Control of vacuolar dynamics and regulation of stomatal aperture by tonoplast potassium uptake

Stomatal movements rely on alterations in guard cell turgor. This requires massive K⁺ bidirectional fluxes across the plasma and tonoplast membranes. Surprisingly, given their physiological importance, the transporters mediating the energetically uphill transport of K⁺ into the vacuole remain to be identified. Here, we report that, in Arabidopsis guard cells, the tonoplast-localized K⁺/H⁺ exchangers NHX1 and NHX2 are pivotal in the vacuolar accumulation of K⁺ and that nhx1 nhx2 mutant lines are dysfunctional in stomatal regulation. Hypomorphic and complete-loss-of-function double mutants exhibited significantly impaired stomatal opening and closure responses. Disruption of K⁺ accumulation in guard cells correlated with more acidic vacuoles and the disappearance of the highly dynamic remodelling of vacuolar structure associated with stomatal movements. Our results show that guard cell vacuolar accumulation of K⁺ is a requirement for stomatal opening and a critical component in the overall K⁺ homeostasis essential for stomatal closure, and suggest that vacuolar K⁺ fluxes are also of decisive importance in the regulation of vacuolar dynamics and luminal pH that underlie stomatal movements.The rapid accumulation and release of K⁺ and of organic and inorganic anions by guard cells controls the opening and closing of stomata and thereby gas exchange and transpiration of plants. The intracellular events that underlie stomatal opening start with plasma membrane hyperpolarization caused by the activation of H⁺-ATPases, which induces K⁺ uptake through voltage-gated inwardly rectifying K⁺_in channels (1). Potassium uptake is accompanied by the electrophoretic entry of the counterions chloride, nitrate, and sulfate, and by the synthesis of malate. These osmolytes, together with sucrose accumulation, increase the turgor in guard cells and thereby drive stomatal opening. Stomatal closure is initiated by activation of the plasma membrane localized chloride and nitrate efflux channels SLAC1 and SLAH3 that are regulated by the SnRK2 protein kinase OST1 and the Ca²⁺-dependent protein kinases CPK21 and 23 (2, 3). CPK6 also activates SLAC1 and coordinately inhibits rectifying K⁺_in channels to hinder stomatal opening (4, 5). Sulfate and organic acids exit the guard cell through R-type anion channels. The accompanying reduction in guard cell turgor results in stomatal closure (1).Despite the established role of plasma membrane transport in guard cell function and stomatal movement, ion influx into the cytosol represents only a transit step to the vacuole, as more than 90% of the solutes released from guard cells originate from vacuoles (6). In contrast to the plasma membrane, knowledge of the transport processes occurring in intracellular compartments of guard cells during stomatal movements is less advanced (7). Only recently, AtALMT9 has been shown to act as a malate-induced chloride channel at the tonoplast that is required for stomatal opening (8). Vacuoles govern turgor-driven changes in guard cell volumes by increases and decreases in vacuolar volume during stomatal opening and closure, respectively, by more than 40% (9, 10). Monitoring the dynamic changes in guard cell vacuolar structures revealed an intense remodeling during stomatal movements (11, 12). Pharmacological and genetic approaches indicated that dynamic changes of the vacuole are crucial for achieving the full amplitude of stomatal movement (12–14). However, so far, no specific tonoplast transport proteins or processes have been functionally linked to vacuolar dynamics during guard cell movements.Cation channel activities mediating K⁺ release and stomatal closure have been characterized at the tonoplast, including fast vacuolar, slow vacuolar, and K⁺-selective vacuolar cation channels (7, 15). Genetic inactivation of K⁺-release channels leads to slower stomatal closure kinetics (7, 16). By contrast, the transporters responsible for the uptake of K⁺ into vacuoles against the vacuolar membrane potential that drive the stomatal aperture have remained unknown. We have recently reported that the tonoplast-localized K⁺,Na⁺/H⁺ exchangers NHX1 and NHX2 from Arabidopsis are involved in the accumulation of K⁺ into the vacuole of plant cells, thereby increasing their osmotic potential and driving the uptake of water that generates the turgor pressure necessary for cell expansion and growth (17). The involvement of K⁺,Na⁺/H⁺ exchangers in the regulation of plant transpiration was also proposed, as the nhx1 nhx2 mutant exhibited enhanced transpirational water loss compared with WT when subjected to osmotic stress. Here, to resolve whether active K⁺ uptake at the tonoplast directly regulates stomatal activity by mediating K⁺ accumulation in the vacuole of guard cells, we analyzed the stomatal movements of nhx1 nhx2 double mutant lines by using a range of physiological, molecular, and imaging-based approaches. Moreover, we have developed a noninvasive, fluorescence ratiometric method to measure vacuolar pH (pHv) in guard cells by using the H⁺-sensitive and cell-permeant dye Oregon green and epidermal peels. Our data establish that (i) the capacity for K⁺ accumulation into guard cell vacuoles is essential for stomatal activity by facilitating not only stomatal aperture but also closure, (ii) K⁺/H⁺ exchange at the guard cell tonoplast mediates the luminal pHv shifts associated to stomata opening, and (iii) the dynamic morphological changes that guard cell vacuoles undergo during stomatal movements are brought about by the uptake of K⁺ into the vacuole.     

