The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel.
To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit.
Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles.
The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function.
The plant vacuole represents a dynamic store for metabolites and ions. The major cations shuttling across the vacuolar membrane are potassium, protons and calcium. Under salt stress large fluxes of sodium into the vacuole lumen are observed, where the increase in Na+ concentration on top of that with K+ provide for maintenance of cell turgor. Early patch-clamp studies with plant vacuoles have shown that elevation of cytoplasmic calcium concentration addresses a vacuolar cation channel activating with slow kinetics (Hedrich & Neher, 1987). This channel, named Slow Vacuolar (SV) channel is, however, blocked when the Ca2+ concentration in the vacuolar lumen increases (Dadacz-Narloch et al., 2011). Peiter et al. (2005) in the model plant Arabidopsis thaliana identified the molecular nature of the SV channel, showing that slow vacuolar channel currents were mediated by the Two Pore Channel 1 (TPC1). Structurally, TPC1 represents a dimer of two Shaker-like entities linked via a cytoplasmic loop containing two EF hand motifs (Ishibashi et al., 2000; see Hedrich & Marten, 2011; for a review). Recent studies in the Dietrich lab demonstrated that each EF hand contributes to the activation of the SV channel individually (Schulze et al., 2011). Given that a TPC1 monomer contains two potential pore-forming peptide stretches, in the vacuolar membrane very likely TPC1 dimers form the functional SV channel. Due to the difference in the amino acid composition of TPC1 compared with the potassium-selective signature of Shaker channels, one would assume that the pore of TPC1 conducts cations. In line with this assumption SV channels from Arabidopsis besides K+ and NH4+ conduct Na+ and even the potassium channel blocker Cs+. Indeed, upon replacement of K+ by Ca2+ in artificial cytoplasmic medium, in patch-clamp studies with a number of plant cell types and species SV currents into the vacuole lumen were observed (see Hedrich & Marten, 2011 for review).
Vacuoles from the Arabidopsis TPC1 null-mutant tpc1-2 lack SV channel activity (Peiter et al., 2005; Hedrich, 2012; for review). When attpc1-2 mutant was transformed with the orthologous gene from the dicot plant Nicotiana tabacum (NtTPC1b) SV channel function was regained (Dadacz-Narloch et al., 2011). Genes encoding two-pore channel 1 (TPC1) are conserved both in dicots and monocots, and a single gene is present in Arabidopsis thaliana and the monocots Oryza sativa and Triticum aestivum. With the latter species OsTPC1 and TaTPC1 have been localized at the subcellular level (Kurusu et al., 2005; Wang et al., 2005). Transient expression of GFP-OsTPC1 and TaTPC1-GFP in epidermal cells of the monocot Allium cepa suggests that rice and wheat TPC1 are localized at the plasma membrane. When OsTPC1 and TaTPC1 were expressed in yeast both channels appeared to complement 45Ca2+ uptake and to rescue growth of the yeast cch1 mutant, defective in a plasma membrane Ca2+ permeable channel (Hashimoto et al., 2004; Kurusu et al., 2004). This seems to indicate that TPC1 from wheat and rice in the heterologous systems were able to either operate as or regulate the activity of a plasma membrane calcium channel. The localization of OsTPC1 was confirmed by studying this channel in its native environment. Using suspension-cultured rice cells with membrane fractionation and immuno-histochemistry the rice SV channel was localized predominantly at the plasma membrane (Hashimoto et al., 2005; Hamada et al., 2012). These results obtained using an antibody directed against OsTPC1 suggested that vacuoles from cultured rice cells basically lack the product of OsTPC1 gene. Consequently, rice should exhibit SV current-free vacuoles; alternatively the monocot SV channel could be encoded by a gene other than OsTPC1. Given the apparent divergence in membrane localization of TPC1s between dicots and monocot rice and wheat, one might thus ask the question whether TPC1 and SV channels may not inevitably be synonymous.
In order to answer this question, we performed patch-clamp studies with vacuoles from: the Arabidopsis loss-of-function mutant attpc1-2 complemented by TPC1 genes from wheat (TaTPC1) and rice (OsTPC1) as well as wild-type rice and wheat plants and the null-mutant for Oryza sativa tpc1. The results gained with these independent approaches showed that the monocot TPC1 proteins exhibit the hallmarks of the SV channel with respect to both localization and function.
