Roles of tandem-pore K+ channels in plants - a puzzle still to be solved.

The group of voltage-independent K(+) channels in Arabidopsis thaliana consists of six members, five tandem-pore channels (TPK1-TPK5) and a single K(ir)-like channel (KCO3). All TPK/KCO channels are located at the vacuolar membrane except for TPK4, which was shown to be a plasma membrane channel in pollen. The vacuolar channels interact with 14-3-3 proteins (also called General Regulating Factors, GRFs), indicating regulation at the level of protein-protein interactions. Here we review current knowledge about these ion channels and their genes, and highlight open questions that need to be urgently addressed in future studies to fully appreciate the physiological functions of these ion channels.

Group IVA phospholipase A2-mediated production of fibronectin by oxidized LDL in mesangial cells.

The deposition of atherogenic lipoproteins such as oxidized low-density lipoprotein (oxLDL) within the mesangium is involved in the overproduction of extracellular matrix proteins, a key event in the progression of glomerular diseases including glomerulosclerosis. To clarify the mechanisms underlying the oxLDL-induced production of extracellular matrix proteins, we examined the possible involvement of group IVA phospholipase A(2) (PLA(2)) using human mesangial cells and group IVA PLA(2)-deficient mouse mesangial cells. oxLDL accelerated the production of fibronectin and collagen (type IV), components of extracellular matrix proteins, with the preceding release of arachidonic acid. Methyl arachidonyl fluorophosphonate (MAFP), known as an inhibitor of group IVA PLA(2), markedly suppressed the oxLDL-induced production of fibronectin as well as the release of arachidonic acid, whereas it did not inhibit the production of collagen. The inhibitory effect of MAFP on the production of fibronectin was reversed by adding arachidonic acid and 12-hydroxyeicosatetraenoic acid. Furthermore, we found that in group IVA PLA(2)-deficient mouse mesangial cells, the production of fibronectin in response to oxLDL was weak as compared with that in wild-type cells. However, the production by oxLDL of collagen was not suppressed in the group IVA PLA(2)-deficient cells. These findings suggest that group IVA PLA(2) is involved in the production of fibronectin in oxLDL-stimulated mesangial cells.

Properties of shaker-type potassium channels in higher plants.

Potassium (K(+)), the most abundant cation in biological organisms, plays a crucial role in the survival and development of plant cells, modulation of basic mechanisms such as enzyme activity, electrical membrane potentials, plant turgor and cellular homeostasis. Due to the absence of a Na(+)/K(+) exchanger, which widely exists in animal cells, K(+) channels and some type of K(+) transporters function as K(+) uptake systems in plants. Plant voltage-dependent K(+) channels, which display striking topological and functional similarities with the voltage-dependent six-transmembrane segment animal Shaker-type K(+) channels, have been found to play an important role in the plasma membrane of a variety of tissues and organs in higher plants. Outward-rectifying, inward-rectifying and weakly-rectifying K(+) channels have been identified and play a crucial role in K(+) homeostasis in plant cells. To adapt to the environmental conditions, plants must take advantage of the large variety of Shaker-type K(+) channels naturally present in the plant kingdom. This review summarizes the extensive data on the structure, function, membrane topogenesis, heteromerization, expression, localization, physiological roles and modulation of Shaker-type K(+) channels from various plant species. The accumulated results also help in understanding the similarities and differences in the properties of Shaker-type K(+) channels in plants in comparison to those of Shaker channels in animals and bacteria.

Protective role for cytosolic phospholipase A2alpha in autoimmune diabetes of mice.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) plays an important role in arachidonate pathway. To investigate the contribution of cPLA(2)alpha to autoimmune diabetes, we established non-obese diabetic (NOD) mouse, an excellent model for human type 1 diabetes, deficient in cPLA(2)alpha. These mice showed severe insulitis and a higher incidence of diabetes. In their macrophages, decreased prostaglandin E(2) (PGE(2)) induced by cPLA(2)alpha deficiency, and the increase in production of tumor necrosis factor (TNF)-alpha were observed. These results suggested that cPLA(2)alpha plays a protective role in progression of insulitis and development of autoimmune diabetes by suppression of TNF-alpha production from macrophages.

Mice deficient in cytosolic phospholipase A2 are less susceptible to cerebral ischemia/reperfusion injury.

