The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K+ channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca2+-activated K+ channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca2+- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.

TPK1 (formerly KCO1) is the founding member of the family of two-pore domain K(+) channels in Arabidopsis (Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca(2+)- and voltage-dependent outwardly rectifying plasma membrane K(+) channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca(2+) and voltage-dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast (Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100 mM KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca(2+), requiring micromolar concentration for activation. However, in contrast to the SV-type channel, TPK1 exhibits strong selectivity for K(+) over Na(+), and its activity turned out to be independent of the membrane voltage over the range of +/-80 mV. Our data clearly demonstrate that TPK1 is a voltage-independent, Ca(2+)-activated, K(+)-selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.

The yeast plasma-membrane potassium channel, Tok1p, is a voltage-dependent outward rectifier, the gating and steady-state conductance of which are conspicuously modulated by extracellular [K+] ([K+]o). Activation is slow at high [K+]o, showing time constants (τa) of ~90 ms when [K+]o is 150 mM (depolarizing step to +100 mV), and inactivation is weak (<30%) during sustained depolarization. Lowering [K+]o accelerates activation, increases peak current, and enhances inactivation, so that at 15 mM [K+]o τa is less than 50 ms and inactivation suppresses ~60% of peak current. Two negative residues, Asp292 and Asp426, near the mouth of the assembled channel, modulate both kinetics and conductance of the channel. Charge neutralization in the mutant Asp292Asn allows fast activation (τa~20 ms) at high [K+]o, peak currents diminishing with decreasing [K+]o, and fast, nearly complete, inactivation. The voltage dependence of τa persists in the mutant, but the [K+]o dependence almost disappears. Similar but smaller changes are seen in the Asp426Asn mutant, implying that pore geometry in the functional channel has twofold, not fourfold, symmetry.

The Arabidopsis tandem-pore K(+) (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K(+) channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K(+) currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K(+) transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K(+) channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium.

SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K(+) channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K(out) channels. Deletion mutants and chimeric proteins generated from SKOR and the K(in) channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K(T) domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K(out) alpha-subunits did not assemble with K(in) alpha-subunits because of the absence of interaction between their assembly sites.

Isolated vacuoles from the yeast Saccharomyces cerevisiae were examined in the whole-vacuole mode of patch recording, to get a detailed functional description of the vacuolar proton pump, the V-ATPase. Functioning of the V-ATPase was characterized by its current-voltage (I-V) relationship, obtained for various levels of vacuolar and cytosolic pH. I-V curves for the V-ATPase were computed as the difference between I-V curves obtained with the pump switched on (ATP, ADP, and Pi present) or off (no ATP). These difference current-voltage relationships usually crossed the voltage axis within the experimental range (from −80 to +80mV), thus measuring the reversal voltage (ER) for the V-ATPase, which could be compared with the standing ion gradients and free energy of ATP hydrolysis, to calculate the apparent pump stoichiometry or coupling ratio: the number of protons transported for each ATP molecule hydrolyzed. This ratio was found to depend strongly upon the pH difference (ΔpH) across the vacuolar membrane, being ∼2H+/ATP at high ΔpH (4 pH units) and increasing to >4H+/ATP for small or zero ΔpH. That result is in quantitative agreement with previous determinations on plant vacuoles. Considerations of purely electrical behavior, together with the physical properties of a recent detailed structural model for V-ATPases, led to a linear equivalent circuit—which quantitatively accounts for all observations of variable coupling ratios in fungal and plant V-ATPases by variations of the conductance for bona fide proton pumping (GP) through the ATPase relative to independent proton shunting (GS) through the same protein.

The activity of the vacuolar H(+)-ATPase has been characterized in isolated vacuoles of the yeast Saccharomyces cerevisiae by means of the patch-clamp technique. With cytosolic calcium at virtually zero (<10(-9) M), Mg-ATP induced a transient, bafilomycin A(1)-sensitive current corresponding to the flow of positive charges from the cytoplasmic surface to the vacuolar lumen. The Mg-ATP-dependent current reached its maximum amplitude (30+/-8 mA m(-2) with 5 mM Mg-ATP, n=34) within 15-20 s and declined slowly over a period of about 15-20 min even in the continuous presence of Mg-ATP. This decline of pumping activity was independent of the cytosolic KCl concentration, suggesting an inhibitory mechanism different from the high salt-induced dissociation of V(0) and V(1) reported for the V-ATPase of plants and fungi. Cytosolic ADP was found to modulate the pump activity since Mg-ATP-induced pump current was smaller if monitored in the presence of 5 mM ADP and addition of 5 mM ADP in the presence of 5 mM Mg-ATP reduced the pump current by more than 50%. Furthermore, reduction of the cytosolic ADP concentration by the ATP-regenerating system creatine phosphate/creatine kinase partially relieved the endogenous inhibition of the V-ATPase, confirming that interaction of cytosolic ADP with the V-ATPase is the reason for the transient nature of the pump current in yeast vacuoles.

