Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells

Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells, fortifying the finding that two highly distinct types or modes of voltage- and second messenger-dependent anion channel currents coexist in the guard cell plasma membrane. Bioassays using anion channel blockers provide evidence that slow anion channel currents play a substantial role in the regulation of stomatal closing. Interestingly, slow anion channels may also function as a negative regulator during stomatal opening under the experimental conditions applied here. The identification of specific blockers of slow anion channels reported here permits detailed studies of cell biological functions, modulation, and structural components of slow anion channels in guard cells and other higher plant cells.     

Malate-induced feedback regulation of plasma membrane anion channels could provide a CO2 sensor to guard cells.

Plants have developed strategies to circumvent limitations in water supply through the adjustment of stomatal aperture in relation to the photosynthetic capacity (water-use efficiency). The CO2 sensor of guard cells, reporting on the metabolic status of the photosynthetic tissue, is, however, as yet unknown. We elucidated whether extracellular malate has the capability to serve as a signal metabolite in regulating the membrane properties of guard cells. Patch-clamp studies showed that slight variations in the external malate concentration induced major alterations in the voltage-dependent activity of the guard cell anion channel (GCAC1). Superfusion of guard cell protoplasts with malate solutions in the physiological range caused the voltage-gate to shift towards hyperpolarized potentials (Km(mal) = 0.4 mM elicits a 38 mV shift). The selectivity sequence of the anion channel NO3- (4.2) > or = I- (3.9) > Br- (1.9) > Cl- (1) >> mal (0.1) indicates that malate is able to permeate GCAC1. The binding site for shifting the gate is, however, located on the extracellular face of the channel since cytoplasmic malate proved ineffective. Single-channel analysis indicates that extracellular malate affects the voltage-dependent mean open time rather than the unitary conductance of GCAC1. In contrast to malate the rise in the extracellular Cl- concentration increases the unit conductance of the anion efflux channel. We suggest that stomata sense changes in the intercellular CO2 concentration and thus the photosynthetic activity of the mesophyll via feedback regulation of anion efflux from guard cells through malate-sensitive GCAC1.     

AtALMT12 represents an R‐type anion channel required for stomatal movement in Arabidopsis guard cells

Stomatal pores formed by a pair of guard cells in the leaf epidermis control gas exchange and transpirational water loss. Stomatal closure is mediated by the release of potassium and anions from guard cells. Anion efflux from guard cells involves slow (S‐type) and rapid (R‐type) anion channels. Recently the SLAC1 gene has been shown to encode the slow, voltage‐independent anion channel component in guard cells. In contrast, the R‐type channel still awaits identification. Here, we show that AtALMT12, a member of the aluminum activated malate transporter family in Arabidopsis, represents a guard cell R‐type anion channel. AtALMT12 is highly expressed in guard cells and is targeted to the plasma membrane. Plants lacking AtALMT12 are impaired in dark‐ and CO₂‐induced stomatal closure, as well as in response to the drought‐stress hormone abscisic acid. Patch‐clamp studies on guard cell protoplasts isolated from atalmt12 mutants revealed reduced R‐type currents compared with wild‐type plants when malate is present in the bath media. Following expression of AtALMT12 in Xenopus oocytes, voltage‐dependent anion currents reminiscent to R‐type channels could be activated. In line with the features of the R‐type channel, the activity of heterologously expressed AtALMT12 depends on extracellular malate. Thereby this key metabolite and osmolite of guard cells shifts the threshold for voltage activation of AtALMT12 towards more hyperpolarized potentials. R‐Type channels, like voltage‐dependent cation channels in nerve cells, are capable of transiently depolarizing guard cells, and thus could trigger membrane potential oscillations, action potentials and initiate long‐term anion and K⁺ efflux via SLAC1 and GORK, respectively.     

A new scheme of symbiosis: ligand- and voltage-gated anion channels in plants and animals.

