GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K(+)-selective, K(+)-sensing ion channel

Here we report on the molecular identification, guard cell expression and functional characterization of AtGORK, an Arabidopsis thaliana guard cell outward rectifying K(+) channel. GORK represents a new member of the plant Shaker K(+) channel superfamily. When heterologously expressed in Xenopus oocytes the gene product of GORK mediated depolarization-activated K(+) currents. In agreement with the delayed outward rectifier in intact guard cells and protoplasts thereof, GORK is activated in a voltage- and potassium-dependent manner. Furthermore, the single channel conductance and regulation of GORK in response to pH changes resembles the biophysical properties of the guard cell delayed outward rectifier. Thus GORK very likely represents the molecular entity for depolarization-induced potassium release from guard cells.     

Mutational analysis of the Shab-encoded delayed rectifier K(+) channels in Drosophila.

K(+) currents in Drosophila muscles have been resolved into two voltage-activated currents (I(A) and I(K)) and two Ca(2+)-activated currents (I(CF) and I(CS)). Mutations that affect I(A) (Shaker) and I(CF) (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (I(K)) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K(+) channel gene, we show that this gene encodes the delayed rectifier K(+) channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K(+) current (I(K)). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivo physiological role for the Shab-encoded K(+) channels in Drosophila. The availability of mutations that affect I(K) opens up possibilities for studying I(K) and its role in larval muscle excitability.     

Temperature-dependent functional expression of a plant K(+) channel in mammalian cells

The Arabidopsis thaliana potassium channel KAT1 was expressed and characterized in Chinese hamster ovary cells. KAT1-GFP fusion protein was successfully targeted to the plasma membrane and electrophysiological analysis revealed functional expression of KAT1 only in cells cultured at 30 degrees C. The main biophysical characteristics of KAT1 are similar to those described for the channel expressed in other systems. CHO cells represent an advantageous expression system and may be the system of choice to study the expression, assembly, function, and regulation of plant potassium channels in general.     

Delayed rectifier outward K+ current mediates the migration of rat cerebellar granule cells stimulated by melatonin

Melatonin (MT) may work as a neuromodulator through the associated MT receptors in the central nervous system. Previously, our studies have shown that MT increased the I(K) current via a G protein-related pathway. In the present study, patch-clamp whole-cell recording, transwell migration assays and organotypic cerebellar slice cultures were used to examine the effect of MT on granule cell migration. MT increased the I(K) current amplitude and migration of granule cells. Meanwhile, TEA, the I(K) channel blocker, decreased the I(K) current and slowed the migration of granule cells. Furthermore, the effects of MT on the I(K) current and cell migration were not abolished by pre-incubation with P7791, a specific antagonist of MT(3)R, but were eliminated by the application of the MT(2)R antagonists K185 and 4-P-PDOT. I(K) current and cell migration were decreased by the application of dibutyryl cyclic AMP (dbcAMP), which was in contrast to the MT effect on the I(K) current and cell migration. Incubation with dbcAMP essentially blocked the MT-induced increasing effect. Moreover, incubation of isolated cell cultures in the MT-containing medium also decreased the cAMP immunoreactivity in the granule cells. It is concluded, therefore, that I(K) current, downstream of a cAMP transduction pathway, mediates the migration of rat cerebellar granule cells stimulated by MT.     

