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 domains͞one 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.

SummaryThe 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, patchclamp studies identified TPK1 as a voltage-independent and Ca 2+ -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 Ca 2+ -and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.

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The vacuolar membrane of the yeast Saccharomyces cerevisiae, which is proposed as a system for functional expression of membrane proteins, was examined by patchclamp techniques. Its most conspicuous feature, in the absence of energizing substrates, is a cation channel with a characteristic conductance of -120 pS for symmetric 100 mM KCI solutions and with little selectivity between K+ and Na' (PNa+/PK+ 1) but strong selectivity for cations over anions (PCI-/PK+ < 0.1). Channel gating is voltage-dependent; open probability, P., reaches maximum (-0.7) at a transmembrane voltage of -80 mV (cytoplasmic surface negative) and declines at both more negative and more positive voltages (i.e., to 0 around +80 mV). The time-averaged current-voltage curve shows strong rectification, with negative currents (positive charges flowing from vacuolar side to cytoplasmic side) much larger than positive currents. The open probability also depends strongly on cytoplasmic Ca2+ concentration but, for ordinary recording conditions, is high only at unphysiologically high (21 mM) Ca2+. However, reducing agents such as dithiothreitol and 2-mercaptoethanol poise the channels so that they can be activated by micromolar cytoplasmic Ca2+. The channels are blocked irreversibly by chloramine T, which is known to oxidize exposed methionine and cysteine residues specifically.Invention of patch-clamp techniques in 1976 (1, 2) has opened up a whole new range of biological preparations to direct electrophysiological analysis. Isolated single channel molecules can be studied in micrometer-sized patches of cell membranes, and thylakoid membranes of individual chloroplasts (3), inner and outer membranes of mitochondria (4, 5), and small microbial cells (6) have become readily accessible. This circumstance, particularly when combined with new developments in molecular biology, greatly enhances the utility of electrophysiological studies on microorganisms, which had until recently been restricted to a few fungi (7-10), slime molds (11, 12), and one species of swollen bacteria (13).The yeast Saccharomyces cerevisiae seems particularly advantageous for investigation with patch electrodes, for two reasons: (i) the electrical properties of active transport systems in its plasma membrane can readily be compared with those already described (from measurements with penetrating electrodes) in another ascomycete fungus, Neurospora (14, 15), and (ii) Saccharomyces is becoming a major system for stable expression and manipulation of both animal and plant genes (e.g., see refs. 16-18).Previous patch-clamp studies on Saccharomyces have reported plasma membrane K+ channels that are voltagedependent (19) and generally fit into an emerging pattern of outward-rectifying channels in surface membranes of plants and plant-like cells (20,21). More recently, K+ channels have been described as voltage-gated, opening beyond + 100 mV in wild-type strains of yeast, but at lower voltages in a mutant, pmal-105 (22). Most intriguing, this mutant is defective in the structura...

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