Materials and Methods
Plant materials and growth conditions
Surface-sterilized seeds of rice, Oryza sativa L. cv Nipponbare, were germinated on MS medium (Murashige & Skoog, 1962) and grown for 7 d in a growth chamber under long day conditions (16 h : 8 h, light : dark cycle, 28°C). To generate cultured cells of the wild-type and the retrotransposon Tos17-insertional ostpc1-knockout cells (Kurusu et al., 2004), seeds were placed onto the callus-inducing medium. The calli were suspension cultured at 25°C initially in liquid AA medium (Su et al., 1992) and then subcultured in L medium (Kuchitsu et al., 1993) containing 2,4-D (0.5 mg l−1). The cells were subcultured in fresh medium every 7 d and filtered through a 20 μm mesh screen every 2 wk to make fine aggregates. Cells at 7th day after subculture were used for isolation of protoplasts followed by patch clamping the released vacuoles. The attpc1-2 mutant (ecotype Col-0) (Peiter et al., 2005), Triticum aestivum and Nicotiana benthamiana plants were grown on soil under short day conditions (8 : 16 h, 22 : 16°C) and a photon flux density of 150 μmol m−2 s−1 in a growth chamber or a glasshouse, respectively. Tobacco BY-2 cells (Nicotiana tabacum L. cv Bright Yellow 2) were subcultured every week in a modified Linsmaier and Skoog medium (LSD medium) as described previously (Sano et al., 2007).
Total RNA was extracted from leaves of 7-d-old T. aestivum plants using the RNeasy Micro Kit (Qiagen). RNA was DNase digested using RNase-free DNase I (Thermo Fisher Scientific, Schwerte, Germany) and reverse transcribed as described previously (Larisch et al., 2012a). TaTPC1 was amplified from cDNA using the primers presented in Supporting Information Table S1. TPC1 clones from Arabidopsis, rice (O. sativa, OsTPC1) (Kurusu et al., 2004), tobacco (N. tabacum, NtTPC1 a and b) (Kadota et al.,2004) and T. aestivum were cloned as N- or C-terminal GFP or YFP fusions into modified vectors: pSAT6-EGFP-C1 (GenBank AY818377.1), pSAT6-EGFP-N1 (GenBank AY818382), pSAT6-YFP-C1 and pSAT-YFP-N1 (GenBank DQ005469) as described previously (Dadacz-Narloch et al., 2011) followed the advanced uracil-excision-based cloning technique (Nour-Eldin et al., 2010). This resulted in 35S:AtTPC1-GFP, 35S:GFP-AtTPC1, UBQ10:AtTPC1-YFP, UBQ10:YFP-AtTPC1, 35S:OsTPC1-GFP, 35S:GFP-OsTPC1, UBQ10:OsTPC1-YFP, UBQ10:YFP-OsTPC1, 35S:GFP-TaTPC1, 35S:GFP-NtTPC1a, 35S:GFP-NtTPC1b. All primer sequences are provided in Table S1.
Transient transformation and imaging of protoplasts
Transient transformation of protoplasts was performed according to the procedure established by Sheen (2002) and Yoo et al. (2007) (for A. thaliana, N. benthamiana and T. aestivum) and Zhang et al. (2011) (for O. sativa plants and cell culture). Protoplasts used for transient expression were isolated from: leaves of 6–7-wk-old attpc1-2 and N. benthamiana plants, 1-wk-old O. sativa and T. aestivum plants and O. sativa (wild-type and ostpc1 knockout), tobacco BY-2 (Bright Yellow 2) cell cultures 7 d after subculture. Intact protoplasts and released vacuoles were used for imaging 48 h after transformation. Onion epidermal cells were transfected via particle bombardment using biolistic delivery of tungsten particles (tungsten M-17, Bio-Rad Laboratories, Hercules, CA, USA) coated with the respective DNA as described by Dunkel et al. (2008). At 12 or 24 h post-transfection the cells were used for imaging.
GFP/YFP fluorescence imaging was performed by using a confocal laser scanning microscope (TCS SP5; Leica, Mannheim, Germany). GFP and YFP were excited with an Argon laser at 490 and 514 nm, respectively. Emission of fluorescence was recorded between 500 and 520 nm (for GFP) and 520–540 nm (for YFP). Red autofluorescence of chlorophyll was excited at 540 nm with emission recorded between 590 and 610 nm.