Anion channels in the plasma membrane of both plant and animal cells participate in a number of important cellular functions such as volume regulation, trans-epithelial transport, stabilization of the membrane potential and excitability. Only very recently attention has turned to the presence of anion channels in higher plant cells. A dominant theme among recent discoveries is the role of Ca2+ in activating or modulating channel current involved in signal transduction. The major anion channel of stomatal guard cell protoplasts is a 32-40 pS channel which is highly selective for anions, in particular NO3-, Cl- and malate. These channels are characterized by a steep voltage dependence. Anion release is elicited upon depolarization and restricted to a narrow voltage span of -100 mV to the reversal potential of anions. During prolonged activation the current slowly inactivates. A rise in cytoplasmic calcium in the presence of nucleotides evokes activation of the anion channels. Following activation they catalyse anion currents 10-20 times higher than in the inactivated state thereby shifting the resting potential of the guard cell from a K(+)-conducting to an anion-conducting state. Patch-clamp studies have also revealed that growth hormones directly affect voltage-dependent activity of the anion channel in a dose-dependent manner. Auxin binding resulted in a shift of the activation potential towards the resting potential. Auxin-dependent gating of the anion channel is side- and hormone-specific. Its action is also channel-specific as K+ channels coexisting in the same membrane patch were insensitive to this ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

The coronatine-insensitive 1 mutation reveals the hormonal signaling interaction between abscisic acid and methyl jasmonate in Arabidopsis guard cells. Specific impairment of ion channel activation and second messenger production

Methyl jasmonate (MeJA) elicits stomatal closing similar to abscisic acid (ABA), but whether the two compounds use similar or different signaling mechanisms in guard cells remains to be clarified. We investigated the effects of MeJA and ABA on second messenger production and ion channel activation in guard cells of wild-type Arabidopsis (Arabidopsis thaliana) and MeJA-insensitive coronatine-insensitive 1 (coi1) mutants. The coi1 mutation impaired MeJA-induced stomatal closing but not ABA-induced stomatal closing. MeJA as well as ABA induced production of reactive oxygen species (ROS) and nitric oxide (NO) in wild-type guard cells, whereas MeJA did not induce production of ROS and NO in coi1 guard cells. The experiments using an inhibitor and scavengers demonstrated that both ROS and NO are involved in MeJA-induced stomatal closing as well as ABA-induced stomatal closing. Not only ABA but also MeJA activated slow anion channels and Ca(2+) permeable cation channels in the plasma membrane of wild-type guard cell protoplasts. However, in coi1 guard cell protoplasts, MeJA did not elicit either slow anion currents or Ca(2+) permeable cation currents, but ABA activated both types of ion channels. Furthermore, to elucidate signaling interaction between ABA and MeJA in guard cells, we examined MeJA signaling in ABA-insensitive mutant ABA-insensitive 2 (abi2-1), whose ABA signal transduction cascade has some disruption downstream of ROS production and NO production. MeJA also did not induce stomatal closing but stimulated production of ROS and NO in abi2-1. These results suggest that MeJA triggers stomatal closing via a receptor distinct from the ABA receptor and that the coi1 mutation disrupts MeJA signaling upstream of the blanch point of ABA signaling and MeJA signaling in Arabidopsis guard cells.     

Anion channels as central mechanisms for signal transduction in guard cells and putative functions in roots for plant-soil interactions

In higher plants anion channels have recently been suggested to play key roles in controlling cellular functions, including turgor- and osmoregulation, stomatal movements, anion transport, signal transduction and possibly also signal propagation. In guard cells and roots, physiological functions of anion channels have been proposed which will be discussed here. In initial investigations it was proposed that anion channels in the plasma membrane of guard cells provide a prominent control mechanism for stomatal closing. The proposed model suggests that anion channel activation and the resulting anion efflux from guard cells cause membrane depolarization, thereby driving K⁺ efflux through outward-rectifying K⁺ channels required for stomatal closing. This article provides a brief review of new and recent insights into the molecular properties and cell biological functions of anion channels in guard cells. Furthermore, recently implicated putative functions of anion channels in roots during salt stress, xylem loading and Al³⁺ tolerance are addressed.     

