Characterization of a Tobacco TPK-type K+ Channel as a Novel Tonoplast K+ Channel Using Yeast Tonoplasts

Abstract

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 ϳ2-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 Ca 2؉ , but a cytosolic concentration of 45 M Ca 2؉ 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.Plants take up potassium (K ϩ ) from the soil and plant cells accumulate K ϩ to regulate the membrane potential and turgor pressure. The cytoplasmic K ϩ concentration is tightly controlled at ϳ100 mM (1). Vacuoles are major subcellular reservoirs for controlling K ϩ homeostasis in plant cells (1). During cell expansion, for instance during stomata opening or cell growth, tonoplast transport system moves K ϩ into the vacuole, whereas, under conditions of salinity stress, K ϩ is replaced by Na ϩ (2-5).Several kinds of genes encoding K ϩ channels and K ϩ 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 ϩ 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 ϩ profile in Xenopus laevis ooctyes and in yeast, and the K ϩ current is inhibited by extracellular Ca 2ϩ 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 ...

Intracellular localization and physiological function of a rice Ca2+-permeable channel OsTPC1

“…15 For the purification of the VM fraction, we used the experimental protocol optimized for BY-2 cells. 26 Proteins of each fraction was analyzed by SDS-PAGE without thermal denaturation, and OsTPC1-GFP was detected both by immunoblot analysis using specific antibody against OsTPC1 15 and fluorescence image analysis of GFP. 24,27 Unlike the localization in the rice cells, the majority of OsTPC1-GFP expressed in tobacco BY-2 cells was detected in the VM fraction (Fig.…”

“…Three-day-old BY-2 cells were treated with FM4-64 for 3 h and washed twice with the culture medium at room temperature to label the VM. 23,25 (B) The PM and the VM fractions of tobacco BY-2 cells were prepared using an aqueous two-phase partitioning 15 and a sucrose/sorbitol method, 26 respectively. To detect the GFP fluorescence in the SDS-PAGE gels, unheated protein samples (20 μg per lane) of each fraction were subjected to SDS-PAGE and the fluorescence of OsTPC1-GFP were detected using a 473 nm excitation laser through a 520 nm LP emission filter 24,27 using the fluoro image analyzer FLA3000G (GE Healthcare Bio-sciences, USA).…”

Rice Two-Pore K+ Channels Are Expressed in Different Types of Vacuoles

“…properties, with K + selectivity, inward rectification, and voltageindependent gating being reminiscent of Arabidopsis and tobacco TPKs (Gobert et al, 2007;Hamamoto et al, 2008). Nevertheless, these proteins appear to localize to different cellular organelles.…”

“…Only TPK1 has been shown to form functional ion channels; TPK1 activity is voltage independent, it has one or two EF hands localized in the C terminus, and it is regulated by intracellular Ca 2+ (Gobert et al, 2007). TPK1 activity is further modulated by cytoplasmic pH, phosphorylation, and 14-3-3 proteins (Gobert et al, 2007;Latz et al, 2007), whereas the activity of Nicotiana tabacum TPK1 was also sensitive to spermidine and spermine (Hamamoto et al, 2008). At TPK1 has been functionally characterized using overexpression, loss-of-function, and heterologous expression analyses; it is ubiquitously expressed and has a role in cellular K + homeostasis and K + release during stomatal functioning (Gobert et al, 2007).…”

Assembly and Sorting of the Tonoplast Potassium Channel AtTPK1 and Its Turnover by Internalization into the Vacuole

“…The observations that AtTPK1 undergoes homotypic interactions (Voelker et al, 2006) and that an active selective pore must be formed by four P domains (Lesage et al, 1996;Daram et al, 1997;Perozo et al, 1998) suggest that TPKs should be dimers (Latz et al, 2007a). However a second model has been proposed where only the first P domain would take part to the formation of the selective pore and thus functional channels would involve four subunits (Hamamoto et al, 2008;Marcel et al, 2010).…”