HEK293 cell transfection and staining
HEK293ad cells were cultured in DMEM (Invitrogen) supplemented with 1% (v/v) PenStrep (VWR) and 5% FCS (Biochrom, Berlin, Germany). Cells were kept at 37°C in a 65% - humidified incubator supplied with 5% (v/v) CO2. Two days before transfection, 2.5-ml cell suspensions (0.5 × 105 cells ml−1) were seeded in a 6-well plate containing cover slips (one per well) coated with poly-D-lysine. Twelve hours after seeding the growth medium was exchanged with a fresh one (2.5 ml). For DNA preparation 2.5 μg (pcDNA3.1:OsTPC1-GFP) or 1.25 μg (pcDNA3.1:GFP-OsTPC1) DNA were mixed with 125 μl OptiMEMI (Invitrogen) by short vortexing. Ten or 5 μl PEI (Polysciences, Eppenheim, Germany) were mixed with 125 μl OptiMEMI. DNA and PEI solutions were mixed together, incubated for 30 min at room temperature, and then the mix was applied to the cells. Twenty-four hours post-transfection the cells were treated with 5 μM vacuolin-1 (Sigma) for 1 h as described previously (Huynh & Andrews, 2005; Schieder et al., 2010) and then stained with LysoTracker Red DND-99 (Invitrogen) or Deep Red (Invitrogen) according to the manufacturer's instructions. After removing the staining solutions and rinsing the cover slips with PBS medium the samples were imaged immediately as described above.
Patch-clamp measurements in the whole vacuole configuration were performed as described by Dadacz-Narloch et al. (2011). All experiments were carried out on the second day after transient transformation. Vacuoles were released from transformed or nontransformed protoplasts by incubation in 5× diluted W5 buffer (Sheen, 2002; Yoo et al., 2007). The composition of the bath (cytosolic) solution was 150 or 30 or 0 mM KCl or 150 mM NaCl, 1 or 15 mM CaCl2, 2 mM DTT, 10 mM HEPES/Tris pH 7.5 with the osmolarity (π) adjusted to 310 mOsmol kg−1 with d-sorbitol. The pipette (vacuolar) solution contained 150 or 30 mM KCl or 150 mM NaCl, 2 mM MgCl2, 0.1 mM EGTA, 10 mM HEPES/Tris pH 7.5; π = 310 mOsmol kg−1 with d-sorbitol. Free Ca2+ concentrations for the pipette and bath media were calculated with WEBMAXC standard (http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm). The patch-clamp solutions are described in detail in the figure legends. To estimate midpoint potentials, V1 and V2, I/V curves recorded in a whole vacuole configuration (the mean of 3–7 measurements ± SE) were fitted by a combination of two Boltzman functions:
as described (Pottosin et al., 2004; Dadacz-Narloch et al., 2011; Gutla et al., 2012). IMax indicates the maximum current, z1 (0.77) and z2 (2.9) are gating charges, V1 and V2 are midpoint potentials, whereas F, R and T have their usual meaning. The gating mechanism consisting of two closed (C2 and C1) and one open (O) states is described in the legend to Fig. 2(b).
Vacuoles from monocots Oryza sativa and Triticum aestivum operate SV channels similar to those in dicots
In order to test whether rice vacuoles predicted to operate OsTPC1 in the plasma membrane (Hashimoto et al., 2005; Hamada et al., 2012) lack SV channel activity, we isolated vacuoles from leaves of 7-d-old rice plants grown in liquid MS-based medium (Murashige & Skoog, 1962). Leaves were harvested and protoplasts isolated following the method established by Zhang et al. (2011). A similar procedure was used to extract mesophyll protoplasts from wheat (Sheen, 2002; Yoo et al., 2007). Vacuoles were released by selective osmotic shock and used for patch-clamp studies within 15 min after liberation from the cellular context of the rice leaf. Patch pipettes were sealed on vacuoles and whole-vacuole configuration was established by a punch-through voltage pulse protocol (Hedrich et al., 2012). Following access to the vacuole lumen and equilibration with pipette solution, ion species on the cytosolic side of the membrane and voltage protocols were chosen to selectively elicit SV currents. Experiments were performed at symmetric K+ concentration (150 mM) on both membrane sides supplemented with 1 mM Ca2+ in the cytoplasmic buffer and the nominal absence of SV channel-blocking Ca2+ ions in the vacuole sap (cf. Dadacz-Narloch et al., 2011). With K+ as the major physiological SV channel substrate and Ca2+ addressing TPC1's cytosolic EF hands typical SV currents could be recorded with vacuoles from the monocots in question. Starting at a holding potential of −60 mV, 1200-ms-long voltage pulses were applied in the range from −100 to +110 mV. This pulse protocol with vacuoles released from the wild-type rice and wheat plants elicited slowly activating outward currents (Figs 1a, S1) with activation half-times (t½) of 168 ± 21 and 66 ± 9 ms at + 65 mV, respectively (Fig. 1c). Moreover, with the same patch-clamp conditions, a typical SV current was measured with vacuoles released from cultured rice cells (Fig. 1b). SV currents were not present in vacuoles from the O. sativa tpc1 knockout mutant (ostpc1) (Fig. 1a,b, right panel). Likewise, buffering cytosolic calcium to nominal zero with EGTA completely suppressed SV currents in rice WT vacuoles (Fig. 1a). Thus the SV channels from rice leaves (Fig. 1a) and cell culture (Fig. 1b), as well as those from wheat leaves (Fig. S1), apparently seem to share the basic features of those so far recorded from dicots.
