 2008
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Characterization of a Tobacco TPK-type K+ Channel as a Novel Tonoplast K+ Channel Using Yeast Tonoplasts*Formula

Shin Hamamotoa, Junichiro Maruib, Ken Matsuokac, Kyohei Higashid, Kazuei Igarashid, Tsuyoshi Nakagawae, Teruo Kurodaf, Yasuo Morig, Yoshiyuki Muratah, Yoichi Nakanishii, Masayoshi Maeshimai, Isamu Yabej, and Nobuyuki Uozumia1

From the aDepartment of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, Japan, the bNew Industry Creation Hatchery Center, Tohoku University, Aobayama 6-6-10, Sendai 980-8579, Japan, the cLaboratory of Plant Nutrition, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, the dGraduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan, the eDepartment of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan, the fDepartment of Genome Applied Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan, the gDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Kyoto 615-8510, Japan, the hGraduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Okayama 700-8530, Japan, the iLaboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan, and the jInstitute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

Received for publication, October 3, 2007 , and in revised form, November 19, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The tonoplast K+ membrane transport system plays a crucial role in maintaining K+ homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K+ channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased ~2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K+ over Na+. NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca2+, but a cytosolic concentration of 45 µM Ca2+ resulted in a 2-fold increase in the amplitude of the K+ current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K+ currents. These results show that NtTPK1 is a novel tonoplast K+ channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plants take up potassium (K+) from the soil and plant cells accumulate K+ to regulate the membrane potential and turgor pressure. The cytoplasmic K+ concentration is tightly controlled at ~100 mM (1). Vacuoles are major subcellular reservoirs for controlling K+ homeostasis in plant cells (1). During cell expansion, for instance during stomata opening or cell growth, tonoplast transport system moves K+ into the vacuole, whereas, under conditions of salinity stress, K+ is replaced by Na+ (2–5).

Several kinds of genes encoding K+ channels and K+ transporters have been identified in the Arabidopsis thaliana genome, and their function and tissue and cellular distribution have been extensively studied. They consist of two families, the Shaker-type channels, with six hydrophobic transmembrane domains and a single pore domain, and the two-pore K+ channel (TPK)2 family, with four transmembrane and two pore domains. Six different genes encoding TPK-type channels are present in A. thaliana. AtTPK4 is targeted to the plasma membrane (6), while the other five, AtTPK1, AtTPK2, AtTPK3, AtTPK5, and AtKCO3, are localized in the vacuolar membrane (7). AtTPK1 and AtTPK4 have been functionally characterized. AtTPK4 shows a voltage-independent K+ profile in Xenopus laevis ooctyes and in yeast, and the K+ current is inhibited by extracellular Ca2+ and reduced by shifting the cytosolic pH from 7.5 to 6.3, but is not affected by the external pH (6). AtTPK1 has different properties to AtTPK4 (7, 8). In the yeast and plant tonoplast membrane, cytosolic Ca2+ enhances AtTPK1 activity, and the optimum cytosolic pH is 6.5 (6, 9, 10).

Stomata opening and closing is closely associated with membrane transport of K+. In a study on guard cells, three kinds of tonoplast channels, SV, FV, and VK, were distinguished by their K+ current profiles (11). On the basis of patch clamp recording, the SV channel was recently shown to be AtTPC1 in Arabidopsis (12), and the VK channel was reported to be AtTPK1 (9, 13). Further studies on FV, detailed data on SV and VK, and the identification of other TPK-type channels are needed to understand vacuolar K+ transport.

To gain insights into the biophysical properties of the TPK-type channels, we isolated the homologous genes from BY-2 cultured tobacco cells and Nicotiana tabacum cv. SR1. The protein sequences revealed a novel feature of the K+ filter in these channels. We also examined promoter activity and the intracellular localization of the channel and performed accurate electrophysiological measurements of NtTPK1 using the vacuolar membrane from enlarged mutant yeast cells with very low background currents, unlike the plant vacuolar membrane. The results showed that NtTPK1 was a novel tonoplast channel with characteristics different from those of previously characterized tonoplast channels.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material—Tobacco BY-2 (N. tabacum cv. Bright Yellow 2) cells were grown in BY-2 growth medium (modified Linsmaier and Skoog medium) (14) at 26 °C in the dark with constant shaking and were maintained by subculturing 1 ml of BY-2 cells into 100 ml of fresh medium every 7 days.

Cloning of NtTPK1 Full-length cDNAs from N. tabacum cv. SR1 and BY-2 Cells—A search of the EST data base of tobacco BY-2 cells for sequence homology with the known TPK family (Transcriptome Analysis of BY-2 revealed a 684-bp partial clone which was a homologue of A. thaliana TPK2 (known as KCO2). Total RNA was isolated from N. tabacum cv. SR1 and BY-2 cells using a standard guanidine thiocyanate method (15). To obtain the 5'-end of the NtTPK1 cDNA, 5'-RACE was performed as described previously (16). Polyadenylated RNA was reverse-transcribed using three different NtTPK1-specific antisense primers, 5'-GGATGCAAAGATACGACCGG-3', 5'-CGCCATACCCCACAGTGGTAAC-3', and 5'-CAAAATGCGTAACCGG-3', and RACE amplification primers. First strand cDNA was synthesized using SuperScriptTM III Reverse Transcriptase (Invitrogen) and was used as template DNA for the isolation of full-length NtTPK1 cDNA by PCR reactions using primer 5'-ATGGAGAAAGAGCCTCTTC-3', starting at the start codon, and primer 5'-TTAATGGTGGTGGCTTTCC-3', starting at the first stop codon.

