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 ...

Formation of giant protoplasts from normal Escherichia coli cells resulted in the formation of giant vacuole-type structures (which we designate as provacuoles) in the protoplasts. Electron microscopic observation revealed that these provacuoles were surrounded by a single membrane. We detected inner (cytoplasmic) membrane proteins in the provacuolar membrane but not outer membrane proteins. Biochemical analyses revealed that the provacuoles consist of everted cytoplasmic membranes. We applied the patch clamp method to the giant provacuoles. We have succeeded in measuring current that represents inward movement of H ؉ because of respiration and to ATP hydrolysis by the F o F 1 -ATPase. Such current was inhibited by inhibitors of the respiratory chain or F o F 1 -ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.The patch clamp technique is an excellent method to measure ion movement across cell membranes as current (1). An extremely small glass pipette (about 1 m in diameter) is attached to the membranes, and activity of ion translocating proteins (ion channels, ion pumps, or ion transporters) is directly measured. So far, however, this important method has been mainly utilized for studies on animal or plant cells but scarcely for bacterial cells (2). Bacterial cells are usually too small to be measured by this method.Escherichia coli, a Gram-negative bacterium, is the best characterized organism from both biochemical and genetical points of view. Ion pumps and ion transporters in E. coli are biochemically well characterized. Many mutant E. coli cells are available. Thus, genetical manipulations are very easy with this microorganism. Therefore, development of a patch clamp method applicable to E. coli membranes must be extremely valuable. Cells of E. coli are surrounded by an outer membrane and an inner membrane (cytoplasmic membrane) separated by a peptidoglycan layer and a periplasmic space. All of the major ion pumps and ion transporters such as the respiratory chain, F o F 1 -ATPase, various ion transporters, and ion-coupled solute transporters are located in the cytoplasmic membrane. To measure ion translocation via such ion pumps or transporters of the cytoplasmic membrane, we have to overcome the following three hurdles: 1) we have to prepare giant vesicles, the diameter of which must be at least 10 m (this is important to get high success rate and accuracy of measurement), 2) the pipette must be directly accessible to the cytoplasmic membrane, and 3) the substrates or effectors of the ion pumps or transporters must be easily accessible to the active site of the proteins and easily removable from the system.It would be essential to prepare giant protoplasts to overcome the first two hurdles. Many attempts have been made by many research groups to prepare giant bacterial cells or giant protoplasts. So far, however, no giant protoplasts surrounded by cytoplasmic membranes and suitable for patch clamp analysis have been prepared....

Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(⌬vma1::TRP)). The K m value for the ATP dose response of the current was 159 M and the H ؉ /ATP ratio estimated from the reversible potential of the V-I curve was 3.5 ؎ 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.

No abstract

Saccharomyces cerevisiae possesses a transient receptor potential (TRP) channel homolog TRPY1 in its vacuolar membrane, considered to be an ancestral TRP channel. So far, studies have focused on the channel properties of TRPY1, but its regulation and physiologic role remained to be elucidated. Here, we investigated TRPY1 channel function in vitro and in vivo. Patch-clamp recording of TRPY1 in yeast vacuolar membranes showed that Ca on the lumen side inhibited TRPY1-mediated channel activity, whereas luminal Zn increased the currents. TRPY1 was activated in the presence of a reducing agent, 2-mercaptoethanol. The cysteine at position 624 was identified as the target for this activating action. This activation was independent of the presence of cytosolic Ca . The amplitude of TRPY1-mediated current was reduced by addition of phosphatidylinositol 3-phosphate on the cytosolic side but not by phosphatidylinositol (PI) or phosphatidylinositol 3,5-phosphate. Measurement of the transient Ca increase in response to hyper-osmotic shock in several yeast mutants defective in different steps of the PI phosphate biogenesis pathway supported this interpretation. Addition of a microtubule inhibitor strongly decreased the transient cytosolic Ca increase upon hyper-osmotic shock. Taken together, the data indicate that the vacuolar TRPY1 Ca channel mediates the perception of cytosolic signals that were induced by external changes in osmolarity, and participates in the modulation of cytosolic calcium signaling through Ca release from the vacuole to maintain intracellular Ca homeostasis in yeast.