Response of rice SV channel to cytosolic and vacuolar cations
Under physiological conditions in its natural environment of the plant cell the SV channel is facing potassium ions, the major inorganic osmolyte of plants. Potassium concentrations in vacuoles are known to vary in response to changing growth conditions, but cytoplasmic K+ remains rather constant (Walker et al., 1996; Leigh, 2001).
With patch-clamp studies under symmetric 150 mM K+ on both sides of the vacuolar membrane the rice SV channel operated as an outward rectifier. Slow vacuolar currents activated c. 5 mV positive of the Nernst potential of K+ steeply increasing upon progressive depolarization (Figs 1a,b, 2a). When fitted by a combination of two Boltzman functions (Fig. S2) taking into account a gating model composed of two closed states, C2 and C1, and one open state, O (Pottosin et al., 2004; Dadacz-Narloch et al., 2011; Gutla et al., 2012), midpoint potentials of V1 = 101 mV and V2 = 53 mV were determined (Fig. 2b). These SV channel features did not alter much, when K+ on both membrane sides was replaced by, for example, Na+ (150 Nasym: V1 = 104 mV and V2 = 59 mV, Fig. 2b). This finding did not come as a surprise as in dicots Na+ permeability of SV channels has been documented previously (Ivashikina & Hedrich, 2005). Upon reduction of the cytosolic K+ concentration from 150 to 30 mM the SV current amplitude dropped to c. 25% and midpoint potentials shifted by c. 20 mV to more positive values (Fig. 2a,b). This shift was unaffected by switching 150 mM K+ to Na+ in the vacuolar lumen (Fig. 2b). It should be noted, however, that in contrast to the situation in Arabidopsis K+ gradient-dependent shifts in the current–voltage curve did not cause SV currents to invert (Fig. 2a; cf. fig. 3 in Beyhl et al., 2009). Shifts of V1 and V2 midpoint potentials of 20 mV to more negative membrane potentials were observed after decreasing the vacuolar potassium concentration from 150 to 30 mM (Fig. 2b). Further, in contrast to the SV channel in Arabidopsis, the half-times for current activation (t½) and characteristic times (τ) for deactivation were found to be independent of cytosolic or vacuolar potassium (Fig. 1c,d).
In the next step and under a 30cyt/150vac mM K+ gradient (cf. fig. 3 in Beyhl et al., 2009) we replaced the 30 mM [K+]cyt by 15 mM [Ca2+]cyt. During solute exchange SV currents decreased continuously and at steady state the outward current in 15 mM [Ca2+]cyt was 40 pA only compared to the situation of 200 pA with 30 mM [K+]cyt (Fig. 2a). To test the response of the rice channel towards high Ca2+ loads in the vacuolar lumen, we added 10 mM Ca2+ to the pipette solution under symmetric K+ conditions (150 mM). This Ca2+ increase inside the vacuole dramatically reduced the SV current. At 50 mV outward K+ currents dropped from 198 to 14 pA (Fig. 2a). This feature with Arabidopsis has been recently associated with a new type of a Ca2+ sensor monitoring the luminal calcium concentration and adjusting the voltage gate accordingly (Dadacz-Narloch et al., 2011).
Dicot SV channels for activation strictly require the presence of cytosolic calcium ions (Hedrich & Neher, 1987, and the papers cited in Hedrich & Marten, 2011). To test the Ca2+ dependent activity of the SV channel from rice and wheat, we challenged vacuoles with calcium-free buffer containing 10 mM EGTA. Vacuoles analyzed like those depicted in Figs 1(a) and S1 (left panel) were characterized by pronounced SV conductance. Following cytoplasmic removal of the signaling cation, SV currents progressively dropped and finally faded away completely (Figs 1a, S1, right panel). These results indicate that the slow vacuolar channel from O. sativa and T. aestivum as their dicot counterparts are activated by cytoplasmic calcium ions. Thus, although previous studies had localized OsTPC1 and TaTPC1 to the plasma membrane of rice and wheat (Hashimoto et al., 2005; Kurusu et al., 2005; Wang et al., 2005; Hamada et al., 2012), vacuoles isolated from the leaf of these monocots have SV channels with properties similar to those of dicots studied so far. Are TPC1 and SV channel synonymous in monocots? We have shown that rice does not represent an SV current-free species (Figs 1a,b, 2a), we therefore in the next step tested whether or not the rice SV channel is encoded by OsTPC1.