Existence of a transient outward K(+) current in guinea pig cardiac myocytes

A novel transient outward K(+) current that exhibits inward-going rectification (I(to.ir)) was identified in guinea pig atrial and ventricular myocytes. I(to.ir) was insensitive to 4-aminopyridine (4-AP) but was blocked by 200 micromol/l Ba(2+) or removal of external K(+). The zero current potential shifted 51-53 mV/decade change in external K(+). I(to.ir) density was twofold greater in ventricular than in atrial myocytes, and biexponential inactivation occurs in both types of myocytes. At -20 mV, the fast inactivation time constants were 7.7 +/- 1.8 and 6.1 +/- 1.2 ms and the slow inactivation time constants were 85.1 +/- 14.8 and 77.3 +/- 10.4 ms in ventricular and atrial cells, respectively. The midpoints for steady-state inactivation were -36.4 +/- 0.3 and -51.6 +/- 0.4 mV, and recovery from inactivation was rapid near the resting potential (time constants = 7.9 +/- 1.9 and 8.8 +/- 2.1 ms, respectively). I(to.ir) was detected in Na(+)-containing and Na(+)-free solutions and was not blocked by 20 nmol/l saxitoxin. Action potential clamp revealed that I(to.ir) contributed an outward current that activated rapidly on depolarization and inactivated by early phase 2 in both tissues. Although it is well known that 4-AP-sensitive transient outward current is absent in guinea pig, this Ba(2+)-sensitive and 4-AP-insensitive K(+) current has been overlooked.     

Root K(+) acquisition in plants: the Arabidopsis thaliana model

K(+) is an essential macronutrient required by plants to complete their life cycle. It fulfills important functions and it is widely used as a fertilizer to increase crop production. Thus, the identification of the systems involved in K(+) acquisition by plants has always been a research goal as it may eventually produce molecular tools to enhance crop productivity further. This review is focused on the recent findings on the systems involved in K(+) acquisition. From Epstein's pioneering work >40 years ago, K(+) uptake was considered to consist of a high- and a low-affinity component. The subsequent molecular approaches identified genes encoding K(+) transport systems which could be involved in the first step of K(+) uptake at the plant root. Insights into the regulation of these genes and the proteins that they encode have also been gained in recent studies. A demonstration of the role of the two main K(+) uptake systems at the root, AtHKA5 and AKT1, has been possible with the study of Arabidopsis thaliana T-DNA insertion lines that knock out these genes. AtHAK5 was revealed as the only uptake system at external concentrations <10 μM. Between 10 and 200 μM both AtHAK5 and AKT1 contribute to K(+) acquisition. At external concentrations >500 μM, AtHAK5 is not relevant and AKT1's contribution to K(+) uptake becomes more important. At 10 mM K(+), unidentified systems may provide sufficient K(+) uptake for plant growth.     

Nectar properties of the sunbird-pollinated plant Impatiens sakeriana: A comparison with six other co-flowering species

Adaptations of the nectar traits in bird-pollinated flowers are amongst the most discussed aspects of floral evolution. In the case of sunbird-pollinated plants, data on nectar traits originate almost exclusively from the South African region and are very scarce for tropical Africa, where paradoxically the highest sunbird diversity occurs. Here we present a study on the nectar properties of a sunbird-pollinated plant, Impatiens sakeriana, growing in the West African mountains, including the nectar production, diurnal changes in the nectar standing crop, the nectar concentrations, the nectar volumes, total sugar amounts and sugar composition. Moreover we compare the nectar traits of I. sakeriana with six other co-flowering insect-visited plant species.Our results showed that many nectar properties, including high volume (approx. 38 μL in flowers unvisited by sunbirds), low sugar concentration (approx. 30% w/w) and high sucrose content (95%), are specific to I. sakeriana, compared to the insect-visited plants. These are in accordance with the most recent theory that nectar properties of the sunbird-pollinated plants are similar to those pollinated by hummingbirds.     