Expression of NtTPK1 in Saccharomyces cerevisiae and Escherichia coli NtTPK1 was amplified by PCR using the EcoRI site-containing sense primer 5'-CAGGAATTCATGGAGAAAGAGCCTCTTC-3' and the SalI site-containing antisense primer 5'-CAGGTCGACTTAATGGTGGTGGCTTTCC-3'. The PCR fragment was then digested with EcoRI and SalI and ligated into the corresponding sites of the yeast expression vector pKT10 (17, 18). NtTPK1 was ligated into the BamHI and PstI cloning sites in pPAB404 (19) and the resulting plasmid introduced into E. coli strain LB2003, which lacks the three K+ uptake systems Trk, Kup, and Kdp (20). Growth tests of the plasmid-containing E. coli LB2003 at different K+ concentrations were carried out as described previously (21–23).

Generation of NtTPK1-overexpressing Tobacco BY-2 Transgenic Lines—Full-length cDNA for NtTPK1 was amplified by PCR using primers 5'-CACCATGGAGAAAGAGCCTCTTC-3' and 5'-TTAATGGTGGTGGCTTTCC-3' and subcloned into the pENTR/D TOPO vector (Invitrogen). The cDNA was then transferred into the GATEWAY destination vector pGWB2 containing the CAMV 35S promoter by an LR Clonase reaction (Invitrogen) (24). The [35S]NtTPK1 construct was introduced into A. tumefaciens EHA101 by electroporation (25). Stable transformation of BY-2 cells mediated by A. tumefaciens (strain EHA101) was carried out as described by Matsuoka and Nakamura (26).

Cloning of the NtTPK1 Promoter Region for Measurement of Promoter GUS Activity—Tobacco genomic DNA was extracted from tobacco leaves by the CTAB method (27) and used as the template for the PCR. To isolate the NtTPK1 promoter region, thermal asymmetric interlaced (TAIL) PCR was performed (28). Three nested primers hybridizing to the NtTPK1 cDNA were used: the 1st PCR primer, 5'-GTCAAGAGCAGTGGCAGAAGCTTC-3'; 2nd PCR primer, 5'-CAGAGGTACCAAAGATAAGGCGTTC-3'; and 3rd PCR primer, 5'-TGTTCTGGTGCTGACATAAGG-3'. To confirm the DNA sequence of the NtTPK1 promoter region in the genomic DNA, three upstream primers, 5'-CGTTTATATCGTGTCAACCTTTG-3',5'-GCGTTCTTGAATCCAAACGAC-3', and 5'-GCTGGGAGAGTCCTATACCGC-3', were used with the downstream primer 5'-TATGGGGTTTCGCCGGAAAACGG-3'. The promoter sequence was subcloned into the pENTR/D TOPO vector and transferred to the GATEWAY destination vector pGWB3 (24). pGWB3 vector harboring the promoter sequence was used to transform BY-2 cells as described above. To measure promoter activity, fluorimetric determination of GUS activity was performed as reported previously (29), using 4-methylumbelliferyl-β-D-glucuronide as substrate. BY-2 cells grown in BY-2 growth medium for 5 days were transferred for 10 min to BY-2 growth medium containing 250 mM NaCl or 500 mM mannitol, harvested by centrifugation, rapidly frozen in liquid nitrogen, and stored at –80 °C until analyzed. Three replicates were used for each treatment. GUS activity was measured at an excitation wavelength of 365 nm and emission wavelength of 455 nm on a spectrofluorometer (JASCO, Japan). Protein was determined by the method of Bradford (30), and the specific GUS activity determined as the rate of increase in fluorescence of 4-methylumbelliferone (4-MU) divided by the protein concentration (pmol of 4-MU/h/µg protein).

Quantitative Real-time PCR Assay—Expression was measured in different tissues of N. tabacum cv. SR1 using One Step SYBR RT-PCR (Takara, Japan), a method that relies on real-time monitoring of the release of a fluorescent reporter dye (SYBR-Green I) as the PCR product accumulates in the reaction mix (31). The cDNA was amplified using an ABI Prism 7500 (Applied Biosystems). Amplification of tubulin cDNA under identical conditions was performed as an internal control to normalize cDNA levels.

Preparation of BY-2 and Yeast Vacuolar Membranes—Vacuolar membranes were prepared from BY-2 cells as described previously (32), except for a few modifications. All steps were performed at 4 °C. The cells were ground in a mortar with liquid nitrogen and grinding medium (0.25 mM sorbitol, 1 mM MgCl2, 2 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 1% (w/v) PVP-40, and 50 mM Tris acetate, pH 7.5). The frozen cell homogenates were thawed and centrifuged at 5,000 x g for 10 min, then the supernatant was centrifuged at 120,000 x g for 50 min, and the final precipitate suspended in 0.75 mM sucrose in 20 mM Tris acetate, pH 7.5, 2 mM dithiothreitol, 1 mM EGTA, and 1 mM MgCl2 (Tris-DEM) (microsomal membrane fraction). Further purification of vacuolar membranes was carried out by floating centrifugation. The microsomal membrane suspension was placed in a centrifuge tube and overlaid with the same volume of Tris-DEM buffer containing 0.25 mM sorbitol. After centrifugation at 120,000 x g for 30 min, the vacuolar membrane vesicles, which formed a band at the interface between the two solutions, were collected and suspended in 15 ml of Tris-DEM buffer containing 0.25 mM sorbitol. The suspension was centrifuged at 120,000 x g for 30 min and the pellet suspended in a small volume of the same buffer. Yeast vacuoles were prepared using the floating centrifugation method (33, 34). For immunoblotting, NtTPK1-transformed S. cerevisae strain BJ5458 (Mat{alpha}, ura3–52, trp1, lys2–801, leu2{Delta}1, his3{Delta}200, pep4::HIS3, prb{Delta}1.6R can1, GAL), lacking two major vacuolar proteases (17), was used.