A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK.

Calcium-Dependent Protein Kinases (CDPKs) in higher plants contain a C-terminal calmodulin-like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+-dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch-clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+-independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl-and vanished in glutamate. Single channel recordings showed a CDPK-activated 34 +/- 5 pS Cl- channel. VCL channels were activated at physiological potentials enabling Cl- uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK-activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase-dependent anion uptake.     

Anion-Channel Blockers Inhibit S-Type Anion Channels and Abscisic Acid Responses in Guard Cells

The effects of anion-channel blockers on light-mediated stomatal opening, on the potassium dependence of stomatal opening, on stomatal responses to abscisic acid (ABA), and on current through slow anion channels in the plasma membrane of guard cells were investigated. The anion-channel blockers anthracene-9-carboxylic acid (9-AC) and niflumic acid blocked current through slow anion channels of Vicia faba L. guard cells. Both 9-AC and niflumic acid reversed ABA inhibition of stomatal opening in V. faba L. and Commelina communis L. The anion-channel blocker probenecid also abolished ABA inhibition of stomatal opening in both species. Additional tests of 9-AC effects on stomatal aperture in Commelina revealed that application of this anion-channel blocker allowed wide stomatal opening under low (1 mM) KCI conditions and increased the rate of stomatal opening under both low and high (100 mM) KCI conditions. These results indicate that anion channels can function as a negative regulator of stomatal opening, presumably by allowing anion efflux and depolarization, which prohibits ion up-take in guard cells. Furthermore, 9-AC prevented ABA induction of stomatal closure. A model in which ABA activation of anion channels contributes a rate-limiting mechanism during ABA-induced stomatal closure and inhibition of stomatal opening is discussed.     

Anion channels: master switches of stress responses

During stress, plant cells activate anion channels and trigger the release of anions across the plasma membrane. Recently, two new gene families have been identified that encode major groups of anion channels. The SLAC/SLAH channels are characterized by slow voltage-dependent activation (S-type), whereas ALMT genes encode rapid-activating channels (R-type). Both S- and R-type channels are stimulated in guard cells by the stress hormone ABA, which leads to stomatal closure. Besides their role in ABA-dependent stomatal movement, anion channels are also activated by biotic stress factors such as microbe-associated molecular patterns (MAMPs). Given that anion channels occur throughout the plant kingdom, they are likely to serve a general function as master switches of stress responses.     

Characterization of anion channels in the plasma membrane of Arabidopsis epidermal root cells and the identification of a citrate-permeable channel induced by phosphate starvation

Organic-acid secretion from higher plant roots into the rhizosphere plays an important role in nutrient acquisition and metal detoxification. In this study we report the electrophysiological characterization of anion channels in Arabidopsis (Arabidopsis thaliana) root epidermal cells and show that anion channels represent a pathway for citrate efflux to the soil solution. Plants were grown in nutrient-replete conditions and the patch clamp technique was applied to protoplasts isolated from the root epidermal cells of the elongation zone and young root hairs. Using SO4(2-) as the dominant anion in the pipette, voltage-dependent whole-cell inward currents were activated at membrane potentials positive of -180 mV exhibiting a maximum peak inward current (I(peak)) at approximately -130 mV. These currents reversed at potentials close to the equilibrium potential for SO4(2-), indicating that the inward currents represented SO4(2-) efflux. Replacing intracellular SO4(2-) with Cl- or NO3(-) resulted in inward currents exhibiting similar properties to the SO4(2-) efflux currents, suggesting that these channels were also permeable to a range of inorganic anions; however when intracellular SO4(2-) was replaced with citrate or malate, no inward currents were ever observed. Outside-out patches were used to characterize a 12.4-picoSiemens channel responsible for these whole-cell currents. Citrate efflux from Arabidopsis roots is induced by phosphate starvation. Thus, we investigated anion channel activity from root epidermal protoplasts isolated from Arabidopsis plants deprived of phosphate for up to 7 d after being grown for 10 d on phosphate-replete media (1.25 mm). In contrast to phosphate-replete plants, protoplasts from phosphate-starved roots exhibited depolarization-activated voltage-dependent citrate and malate efflux currents. Furthermore, phosphate starvation did not regulate inorganic anion efflux, suggesting that citrate efflux is probably mediated by novel anion channel activity, which could have a role in phosphate acquisition.     