OsTPC1 represents the rice SV channel
The rice genome harbors a single TPC1 gene. To gain insights into the molecular nature of the rice TPC1/SV channel we took advantage of an O. sativa mutant ostpc1 featuring a transposon insertion in the coding region of the rice TPC1 gene (Kurusu et al., 2004). When grown under the same conditions as wild-type rice plants the ostpc1 mutant exhibited no obvious phenotype in this study. The ostpc1 mutant in patch-clamp studies, however, did not show any SV channel activity (Fig. 1a). In addition, lack of SV currents was observed in vacuoles released from suspension-cultured cells derived from the ostpc1 mutant (Fig. 1b, right panel). These results indicate that the SV channel or at least a major component is encoded by OsTPC1.
How can we distinguish between these two possibilities? With the model plant Arabidopsis, Peiter et al. (2005) discovered that the TPC1 gene encodes the SV channel. The Arabidopsis mutant fou2 expresses a hyperactive AtTPC1/SV channel (Beyhl et al., 2009) characterized by a wounding-related high jasmonate phenotype (Bonaventure et al., 2007a,b). A recent screen for 2nd-site-mutations that suppressed the fou2 phenotype identified a set of new mutants named oufs. These mutations also mapped to the TPC1 gene that is mutated in fou2 (A. Lenglet & E. Farmer, unpublished). This fact indicates that in Arabidopsis AtTPC1 and the SV channel are very likely to be synonymous.
In order to revisit the localization and function of OsTPC1 we transiently transformed the Arabidopsis loss-of-tpc-1-2 mutant with OsTPC1. In previous studies N-terminal GFP labeled OsTPC1 was localized in the plasma membrane of onion epidermal cells as well as the rice cell culture, but very recently C-terminal GFP tagged OsTPC1 in the vacuolar membrane of tobacco BY-2 cells (Kurusu et al., 2005, 2012; Hamada et al., 2012). In all of these experiments expression of OsTPC1 was driven by the CaMV 35S promoter. In order to get a comprehensive picture on the localization and function of plant TPC1 channels in general and the rice TPC1 in particular, we investigated channel targeting and function by employing cell biology in combination with the patch-clamp technique. To account for the possible impact of expression strength on channel localization we expressed TPC1 genes under the control of constitutively strong as well as moderately active promoters, reflected by the CaMV 35S and Arabidopsis Ubiquitin-10 (UBQ10, Grefen et al., 2010) promoter, respectively. Further, it has recently been proven that a conserved N-terminal dileucine motif in the TPC1 channel protein (EXXL[LI]) is required for proper tonoplast/endolysosomal sorting of plant and animal TPC1 channels (Brailoiu et al., 2010; Larisch et al., 2012b). Thus, an important concern during transient expression of TPC1 channels is reflected by the tag position of fluorescent proteins. To address this issue we tested N- as well as C-terminal GFP/YFP tagged rice and Arabidopsis TPC1 proteins. Irrespective of the promoter used 2 d after transformation in attpc1-2 mesophyll protoplasts tag fluorescence was observed. The 35S promoter driven GFP-OsTPC1 and OsTPC1-GFP signals were emitted from the vacuolar membrane; however, vacuolar targeting always appeared more efficient with C-terminally tagged OsTPC1 constructs (Fig. 3a,b, middle panel). In addition, vacuolar localization signals from N-terminally-labeled OsTPC1 were associated with the cytoplasm as well as endomembrane compartments, but not with the plasma membrane (Fig. 3a,b, left panel). These results were similar to the observed targeting and localization of AtTPC1 (Fig. S3). Moreover, Triticum TPC1 (GFP-TaTPC1) expressed in attpc1-2 protoplasts under the same promoter (35S) localized to the tonoplast, too, but not to the plasma membrane (Fig. 3c, left and middle panel). We made comparable observations using the rice tpc1 knockout mutant expressing either C-terminally labeled OsTPC1 or AtTPC1 (Fig. 3d,e). Again, N-terminally tagged GFP-OsTPC1/AtTPC1 fusion proteins were localized mostly in the endomembrane containing cytoplasm (Fig. S4a,b). We obtained essentially the same results using the UBQ10 promoter. Whilst the OsTPC1/AtTPC1-YFP clearly showed vacuolar localization either in attpc1-2 (Figs S3c, S5a) or ostpc1 (Fig. S4c,e) protoplasts, the N-terminal YFP tagged TPCs were present predominantly in the cytosol (Figs S3d, S4d,f, S5b). As an additional monocot expression system we tested onion epidermal cells and confirmed our results obtained in protoplasts (Fig. S6). Upon plasmolysis of YFP-OsTPC1 expressing onion epidermal cells in addition to the tonoplast, bright fluorescence of Hechtian strands became visible (Fig. S5e). Because Hechtian strands contain endomembrane structures in a highly condensed cytoplasm (Buer et al., 2000; Lang-Pauluzzi & Gunning, 2000) in accordance with our previous findings fluorescence likely reflects YFP-OsTPC1 protein stuck in ER and/or Golgi rather than plasma membrane localization. While the two promoters differed only marginally with respect to sub-cellular TPC1 localization, our results suggest that N-terminal fusions to TPC1 have to be viewed with caution (see the 'Discussion' section).