To determine the role of cytosolic phospholipase A2 (cPLA2) in infarct development, wild-type and cPLA2 knock-out mice were subjected to focal cerebral ischemia for 75 min by occluding the middle cerebral artery using nylon filament and subsequent reperfusion by withdrawing the filament. The neurological deficit severity was evaluated by a modified 4-point scale. After the reperfusion period (72 h), mice were killed, and the brains were cut into four 2 mm coronal sections using a rodent brain matrix. Sections were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC). The infarct volume was 87.19 +/- 27.54 mm3 (mean +/- SD, n = 11) in the wild-type mice and 48.20 +/- 31.32 mm3 (n = 10; P < 0.01 vs. wild-type) in the knock-out mice. Less severe functional neurological deficits were observed in knock-out mice at 72 h after ischemia when compared with wild-type. Thus, disruption of cPLA2 resulted in significant reduction of infarct area and neurological deficit severity in the MCA occlusion model. These data indicate a critical role for cPLA2 in the pathogenesis of cerebral ischemia/ reperfusion injury.

Residue aspartate-147 from the third transmembrane region of Na(+)/H(+) antiporter NhaB of Vibrio alginolyticus plays a role in its activity.

NhaB is a bacterial Na(+)/H(+) antiporter with unique topology. The pH dependence of NhaB from Vibrio alginolyticus differs from that of the Escherichia coli NhaB homolog. Replacement of Asp-147 with Glu made high H(+) concentrations a requirement for the NhaB activity. Replacement of Asp-147 with neutral amino acids inactivated NhaB.

Escherichia coli as an expression system for K(+) transport systems from plants.

The value of the Escherichia coli expression system has long been established because of its effectiveness in characterizing the structure and function of exogenously expressed proteins. When eukaryotic membrane proteins are functionally expressed in E. coli, this organism can serve as an alternative to eukaryotic host cells. A few examples have been reported of functional expression of animal and plant membrane proteins in E. coli. This mini-review describes the following findings: 1) homologous K(+) transporters exist in prokaryotic cells and in eukaryotic cells; 2) plant K(+) transporters can functionally complement mutant K(+) transporter genes in E. coli; and 3) membrane structures of plant K(+) transporters can be elucidated in an E. coli system. These experimental findings suggest the possibility of utilizing the E. coli bacterium as an expression system for other eukaryotic membrane transport proteins.

Sodium blocking induced by a point mutation at the C-terminal end of the pore helix of the KAT1 channel.

A plant hyperpolarization-activating K+ channel, KAT1, is highly selective for K+ over Na+ and is little affected by external Na+, which is crucial to take up K+ effectively in a Na+-containing environment. It has been shown that a mutation at the location (Thr256) preceding the selectivity signature sequence dramatically enhanced the sensitivity of the KAT1 channel to external Na+. We report here electrophysiological experiments for the mechanism of action of external Na+ on KAT1 channels. The Thr256 residue was substituted with either glutamine (Q) or glutamate (E). The wild-type channel was insensitive to external Na+. However, the activity of both mutant channels was significantly depressed by Na+ with apparent dissociation constants of 6.7 mm and 11.3 mm for T256Q and T256E, respectively. The instantaneous current-voltage relationships revealed distinct blocking mechanisms for these mutants. For T256Q a typical voltage-dependent fast blocking was shown. On the other hand, the blocking for the T256E mutant was voltage-independent at low Na+ concentrations and became voltage-dependent at higher concentrations. At extreme hyperpolarization the blocking was relieved significantly. These data strongly suggest that the mutation at the end of the pore helix rearranged the selectivity filter and allows Na+ to penetrate into the pore.

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters.

The Arabidopsis thaliana AtHKT1 protein, a Na(+)/K(+) transporter, is capable of mediating inward Na(+) currents in Xenopus laevis oocytes and K(+) uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K(+) transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K(+) channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na(+) currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli-alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135-142 and 377-384, face the cytosol, whereas the region of residues 55-62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.

Aromatic monoamine-induced immediate oxidative burst leading to an increase in cytosolic Ca2+ concentration in tobacco suspension culture.