The number of K(+) channels in the plasma membrane of guard cell protoplasts changes in parallel with the surface area

The activity of the two dominant K(+) channels in the plasma membrane of Vicia faba guard cell protoplasts was examined during pressure-driven swelling. For this purpose, the K(+) currents and the membrane capacitance (C(m)) of guard cell protoplasts were recorded in parallel. A rise in C(m), reflecting an increase of the membrane surface area, was coupled to a proportional rise in conductance of both the K(+) inward and K(+) outward rectifier. The activation kinetics of the K(+) channels were not affected during this process. The quantitative and temporal coupling of C(m) and K(+) conductance can hence be interpreted as the result of the addition of active inward and outward rectifier K(+) channels to the plasma membrane during an increase in surface area.     

Electrophysiological properties of human coronary endothelial cells

The electrophysiological properties of human coronary endothelial cells (HCEC) of macro-and microvascular origin were studied using the whole-cell configuration of the patch-clamp technique. The membrane potential of confluent HCEC(–41.9±3.9 mV (mean±SEM, n=32) for macro-and –33.6±22.6 mV (n=64) for microvascular cells, respectively) was less negative than the K⁺ equilibrium potential. Inward currents of isolated cells at potentials below the K⁺ equilibrium potential were blocked by external Ba²⁺ (1 mM), inactivated due to time- and voltage-dependent block caused by external Na⁺, and their amplitudes were enhanced by increasing extracellular [K⁺]; these currents were identified as inwardly rectifying K⁺ currents. Some isolated cells displayed outwardly directed K⁺ currents which were abolished after replacement of Cs⁺ for K⁺ on both sides of the membrane. Voltage-dependent Ca²⁺ currents could not be observed in isolated HCEC. Hyperpolarizations induced by vasoactive agonists have been observed in some endothelial cells from different species. In contrast, extracellularly applied ATP (adenosine-5-triphosphate) and ADP (adenosine-5-diphosphate) at micromolar concentrations depolarized confluent HCEC, whereas adenosine had no effect on resting potentials (RP), indicating that the nucleotide-induced depolarizations were mediated via P₂-purinoceptors. These depolarizations occurred even after replacement of N-methyl-D-glucamine for extracellular Na⁺, indicating that Ca²⁺-influx was involved. There were no marked differences in the electrophysiological properties between cells of macro-and microvascular origin.     

Trimebutine maleate has inhibitory effects on the voltage-dependent Ca2+ inward current and other membrane currents in intestinal smooth muscle cells

We examined effects of trimebutine maleate on the membrane currents of the intestinal smooth muscle cells by using the tight-seal whole cell clamp technique. Trimebutine suppressed the Ba²⁺ inward current through voltage-dependent Ca²⁺ channels in a dose-dependent manner. The inhibitory effect of trimebutine on the Ba²⁺ inward current was not use-dependent. It shifted the steady-state inactivation curve to the left along the voltage axis. Trimebutine also had inhibitory effects on the other membrane currents of the cells, such as the voltage-dependent K⁺ current, the Ca²⁺-activated oscillating K⁺ current and the acetylcholine-induced inward current. These relatively non-specific inhibitory effects of trimebutine on the membrane currents may explain, at least in part, the dual actions of the drug on the intestinal smooth muscle contractility, i.e. inhibitory as well as excitatory.     