Immunoblotting—An anti-NtTPK1 antibody was raised against a synthetic peptide containing the sequence NH2-CGRITLADLMESHHH-COOH from the C terminus of NtTPK1 (Operon Biotechnologies, Japan). Antibodies against YVC1, a marker for the S. cerevisiae tonoplast, were raised against the peptide NH2-CNLTAVITDLLEKLDIKDKKECOOH located at the C terminus of YVC1 (35). Polyclonal antibodies raised against v-PPase (34), Sec61 alpha (36), or PAQ2 (plasma membrane aquaporin of Raphanus sativus) (37) were used to identify the BY-2 cell tonoplast, plasma membrane, or endoplasmic reticulum (ER) membrane, respectively. Proteins were electrophoresed on SDS-10% polyacrylamide gels, then transferred to polyvinylidene fluoride membranes. The polyvinylidene difluoride membrane was blocked with 10% skim milk in PBS-T (140 mM NaCl, 16.0 mM Na2HPO4, 2.00 mM KH2PO4, 3.75 mM KCl, 0.1% Tween 20) (blocking buffer) for 1 h at room temperature, then incubated for 1 h at room temperature with primary antibody (1:4000 in blocking buffer), washed three times with PBS-T, incubated for 30 min at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare) (1:5000 in blocking buffer), and subjected to chemiluminescence detection (ECL, Amersham Biosciences). Chemiluminescence signals on the polyvinylidene difluoride membrane were recorded using an LAS 3000 imaging system (Fujifilm, Japan).

Generation of the YVC1 Knock-out Strain—Deletion of the YVC1 gene in the strain BJ5458 was achieved by homologous recombination of the integration cassette with the LEU2 marker, amplified from the plasmid pUG73 (38). The linearized LEU2 PCR product was used for yeast transformation as described previously (39). We named the YVC1-deleted strain SH1006.

Preparation of Giant Yeast Cells and Isolation of Giant Yeast Vacuoles—Preparation of giant yeast from the yvc1 mutant. SH1006 was performed as described previously (34, 40). Briefly, at the early log phase (OD660 of 0.2), 1 ml of cells was incubated for 10 min at 30 °C in 0.1 M Tris-HCl, pH 9.4, 50 mM 2-mercaptoethanol and 0.1 M glucose, then for 15 min at 30 °C with 1 mg/ml of Zymolyase 20T in 1 M sorbitol, 2% (w/v) glucose, 50 mM Tris-HCl, pH 7.5, 0.17% yeast nitrogen base without amino acids and ammonium sulfate, and 0.25x dropout solution composed of all amino acids and adenines (17). The spheroplasts formed were obtained as described previously (34). Giant vacuoles were prepared from giant spheroplasts by the osmotic shock method with the following modifications (34, 40). The spheroplasts were concentrated by floating centrifugation at 1,000 x g for 5 min, then diluted with 5 volumes of a hypotonic solution (0.15 M sorbitol, 0.1 M KCl, 1 mM MgCl2 20 mM Tris-MES, pH 7.5) in the recording chamber of the patch-clamp apparatus.


Figure 1
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  		FIGURE 1.
Amino acid sequences of four TPK family channels from tobacco plant and cultured BY-2 tobacco cells. NtTPK1 cDNA was isolated from N. tabacum cv. SR1 and BY2 cells. The four predicted transmembrane segments (M1–M4) and two putative pore domains (P1 and P2) are shown. The EF-hand at the C terminus is shown as an oval. NtTPK1a, NtTPK1b, and NtTPK1c cDNAs were isolated from BY2 cells. The K+ channel signature sequence GYG in P2 is replaced by GHG or VHG in NtTPK1b or NtTPK1c, respectively.

 
Electrophysiological Experiments—For electrophysiological analysis, NtTPK1 was expressed from the pKT10 plasmid in the YVC1-lacking yeast strain SH1006. Currents were recorded in the whole vacuole mode, as described previously (40, 41). All experiments were carried out using the standard patch-clamp technique at 25 °C.