The multi-protein family of Arabidopsis sulphotransferases and their relatives in other plant species

All members of the sulphotransferase (SOT, EC 2.8.2.-) protein family use 3'-phosphoadenosine 5'-phosphosulphate (PAPS) as the sulphuryl donor and transfer the sulphonate group to an appropriate hydroxyl group of several classes of substrates. These enzymes have highly conserved domains and can be found in eubacteria and eukaryotes. In mammals, sulphate conjugation catalysed by SOTs constitutes an important reaction in the transformation of xenobiotics, and in the modulation of the biological activity of steroid hormones and neurotransmitters. In plants, sulphate-conjugation reactions seem to play an important role in plant growth, development, and adaptation to stress. To date only a few plant SOTs have been characterized in detail. The flavonol 3- and 4'-SOTs from Flaveria species (Asteraceae), which catalyse the sulphonation of flavonol aglycones and flavonol 3-sulphates, respectively, were the first plant SOTs for which cDNA clones were isolated. The plasma membrane associated gallic acid SOT of Mimosa pudica L. pulvini cells may be intrinsic to signalling events that modify the seismonastic response. In Brassica napus L. a SOT catalyses the O-sulphonation of brassinosteroids and thereby abolishes specifically the biological activity of 24-epibrassinolide. The fully sequenced genome of Arabidopsis thaliana Heynh. contains in total 18 genes that are likely to encode SOT proteins based on sequence similarities of the translated products with an average identity of 51.1%. So far only one SOT from A. thaliana (At5g07000) was functionally characterized: the protein was shown to catalyse the sulphonation of 12-hydroxyjasmonate and thereby inactivate excess jasmonic acid in plants. The substrates and, therefore, the physiological roles of SOTs are very diverse. By using the numerous informative databases and methods available for the model plant A. thaliana, the elucidation of the functional role of the SOT protein family will be accelerated.     

Fumaric acid: an overlooked form of fixed carbon in Arabidopsis and other plant species

Photoassimilates are used by plants for production of energy, as carbon skeletons and in transport of fixed carbon between different plant organs. Many studies have been devoted to characterizing the factors that regulate photoassimilate concentrations in different plant species. Most studies examining photoassimilate concentrations in C₃ plants have focused on analyzing starch and soluble sugars. However, work presented here demonstrates that a number of C₃ plants, including the popular model organism Arabidopsis thaliana (L.) Heynh., and agriculturally important plants, such as soybean, Glycine max (L.) Merr., contain significant quantities of fumaric acid. In fact, fumaric acid can accumulate to levels of several milligrams per gram fresh weight in Arabidopsis leaves, often exceeding those of starch and soluble sugars. Fumaric acid is a component of the tricarboxylic acid cycle and, like starch and soluble sugars, can be metabolized to yield energy and carbon skeletons for production of other compounds. Fumaric acid concentrations increase with plant age and light intensity in Arabidopsis leaves. Moreover, Arabidopsis phloem exudates contain significant quantities of fumaric acid, raising the possibility that fumaric acid may function in carbon transport. Received: 11 February 2000 / Accepted: 1 April 2000     

Isoenzyme analysis of Echinostoma liei: comparison and hybridization with other African species

Isoenzyme analysis was carried out on the laboratory strain of Echinostoma liei. The results were compared with those from a preliminary study on Echinostoma caproni, Echinostoma togoensis, and Echinostoma sp. (A. Voltz, J. Richard, B. Pesson, and J. Jourdane, 1986, Annales de Parasitologie Humaine et Comparée, 61, 617-623). Isoelectrofocusing showed characteristic phenotypes for phosphoglucomutase (PGM, EC 5.4.2.2) and glucosephosphate isomerase (GPI, EC 5.3.1.9). The four experimental strains were monomorphic. Their genotypes were defined. Isoenzyme analysis of F1-hybrids and their F2 descendants indicated the subunit structure of both isoenzymes and showed that they were encoded by independent genes. Finally, it also suggested that the four strains corresponded to variants of the same species.     