Repetitive increases in cytosolic Ca2+ of guard cells by abscisic acid activation of nonselective Ca2+ permeable channels.

Many signal-transduction processes in higher plant cells have been suggested to be triggered by signal-induced opening of Ca2+ channels in the plasma membrane. However, direct evidence for activation of plasma-membrane Ca2+ channels by physiological signals in higher plants has not yet been obtained. In this context, several lines of evidence suggest that Ca2+ flux into the cytosol of guard cells is a major factor in the induction of stomatal closing by abscisic acid (ABA). ABA closes stomatal pores, thereby reducing transpirational loss of water by plants under drought conditions. To directly investigate initial events in ABA-induced signal transduction in guard cells, we devised an experimental approach that allows simultaneous photometric measurements of cytosolic Ca2+ and patch-clamp recordings of ion currents across the plasma membrane of single Vicia faba guard cells. Using this approach, we found that the resting cytosolic Ca2+ concentration was 0.19 +/- 0.09 microM (n = 19). In responsive guard cells, external exposure to ABA produced transient repetitive increases in the cytosolic free Ca2+ concentration. These Ca2+ transients were accompanied by concomitantly occurring increases in an inward-directed ion current. Depolarization of the membrane terminated both repetitive elevations in cytosolic Ca2+ and inward-directed ion currents, suggesting that ABA-mediated Ca2+ transients were produced by passive influx of Ca2+ from the extracellular space through Ca2(+)-permeable channels. Detailed voltage-clamp measurements revealed that ABA-activated ion currents could be reversed by depolarizations more positive than -10 mV. Interestingly, reversal potentials of ABA-induced currents show that these currents are not highly Ca2(+)-selective, thereby permitting permeation of both Ca2+ and K+. These results provide direct evidence for ABA activation of Ca2(+)-permeable ion channels in the plasma membrane of guard cells. ABA-activated ion channels allow repetitive elevations in the cytosolic Ca2+ concentration, which, in turn, can modulate cellular responses promoting stomatal closure.     

An inward rectifier and a voltage-dependent K+ current in single, cultured pericytes from bovine heart

OBJECTIVE: The purpose of this study was to describe passive electrical properties and major membrane currents in coronary pericytes. METHODS: 78 single, cultured bovine pericytes were studied with the patch-clamp technique in the whole-cell mode. RESULTS: The membrane potential of the cells was -48.9+/-9.6 mV (mean+/-S.D.) with 5 mM and -23.2+/-2.2 mV with 60 mM extracellular K+. The membrane capacitance was 150.2+/-123.2 pF. The current-voltage relation of the pericytes was dominated by an inward current at hyperpolarized potentials and an outward current at depolarized potentials. Increasing extracellular K+ from 5 to 60 mM led to an increase of the inward current and to a shift of this current to more depolarized potentials. The inward current was very sensitive to extracellular barium (50 microM). The maximum slope conductance of the cells at hyperpolarized potentials was 2.9+/-2.8 nS. Inward rectification of whole-cell currents was steep (slope factor = 6.8 mV). With elevated external K+ the outward current reversed near the potassium equilibrium potential. Onset of the outward current was sigmoid and inactivation of this current was monoexponential, slow (time constant = 12.8 s) and incomplete. Voltage-dependence of outward current steady-state activation was steep (slope factor = 4.6 mV). The outward current was very sensitive to 4-aminopyridine (dissociation constant = 0.1 mM). The maximum slope conductance at depolarized potentials was 16.6+/-15.6 nS. CONCLUSION: We report for the first time, patch-clamp recordings from coronary pericytes. An inward rectifier and a voltage-dependent K+ current were identified and characterized. Regulation of these currents may influence coronary blood flow.     