Aromatic monoamines may contribute to both chemical and physical protection of plants. Addition of phenylethylamine (PEA) and benzylamine to tobacco suspension culture (cell line BY-2) induced a very rapid and transient generation of two active oxygen species (AOS), H2O2 and superoxide anion, both detected with chemiluminescence. Electron spin resonance spectroscopy revealed that hydroxy radicals are also produced. With laser-scanning confocal microscopy, fluorescence spectroscopy and microplate fluorescence reading, intracellular H2O2 production was detected using dichlorofluorescin diacetate as a fluorescent probe. Following AOS production, cytosolic Ca2+ concentration ([Ca2+]c) of the tobacco cells, monitored with luminescence of transgenic aequorin, increased and attained to a peak level 12 s after PEA addition. The PEA-induced increase in [Ca2+]c was inhibited by a Ca2+ chelator, Ca2+ antagonists and AOS scavengers, suggesting that PEA-induced AOS triggered a Ca2+ influx across the plasma membrane.

Phenylethylamine-induced generation of reactive oxygen species and ascorbate free radicals in tobacco suspension culture: mechanism for oxidative burst mediating Ca2+ influx.

In the previous paper [Kawano et al. (2000a) Plant Cell Physiol. 41: 1251], we demonstrated that addition of phenylethylamine (PEA) and benzylamine can induce an immediate and transient burst of active oxygen species (AOS) in tobacco suspension culture. Detected AOS include H2O2, superoxide anion and hydroxyl radicals. Use of several inhibitors suggested the presence of monoamine oxidase-like H2O2-generating activity in the cellular soluble fraction. It was also suggested that peroxidase(s) or copper amine oxidase(s) are involved in the extracellular superoxide production as a consequence of H2O2 production. Since more than 85% of the PEA-dependent AOS generating activity was localized in the extracellular space (extracellular fluid + cell wall), extracellularly secreted enzymes, probably peroxidases, may largely contribute to the oxidative burst induced by PEA. The PEA-induced AOS generation was also observed in the horseradish peroxidase (HRP) reaction mixture, supporting the hypothesis that peroxidases catalyze the oxidation of PEA leading to AOS generation. In addition to AOS production, we observed that PEA induced an increase in monodehydroascorbate radicals (MDA) in the cell suspension culture and in HRP reaction mixture using electron spin resonance spectroscopy and the newly invented MDA reductase-coupled method. Here we report that MDA production is an indicator of peroxidase-mediated generation of PEA radical species in tobacco suspension culture.

Role of cytosolic phospholipase A2 in the production of lipid mediators and histamine release in mouse bone-marrow-derived mast cells.

Cytosolic phospholipase A(2) (cPLA(2)) plays a critical role in mast-cell-related allergic responses [Uozumi, Kume, Nagase, Nakatani, Ishii, Tashiro, Komagata, Maki, Ikuta, Ouchi et al. (1997) Nature (London) 390, 618-622]. Bone-marrow-derived mast cells from mice lacking cPLA(2) (cPLA(-/-)(2) mice) were used in order to better define the role of cPLA(2) in the maturation and degranulation of such cells. Cross-linking of high-affinity receptors for IgE (FcepsilonRI) on cells from cPLA(-/-)(2) mice led to the release of negligible amounts of arachidonic acid or its metabolites, the cysteinyl leukotrienes and prostaglandin D(2), indicating an essential role for cPLA(2) in the production of these allergic and pro-inflammatory lipid mediators. In addition, the histamine content of the mast cells and its release from the cells were reduced to 60%. While these results are in agreement with a reduced anaphylactic phenotype of cPLA(-/-)(2) mice, the ratios of release of histamine and beta-hexosaminidase were, paradoxically, significantly higher for cells from cPLA(-/-)(2) mice than for those from wild-type mice. Consistently, IgE-induced calcium influx in mast cells was greater and more prolonged in cells from cPLA(-/-)(2) mice than in those from wild-type mice. Thus the loss of cPLA(2) not only diminishes the release of lipid mediators, but also alters degranulation. While the overall effect is still a decrease in the release of mast cell mediators, explaining the in vivo findings, the present study proposes a novel link between cPLA(2) and the degranulation machinery.

A novel carbohydrate binding activity of annexin V toward a bisecting N-acetylglucosamine.