Measurement of Polyamine Levels in Tobacco Plants—Different tissues (leaf, shoot, and root) collected from 3–4-week-old tobacco plants were ground in an Eppendorf tube and extracted for 2 h at 70°C with 5% (w/v) trichloroacetic acid. Levels of different polyamines were determined by HPLC as described previously (42). Protein was determined using the Bradford assay (30).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of NtTPK1—We searched the EST data base of tobacco BY-2 cells for sequence homology with the known TPK family (43) and identified a partial cDNA homologous sequence in the data base and isolated the upstream cDNA by 5'-RACE and PCR. The isolated tobacco homologous gene was named NtTPK1. The NtTPK1 cDNA contained an open reading frame of 1284 bp encoding a 428-amino acid protein (47.6 kDa) (GenBankTM accession number EU161633 [GenBank] ) (Fig. 1). In a further cloning approach, we isolated three more cDNAs from BY-2 cells and one from N. tabacum cv. SR1. The sequence of the cDNA from N. tabacum cv. SR1 was identical to the NtTPK1 sequence in the data base. The three sequences from BY-2 cells contained some amino acid differences compared with that of NtTPK1 and were named NtTPK1a, NtTPK1b, and NtTPK1c (Fig. 1). These different sequences were caused by an amphidiploid nature of tobacco plant. N. tabacum cv. SR1 is an amphidiploid species derived from ancestors related the Nicotiana sylvestris and Nicotiana tomentosiformis, which are both diploid species. Analysis of the predicted amino acid sequence for NtTPK1 showed 62, 60, and 60% identity with those of A. thaliana AtTPK2 (formerly KCO2), AtTPK3 (formerly KCO6), and AtTPK5 (formerly KCO5), respectively; in contrast, the percentage identity with AtTPK1 and AtTPK4 was 39 and 40%, respectively (supplemental Fig. 1. The phylogenetic tree is shown in supplemental Fig. 2.) The hydropathy profile of NtTPK1, generated using the Kyte and Doolittle algorithm (44) predicted four potential transmembrane domains, designated M1–M4 (Fig. 1). The predicted protein sequence contained two pore (P) domains, P1, located between M1 and M2, and P2, located between M3 and M4. In the two P domains of NtTPK1, the K+ channel-selective motif (TXGYGD) (45, 46) was conserved, whereas in NtTPK1, NtTPK1b, and NtTPK1c, several amino acid substitutions were found. Interestingly, NtTPK1b and NtTPK1c contained, respectively, VHG and GHG at the GYG site in P2. Analysis of the NtTPK1 sequence using the PROSITE pattern search data base showed a single EF-hand motif in its C-terminal region. The EF-hand, a structure common to several calcium-binding proteins, consists of two perpendicular 10–12-residue {alpha} helices separated by a 12-residue loop region, forming a single calcium-binding site (helix-loop-helix) (47). AtTPK1, AtTPK2, and AtTPK3 contain one (AtTPK2 and 3) or two (AtTPK1) EF-hands located in the C-terminal domain, while AtTPK4 has none (6).


Figure 2
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  		FIGURE 2.
Complementation of the K+ uptake-deficient E. coli strain by NtTPK1s. A, growth of E. coli LB2003 harboring NtTPK1, NtTPK1a, NtTPK1b, NtTPK1c, or the Arabidopsis K+ channel KAT1 on synthetic solid medium containing 15 mM KCl. B, top, sequence comparison of P1 of NtTPK1, Y193H, and P1-AAA and P2 of NtTPK1, Y309H, P2-AAA. Bottom, growth of E. coli LB2003 harboring NtTPK1, Y193H, P1-AAA, Y309H, or the double replacements P1-Y193H/P2-Y309H, and P1-AAA/P2-AAA on synthetic solid medium containing 15 mM KCl. C, model of the dimerization of NtTPK1. P1 containing AAA in the signature sequence results in lack of K+ transport, but P2 containing AAA gives a functional K+ channel.

 
Heterologous Expression of NtTPK1 in E. coli—We expressed NtTPK1 in X.laevis oocyte and the K+ uptake-deficient S. cerevisiae CY162, but did not observe any K+ transport activity (data not shown). This suggested that NtTPK1 might not be targeted to the plasma membrane, but to a subcellular membrane. Functional expression of plant K+ transporters in E. coli has been described previously (21). This expression system allows measurement of the K+ uptake activity of plant endosome membrane-associated K+ channels/transporters (23). NtTPK1, NtTPK1a, NtTPK1b, and NtTPK1c were expressed in E. coli strain LB2003, which is deficient in all three major K+ uptake systems (20) and the growth of the cells was examined at limiting K+ concentrations. As shown in Fig. 2A, all four sets of cells grew as well as LB2003 cells expressing the Arabidopsis K+ channel KAT1 (pPAB404-KAT1), whereas cells transformed with empty vector (pPAB404) did not grow. These data suggested that NtTPK1, NtTPK1a, NtTPK1b, and NtTPK1c could take up K+ in the E. coli mutant.


Figure 3
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  		FIGURE 3.
Expression profile of NtTPK1. A, schematic representation of the NtTPK1 promoters isolated by TAIL-PCR from N. tabacum cv.SR1 genomic DNA. B, NtTPK1pro:GUS-transformed BY2 cells and non-transgenic BY2 cells were collected after 5 days' incubation and GUS activity was measured. C, NtTPK1pro:GUS transformed BY2 cells were transferred for 10 min to hyperosmotic medium containing 0.25 M NaCl or 0.5 M mannitol, then GUS activity was measured. D, quantitative real-time PCR was used to determine the level of expression of NtTPK1 in different tissues of N. tabacum cv. SR1. NtTUBLIN was used as an internal control. All data are expressed as the mean ± S.E. (n = 3).

 
The ability of NtTPK1b and NtTPK1c to rescue the K+ uptake deficiency of E. coli suggested that the second signature sequence was not necessary for K+ uptake by two-pore K+ channels. To assess this hypothesis, we introduced substitutions in the GYG sequence in P1 and/or P2 of NtTPK1 (Fig. 1A); these were Y193H (P1-Y193H), G192A+Y193A+G194A (P1-AAA), and Y309H in P1 (P2-Y309H) and G308A+Y309A+G310A in P2 (P2-AAA), and the two double replacements, P1-Y193H/P2-Y309H and P1-AAA/P-2AAA (Fig. 2, B and C). Fig. 2B shows that P2-Y309H and P2-AAA allowed growth of LB2003, whereas P1-Y193H, P1-AAA, P1-Y193H/P2-Y309H, and P1-AAA/P-2AAA did not complement the K+ uptake deficiency. These results indicated that the first selectivity filter of the GYG sequence is absolutely required, but the second less important for K+ passage in NtTPK1 (Fig. 2C).

