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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
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA are implicated in signal transduction pathways. DGKs have been widely studied in animals, but their analysis in plants is fragmentary. Here, we report the cloning and biochemical characterization of AtDGK2, encoding DGK from Arabidopsis thaliana. AtDGK2 has a predicted molecular mass of 79.4 kDa and, like AtDGK1 previously reported, harbors two copies of a phorbol ester/DAG-binding domain in its N-terminal region. AtDGK2 belongs to a family of seven DGK genes in A. thaliana. AtDGK3 to AtDGK7 encode similar to55-kDa DGKs that lack a typical phorbol ester/DAG-binding domain. Phylogenetically, plant DGKs fall into three clusters. Members of all three clusters are widely expressed in vascular plants. Recombinant AtDGK2 was expressed in Escherichia coli and biochemically characterized. The enzyme phosphorylated 1,2-dioleoyl-sn-glycerol to yield PA, exhibiting Michaelis-Menten type kinetics. Estimated K-m and V-max values were 125 muM for DAG and 0.25 pmol of PA min(-1) mug(-1), respectively. The enzyme was maximally active at pH 7.2. Its activity was Mg2+-dependent and affected by the presence of detergents, salts, and the DGK inhibitor R59022, but not by Ca2+. AtDGK2 exhibited substrate preference for unsaturated DAG analogues (i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2- dioleoyl-sn-glycerol). The AtDGK2 gene is expressed in various tissues of the Arabidopsis plant, including leaves, roots, and flowers, as shown by Northern blot analysis and promoter-reporter gene fusions. We found that AtDGK2 is induced by exposure to low temperature (4degreesC), pointing to a role in cold signal transduction
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 thatchannel a-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