Characterization of ionic currents and electrophysiological properties of goldfish somatotropes in primary culture

Growth hormone release in goldfish is partly dependent on voltage-sensitive Ca²⁺ channels but somatotrope electrophysiological events affecting such channel activities have not been elucidated in this system. The electrophysiological properties of goldfish somatotropes in primary culture were studied using the whole-cell and amphotericin B-perforated patch-clamp techniques. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) of identified somatotropes was measured using Fura-2/AM dye. Goldfish somatotropes had an average resting membrane potential of −78.4 ± 4.6 mV and membrane input resistance of 6.2 ± 0.2 GΩ. Voltage steps from a holding potential of −90 mV elicited a non-inactivating outward current and transient inward currents at potentials more positive than 0 and −30 mV, respectively. Isolated current recordings indicate the presence of 4-aminopyridine- and tetraethylammonium (TEA)-sensitive K⁺, tetrodotoxin (TTX)-sensitive Na⁺, and nifedipine (L-type)- and ω-conotoxin GVIA (N-type)-sensitive Ca²⁺ channels. Goldfish somatotropes rarely fire action potentials (APs) spontaneously, but single APs can be induced at the start of a depolarizing current step; this single AP was abolished by TTX and significantly reduced by nifedipine and ω-conotoxin GVIA. TEA increased AP duration and triggered repetitive AP firing resulting in an increase in [Ca²⁺]_i, whereas TTX, nifedipine and ω-conotoxin GVIA inhibited TEA-induced [Ca²⁺]_i pulses. These results indicate that in goldfish somatotropes, TEA-sensitive K⁺ channels regulate excitability while TTX-sensitive Na⁺ channels together with N- and L-type Ca channels mediates the depolarization phase of APs. Opening of voltage-sensitive Ca²⁺ channels during AP firing leads to increases in [Ca²⁺]_i.     

Cesium Abolishes the Barium-Induced Pacemaker Potential and Current in Guinea Pig Ventricular Myocytes

Cesium Abolishes Barium-Induced PM Current. Introduction: The ability of cesium to block barium-induced diastolic depolarization (“Ba-DD”) and pacemaker current was tested in isolated ventricular myocytes. Because Ba-DD is due to decreasing k conductance and there is no I_f at the resting potential, this approach permits verification of whether Cs⁺ is a specific blocker of I, or if it instead also blocks a K⁺ pacemaker current. Methods and Results: Guinea pig isolated ventricular myocytes were studied by a discontinuous, single electrode, voltage clamp method. During hyperpolarizing voltage clamp steps from -80 up to -140 mV in Tyrode's solution, the inward current increased as a function of voltage but did not change us a function of time (no I_f or K⁺ depletion). Cesium (4mM) reduced the current size during the hyperpolarizing steps hut did not induce or unmask time-dependent currents. Barium (0.05 to 0.1 mM) induced diastolic depolarization, and, in its presence, depolarizing voltage clamp steps were followed by an outward tail current that reversed at -92.0 ± 1.3 mV. Outward tail currents were larger at -50 mV than at the resting potential, and inward tail currents decayed more rapidly and to a larger extent during larger hyperpolarizing steps. In the presence of Ba²⁺, Cs⁺ (4 mM) had little effect on the steady-state current but markedly reduced or abolished undershoot, Ba-DD, and time-dependent tail currents at potentials both positive and negative to the resting potential. Cs⁺ had a smaller effect on the steady-state current-voltage (I-V) relation in the presence than in the absence of Ba²⁺, as part of the I_kl channels were already blocked by Ba²⁺ and the time-dependent changes induced by Ba²⁺ were not present. Both Ba²⁺ and Cs⁺ had little blocking effect on the steady-state current positive to the negative slope region of the I-V relation. Conclusion: In ventricular myocytes, Cs⁺ abolishes the Ba²⁺-induced pacemaker current by blocking the time-dependent change in K⁺ conductance, not by blocking I_f. Because Cs⁺ can also block a decaying K⁺ pacemaker current, the abolition of a pacemaker current by Cs⁺ in other cardiac tissues cannot be taken as unequivocal proof that the blocked current is I_f     

Electrophysiological Characteristics of Enteric Neurons Isolated from the Immortomouse

Background

Recently, two enteric neuronal cell lines, one fetal and the other post-natal (IM-PEN), have been developed from the H-2K^b-tsA58 transgenic mouse (immortomouse). However, their electrophysiological properties are not known. The goal of this study was to determine the electrical excitability and ionic conductance of the immortalized postnatal enteric neuronal (IM-PEN) cell line.

