Heteromeric K+ channels in plants

Voltage-gated potassium channels of plants are multimeric proteins built of four α-subunits. In the model plant Arabidopsis thaliana , nine genes coding for K⁺ channel α-subunits have been identified. When co-expressed in heterologous expression systems, most of them display the ability to form heteromeric K⁺ channels. Till now it was not clear whether plants use this potential of heteromerization to increase the functional diversity of potassium channels. Here, we designed an experimental approach employing different transgenic plant lines that allowed us to prove the existence of heteromeric K⁺ channels in plants. The chosen strategy might also be useful for investigating the activity and function of other multimeric channel proteins like, for instance, cyclic-nucleotide gated channels, tandem-pore K⁺ channels and glutamate receptor channels.     

The Kv4.2 mediates excitatory activity-dependent regulation of neuronal excitability in rat cortical neurons

Neuronal excitability can cooperate with synaptic transmission to control the information storage. This regulation of neuronal plasticity can be affected by alterations in neuronal inputs and accomplished by modulation of voltage-dependent ion channels. In this study, we report that enhanced excitatory input negatively regulated neuronal excitability. Enhanced excitatory input by glutamate, electric field stimulation or high K⁺ increased transient outward K⁺ current, whereas did not affect the delayed rectifier K⁺ current in rat cultured cortical neurons. Both the voltage-dependent K⁺ channel 4.2 and 4.3 subunits contributed to the increase. The increase in the K⁺ current density by Kv4.2 was ascribed to its cytoplasmic membrane translocation, which was mediated by NMDA type of glutamate receptor. Furthermore, enhanced excitatory input inhibited neuronal excitability. Taken together, our results suggest that excitatory neurotransmission affects neuronal excitability via the regulation of the K⁺ channel membrane translocation.     

Effect of ion channel blockers on germination of Bacillus megaterium spores

Abstract We surveyed 23 drugs that can interact with membrane components, such as ion channels, for their effect on spore germination. The results showed that triggering of spore germination was inhibited by specific calcium (Ca²⁺) potassium (K⁺) and sodium (Na⁺) channel blockers.     

Molecular determinants of the Arabidopsis AKT1 K+ channel ionic selectivity investigated by expression in yeast of randomly mutated channels

The Arabidopsis thaliana K⁺ channel AKT1 was expressed in a yeast strain defective for K⁺ uptake at low K⁺ concentrations (<3 m M ). Besides restoring K⁺ transport in this strain, AKT1 expression increased its tolerance to salt (NaCl or LiCl), whatever the external K⁺ concentration used (50 μ M , 5 m M , or 50 m M ). We took advantage of the latter phenomenon for screening a library of channels randomly mutated in the region that shares homologies with the pore forming domain (the so-called P domain) of animal K⁺ channels (Shaker family). Cassette mutagenesis was performed using a degenerate oligonucleotide that was designed to ensure, theoretically, a single mutation per P cassette. The mean number of amino acid exchanges per cassette turned out to be 1.4. Mutant channels that conferred on the transformed cells a reduction in salt tolerance (increased Na⁺ content, decreased K⁺ content, and lower growth rate, as compared to control cells expressing the wild-type channel) were selected. By co-expressing them with the wild-type AKT1 cDNA, it was shown that the mutated polypeptides were expressed, stable and correctly targeted to the cell membrane where they formed channels with altered properties. Analysis of the mutation distribution in these channels suggests that the AKT1 P domain has a structure similar to that of animal Shaker channels (a strongly constrained central region lining the tunnel that includes the highly conserved consensus motif TXXTXGYGD, and flanking regions forming the outer mouth of the pore), with an additional selectivity filter located upstream from the tunnel and formed by residues present in the N-terminal flanking region.     