Osmotic Stress-dependent Expression of NtTPK1 and Tissue Specificity of Expression—Three distinct NtTPK1 promoter sequences were isolated from N. tabacum cv. SR1 genomic DNA using the TAIL-PCR method. The length of the three sequences upstream of the NtTPK1 initiation codon was 1,101, 1,296, and 1,964 bp, and they were designated as promoter-A, promoter-B, and promoter-C, respectively. Promoter-A and promoter-B shared the same 327 bp sequence (–327 to –1) and all three promoters contained the same 168 bp (–168 to –1) of the genomic DNA sequence lying immediately upstream of the initiation codon (Fig. 3A). To evaluate their promoter activity, all three were individually inserted into a plant expression vector containing the GUS reporter gene and the resultant constructs introduced into BY-2 cells. As shown in Fig. 3B, promoter-A resulted in 3 times more GUS activity than promoter-B, while promoter C resulted in GUS activity comparable to the background levels seen in untransformed BY-2. Next, we assessed the gene expression pattern in response to salt stress and hyperosmotic shock. When transgenic BY-2 cells harboring promoter-A or promoter-B were treated with 0.25 M NaCl or 0.5 M mannitol, both sets of cells showed a 2–3-fold increase in GUS activity compared with the untreated transgenic lines (Fig. 3C), showing that both promoters could be activated in response to salt stress or osmotic shock. We also examined the tissue-specific expression pattern of NtTPK1s by quantitative RT-PCR using a primer which does not discriminate between the 4 forms and found that mRNAs coding for NtTPK1s were expressed in flower, leaves, shoot, and roots of N. tabacum cv. SR1. The highest expression was seen in shoot tissue, but there was not a large difference between tissues (Fig. 3D).


Figure 4
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  		FIGURE 4.
Localization of NtTPK1. A, cell fractions were prepared from BY-2 cells overexpressing NtTPK1 under the control of the CaMV 35S promoter (Pro35S:NtTPK1). NtTPK1, v-PPase, PAQ2, and Sec61 were detected on Western blots using the corresponding antibodies. S, soluble fraction; T, tonoplast fraction; PM/E, plasma membrane and ER membrane fraction. B, cell fractions were prepared from NtTPK1-transformed S. cerevisiae strain BJ5458 and NtTPK1 and YVC1 were detected on Western blots using the corresponding antibodies. W, whole cell lysate; PM, plasma membrane; T, tonoplast.

 
Subcellular Distribution of NtTPK1 by Immunoblotting—In Arabidopsis, a plasma membrane-targeted TPK-type channel (AtTPK4) and tonoplast-targeted TPK-type channels (AtTPK1, 2, 3, 5, and ATKCO3) have been identified (6, 7). Our results showing functional expression of NtTPK1 in E. coli, but not in yeast or in X. laevis oocytes (Fig. 2) suggest that NtTPK1 might be located in the subcellular membrane. To determine the cellular localization of NtTPK1, we performed biochemical analysis of fractionated membrane proteins. First, we tried to detect NtTPK1s in tobacco and BY-2 cells by Western blotting, but did not see any bands, showing that endogenous expression of NtTPK1s is low. We then collected BY-2 cells overexpressing NtTPK1 under the control of the CaMV 35S promoter and performed SDS-PAGE and immunoblotting with anti-NtTPK1 antibodies. As shown in Fig. 4A, a single protein band at 41 kDa was seen in the tonoplast fraction, identified by the presence of v-PPase (vacuolar proton pumping pyrophosphatase), the tonoplast marker (32). The apparent molecular mass of the protein band did not match that calculated for NtTPK1 protein (47.6 kDa). However, abnormal electrophoretic mobility has been reported for many membrane proteins (48, 49). No band was seen in the soluble fraction and plasma membrane (PAQ2 marker)/ER membrane fraction (Sec61 marker) (36, 50). We therefore concluded that NtTPK1 is localized in the tonoplast in tobacco cells. Moreover, when NtTPK1 was expressed in S. cerevisiae strain BJ5458, which is deficient in major vacuolar proteinases (17), a single band with an approximate molecular mass of 41 kDa was seen in the tonoplast fraction in the same fraction as the band for the Ca2+ activated cation channel YVC1, a tonoplast marker protein (8). These results show that heterologously expressed NtTPK1 was also localized to the yeast vacuolar membrane.

Patch Clamp Recording of NtTPK1 in the Yeast Tonoplast—Because the endogenous currents of the vacuolar membrane in yeast can be removed by inactivation of the genes, we used the yeast expression system to evaluate NtTPK1 function. The yeast vacuolar membrane contains a cation channel, which is modulated by membrane voltage, cytosolic Ca2+, redox state, and pH (51), and the YCV1 channel, which exhibits a large conductance for K+ (35). We generated a S. cerevisiae mutant lacking the yvc1 gene, named SH1006, and confirmed the absence of yvc1 gene products by Western blotting (data not shown). We then enlarged S. cerevisiae SH1006 using the spheroplast incubation method (Fig. 5A, panels a and b) (34, 40) and fixed the giant yeast under hypotonic conditions, causing breakage of the plasma membrane of the protoplast and rapid release of the intact vacuole into the medium (Fig. 5A, panels c and d). When we performed whole cell clamp recording on the isolated vacuolar membrane, it did not show any intrinsic tonoplast channel activity, a finding reported by Palmer et al. (35). This strain was therefore used for patch clamp recording.