A bisecting GlcNAc-binding protein was purified from a Triton X-100 extract of a porcine spleen microsomal fraction using affinity chromatography, in conjunction with an agalacto bisected biantennary sugar chain-immobilized Sepharose. Since the erythroagglutinating phytohemagglutinin (E-PHA) lectin preferentially binds to sugar chains which contain the bisecting GlcNAc, during purification the binding activity of the protein was evaluated by monitoring the inhibition of lectin binding to the N-acetylglucosaminyltransferase III (GnT-III)-transfected K562 cells which express high levels of the bisecting GlcNAc. The molecular mass of the purified protein was found to be 33 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By sequencing analysis, the isolated protein was identified as annexin V. Flow cytometric analysis showed that fluorescein-labeled annexin V binds to the GnT-III-transfected cells but not to mock cells, and that the binding was not affected by the addition of phospholipids. Furthermore, surface plasmon resonance measurements indicated that annexin V binds to the agalacto bisected biantennary sugar chain with a K(d) of 200 microM while essentially no binding was observed in the case of the corresponding non-bisected sample. These results suggest that annexin V has a novel carbohydrate binding activity and may serve as an endogenous lectin for mediating possible signals of bisecting GlcNAc, which have been implicated in a variety of biological functions.

Suppression of intestinal polyposis in Apc(delta 716) knockout mice by an additional mutation in the cytosolic phospholipase A(2) gene.

Arachidonic acid is a precursor for biosynthesis of eicosanoids, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. Cytosolic phospholipase A(2) (cPLA(2)) plays a key role in the release of arachidonic acid as the substrate of cyclooxygenase-1 (COX-1) or COX-2. We found that the level of cPLA(2) mRNA was markedly elevated in the polyps and correlated with the polyp size in the small intestine of the Apc(delta)(716) knockout mouse, a model for human familial adenomatous polyposis. To determine the role of cPLA(2) in intestinal tumorigenesis, we then introduced a cPLA(2) gene mutation into Apc(delta)(716) mice. In the compound mutant mice, the size of the small intestinal polyps was reduced significantly, although the numbers remained unchanged. These results provide direct genetic evidence that cPLA(2) plays a key role in the expansion of polyps in the small intestine rather than in the initiation process. In contrast, colonic polyps were not affected in either size or number. Interestingly, group X sPLA(2) was constitutively expressed in the colon at much higher levels than in the small intestine. These results suggest that in the colon, group X sPLA(2) supplies arachidonic acid in both the normal epithelium and the polyps even in the absence of cPLA(2).

Phosphorylation of the inward-rectifying potassium channel KAT1 by ABR kinase in Vicia guard cells.

A 48-kDa protein kinase was detected in Vicia faba guard cell protoplasts by an in-gel protein kinase assay using a recombinant peptide (KAT1C) of the carboxyl-terminus of an inward-rectifying voltage-dependent K+ channel cloned from Arabidopsis thaliana, KAT1. This protein kinase (ABR* kinase) was activated by pretreatment of guard cell protoplasts with ABA, but not by pretreatment with IAA, 2,4-D, kinetin or GA3. The activation of ABR* kinase was dependent on the time and concentration of ABA. The kinase activity was sensitive to staurosporine and K-252a, protein kinase inhibitors, and insensitive to Ca2+. No ABR* kinase activity was detected in mesophyll cell protoplasts. These characteristics of ABR* kinase are consistent with those of an ABA-responsive protein kinase (ABR kinase) reported previously [Mori and Muto (1997), Plant Physiol. 113: 833]. These results indicate that ABR* kinase phosphorylates the inward-rectifying K+ channel in response to treatment of stomatal guard cells with ABA. The data reported here provide evidence that this ABA-responsive protein kinase may promote ABA signaling by directly phosphorylating guard cell ion channels.

Roles of cytosolic phospholipase A(2) and platelet-activating factor receptor in the Ca-induced biosynthesis of PAF.

Casein-elicited peritoneal exudate cells (PEC), mainly consisted of neutrophils, were collected from platelet-activating factor receptor-knock-out (PAFR-KO), cytosolic phospholipase A(2) knock-out (cPLA(2)-KO), and wild-type (WT) mice. After stimulation of PEC with calcium ionophore A 23187, PAF levels were measured by radio-ligand binding assay using receptor-rich membrane fraction prepared from the PAF receptor transgenic mice. We found that the level of PAF production by PEC was not different between WT and PAFR-KO mice. On the other hand, cPLA(2)-KO mice were deficient in the PAF production. These results provide the direct evidence while cPLA(2) is essential in the production of PAF, PAF receptor deficiency has little effect on the PAF production.