Electrophysiological characterization of pathways for K+ uptake into growing and non-growing leaf cells of barley

Potassium is a major osmolyte used by plant cells. The accumulation rates of K⁺ in cells may limit the rate of expansion. In the present study, we investigated the involvement of ion channels in K⁺ uptake using patch clamp technique. Ion currents were quantified in protoplasts of the elongation and emerged blade zone of the developing leaf 3 of barley ( Hordeum vulgare L.). A time-dependent inward-rectifying K⁺-selective current was observed almost exclusively in elongation zone protoplasts. The current showed characteristics typical of Shaker-type channels. Instantaneous inward current was highest in the epidermis of the emerged blade and selective for Na⁺ over K⁺. Selectivity disappeared, and currents decreased or remained the same, depending on tissue, in response to salt treatment. Net accumulation rates of K⁺ in cells calculated from patch clamp current–voltage curves exceeded rates calculated from membrane potential and K⁺ concentrations of cells measured in planta by factor 2.5–2.7 at physiological apoplastic K⁺ concentrations (10–100 m m ). It is concluded that under these conditions, K⁺ accumulation in growing barley leaf cells is not limited by transport properties of cells. Under saline conditions, down-regulation of voltage-independent channels may reduce the capacity for growth-related K⁺ accumulation.     

Potassium ion channels in the plasmalemma

The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K⁺ transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K⁺ equilibrium potential (E_K) – membrane potential (V_m), or secondary active by coupling through a carrier to the inward driving force of H⁺ or Na⁺. Known K⁺ channels are permeable to monovalent cations, a permeability order being K⁺ > Rb⁺ > NH₄⁺ > Na⁺≥ Li⁺ > Cs⁺. The macroscopic K⁺ currents across a cell or protoplast surface commonly show rectification, i.e. a V^m-dependent conductance which in turn, may be controlled by the cytosolic activity of Ca²⁺, of K⁺, of H⁺, or by the K⁺ driving force. Analysis by the patch clamp technique reveals that plant K⁺ channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K⁺ channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.     

Potassium channels in barley: cloning, functional characterization and expression analyses in relation to leaf growth and development

It is not known how the uptake and retention of the key osmolyte K⁺ in cells are mediated in growing leaf tissue. In the present study on the growing leaf 3 of barley, we have cloned the full-length coding sequence of three genes which encode putative K⁺ channels ( HvAKT1 , HvAKT2 , HvKCO1 / HvTPK1 ), and of one gene which encodes a putative K⁺ transporter ( HvHAK4 ). The functionality of the gene products of HvAKT1 and HvAKT2 was tested through expression in Xenopus laevis oocytes. Both are inward-rectifying K⁺ channels which are inhibited by Cs⁺. Function of HvAKT1 in oocytes requires co-expression of a calcineurin-interacting protein kinase ( At CIPK23) and a calcineurin B-like protein (AtCBL9) from Arabidopsis , showing cross-species complementation of function. In planta , HvAKT1 is expressed primarily in roots, but is also expressed in leaf tissue. HvAKT2 is expressed particularly in leaf tissue, and HvHAK4 is expressed particularly in growing leaf tissue. Within leaves, HvAKT1 and HvAKT2 are expressed predominantly in mesophyll. Expression of genes changes little in response to low external K⁺ or salinity, despite major changes in K⁺ concentrations and osmolality of cells. Possible contributions of HvAKT1 , HvAKT2 , HvKCO1 and HvHAK4 to regulation of K⁺ relations of growing barley leaf cells are discussed.     

AtKC1, a conditionally targeted Shaker-type subunit, regulates the activity of plant K+ channels

Amongst the nine voltage-gated K⁺ channel (Kv) subunits expressed in Arabidopsis, AtKC1 does not seem to form functional Kv channels on its own, and is therefore said to be silent. It has been proposed to be a regulatory subunit, and to significantly influence the functional properties of heteromeric channels in which it participates, along with other Kv channel subunits. The mechanisms underlying these properties of AtKC1 remain unknown. Here, the transient (co-)expression of AtKC1 , AKT1 and/or KAT1 genes was obtained in tobacco mesophyll protoplasts, which lack endogenous inward Kv channel activity. Our experimental conditions allowed both localization of expressed polypeptides (GFP-tagging) and recording of heterologously expressed Kv channel activity (untagged polypeptides). It is shown that AtKC1 remains in the endoplasmic reticulum unless it is co-expressed with AKT1. In these conditions heteromeric AtKC1-AKT1 channels are obtained, and display functional properties different from those of homomeric AKT1 channels in the same context. In particular, the activation threshold voltage of the former channels is more negative than that of the latter ones. Also, it is proposed that AtKC1-AKT1 heterodimers are preferred to AKT1-AKT1 homodimers during the process of tetramer assembly. Similar results are obtained upon co-expression of AtKC1 with KAT1 . The whole set of data provides evidence that AtKC1 is a conditionally-targeted Kv subunit, which probably downregulates the physiological activity of other Kv channel subunits in Arabidopsis.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