In the next step we used the vacuoles presented in Fig. 3 (middle panel) for patch-clamp studies, and could unequivocally assign OsTPC1 as well as TaTPC1 the potential to rescue the SV channel defect of the attpc1-2 mutant (Fig. 3a–c, right panel). These data support the notion that in rice as in Arabidopsis TPC1 represents the SV channel. Likewise, we could show that vacuoles from the ostpc1-deficient rice plants and suspension-cultured cells lack SV channel currents (Fig. 1a,b). In a complementary approach vacuoles isolated from rice ostpc1 mutant protoplasts regained SV channel activity too following transient transformation of with AtTPC1 (Fig. 3d,e). Likewise tobacco BY2-cells exhibit SV-type vacuolar currents (Fig. S7c) and Arabidopsis tpc1-2 vacuoles could be rescued by functional complementation with either tobacco NtTPC1a or b (Fig. S7c). Transient expression of Nicotiana benthamiana (Fig. S7a) as well as Arabidopsis tpc1-2 (Fig. S7b) protoplasts with GFP-NtTPC1a and GFP-NtTPC1b under control of the CaMV 35S promoter showed vacuolar localization.
In animal cells OsTPC1 is associated with the lyso-vacuole membrane
Recently Hamada et al. (2012) expressed Ostpc1 in Human Embryonic Kidney (HEK293) cells. From whole-cell patch-clamp analyses OsTPC1 was characterized as a voltage-dependent Ca2+-permeable ion channel. These studies were conducted using media promoting Ca2+ currents and suppressing K+ channel activity. Therefore, the pipette solution (artificial cytosol) contained 120 mM Cs+, impermeable to K+-selective channels. At the same time the extra-cytoplasmic solution was dominated by 40 mM Ba2+, a known Ca2+ channel substrate that blocks K+ channels (Jiang & MacKinnon, 2000). Then we studied the performance of OsTPC1 in rice vacuoles under ionic conditions used before with OsTPC1-expressing HEK293T cells (Hamada et al., 2012). For whole vacuole patch-clamp recordings we exposed isolated vacuoles with artificial HEK293T cell cytoplasm and loaded patch pipettes with extra-cytoplasmic HEK293T medium (Fig. 4a). Because activation of the SV channel requires cytosolic calcium ions, we initially replaced 10 mM EGTA by 1 mM Ca2+ in the extra-vacuole medium (intra-HEK293T cell solution). Right after establishment of the whole-vacuole configuration pronounced outward SV currents of c. 140 pA/pF were recorded at a test potential of 70 mV (Fig. 4b). Within 5 min, however, the SV current dropped to a residual state of 20 pA/pF only (Fig. 4b, grey trace). A similar inhibition of SV currents was observed when replacing the vacuolar Ba2+ with Ca2+ ions (Fig. 4b, right panel). In addition, these divalents not only blocked the SV channel, but shifted midpoint activation potentials to more positive membrane potential (Fig. 2b). Taken together, this behavior is in line with the fact that the SV channel conducts Cs+ but is blocked by luminal divalent cations such as Ca2+ and Ba2+ (Ranf et al., 2008; Dadacz-Narloch et al., 2011). Our results hence indicate that OsTPC1 in the HEK293T cell plasma membrane (Hamada et al., 2012) and the two-pore channel operating as the SV channel display different properties.