Barley mildew and its elicitor chitosan promote closed stomata by stimulating guard-cell S-type anion channels

Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe-associated molecular patterns (MAMPs). However, the molecular mechanisms underlying these guard-cell responses have not yet been elucidated. We therefore studied pathogen-induced changes in ion channel activity in Hordeum vulgare guard cells. Barley mildew (Blumeria graminis) hyphae growing on leaves inhibited light-induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed. Alternatively, stomatal closure was induced by nano-infusion of chitosan via open stomata into the sub-stomatal cavity. Experiments using intracellular double-barreled micro-electrodes revealed that mildew stimulated S-type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K(+) extrusion by altering the plasma membrane potential. Stimulation of S-type anion channels was also provoked by nano-infusion of chitosan. These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S-type anion channels in guard cells. In response, guard cells extrude K(+) salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types.     

The phosphoinositide 3-kinase Vps34p is required for pexophagy in Saccharomyces cerevisiae

Stomatal guard cells play a key role in gas exchange for photosynthesis and in minimizing transpirational water loss from plants by opening and closing the stomatal pore. The bulk of the osmotic content driving stomatal movements depends on ionic fluxes across both the plasma membrane and tonoplast, the metabolism of organic acids, primarily Mal (malate), and its accumulation and loss. Anion channels at the plasma membrane are thought to comprise a major pathway for Mal efflux during stomatal closure, implicating their key role in linking solute flux with metabolism. Nonetheless, little is known of the regulation of anion channel current (I(Cl)) by cytosolic Mal or its immediate metabolite OAA (oxaloacetate). In the present study, we have examined the impact of Mal, OAA and of the monocarboxylic acid anion acetate in guard cells of Vicia faba L. and report that all three organic acids affect I(Cl), but with markedly different characteristics and sidedness to their activities. Most prominent was a suppression of ICl by OAA within the physiological range of concentrations found in vivo. These findings indicate a capacity for OAA to co-ordinate organic acid metabolism with I(Cl) through the direct effect of organic acid pool size. The findings of the present study also add perspective to in vivo recordings using acetate-based electrolytes.     

Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology

We have used the whole-cell patch clamp recording technique to characterize a swelling-activated chloride current in guinea pig atrial and ventricular myocytes and to compare the electrophysiological and pharmacological properties of this current with the isoprenaline- activated chloride current in the same cell types. Osmotic swelling of guinea pig cardiac myocytes caused activation of an outwardly rectifying, anion-selective current with a conductance and permeability sequence of I- approximately NO3- > Br- > Cl- > Asp-. This current was inhibited by tamoxifen, 4,4''-diisothiocyano-stilbene-2,2''-disulphonate and anthracene-9-carboxylic acid, in decreasing order of potency. The isoprenaline-activated anion current, like the swelling-activated current, had a higher permeability to I- relative to Cl-, but it had a markedly reduced conductance for I- compared to Cl-. The isoprenaline- activated current was insensitive to inhibition by tamoxifen, 4,4''- diisothiocyanostilbene-2,2''-disulphonate and anthracene-9-carboxylic acid. The swelling-activated current could be elicited in > 90% atrial myocytes studied but only 34% ventricular myocytes. Conversely, the isoprenaline-activated current was elicited in < 10% atrial myocytes and > 90% ventricular myocytes. In those ventricular myocytes where it was possible to elicit swelling-activated and isoprenaline-activated currents simultaneously, the currents retained the same distinguishing characteristics as when they were elicited in isolation. Thus, while guinea pig atrial cells appear to preferentially express swelling- activated chloride channels and guinea pig ventricular myocytes preferentially express isoprenaline-activated chloride channels, the presence of these two channel types are not necessarily mutually exclusive. This raises the possibility that there may be coordinated regulation of the expression of different Cl- channels within the heart.     

Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants.

Abscisic acid (ABA) regulates vital physiological responses, and a number of events in the ABA signaling cascade remain to be identified. To allow quantitative analysis of genetic signaling mutants, patch-clamp experiments were developed and performed with the previously inaccessible Arabidopsis guard cells from the wild type and ABA-insensitive (abi) mutants. Slow anion channels have been proposed to play a rate-limiting role in ABA-induced stomatal closing. We now directly demonstrate that ABA strongly activates slow anion channels in wild-type guard cells. Furthermore, ABA-induced anion channel activation and stomatal closing were suppressed by protein phosphatase inhibitors. In abi1-1 and abi2-1 mutant guard cells, ABA activation of slow anion channels and ABA-induced stomatal closing were abolished. These impairments in ABA signaling were partially rescued by kinase inhibitors in abi1 but not in abi2 guard cells. These data provide cell biological evidence that the abi2 locus disrupts early ABA signaling, that abi1 and abi2 affect ABA signaling at different steps in the cascade, and that protein kinases act as negative regulators of ABA signaling in Arabidopsis. New models for ABA signaling pathways and roles for abi1, abi2, and protein kinases and phosphatases are discussed.     

Anion channels in plant cells

Plant anion channels allow the efflux of anions from cells. They are involved in turgor pressure control, changes in membrane potential, organic acid excretion, tolerance to salinity and inorganic anion nutrition. The recent molecular identification of anion channel genes in guard cells and in roots allows a better understanding of their function and of the mechanisms that control their activation.     

Alternation of the slow with the quick anion conductance in whole guard cells effected by external malate

In previous investigations two anion conductances were discovered in guard-cell protoplasts: the quickly activating anion conductance (QUAC, R-type) and the slowly activating anion conductance (SLAC, S-type). In this investigation, effects of malate on the two anion conductances were tested in whole guard cells of Vicia faba L. by the use of the discontinuous single-electrode voltage-clamp method. Application of 1-s voltage ramps proved that QUAC displayed the malate shift of the activation threshold toward hyperpolarization also in complete guard cells. The sensitivity of SLAC to external malate was determined by responses to voltage pulses of 20 s duration at Cl- concentrations of 0.1, 3 or 50 mM. At no voltage were the currents measured at the end of the pulses in the presence and absence of malate significantly different from each other; the current-voltage relationship of SLAC appeared not to be affected by malate. However, in 32% of the cells exposed to malate, current activation in response to voltage steps occurred within 0.1 s, faster than was typical for SLAC, and activation was followed by inactivation with a half-time similar to 10 s: SLAC apparently had changed to QUAC. Simultaneously, the free-running membrane voltage depolarized at 0.1 mM Cl-, did not change at 3 mM Cl- and polarized at 50 mM Cl-, indicating that activation of QUAC increased the membrane conductance for anions and thereby drove the membrane voltage toward the equilibrium voltage of Cl-. The malate-induced changes were fully reversible at Cl- concentrations of 0.1 and 3 mM. These results reinforce the proposition that SLAC and QUAC represented two switching modes of the same anion channel (however, they do not suffice as proof); they also show that this interconvertibility can enable guard cells to control their membrane voltage rapidly.     

ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells

In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells.     