Phytochrome effects on K+ fluxes in guard cells of Commelina communis

The effect of phytochrome on K⁺ transport in guard cells of Commelina communis L. was studied following stomatal movement and using the K⁺−channel blockers tetraethylammonium (TEA), Cs⁺ and quinidine. TEA and quinidine prevented stomatal opening and closure in red light, but not when it was supplemented with far-red. This indicates that channels that can be blocked by TEA and quinidine are regulated by phytochrome. Evidence for a phytochrome effect on K⁺ leakage through other membranal compartments was also found. These phytochrome effects are modified by temperature. Elevated temperature decreases the involvement of channels and increases K⁺ transport through other membrane compartments, while low temperature causes channel opening and diminishes K⁺ leakage. The interaction between phytochrome effects and those of temperature is discussed.     

Fast-activating cation channel in barley mesophyll vacuoles. Inhibition by calcium

In contrast to the vacuolar ion channels which are gated open by an increase of cytosolic Ca²⁺ the vacuolar ion currents at resting cytosolic Ca²⁺are poorly explored. Therefore, this study was performed to investigate the properties of the so-called fast-activating vacuolar (FV) current which dominates the electrical characteristics of the tonoplast at physiological free Ca²⁺ concentrations. Patch—clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. Single ion channels were identified, which, based on their selectivity, activation kinetics, Ca²⁺- and voltage-dependence, carry the whole-vacuole FV current. Reversal potential determinations indicated a K⁺ overs C⁻ permeability ratio of about 30. Both inward and outward whole-vacuole currents as well as the activity of single FV channels were inhibited by an increase of cytosolic Ca²⁺, with a K_d≈ 6 µM. At physiological vacuolar Ca²⁺ activities, the FV channel is an outward-rectifying potassium channel. The FV channel was activated in less than a few milliseconds both by negative and positive potential steps, having a minimal activity that is 40 mV negative of the K⁺ equilibrium potential. It is proposed that transport of K⁺ through this cation channel controls the electrical potential difference across the tonoplast.     

Antibodies Against Rat Brain Vesicle-Associated Membrane Protein (Synaptobrevin) Prevent Inhibition of Acetylcholine Release by Tetanus Toxin or Botulinum Neurotoxin Type B

Abstract: The Shaw-type K⁺ channel K_v3.1 was stably transfected in human embryonic kidney cells. Voltage dependence of activation, K⁺ permeability, sensitivity to external tetraethylammonium, and unitary conductance were similar to K_v 3.1 channels expressed transiently in Xenopus oocytes. K_v 3.1 channels appear to be regulated because the protein kinase C activator phorbol 12,13-dibutyrate decreased K_v 3.1 currents. Based on these results, we find that the stable expression of voltage-gated K⁺ channels in human embryonic kidney cells appears to be well suited for analysis of both biophysical and biochemical regulatory processes.     

Signalling gates in abscisic acid-mediated control of guard cell ion channels

Multiple signalling pathways and their messengers – entailing changes in cytosolic-free Ca²⁺([Ca²⁻]). pH (pH) and protein phosphorylation – underpin K⁺and anion channel control during stomatal movements. This redundancy is wholly consistent with the ability of the guard cells to integrate the wide range of environmental and hormonal stimuli that affect stomatal aperture. Signal redundancy effects a spectrum of graded responses by linking pathways to gate signal transmission, and so boosts or mutes the final 'integrated signal' that reaches each ion channel. All evidence supports a role for the AB11 protein phosphatase and protein kinase elements in gating K⁺channel sensitivity to pH and ABA. Changes in [Ca²⁺] I . in turn, are demonstrably sensitive to pH₁. Because each of these signal elements modulate and, in turn, are influenced by the activity of different sets of ion channels, the additional couplings engender a remarkably complex network, layering positive and negative controls with the ion channels that facilitate ion fluxes for stomatal movement.     