A K(+)-selective channel in the colonic carcinoma cell line: CaCo-2 is activated with membrane stretch

CaCo-2 is a human colonic carcinoma cell line which becomes differentiated in culture to form a polarized epithelium exhibiting several of the functional characteristics of native colonic tissue. In the present study, CaCo-2 cells have been used for a patch-clamp study of colonic ion conductance pathways. A large, 120 pS K(+)-selective channel was found in cells forming subconfluent monolayers in culture. Unlike Maxi-K+ channels found in other epithelial cells, this channel was not activated with elevations in cytosolic Ca2+. Channel activity was stimulated with membrane depolarization and most markedly with membrane stretch. The application of negative pressure (20 mm-Hg) to both cell-attached and excised, inside-out membrane patches caused a burst of channel activity which disappeared within seconds of suction removal. Single-channel conductance of the pressure-activated channel was decreased when quinine (100 microM) was present in the patch pipette.     

Interactions of cations with the cytoplasmic pores of inward rectifier K(+) channels in the closed state

Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K(+) (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg(2+) by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg(2+) when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues by Mg(2+) indicates that the mode of binding of Mg(2+) is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore.     

The distribution of homologues of the Escherichia coli KefC K(+)-efflux system in other bacterial species

Using a variety of techniques the distribution of the glutathione-regulated KefC K(+)-transport system among bacterial species was investigated. The presence of similar systems in a number of Gram-negative bacteria was demonstrated. In contrast, the system appeared to be absent from most Gram-positive bacteria tested with the exception of Staphylococcus aureus. Using the cloned Escherichia coli kefC gene as a probe for Southern hybridization it was shown that only limited DNA sequence homology exists with other bacteria, even when closely related members of the enteric group were examined.     

Developmental regulation of a Na(+)-activated fast outward K+ current in rat myoblasts.

BACKGROUND/AIMS: Myoblasts undergoing differentiation sequentially express multiple K+ channels, and that ion channel expression varies depending on species and state of development. In this report, we reported a developmental regulation of fast activated and fast inactivated outward current in rat myoblasts. METHODS: The kinetic and pharmacological property of the outward current was investigated by using the patch-clamp technique. RESULTS: The outward current was elicited by a depolarizing step from -100 mV holding potential to +40 mV- +80 mV. The activation properties of this channel changed with days in culture. The outward current was blocked by 4-AP in a concentration dependent manner, with 0.5 mM and 2 mM 4-AP inhibiting the current by 10 +/- 3% and 56 +/- 3%, respectively. When 1 mM tetrodotoxin (TTX) was added to the bath solution or the membrane potential was depolarized to -50 mV, the fast outward current was aborted. Na+ dependent inhibition was observed when Na+ in the bath solution was replaced by Li+. In addition, replacement of K+ in the pipette solution by Cs+ almost completely eliminated the outward current. CONCLUSION: The developmentally regulated outward current recorded in rat myoblasts is a Na+ influx-dependent outward K+ current, which may contribute to myoblast membrane firing of action potential or myoblast fusion.     

Transfer of gases and metabolites in the equine placenta: a comparison with other species.

Mares and fetuses with indwelling catheters in the umbilical and uterine vessels have been used to monitor transplacental blood gas tensions, pH, O2 affinities and the concentration of various metabolites in fetal and maternal blood during late gestation. Measurements of umbilical and uterine blood flows and arterio-venous differences enabled the uptake of O2 and glucose by the fetus and the uterus to be estimated. The present findings are compared with those from other species in comparable conditions.     

Contributions of a negatively charged residue in the hydrophobic domain of the IRK1 inwardly rectifying K+ channel to K(+)-selective permeation.

Inwardly rectifying K+ channels are highly selective for K+ ions and show strong interaction with ions in the pore. Both features are important for the physiological functions of these channels and pose intriguing mechanistic questions of ion permeation. The aspartate residue in the second putative transmembrane segment of the IRK1 inwardly rectifying K+ channel, previously implicated in inward rectification gating due to cytoplasmic Mg2+ and polyamine block, is found in this study to be crucial for the channel's ability to distinguish between K+ and Rb+ ions. Mutation of this residue also perturbs the interaction between the channel pore and the Sr2+ blocking ion. Our studies suggest that this aspartate residue contributes to a selectivity filter near the cytoplasmic end of the pore.     

