The Arabidopsis thaliana genome contains five genes that encode two pore K ؉ (TPK) channels. The most abundantly expressed isoform of this family, TPK1, is expressed at the tonoplast where it mediates K ؉ -selective currents between cytoplasmic and vacuolar compartments. TPK1 open probability depends on both cytoplasmic Ca 2؉ and cytoplasmic pH but not on the tonoplast membrane voltage. The channel shows intrinsic rectification and can be blocked by Ba 2؉ , tetraethylammonium, and quinine. TPK1 current was found in all shoot cell types and shows all of the hallmarks of the previously described vacuolar K (VK) tonoplast channel characterized in guard cells. Characterization of TPK1 loss-of-function mutants and TPK1-overexpressing plants shows that TPK1 has a role in intracellular K ؉ homeostasis affecting seedling growth at high and low ambient K ؉ levels. In stomata, TPK1 function is consistent with vacuolar K ؉ release, and removal of this channel leads to slower stomatal closure kinetics. During germination, TPK1 contributes to the radicle development through vacuolar K ؉ deposition to provide expansion growth or in the redistribution of essential minerals.

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 Arabidopsis thaliana K + channel family of AtTPK/KCO proteins consists of six members including a 'singlepore' (K ir -type) and five 'tandem-pore' channels. AtTPK4 is currently the only ion channel of this family for which a function has been demonstrated in planta. The protein is located at the plasma membrane forming a voltage-independent K + channel that is blocked by extracellular calcium ions. In contrast, AtTPK1 is a tonoplast-localized protein, that establishes a K + -selective, voltage-independent ion channel activated by cytosolic calcium when expressed in a heterologous system, i.e. yeast. Here, we provide evidence that other AtTPK/KCO channel subunits, i.e. AtTPK2, AtTPK3, AtTPK5 and AtKCO3, are also targeted to the vacuolar membrane, opening the possibility that they interact at the target membrane to form heteromeric ion channels. However, when testing the cellular expression patterns of AtTPK/KCO genes we observed distinct expression domains that overlap in only a few tissues of the Arabidopsis plant, making it unlikely that different channel subunits interact to form heteromeric channels. This conclusion was substantiated by in planta expression of combinations of selected tonoplast AtTPK/KCO proteins. Fluorescence resonance energy transfer assays indicate that protein interaction occurs between identical channel subunits (most efficiently between AtTPK1 or AtKCO3) but not between different channel subunits. The finding could be confirmed by bimolecular fluorescence complementation assays. We conclude that tonoplast-located AtTPK/KCO subunits form homomeric ion channels in vivo.

Potassium (K+) channels mediating important physiological functions are characterized by a common pore–forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of ‘two‐pore’ K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium‐binding EF‐hand motifs. Heterologous expression of KCO1 in baculovirus‐infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+‐selective currents elicited by depolarizing voltage pulses in whole‐cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at ∼300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+‐dependent activation. These data suggest a direct link between calcium‐mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure–function studies in plant ion channels.

SummaryPotassium (K + ) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K + channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K + channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus-infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5¢ region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying b-glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5¢ leader intron. The same overall structure was identi®ed in two genes encoding AtKCO1-like K + channels from Solanum tuberosum (StKCO1a and StKCO1b). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green¯uorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co-fractionate with tonoplast markers (NtTIPa, vATPase). In¯uorescence images from transgenic AtKCO1±GFP BY2 cells¯uorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the ®rst cloned K + channel demonstrated to be a vacuolar K + channel.