The Kv2.1 channels mediate neuronal apoptosis induced by excitotoxicity

Chronic loss of intracellular K⁺ can induce neuronal apoptosis in pathological conditions. However, the mechanism by which the K⁺ channels are regulated in this process remains largely unknown. Here, we report that the increased membrane expression of Kv2.1 proteins in cortical neurons deprived of serum, a condition known to induce K⁺ loss, promotes neuronal apoptosis. The increase in I _K current density and apoptosis in the neurons deprived of serum were inhibited by a dominant negative form of Kv2.1 and MK801, an antagonist to NMDA receptors. The membrane level of Kv2.1 and its interaction with SNAP25 were increased, whereas the Kv2.1 phosphorylation was inhibited in the neurons deprived of serum. Botulinum neurotoxin, an agent known to prevent formation of soluble N -ethylmaleimide-sensitive factor attachment protein receptor complex, suppressed the increase in I _K current density. Together, these results suggest that NMDA receptor-dependent Kv2.1 membrane translocation is regulated by a soluble N -ethylmaleimide-sensitive factor attachment protein receptor-dependent vesicular trafficking mechanism and is responsible for neuronal cell death induced by chronic loss of K⁺.     

Prokaryotic K(+) channels: from crystal structures to diversity

The deep roots and wide branches of the K⁺-channel family are evident from genome surveys and laboratory experimentation. K⁺-channel genes are widespread and found in nearly all the free-living bacteria, archaea and eukarya. The conservation of basic structures and mechanisms such as the K⁺ filter, the gate, and some of the gate's regulatory domains have allowed general insights on animal K⁺ channels to be gained from crystal structures of prokaryotic channels. Since microbes are the great majority of life's diversity, it is not surprising that microbial genomes reveal structural motifs beyond those found in animals. There are open-reading frames that encode K⁺-channel subunits with unconventional filter sequences, or regulatory domains of different sizes and numbers not previously known. Parasitic or symbiotic bacteria tend not to have K⁺ channels, while those showing lifestyle versatility often have more than one K⁺-channel gene. It is speculated that prokaryotic K⁺ channels function to allow adaptation to environmental and metabolic changes, although the actual roles of these channels in prokaryotes are not yet known. Unlike enzymes in basic metabolism, K⁺ channel, though evolved early, appear to play more diverse roles than revealed by animal research. Finding and sorting out these roles will be the goal and challenge of the near future.     

Effect of desiccation on potassium and anion currents from young root hairs: Implication on tip growth

Little is known about the early response of roots to desiccation. Young growing root hairs of Arabidopsis thaliana , Vigna unguiculata and Phaseolus vulgaris were used to study the early response of roots to desiccation since they behave like sensors that are able to perceive environmental signals. In control conditions, root hairs were polarized around −120 mV and displayed inward rectifying K⁺ currents. When submitted to short-term desiccation, root hairs stopped their tip growth and their membrane became depolarized. Under these conditions, the K⁺ influx carried by the inward rectifying K⁺ channels was not maintained and instead slow deactivating anion channels were recorded. The inhibition of K⁺ influx and the large anion efflux due to the activation of slow anion currents could participate in the inhibition of tip growth.     

The effect of xanthoxylin (2-oxy, 4-6-dimethoxyacetophenone) on potassium-dependent acid: extrusion in wheat and maize root segments

Abstract. Xanthoxylin is a cytotoxic and fungicidal compound with the characteristics of a typical phytoalexin. At a concentration of 0.3 mol m⁻³ it inhibits K⁺-dependent acid extrusion and K⁺ net uptake (or uptake of equivalent alkaline cations such as Rb⁺ and CS⁺) by up to about 80% and hyperpolarizes by about 20% the membrane electrical potential. Its inhibition of the acid extrusion does not depend on altered ion exchange involving the anions in the media, a reduction of the metabolic energy available, or detectable changes in the permeability of the cell membrane to H⁺ ions. The drop in K⁺ net uptake depends on a decrease in the influx of K⁺ into the cell. In functional terms, xanthoxylin is an inhibitor of the K⁺ permeation mechanism and does not appear to interact with the mechanisms creating the electrochemical energy gradient.