K+ transport properties of K+ channels in the plasma membrane of Vicia faba guard cells

Electrical properties of the plasma membrane of guard cell protoplasts isolated from stomates of Vicia faba leaves were studied by application of the whole-cell configuration of the patch-clamp technique. The two types of K+ currents that have recently been identified in guard cells may allow efflux of K+ during stomatal closing, and uptake of K+ during stomatal opening (Schroeder et al., 1987). A detailed characterization of ion transport properties of the inward-rectifying (IK+,in) and the outward-rectifying (IK+,out) K+ conductance is presented here. The permeability ratios of IK+,in and IK+,out currents for K+ over monovalent alkali metal ions were determined. The resulting permeability sequences (PK+ greater than PRb+ greater than PNa+ greater than PLi+ much greater than PCs+) corresponded closely to the ion specificity of guard cell movements in V. faba. Neither K+ currents exhibited significant inactivation when K+ channels were activated for prolonged periods (greater than 10 min). The absence of inactivation may permit long durations of K+ fluxes, which occur during guard cell movements. Activation potentials of inward K+ currents were not shifted when external K+ concentrations were changed. This differs strongly from the behavior of inward-rectifying K+ channels in animal tissue. Blue light and fusicoccin induce hyperpolarization by stimulation of an electrogenic pump. From slow-whole-cell recordings it was concluded that electrogenic pumps require cytoplasmic substrates for full activation and that the magnitude of the pump current is sufficient to drive K+ uptake through IK+,in channels. First, direct evidence was gained for the hypothesis that IK+,in channels are a molecular pathway for K+ accumulation by the finding that IK+,in was blocked by Al3+ ions, which are known to inhibit stomatal opening but not closing. The results presented in this study strongly support a prominent role for IK+,in and IK+,out channels in K+ transport across the plasma membrane of guard cells.     

Characterization of the TaALMT1 protein as an Al3+-activated anion channel in transformed tobacco (Nicotiana tabacum L.) cells

TaALMT1 encodes a putative transport protein associated with Al(3+)-activated efflux of malate from wheat root apices. We expressed TaALMT1 in Nicotiana tabacum L. suspension cells and conducted a detailed functional analysis. Protoplasts were isolated for patch-clamping from cells expressing TaALMT1 and from control cells (empty vector transformed). With malate(2-) as the permeant anion in the protoplast, an inward current (anion efflux) that reversed at positive potentials was observed in protoplasts expressing TaALMT1 in the absence of Al(3+). This current was sensitive to the anion channel antagonist niflumate, but insensitive to Gd(3+). External AlCl(3) (50 microM), but not La(3+) and Gd(3+), increased the inward current in TaALMT1-transformed protoplasts. The inward current was highly selective to malate over nitrate and chloride (P(mal) > P(NO3) >or= P(Cl), P(mal)/P(Cl) >or=18, +/-Al(3+)), under conditions with higher anion concentration internally than externally. The anion currents displayed a voltage and time dependent deactivation at negative voltages. Voltage ramps revealed that inward rectification was caused by the imposed anion gradients. Single channels with conductances between 10 and 17 pS were associated with the deactivation of the current at negative voltages, agreeing with estimates from voltage ramps. This study of the electrophysiological function of the TaALMT1 protein in a plant heterologous expression system provides the first direct evidence that TaALMT1 functions as an Al(3+)-activated malate(2-) channel. We show that the Al(3+)-activated currents measured in TaALMT1-transformed tobacco cells are identical to the Al(3+)-activated currents observed in the root cells of wheat, indicating that TaALMT1 alone is likely to be responsible for those endogenous currents.     

A slow anion channel in guard cells, activating at large hyperpolarization, may be principal for stomatal closing.

Slowly activating anion channel currents were discovered at micromolar 'cytoplasmic' Ca2+ during patch-clamp measurements on guard-cell protoplasts of Vicia faba and Xanthium strumarium. They activated at potentials as low as -200 mV, with time constants between 5 and 60 s, and no inactivation. The broad voltage dependence exhibited a current maximum near -40 mV. The single-channel open time was in the order of seconds, and the unitary conductance was 33 ps, similar to that of the already described 'quick' anion channel of guard cells. Because of its activity at low potentials, the slow anion channel may be essential for the depolarization of the plasmalemma that is required for salt efflux during stomatal closing.