Saccharomyces cerevisiae cells express three defined potassium-specific transport systems en-coded by TRK1, TRK2 and TOK1. To gain a more complete understanding of the physiological function of these transport proteins, we have constructed a set of isogenic yeast strains carrying all combinations of trk1delta, trk2delta and tok1delta null mutations. The in vivo K+ transport characteristics of each strain have been documented using growth-based assays, and the in vitro biochemical and electrophysiological properties associated with K+ transport have been determined. As has been reported previously, Trk1p and Trk2p facilitate high-affinity potassium uptake and appear to be functionally redundant under a wide range of environmental conditions. In the absence of TRK1 and TRK2, strains lack the ability specifically to take up K+, and trk1deltatrk2delta double mutant cells depend upon poorly understood non-specific cation uptake mechanisms for growth. Under conditions that impair the activity of the non-specific uptake system, termed NSC1, we have found that the presence of functional Tok1p renders cells sensitive to Cs+. Based on this finding, we have established a growth-based assay that monitors the in vivo activity of Tok1p.

Previous descriptions by whole-cell patch clamping of the calcium-inhibited non-selective cation channel (NSC1) in the plasma membrane of Saccharomyces cerevisiae (H. Bihler, C.L. Slayman, A. Bertl, FEBS Lett. 432 (1998); S.K. Roberts, M. Fischer, G.K. Dixon, D.Sanders, J. Bacteriol. 181 (1999)) suggested that this inwardly rectifying pathway could relieve the growth inhibition normally imposed on yeast by disruption of its potassium transporters, Trk1p and Trk2p. Now, demonstration of multiple parallel effects produced by various agonists and antagonists on both NSC1 currents and growth (of trk1Δtrk2Δ strains), has identified this non-selective cation pathway as the primary low-affinity uptake route for potassium ions in yeast. Factors which suppress NSC1-mediated inward currents and inhibit growth of trk1Δtrk2Δ cells include (i) elevating extracellular calcium over the range of 10 μM–10 mM, (ii) lowering extracellular pH over the range 7.5–4, (iii) blockade of NSC1 by hygromycin B, and (iv) to a lesser extent by TEA+. Growth of trk1Δtrk2Δ cells is also inhibited by lithium and ammonium; however, these ions do not inhibit NSC1, but instead enter yeast cells via NSC1. Growth inhibition by lithium ions is probably a toxic effect, whereas growth inhibition by ammonium ions probably results from competitive inhibition, i.e. displacement of intracellular potassium by entering ammonium.

Ionic currents related to the major potassium uptake systems in Saccharomyces cerevisiae were examined by whole cell patch‐clamping, under K + replete conditions. Those currents have the following properties. They (1) are inward under all conditions investigated, (2) arise instantaneously with appropriate voltage steps, (3) depend solely upon the moderate affinity transporter Trk2p, not upon the high affinity transporter Trk1p. They (4) appear to be independent of the extracellular K + concentration, (5) are also independent of extracellular Ca 2+ , Mg 2+ and Cl − but (6) are strongly dependent on extracellular pH, being large at low pH (up to several hundred pA at −200 mV and pH 4) and near zero at high pH (above 7.5). They (7) increase in proportion to log[H + ] o , rather than directly in proportion to the proton concentration and (8) behave kinetically as if each transporter cycle moved one proton plus one (high pH) or two (low pH) other ions, as yet unidentified. In view of background knowledge on K + transport related to Trk2p, the new results suggest that the K + status of yeast cells modulates both the kinetics of Trk2p‐mediated transport and the identity of ions involved. That modulation could act either on the Trk2 protein itself or on interactions of Trk2 with other proteins in a hypothetical transporter complex. Structural considerations suggest a strong analogy to the KtrAB system in Vibrio alginolyticus and/or the TrkH system in Escherichia coli .

The plasma membrane of the yeast Saccharomyces cerevisiae possesses a non‐specific cation ‘channel', tentatively dubbed NSC1, which is blocked by normal (mM) calcium and other divalent metal ions, is unblocked by reduction of extracellular free divalents below ∼10 μM, and is independent of the identified potassium channel and porters in yeast, Duk1p, Trk1p, and Trk2p. Ion currents through NSC1, observed by means of whole‐cell patch recording, have the following characteristics: Large amplitude , often exceeding 1 nA of K + /cell at −200 mV, in tetraploid yeast, sufficient to double the normal intracellular K + concentration within 10 s; non‐saturation at large negative voltages; complicated activation kinetics , in which ∼50% of the total current arises nearly instantaneously with a voltage‐clamp step, while the remainder develops as two components, with time constants of ∼100 ms and ∼1.3 s; and voltage independence of both the activation time constants and the associated fractional current amplitudes.

The major voltage-dependent ion channel in the plasma membrane of Saccharomyces cerevisiae, a conspicuous outwardly rectifying K+ channel, was first dubbed YPK1 and later renamed according to its registered gene names (DUK1, TOK1). It has proven novel in both structure and function. Whole-cell patch-clamp studies of the channel directly on yeast protoplasts now extend our earlier description obtained from isolated patches of yeast membrane (Bertl & Slayman, 1992; Bertl et al., 1993), and provide new data both on the contributions of channel properties to yeast physiology and on possible contributions of molecular structure to channel properties. Three recording tactics produce completely equivalent results and thereby allow great flexibility in the design of experiments: whole-cell voltage clamp with sustained voltage steps (∼2.5 sec), whole-cell voltage clamp with slow voltage ramps (5 sec, −40 to +100 mV), and time-averaging of single-channel currents.