Methods

Whole cell patch clamp studies, immunohistochemistry and RT-PCR were performed on differentiated IM-PEN cells following propagation at 33 °C and differentiation at 37 °C.

Results

Differentiated IM-PEN cells stained positively for the neuron specific markers βIII-tubulin and PGP9.5. The mRNA for several ion channels expressed in enteric neurons were detected by RT-PCR. In current clamp, the resting membrane potential was ?24.6 ± 2.1 mV (n = 6) for IM-FEN and ?29.8 ± 0.9 mV (n = 30) for IM-PEN. Current injections from Vh ?80 mV resulted in passive responses but not action potentials. Depolarizing pulses in the whole cell voltage clamp configuration from Vh ?80 mV elicited small nifedipine-sensitive inward currents. Additionally, outward currents with slow deactivating tail currents were blocked by niflumic acid and low chloride solution. A volume-regulated anion current was elicited by hypo-osmotic solution and inhibited by 10 μM DCPIB. Growth with rabbit gastrointestinal smooth muscle did not yield significant differences in the active properties of the IM-PEN cell line. Transient expression of L-type Ca²⁺ channels produced large inward currents demonstrating a working mechanism for protein folding and transport.

Conclusion

The electrophysiological characteristics of IM-PEN cells suggest that chloride channels in IM-PEN cells play an important role in their resting state, and membrane trafficking of some of the ion channels may preclude their electrical excitability.     

Molecular basis of plant-specific acid activation of K+ uptake channels

During stomatal opening potassium uptake into guard cells and K⁺ channel activation is tightly coupled to proton extrusion. The pH sensor of the K⁺ uptake channel in these motor cells has, however, not yet been identified. Electrophysiological investigations on the voltage-gated, inward rectifying K⁺ channel in guard cell protoplasts from Solanum tuberosum (KST1), and the kst1 gene product expressed in Xenopus oocytes revealed that pH dependence is an intrinsic property of the channel protein. Whereas extracellular acidification resulted in a shift of the voltage-dependence toward less negative voltages, the single-channel conductance was pH-insensitive. Mutational analysis allowed us to relate this acid activation to both extracellular histidines in KST1. One histidine is located within the linker between the transmembrane helices S3 and S4 (H160), and the other within the putative pore-forming region P between S5 and S6 (H271). When both histidines were substituted by alanines the double mutant completely lost its pH sensitivity. Among the single mutants, replacement of the pore histidine, which is highly conserved in plant K⁺ channels, increased or even inverted the pH sensitivity of KST1. From our molecular and biophysical analyses we conclude that both extracellular sites are part of the pH sensor in plant K⁺ uptake channels.     

Remodeling of outward K+ currents in pressure-overload heart failure

Objectives: Outward K⁺ currents are critical determinants of action potential repolarization and the site of action of a number of electrophysiologically active drugs. Further, expression and processing of the channels underlying these currents is altered in heart disease. Here, we investigated the native transmural gradient of outward K⁺ currents in murine left ventricle (LV) and delineated disease‐related remodeling of these currents in heart failure (HF). Methods: Pressure‐overload heart failure was induced in mice by thoracic aortic constriction. Outward K⁺ currents were recorded using the whole‐cell patch clamp technique in acutely dissociated ventricular myocytes. Results: Unambiguous gradients of outward K⁺ current density and Kv4.2 protein abundance were observed across the wall of the LV, with significantly larger current density and protein levels in subepicardial (SEP) myocytes, compared with subendocardial (SEN) myocytes. Voltage dependences of current activation and inactivation were similar in SEP and SEN myocytes. In failing LV, however, outward K⁺ current density was significantly decreased in SEP but not in SEN cells leading to elimination of the native transmural gradient. In failing LV, the voltage dependences of K⁺ current activation and inactivation were not altered. However, current inactivation (decay) was significantly accelerated and recovery from inactivation was significantly slowed. Consistent with this, Western blot analysis revealed a decrease in KChIP2 protein abundance in failing LV. Conclusions: This is the first report of HF‐related remodeling of outward K⁺ currents in murine LV. Similar to humans, disease‐related remodeling occurs differentially across the murine ventricular wall, leading to loss of the native gradient of repolarization. Together with slowed recovery from inactivation, these alterations likely promote abnormal impulse conduction, a major proarrhythmic mechanism.     