Genomic structure and promoter analysis of the human alpha1, 6-fucosyltransferase gene (FUT8).

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1,6-fucosyltransferase (alpha1,6FucT) catalyzes the transfer of a fucosyl moiety from GDP-fucose to the asparagine-linked GlcNAc residue of complex N-glycans via alpha1,6-linkage. We have cloned the genomic DNA which encodes the human alpha1,6FucT gene ( FUT8 ) and analyzed its structure. It was found that the gene consists of at least nine exons spanning more than a 50 kbp genomic region, and the coding sequence is divided into eight exons. The translation initiation codon was located at exon 2, and thus exon 1 encodes only 5'-untranslated sequences. Transcription initiation site of FUT8 was determined by 5'-rapid amplification of the cDNA end and a primer-extension analysis using the total RNA isolated from SK-OV-3 cells, which have a high level of alpha1,6FucT activity. We then characterized the FUT8 promoter region by a reporter gene assay. The luciferase reporter assay indicated that the 5'-flanking region of exon 1, which covered about 1 kbp, conferred the promoter activity in SK-OV-3 cells. This region contains potential binding sites for some transcription factors, such as bHLH, cMyb, GATA-1, as well as a TATA-box, but not a CCAAT motif. 5'-Untranslated sequences found in ESTs and the cDNA for the FUT8 suggest the presence of an additional exon(s) at the upstream of the first exon identified in this study, and therefore, the transcription of the gene would be regulated by multiple promoters.

The Arabidopsis HKT1 gene homolog mediates inward Na(+) currents in xenopus laevis oocytes and Na(+) uptake in Saccharomyces cerevisiae.

The Na(+)-K(+) co-transporter HKT1, first isolated from wheat, mediates high-affinity K(+) uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na(+) uptake transporter in Xenopus laevis oocytes, and the presence of external K(+) did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na(+), which correlates to the large inward Na(+) currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K(+) uptake when cultured in K(+)-limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K(+) transporters. The rescue was associated with a less than 2-fold stimulation of K(+) uptake into K(+)-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na(+) and, to a small degree, K(+) transport in heterologous expression systems.

yam8(+), a Schizosaccharomyces pombe gene, is a potential homologue of the Saccharomyces cerevisiae MID1 gene encoding a stretch-activated Ca(2+)-permeable channel.

The Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-permeable channel. In a protein database, we found a Schizosaccharomyces pombe gene whose predicted protein shows 26% identical and 62% similar to the Mid1 channel in amino acid sequence. cDNA derived from this gene, designated yam8(+), was isolated by reverse transcription-polymerase chain reaction (RT-PCR). Further analysis showed that the Yam8 protein consists of 486 amino acids and has 6 hydrophobic segments. The yam8(+) cDNA, placed under the S. cerevisiae TDH3 promoter, partially complemented the mating pheromone-induced death (mid) phenotype of the S. cerevisiae mid1 mutant. The expression of the yam8(+) cDNA in the mid1 mutant cells partially remediated the mid phenotype and resulted in a slight increase in Ca(2+) uptake activity. These findings suggest that Yam8 is a potential homologue of Mid1.

Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2.

Adult respiratory distress syndrome (ARDS) is characterized by acute lung injury with a high mortality rate and yet its mechanism is poorly understood. Sepsis syndrome and acid aspiration are the most frequent causes of ARDS, leading to increased lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration and respiratory failure. Using a murine model of acute lung injury induced by septic syndrome or acid aspiration, we investigated the role of cytosolic phospholipase A2 (cPLA2) in ARDS. We found that disruption of the gene encoding cPLA2 significantly reduced pulmonary edema, PMN sequestration and deterioration of gas exchange caused by lipopolysaccharide and zymosan administration. Acute lung injury induced by acid aspiration was similarly reduced in mice with a disrupted cpla2 gene. Our observations suggest that cPLA2 is a mediator of acute lung injury induced by sepsis syndrome or acid aspiration. Thus, the inhibition of cPLA2-initiated pathways may provide a therapeutic approach to acute lung injury, for which no pharmaceutical agents are currently effective.