To analyze the ion selectivity of the NtTPK1 channels, we tested various bath solutions. The voltage was first applied at –60 to 80 mV in 20 mV steps in 100 mM KCl bath solution, then the same voltage protocol was used in bath solution containing 30 mM KCl, 10 mM KCl, 30 mM KCl + 70 mM NaCl, or 10 mM KCl + 90 mM NaCl, with 100 mM K+ in the pipette solution (Fig. 5B). The reversal potential shift of the whole vacuole current on going from 100 mM KCl to 10 mM was +56 mV, which is close to the Nernst potential for K+ (+58 mM). This value of the reversal potential gave a PCl/PK ratio of 0.016 and a PNa/PK ratio of 0.05. The data show that NtTPK1 is highly selective for K+ over Na+ and Cl–.

Cytosolic Acidification Increases the NtTPK1 Current—Modulation of K+ channels by alteration of pH has been shown in several reports. Using a symmetrical KCl concentration (100 mM), the pH of the cytosolic side (bath solution) was set at 5.5, 6.5, 7.5, or 8.5, and that of the vacuolar side (pipette solution) at 6.5. A 26-fold increase in the K+ current was seen at 80 mV when the cytosolic pH was changed from 7.5 to 5.5 (Fig. 6). A current amplitude increase by intracellular acidification was also seen when the vacuolar side pH was changed from 6.5 to 7.5. The plasma membrane AtTPK4 has been shown to be sensitive to intracellular acidosis (6) and the tonoplast AtTPK1 shows maximum activity at pH 6.7 (9). The NtTPK1 pH profile was different from those of AtTPK1 and AtTPK4, as AtTPK1 shows maximal activity at pH 6.7 (9), and the AtTPK4 current is reduced when the pH changed from 7.5 to 6.3.


Figure 5
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  		FIGURE 5.
Patch clamp assay on NtTPK1 expressed in giant yeast vacuoles. A, images of normal cells and giant cells of S. cerevisiae SH1006 (lacking YVC) expressing NtTPK1. Panel a, normal-sized cell expressing NtTPK1; panel b, giant yeast cells expressing NtTPK1; panel c, vacuoles isolated from giant yeast cells by hypoosmotic shock; and panel d, patch pipette attached to exposed vacuole. B, ion selectivity of NtTPK1. Whole vacuole current elicited by a series of voltage steps ranging from –60 to 80 mV in 20-mV steps recorded in symmetrical 100 mM KCl ({circ}), then the experiment was repeated after replacement of the cytosolic KCl with 30 mM KCl ({square}), 10 mM KCl ({triangleup}), 30 mM KCl + 70 mM NaCl (x), and 10 mM KCl + 90 mM NaCl (+). The pipette solution contained 100 mM KCl, 1 mM MgCl2, 0.01 mM CaCl2, 10 mM Tris-MES pH 6.5, 200 mM sorbitol. The reversal potential was estimated by fitting the Goldman-Hodgkin-Katz current equation.

 
Effect of Ca2+ on NtTPK1 Channels—Based on the data of computational analysis using the PROSITE pattern search data base, a single EF-hand motif was found at position 371–381 in the C-terminal region of NtTPK1. Such domains are thought to be involved in Ca2+-mediated regulation of TPK channel activity. Of the Arabidopsis TPK channels, AtTPK1, AtTPK2, and AtTPK3 have one or two EF-hand motifs (7, 52), whereas AtTPK4 does not (6). The effect of luminal or cytosolic Ca2+ on NtTPK1 was analyzed. As shown in Fig. 7, the amplitude of the NtTPK1-mediated K+ current in test solution without added Ca2+ was the same as that in presence of the calcium chelating agent, EGTA (final concentration1 mM) (less than 0.1 µM Ca2+), showing that NtTPK1 activity was not affected by any low levels of contaminating Ca2+ in the solution. Addition of 45 µM Ca2+ to the cytosolic side of the membrane (bath solution) caused an ~2-fold increase in the K+ current at cytosolic pH of 6.5 and of about 1.15-fold at a cytosolic pH of 5.5 (Fig. 7). In contrast, application of 45 µM Ca2+ to the vacuolar side of the membrane (pipette solution) had no effect (data not shown). The absence of Ca2+ (less than 0.1 µM) in the cytosolic solution gave significant K+ currents. The Ca2+ dependence of NtTPK1 was relatively small compared with that of AtTPK1 (9).


Figure 6
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  		FIGURE 6.
Activation of NtTPK1 by cytosolic acidification. The pH of the bath solution containing 100 mM KCl, 1 mM MgCl2, 200 mM sorbitol was adjusted with Tris-MES to different pH values. The pipette solution was 100 mM KCl, 1 mM MgCl2, 0.01 mM CaCl2, 200 mM sorbitol, 10 mM Tris-MES, pH 6.5. Bath solution was the same except for pH, which were pH 5.5 ({circ}), pH 6.5 ({triangleup}), pH 7.5 ({square}), pH 8.5 (x). A, whole vacuole current elicited by a series of voltage steps ranging from –60 to 80 mV in 20-mV steps. B, IV relationship of the NtTPK1 current is altered by the pH. C, pH dependence of the NtTPK1 current normalized to that at pH 5.5.