Mitogen-activated protein kinase is involved in the progesterone-mediated induction of baboon glycodelin

Diabetes mellitus is complicated with the development of cardiac contractile dysfunction and electrical instability, which contributes to high morbidity and mortality in diabetic patients. This study examined the possible roles of enhanced endothelin-1 (ET-1) on diabetes-induced alterations in ventricular myocyte electrophysiology. Type 1 diabetic rats were induced by single dose injection of streptozotocin (STZ) and treated with or without ET-1 receptor antagonist bosentan for 8 wk before myocyte isolation. Action potential, outward K⁺ currents, and inward Ca²⁺ currents in ventricular myocytes were recorded using whole-cell patch clamp technique. STZ-injected rats exhibited hyperglycemia, reduced body weight gain, and elevated plasma ET-1 concentration, indicative of diabetes induction. Ventricular myocytes isolated from diabetic rats exhibited prolonged action potential and reduced all three types of outward K⁺ currents. Resting membrane potential, height of action potential, and L-type Ca²⁺ current were not altered in diabetic myocytes. In vivo chronic treatment of diabetic rats with bosentan significantly augmented K⁺ currents and reversed action potential prolongation in ventricular myocytes. On the other hand, bosentan treatment had no detectable effect on the electrophysiological properties in control myocytes. In addition, bosentan had no effect on L-type Ca²⁺ currents in both control and diabetic myocytes. Our data suggest that altered electrophysiological properties in ventricular myocytes were largely resulted from augmented ET-1 system in diabetic animals.     

Potassium channels in cardiac cells

K⁺ channels in heart can be subdivided into two groups, voltage-operated and ligand-operated channels. Only the voltage-operated channels—i_K, i_to, and i_K1—and one ligand-operated channel—i_K(ACh)—are activated under physiological conditions; the i_K1 only carries large currents at the resting potential. The delayed K⁺ current, i_K, and the transient outward current, i_to, are important for the plateau and repolarization phase of the action potential, and thus affect the refractory period in atrial and ventricular cells and also the frequency in the sinoatrial node. A high density of i_to and i_Ca is responsible for the spike-dome appearance of the plateau phase in adult atrial cells, epicardial myocytes, and Purkinje cells stimulated by catecholamines. The action-potential duration in these cells is more sensitive to K_e ⁺, frequency, and drugs. The i_K(ACh) can also be activated by adenosine and somatostatin. Its density is high in nodal and atrial tissue. Under pathological conditions, K⁺ channels dependent on ATP, Na_i ⁺, and fatty acids are activated and can carry large currents at depolarized levels. Local changes in the concentration of ATP or Na close to the membrane are probably important for the activation process. The study of pharmacological modulation of K⁺ currents should include frequency effects for the voltage-activated channels.     

Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells

myo-Inositol hexakisphosphate (InsP6) is the most abundant inositol phosphate in cells, yet it remains the most enigmatic of this class of signaling molecule. InsP6 plays a role in the processes by which the drought stress hormone abscisic acid (ABA) induces stomatal closure, conserving water and ensuring plant survival. Previous work has shown that InsP6 levels in guard cells are elevated in response to ABA, and InsP6 inactivates the plasma membrane inward K+ conductance (IK,in) in a cytosolic calcium-dependent manner. The use of laser-scanning confocal microscopy in dye-loaded patch-clamped guard cell protoplasts shows that release of InsP6 from a caged precursor mobilizes calcium. Measurement of calcium (barium) currents ICa in patch-clamped protoplasts in whole cell mode shows that InsP6 has no effect on the calcium-permeable channels in the plasma membrane activated by ABA. The InsP6-mediated inhibition of IK,in can also be observed in the absence of external calcium. Thus the InsP6-induced increase in cytoplasmic calcium does not result from calcium influx but must arise from InsP6-triggered release of calcium from endomembrane stores. Measurements of vacuolar currents in patch-clamped isolated vacuoles in whole-vacuole mode showed that InsP6 activates both the fast and slow conductances of the guard cell vacuole. These data define InsP6 as an endomembrane-acting calcium-release signal in guard cells; the vacuole may contribute to InsP6-triggered Ca2+ release, but other endomembranes may also be involved.     

Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange between plants and the atmosphere in response to light, CO₂, and the plant hormone abscisic acid. Light-induced stomatal opening is mediated by at least three key components: the blue light receptor phototropin (phot1 and phot2), plasma membrane H⁺-ATPase, and plasma membrane inward-rectifying K⁺ channels. Very few attempts have been made to enhance stomatal opening with the goal of increasing photosynthesis and plant growth, even though stomatal resistance is thought to be the major limiting factor for CO₂ uptake by plants. Here, we show that transgenic Arabidopsis plants overexpressing H⁺-ATPase using the strong guard cell promoter GC1 showed enhanced light-induced stomatal opening, photosynthesis, and plant growth. The transgenic plants produced larger and increased numbers of rosette leaves, with ∼42–63% greater fresh and dry weights than the wild type in the first 25 d of growth. The dry weights of total flowering stems of 45-d-old transgenic plants, including seeds, siliques, and flowers, were ∼36–41% greater than those of the wild type. In addition, stomata in the transgenic plants closed normally in response to darkness and abscisic acid. In contrast, the overexpression of phototropin or inward-rectifying K⁺ channels in guard cells had no effect on these phenotypes. These results demonstrate that stomatal aperture is a limiting factor in photosynthesis and plant growth, and that manipulation of stomatal opening by overexpressing H⁺-ATPase in guard cells is useful for the promotion of plant growth.In the present era of global climate changes and the threat of food insufficiency, finding ways to improve the uptake of CO₂ by terrestrial plants is an increasingly important problem. Stomata, key organs in the uptake of CO₂, are microscopic pores surrounded by two specialized cells in the epidermis (named guard cells) and are mainly found on the leaf surface in terrestrial plants. Because the leaf surface is nearly impermeable to air and water, stomata provide the major pathway for the diffusion of CO₂, O₂, and water vapor between the ambient atmosphere and the interior of the leaf. This facilitation of gas exchange by stomatal opening is one of the most essential processes in plant photosynthesis and transpiration (1, 2). A recent study indicated that stomatal transpiration limited photosynthesis in rice (3). Therefore, increased stomatal opening/transpiration is expected to promote photosynthesis and thereby increase plant growth. Condon et al. (4) examined diverse wheat genotypes and showed that increased stomatal conductance, especially abaxial stomatal conductance, may have a positive effect on crop biomass. However, to our knowledge, no previous studies have determined stomatal effects on plant growth by manipulating stomatal aperture via gene regulation in guard cells, perhaps because of the difficulty in balancing the counteracting effects of taking up CO₂ while losing water vapor through the stomata (5).Light is one of the principal factors that stimulates stomatal opening, and various mechanisms underlie stomatal opening in response to different light wavelengths (6–8). Red light is thought to induce stomatal opening via photosynthesis in the mesophyll and guard cell chloroplasts, as well as the reduction of the intercellular CO₂ concentration (Ci) (5, 9, 10). However, the detailed mechanisms of stomatal responses to red light are under debate (11, 12). In contrast, blue light acts as a signal and exerts the most pronounced effect on stomatal opening. The blue light receptors phototropins (phot1 and phot2) activate plasma membrane H⁺-ATPase through the phosphorylation of the penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated threonine (13–15). Blue light-activated H⁺-ATPase induces hyperpolarization of the plasma membrane, which allows K⁺ uptake through inward-rectifying K⁺ (K⁺_in) channels (16–21). Accumulation of K⁺ induces the swelling of guard cells and stomatal opening. Thus, these three components (phototropins, plasma membrane H⁺-ATPase, and K⁺_in channels) have important roles in blue light-induced stomatal opening. In addition to these components, FLOWERING LOCUS T (FT) is suggested to be a positive regulator for stomatal opening via its effect on the activation status of the plasma membrane H⁺-ATPase (22).In this study, we produced transgenic plants expressing key components active in stomatal opening under the control of the strong guard cell promoter GC1 to promote stomatal opening in Arabidopsis thaliana (23). We showed that transgenic Arabidopsis plants overexpressing H⁺-ATPase in guard cells exhibited enhanced light-induced stomatal opening, photosynthesis, and plant growth, and that stomatal aperture is a limiting factor in photosynthesis and plant growth.     

Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.     

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K⁺ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K⁺ currents, an inactivating outward-rectifying K⁺ current was characterized (plant A-type current: I_AP). I_AP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. I_AP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward I_AP was mainly carried by K⁺ ions. In contrast to the sustained outward-rectifying K⁺ currents, cytosolic alkaline pH was found to inhibit I_AP and extracellular K⁺ was required for I_AP activity. Furthermore, increasing cytosolic free Ca²⁺ in the physiological range strongly inhibited I_AP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K⁺ current in a higher plant cell. Regulation of I_AP by diverse factors including membrane potential, cytosolic Ca²⁺ and pH, and extracellular K⁺ and Ca²⁺ implies that the inactivating I_AP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.     

Regulation of the human ether-a-gogo related gene (HERG) K+ channels by reactive oxygen species

Human ether-a-gogo related gene (HERG) K⁺ channels are key elements in the control of cell excitability in both the cardiovascular and the central nervous systems. For this reason, the possible modulation by reactive oxygen species (ROS) of HERG and other cloned K⁺ channels expressed in Xenopus oocytes has been explored in the present study. Exposure of Xenopus oocytes to an extracellular solution containing FeSO₄ (25–100 μM) and ascorbic acid (50–200 μM) (Fe/Asc) increased both malondialdehyde content and 2′,7′-dichlorofluorescin fluorescence, two indexes of ROS production. Oocyte perfusion with Fe/Asc caused a 50% increase of the outward K⁺ currents carried by HERG channels, whereas inward currents were not modified. This ROS-induced increase in HERG outward K⁺ currents was due to a depolarizing shift of the voltage-dependence of channel inactivation, with no change in channel activation. No effect of Fe/Asc was observed on the expressed K⁺ currents carried by other K⁺ channels such as bEAG, rDRK1, and mIRK1. Fe/Asc-induced stimulation of HERG outward currents was completely prevented by perfusion of the oocytes with a ROS scavenger mixture (containing 1,000 units/ml catalase, 200 ng/ml superoxide dismutase, and 2 mM mannitol). Furthermore, the scavenger mixture also was able to reduce HERG outward currents in resting conditions by 30%, an effect mimicked by catalase alone. In conclusion, the present results seem to suggest that changes in ROS production can specifically influence K⁺ currents carried by the HERG channels.     

Characterization of a G-protein-regulated outward K+ current in mesophyll cells of vicia faba L.

Whole-cell voltage-dependent currents in isolated mesophyll protoplasts of Vicia faba were investigated by patch-clamp techniques. With 104 mM K+ in the cytosol and 13 mM K+ in the external solution, depolarization of the plasma membrane from -47 mV to potentials between -15 and +85 mV activated a voltage- and time-dependent outward current (Iout). The average magnitude of Iout at +85 mV was 28.5 +/- 3.3 pA.pF-1. No inward voltage-dependent current was observed upon hyperpolarization of the plasma membrane from -55 mV to potentials as negative as -175 mV. Time-activated outward current was blocked by Ba2+ (1 mM BaCl2) and was not observed when K+ was eliminated from the external and internal solutions, indicating that this outward current was carried primarily by K+ ions. The voltage dependency of outward K+ current revealed a possible mechanism for K+ efflux from mesophyll cells. A GDP analogue guanosine 5'-[beta-thio]diphosphate (500 microM) significantly enhanced outward K+ current. The outward K+ current was inhibited by the GTP analogue guanosine 5'-[gamma-thio]triphosphate (500 microM) and by an increase in cytoplasmic free Ca2+ concentrations. Cholera toxin, which ADP-ribosylates guanine nucleotide-binding regulatory proteins, also inhibited outward K+ current. These findings illustrate the presence in mesophyll cells of outward-rectifying K+ channels that are regulated by GTP-binding proteins and calcium.