A/C(i) curve analysis across a range of woody plant species: influence of regression analysis parameters and mesophyll conductance

The analysis and interpretation of A/C(i) curves (net CO(2) assimilation rate, A, versus calculated substomatal CO(2) concentration, C(i)) is dependent upon a number of underlying assumptions. The influence of the C(i) value at which the A/C(i) curve switches between the Rubisco- and electron transport-limited portions of the curve was examined on A/C(i) curve parameter estimates, as well as the effect of mesophyll CO(2) conductance (g(m)) values on estimates of the maximum rate of Rubisco-mediated carboxylation (V(cmax)). Based on an analysis using 19 woody species from the Pacific Northwest, significant variation occurred in the C(i) value where the Rubisco- and electron transport-limited portions of the curve intersect (C(i_t)), ranging from 20 Pa to 152 Pa and averaging c. 71 Pa and 37 Pa for conifer and broadleaf species, respectively. Significant effects on estimated A/C(i) parameters (e.g. V(cmax)) may arise when preliminary estimates of C(i_t), necessary for the multiple regression analyses, are set either too high or too low. However, when the appropriate threshold is used, a significant relationship between A/C(i) and chlorophyll fluorescence estimates of carboxylation is achieved. The use of the V(cmax) parameter to describe accurately the Rubisco activity from the A/C(i) curve analysis is also dependent upon the assumption that C(i) is approximately equal to chloroplast CO(2) concentrations (C(c)). If leaf mesophyll conductance is low, C(c) will be much lower than C(i) and will result in an underestimation of V(cmax) from A/C(i) curves. A large range of mesophyll conductance (g(m)) values was observed across the 19 species (0.005+/-0.002 to 0.189+/-0.011 mol m(-2) s(-1) for Tsuga heterophylla and Quercus garryana, respectively) and, on average, g(m) was 1.9 times lower for the conifer species (0.058+/-0.017 mol m(-2) s(-1) for conifers versus 0.112+/-0.020 mol m(-2) s(-1) for broadleaves). When this mesophyll limitation was accounted for in V(cmax) estimates, considerable variation still existed between species, but the difference in V(cmax) between conifer and broadleaf species was reduced from c. 11 micromol m(-2) s(-1) to 4 micromol m(-2) s(-1). For example, A/C(i) curve estimates of V(cmax) were 31.2+/-6.2 and 42.2+/-4.4 micromol m(-2) s(-1), and A/C(c) curve estimates were 41.2+/-7.1 micromol m(-2) s(-1) and 45.0+/-4.8 micromol m(-2) s(-1), for the conifer and broadleaf species, respectively.     

The existence of the K(+) channel in plant mitochondria.

In this study, evidence is given that a number of isolated coupled plant mitochondria (from durum wheat, bread wheat, spelt, rye, barley, potato, and spinach) can take up externally added K(+) ions. This was observed by following mitochondrial swelling in isotonic KCl solutions and was confirmed by a novel method in which the membrane potential decrease due to externally added K(+) is measured fluorimetrically by using safranine. A detailed investigation of K(+) uptake by durum wheat mitochondria shows hyperbolic dependence on the ion concentration and specificity. K(+) uptake electrogenicity and the non-competitive inhibition due to either ATP or NADH are also shown. In the whole, the experimental findings reported in this paper demonstrate the existence of the mitochondrial K(+)(ATP) channel in plants (PmitoK(ATP)). Interestingly, Mg(2+) and glyburide, which can inhibit mammalian K(+) channel, have no effect on PmitoK(ATP). In the presence of the superoxide anion producing system (xanthine plus xanthine oxidase), PmitoK(ATP) activation was found. Moreover, an inverse relationship was found between channel activity and mitochondrial superoxide anion formation, as measured via epinephrine photometric assay. These findings strongly suggest that mitochondrial K(+) uptake could be involved in plant defense mechanism against oxidative stress due to reactive oxygen species generation.