Animal cells possess intrinsic TPC type channels that localize to the lyso-vacuolar compartment (Calcraft et al., 2009; Brailoiu et al., 2010; Ruas et al., 2010; Zhu et al., 2010). A very recent study showed that human TPCs function as endolysosomal Na+ channels (Wang et al., 2012; Cang et al., 2013). Direct patch-clamping studies with human HsTPC1 were feasible after vacuolin-1 induced endosome swelling (Wang et al., 2012). To see whether animal cells properly targeting HsTPC1 would sort rice OsTPC1 in a similar manner we expressed GFP-OsTPC1 and OsTPC1-GFP in HEK293 cells, too. Twenty-four hours post-transfection GFP-tagged fluorescence was present in endosomes. To increase the size of these organelles and to study OsTPC1 localization in detail, HEK293 cells were treated with 5 μM vacuolin-1 for 1 h (Huynh & Andrews, 2005; Schieder et al., 2010; Wang et al., 2012). Subsequently cells were stained with LysoTracker Red DND-99, a red-fluorescent dye for selective labeling of acidic organelles, or with the plasma membrane dye Deep Red. Indeed, after vacuolin-1 treatment we could confirm GFP-positive endolysosomes including those counterstained with LysoTracker Red (Fig. 5a, S8a). It should be noted that HEK293 cells expressing N- and C-terminally GFP-tagged OsTPC1 emitted fluorescence that was not associated with the Deep Red-stained plasma membranes (Figs 5b, S8b).
From past work a picture concerning the plant two-pore channel TPC1 has emerged. The dicot channel is engaged in slow vacuolar SV-type membrane K+ transport and in cellular K+ homeostasis. By contrast, rice and wheat TPC1 seemed to operate plasma membrane Ca2+ fluxes in response to effector mediated signaling (Hashimoto et al., 2005; Kurusu et al., 2005; Wang et al., 2005; Hamada et al., 2012). In the present study we have addressed the question whether the monocots Oryza sativa as well as Triticum aestivum harbor a slow vacuolar channel of the SV type encoded by the TPC1 gene. As in all plant species and cell types looked at so far (Hedrich et al., 1988; Pottosin & Schönknecht, 2007; Hedrich & Marten, 2011 for review), our patch-clamp studies with rice and wheat vacuoles also identified the hallmark properties of the SV channel: calcium- and voltage-dependent, depolarization activated, highly permeable to K+ and Na+ (Figs 1, 2, 3b–e, 4b, S1).
It should be noted that the slow vacuolar channel was first discovered in 1986 during the first patch-clamp studies on vacuoles from the grass Hordeum vulgare (Hedrich et al., 1986). In subsequent studies with Beta vulgaris it was characterized as an inward rectifier and named the SV channel (Hedrich & Neher, 1987). After the electrophysiological community agreed in changing sign convention for the vacuolar membrane (Bertl et al., 1992) the SV channel was classified as outward rectifying. Besides the early work with barley aleurone (Bethke & Jones, 1994, 1997) and barley mesophyll (Pottosin et al., 1997), SV channels were recorded in vacuoles from the monocots Allium cepa (Amodeo et al., 1994) and T. aestivum (Wherrett et al., 2005). After it was shown for Arabidopsis that the SV channel is the gene product of AtTPC1 (Peiter et al., 2005), homologs were identified in genomes of all land plants including bryophytes (Dadacz-Narloch et al., 2011; Hedrich & Marten, 2011). From the historical point of view one would thus not have assumed that rice would harbor the TPC1 gene but lack the SV channel.