 


Figure 7
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  		FIGURE 7.
Effect of Ca2+ on NtTPK1 activity. The pipette solution was 100 mM KCl, 1 mM MgCl2, 0.01 mM CaCl2 [Ca2+]vac, 200 mM sorbitol, 10 mM Tris-MES, pH 6.5. The bath solution was the same except for the pH and Ca2+ concentration, which were pH 5.5 with 0.045 mM CaCl2 [Ca2+]cyt ({circ}), pH 5.5 without CaCl2 [Ca2+]cyt (•) pH 6.5 with 0.045 mM CaCl2 [Ca2+]cyt ({square}), and pH 6.5 without CaCl2 [Ca2+]cyt ({blacksquare}).

 
Effect of Polyamines on NtTPK1 Channels—In animal cells, polyamines have been shown to block inwardly rectifying K+ (Kir) channels, simple structures with two membrane spanning domains separated by a pore region (53, 54). Subsequent studies identified the residues blocked by polyamine at the cytosolic face in Kir channels (55, 56). Moreover, polyamines regulate the plasma membrane K+ channel of the cells and modulate the stomatal aperture (57). The analogous structure of the TPK1-type channels and Kir channels prompted us to test whether polyamines were involved in the regulation of NtTPK1. Because in Vicia fava leaves polyamine concentrations in plant tissues reach the millimolar range under unstressed and stress conditions (57), submillimolar concentrations of polyamines were used in the patch clamp experiment. As shown in Fig. 8, A and B, addition of putrescine (0.5 mM) to the cytosolic side caused a slight decrease (7%) in the K+ current at –60 mV, but had no effect at positive potentials. Spermidine (0.5 mM) on the cytosolic side caused a decrease in the K+ current of 18% at –60 mV and 28% at 80 mV, while spermine (0.5 mM) caused a decrease of 30% at both –60 mV and 80 mV. These results showed that NtTPK1 is sensitive to polyamines. To our knowledge, no information on the putrescine, spermidine, and spermine contents of tobacco cells is available. We therefore measured free polyamine contents in different tissues of N. tabacum cv. SR1. All three polyamines were detected in tobacco leaf, shoot, and root (Fig. 8C), with spermidine being the most abundant. These results suggest that submillimolar concentrations of endogenous polyamines can affect the activities of tonoplast TPK-type channels in tobacco cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The sequence of the two-pore K+ channel NtTPK1 from N. tabacum cv. SR1 and tobacco BY-2 cells provided new evidence on the signature sequence amino acids of K+ channels (Fig. 1). K+ channels have the signature sequence GYG as the selectivity filter (58) and the K+ conducting pore is made up of four pore domains (59) formed by a dimer of NtTPK1 (7, 60). In this study, we found that NtTPK1b, containing His, or NtTPK1c, containing Val-His, in the signature sequence in the second pore region was able to function in the E. coli membrane. This is the first report of a non-GYG signature sequence in natural K+ channels. It has been reported that animal and plant cyclic nucleotide-gated channels do not possess the GYG sequence, but some allow the passage of K+ or rescue mutations of K+ uptake systems in yeast (61). In some channels, the GYG triplet sequence is not essential for K+ passage (62). Experiments using artificial replacement with GHG or AAA in the first or second signature sequence showed that the first GYG was essential for the K+ transport activity of NtTPK1, whereas the second was not (Fig. 2).


Figure 8
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  		FIGURE 8.
Effect of polyamines on NtTPK1 currents. A, IV relationship of the NtTPK1 current is altered by various polyamines. Whole vacuole currents in the absence of polyamines (•) or in the presence of 0.5 mM putrescine (PUT, {square}), spermidine (SPD, {circ}), or spermine (SPM, {triangleup}). B, whole vacuole currents recorded in the presence of 0.5 mM putrescine, spermidine, or spermine at +80 mV (upper columns) or at –60 mV (lower columns) were normalized to the control in the absence of polyamines. The pipette solution was 100 mM KCl, 1 mM MgCl2, 200 mM sorbitol, 10 mM Tris-MES, pH 6.5. The bath solution was the same except for the presence and absence of polyamines. C, polyamine contents in different tissues of N. tabacum cv. SR1. All data are expressed as the mean ± S.E. (n = 3).

 
Our data on the cellular location of NtTPK1 revealed that it was targeted to the vacuolar membrane in BY-2 cells (Fig. 4). This accounted for the failure of the functional expression of NtTPK1 in the plasma membrane of Xenopus oocytes and yeasts (data not shown). To determine the characteristics of NtTPK1, we used the yeast tonoplast, an ideal tool for studying the electrophysiological properties of subcellular membrane-associated channels, as, in plant tonoplast, several types of cation currents have been reported (8, 63), which sometimes interfere with electrophysiological measurements. In yeast, the endogenous K+ transport system can also be eliminated by genetic disruption to reduce background currents from the vacuolar membrane. We confirmed that the remaining background K+ currents of the ycv1 mutant were extremely lower compared with the NtTPK1-mediated currents (8) and that NtTPK1 was targeted to the yeast tonoplast (Fig. 4). We were able to enlarge the yeast tonoplast for patch clamp recording on the NtTPK1-mediated currents (34, 40, 64) and thus to obtain detailed electrophysiological measurement of NtTPK1 in this study (Figs. 4, 6, 7, 8).