Given that TPC1 from rice and wheat was, however, predominantly localized to the plasma membrane and shown to trigger distinct calcium-dependent cellular processes in homologous and heterologous systems (Hashimoto et al., 2004; Kurusu et al., 2004, 2005; Wang et al., 2005; Hamada et al., 2012), one is faced with the question of whether TPC1 and SV channel are synonyms. TPC1 null-mutants in Arabidopsis as well as rice lack SV currents (Fig. 1a,b; Peiter et al., 2005; Dadacz-Narloch et al., 2011). Further, by cross-species complementation of the SV channel-deficient Arabidopsis tpc1-2 mutant with OsTPC1 and patch clamping we could document that the rice TPC1 ortholog restores the slow vacuolar channel function in the dicot (Figs 3a,b, S5). In a complementary approach, a transient transformation of ostpc1 mutant with rice and Arabidopsis TPC1 rescued the SV channel activity (Fig. 3d,e, S4). These lines of evidence suggest that the SV channel is encoded by OsTPC1. Previous patch-clamp studies of liverwort vacuoles (Hedrich et al., 1988; Schönknecht & Trebacz, 2008) together with a cross-species complementation of attpc1-2 by moss and tobacco TPC1s, (Dadacz-Narloch et al., 2011; Fig. S7) as well as transient expression of Arabidopsis, wheat and rice TPC1s in Allium cepa epidermal cells (Fig. S6) indicate that the overall localization and function of slow vacuolar channels is conserved from bryophytes to vascular plants. Just like in Arabidopsis, the rice SV channel is permeable to the major plant cation osmolyte K+ and salt stress associated Na+ ions (Figs 1c, 2a,b). In the context of this Na+ permeability it should be mentioned that the human HsTPC1s have recently been characterized as Na+ channels expressed in endolysosomes (Wang et al., 2012; Cang et al., 2013). Previously proposed to be activated by NAADP (Calcraft et al., 2009), current work by Cang et al. (2013) identified mice TPC1 and TPC2 constituting a ‘lysoNaATP’ channel, based on its susceptibility towards changes in cytosolic ATP and thus the cell's energy and nutrient status. Remarkably, plant SV channels were shown to be inhibited by ATPcyt, too, so they share this pharmacological fingerprint with their mammalian orthologs (Pottosin et al., 2009). Channel localization in mammalian expression systems was accomplished employing HEK293 cells with vacuolin-1 swollen endosomes expressing GFP-labeled HsTPC1 (Wang et al., 2012). When OsTPC1 was introduced into the HEK293 system the rice TPC1 just like that from human was predominantly targeted to the endosomes (Figs 5, S8). This indicates that the animal cells just like cells from, for example, suspension-cultured rice recognize OsTPC1, target domains and sort the channel into the lyso-vacuolar compartment. Our observation showing more efficient vacuolar targeting of carboxy-terminal tagged TPC1 compared to amino-terminal tagging is well in line with the existence of an N-terminal dileucine motif essential for tonoplast/endolysosomal sorting (Brailoiu et al., 2010; Larisch et al.,2012b). In order to strengthen this point we performed experiments which let us conclude that under control of the UBQ10 promoter any TPC1 construct (from rice or Arabidopsis) possessing an N-terminally fused fluorophore is not properly targeted to the vacuolar membrane. This observation was made following expression of the respective constructs in either rice or Arabidopsis protoplasts as well as onion epidermal cells (Figs S3d, S4d–f, S5b). These experiments further strongly suggest that N-terminally tagged TPC1 channels reside in cytosolic (endomembrane) compartments. This finding is supported when comparing the fluorescence appearance of known cytosol localized control constructs like Arabidopsis PP2C ABI1 and SnRK2.6 OST1 (Geiger et al., 2009) or plasma membrane markers such as Arabidopsis remorin AtREM1.3 (Demir et al., 2013) and ATPase AtAHA9 (Houlné & Boutry, 1994) with that of the respective YFP-TPC1 patterns (Fig. S9). In addition, employing two different well characterized promoters (CaMV 35S and UBQ10) provided additional means to study the expression level dependence of channel targeting. Under all conditions tested we recorded TPC1-mediated SV-type currents across the vacuolar membrane. This strongly suggests that the vacuolar membrane represents the default compartment of plant TPC1 channels. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane of cultured rice cells (Hamada et al., 2012). Proteomics studies on rice cells performed so far failed to assign the OsTPC1 channel protein to its subcellular compartment (Tanaka et al., 2004; Whiteman et al., 2008). By contrast, many proteomics reports have confirmed the vacuolar localization of TPC1 channels from Arabidopsis and barley (Carter et al., 2004; Szponarski et al., 2004; Endler et al., 2006; Jaquinod et al., 2007). Future studies will thus have to solve the vacuole–plasma membrane controversy by answering the two major questions: How is OsTPC1 in certain systems targeted to the plasma membrane?; and How can the environment shape OsTPC1 in the plasma membrane to gain function as an inward rectifying Ca2+ channel and in the vacuole as a calcium-dependent outward rectifying K+ permeable SV channel. A recent study using photo-affinity labeling implies that TPC channels may exist as a protein complex with regulatory NAADP-binding proteins (Lin-Moshier et al., 2012). Other unknown components that interact with TPC channels may affect their intracellular localization and properties. Searches for interactors of TPC proteins in vivo may provide new insights.
We thank Stefanie Hüttl for great help in HEK cells transfection, Dr Armando Carpaneto for providing the double Boltzman fitting procedure, Dr Kai Konrad for the AtAHA9 construct and Claudia Horntrich for excellent help with confocal imaging. This work was supported by DFG grants of the research group FOR964 to D.B. and R.H.