The cytosolic pH is reported to regulate activity. The plasma membrane AtTPK4 is inhibited by intracellular acidification (6), as are animal two-pore K+ channel TASK currents (65). In contrast, in the case of the human channel TWIK-1 and mouse channel TREK-1, intracellular acidification is reported to activate K+ currents (66, 67). The KcsA K+ channel from Streptomyces lividans shows similar activation and the external pH does not affect channel gating (68). NtTPK1 showed a pH dependence similar to the latter channels (Fig. 6). In maize, propionic acid and auxin treatments resulted in cytosolic acidification (69). In tobacco BY-2 cells, cytosolic acidification was observed after the addition of high concentration of auxin and other lipophilic acid (70). In plant cells, changes in the cytosolic pH are closely related to tonoplast K+ influx and efflux (11). A strong shift of the cytosolic pH toward a more acidic pH, such as pH 5.5, shown in Fig. 6, increased vacuole proton pumping and altered the vacuolar membrane potential and this was followed by K+ release from the luminal side. The steep increase in the amplitude of the NtTPK1 currents caused by cytosolic acidification implied that NtTPK1 contributes to vacuolar K+ release (Fig. 6). In turn, the sustainable pumping of protons into vacuole causes alkalinization of the cytosol. Alkalinization has been reported in guard cells during stomatal closure (71). The shift to a higher pH in the cytosol led to cessation of the transport function of NtTPK1 (Fig. 6).

To date, three tonoplast channels, SV, FV, and VK, have been identified in Vicia and Arabidopsis guard cells (11). Recent reports indicate that AtTPK1 is the VK channel (8, 9). NtTPK1 was similar to AtTPK1 in terms of the high ionic selectivity. However, some of the properties of NtTPK1 differed from those of AtTPK1 and the VK channel. NtTPK1 displayed significant K+ conductance in solutions containing very low Ca2+ concentrations (less than 0.1 µM Ca2+), whereas, under the same conditions, Arabidopsis AtTPK1 currents are not seen (9). The conductance of the NtTPK1 channel was steeply increased at more acidic pHs (Fig. 6), which is different from the situation with AtTPK1. The difference in sequence homology of the N- and C-terminal regions between NtTPK1 and AtTPK1 is larger than that between NtTPK1 and AtTPK2, AtTPK3, and AtTPK5 (supplemental Fig. 1). The above results show that NtTPK1 plays a different role from the VK channel. Because the SV channel is activated by cytosolic Ca2+ concentrations of higher than 0.5 µM Ca2+, while the FV channel is inhibited by cytosolic Ca2+ concentrations higher than 0.1 µM Ca2+ (2, 11, 72), NtTPK1 is not a SV channel or an FV channel.


Figure 9
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  		FIGURE 9.
Possible mechanism and physiological role of NtTPK1 in plant cells. Hyperosmotic-shock up-regulates NtTPK1expression. Cytosolic acidification and an increase in cytosolic Ca2+ enhance NtTPK1 activity in the tonoplast membrane. NtTPK1 is sensitive to polyamines.

 
In BY-2 cells, NtTPK1 expression was increased by ~2-fold when 250 mM NaCl or 500 mM mannitol was added to the medium (Fig. 3C). Under salinity conditions, control of K+ homeostasis is critical for cell survival (1). Loss of K+ from the cells causes a decrease in both vacuolar and cytosolic osmotic pressure (1). Overexpression of K+ uptake channels confers salt tolerance on rice cell lines (73). In cyanobacterium, the K+ uptake transporter is essential for the early phase of cell adaptation to hyperosmotic shock (74). The finding of induction of the NtTPK1 expression by salinity stress and high osmolarity change indicates that NtTPK1 may be involved in transporting K+ into the cytosol (Fig. 9). Our results using hyperosmotic shock were reminiscent of the transient increase in cytosolic Ca2+ elicited by stress (75). When intracellular Ca2+ is increased, free Ca2+ acts as a second messenger and binds to cellular proteins with a Ca2+ binding motif and Ca2+ binding proteins, e.g. calmodulin. NtTPK1 is probably involved in the Ca2+ signal transduction pathway (10) (Fig. 9).

The sensitivity of animal plasma membrane-associated inward rectifiers to polyamines is well known (55, 56). In the mouse Kir 2.1 channel, negatively charged residues in the second transmembrane region and the cytoplasmic region associate with polyamines, causing blockage of the channels. The overall membrane structure of NtTPK1 is equivalent to that of a tandemly duplicated Kir channel. Spermidine or spermine inhibited the channel activities of NtTPK1 (Fig. 8). This indicates that polyamines probably function as channel regulators in tobacco cells, as in animal cells. From another aspect, polyamines are required for the normal development of both prokaryotes and eukaryotes (76). In higher plants, polyamines are involved in physiological processes, such as growth, development, and adaptation to various types of environmental stress (77–81). Millimolar levels of polyamines are found in Vicia fava guard cells (57). Salt stress, osmotic shock, and drought increase the synthesis of polyamines in plant cells and result in tolerance of the plant cells by an unknown mechanism (57, 82–84). In this study, the presence of putrescine, spermidine, and spermine was confirmed in tobacco tissues (Fig. 8). Increases in polyamines may be relevant to controlling K+ flux in the tobacco vacuolar membrane, but further study is necessary to understand the physiological role of NtTPK1 blockage by polyamines.


   FOOTNOTES
 
* This work was supported by grants-in-aid for scientific research (17078005 and 19380058) (to N. U.) from MEXT and JSPS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) EU161633 [GenBank] . Back

1 To whom correspondence should be addressed. Fax: 81-22-795-7293; E-mail: uozumi{at}biophy.che.tohoku.ac.jp.

2 The abbreviations used are: TPK, two-pore K+ channel; RACE, rapid amplification of cDNA ends; MES, 4-morpholineethanesulfonic acid; GUS, β-glucuronidase. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Souichiro Noguchi and Hidenori Ozawa (Leaf Tobacco Research Center, Japan Tobacco Inc.) for generously supplying the N. tabacum cv. SR1 seeds.



   REFERENCES
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 DISCUSSION
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