Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.     

Rundown of the hyperpolarization-activated KAT1 channel involves slowing of the opening transitions regulated by phosphorylation.

Disappearance of the functional activity or rundown of ion channels upon patch excision in many cells involves a decrease in the number of channels available to open. A variety of cellular and biophysical mechanisms have been shown to be involved in the rundown of different ion channels. We examined the rundown process of the plant hyperpolarization-activated KAT1 K+ channel expressed in Xenopus oocytes. The decrease in the KAT1 channel activity on patch excision was accompanied by progressive slowing of the activation time course, and it was caused by a shift in the voltage dependence of the channel without any change in the single-channel amplitude. The single-channel analysis showed that patch excision alters only the transitions leading up to the burst states of the channel. Patch cramming or concurrent application of protein kinase A (PKA) and ATP restored the channel activity. In contrast, nonspecific alkaline phosphatase (ALP) accelerated the rundown time course. Low internal pH, which inhibits ALP activity, slowed the KAT1 rundown time course. The results show that the opening transitions of the KAT1 channel are enhanced not only by hyperpolarization but also by PKA-mediated phosphorylation.     

Guard cell inward K+ channel activity in arabidopsis involves expression of the twin channel subunits KAT1 and KAT2

Stomatal opening, which controls gas exchanges between plants and the atmosphere, results from an increase in turgor of the two guard cells that surround the pore of the stoma. KAT1 was the only inward K(+) channel shown to be expressed in Arabidopsis guard cells, where it was proposed to mediate a K(+) influx that enables stomatal opening. We report that another Arabidopsis K(+) channel, KAT2, is expressed in guard cells. More than KAT1, KAT2 displays functional features resembling those of native inward K(+) channels in guard cells. Coexpression in Xenopus oocytes and two-hybrid experiments indicated that KAT1 and KAT2 can form heteromultimeric channels. The data indicate that KAT2 plays a crucial role in the stomatal opening machinery.     

Regulation by external K+ in a maize inward shaker channel targets transport activity in the high concentration range

An inward Shaker K(+) channel identified in Zea mays (maize), ZmK2.1, displays strong regulation by external K(+) when expressed in Xenopus laevis (African clawed frog) oocytes or COS cells. ZmK2.1 is specifically activated by K(+) with an apparent K(m) close to 15 mM independent of the membrane hyperpolarization level. In the absence of K(+), ZmK2.1 appears to enter a nonconducting state. Thus, whatever the membrane potential, this maize channel cannot mediate K(+) influx in the submillimolar concentration range, unlike its relatives in Arabidopsis thaliana. Its expression is restricted to the shoots, the strongest signal (RT-PCR) being associated with vascular/bundle sheath strands. Based on sequence and gene structure, the closest relatives of ZmK2.1 in Arabidopsis are K(+) Arabidopsis Transporter 1 (KAT1) (expressed in guard cells) and KAT2 (expressed in guard cells and leaf phloem). Patch-clamp analyses of guard cell protoplasts reveal a higher functional diversity of K(+) channels in maize than in Arabidopsis. Channels endowed with regulation by external K(+) similar to that of ZmK2.1 (channel activity regulated by external K(+) with a K(m) close to 15 mM, regulation independent of external Ca(2+)) constitute a major component of the maize guard cell inward K(+) channel population. The presence of such channels in maize might reflect physiological traits of C4 and/or monocotyledonous plants.     

Co-expression of calcium-dependent protein kinase with the inward rectified guard cell K+ channel KAT1 alters current parameters in Xenopus laevis oocytes

Increased guard cell cytosolic [Ca2+] is known to be involved in signal transduction pathways leading to stomatal closure, and inhibit the inward rectifying guard cell K+ channel KAT1. Guard cell calcium-dependent protein kinase (CDPK) has been shown to phosphorylate KAT1; such phosphorylation is known to modulate other K+ channels involved in signal transduction cascades. The work reported here focused on demonstrating CDPK-dependent inhibition of KAT1 currents. A cDNA encoding soybean CDPK was generated and it's translation product was shown to be functional; demonstrating Ca2+-dependent autophosphorylation and phosphorylation of a target protein. Ion currents were monitored using voltage clamp techniques upon expression of KAT1 in Xenopus laevis oocytes. Coexpression of recombinant CDPK with KAT1 in oocytes altered the kinetics and magnitude of induced K+ currents; at a given hyperpolarizing command voltage, the magnitude of KAT1 currents was reduced and the half-time for channel activation was increased. This finding supports a model of Ca2+-dependent ABA inhibition of inward K+ currents in guard cells as being mediated by CDPK phosphorylation of KAT1.     

Inward potassium channel in guard cells as a target for polyamine regulation of stomatal movements

A number of studies show that environmental stress conditions such as drought, high salt, and air pollutants increase polyamine levels in plant cells. However, little is understood about the physiological function of elevated polyamine levels. We report here that polyamines regulate the voltage-dependent inward K(+) channel in the plasma membrane of guard cells and modulate stomatal aperture, a plant "sensor" to environmental changes. All natural polyamines, including spermidine, spermine, cadaverine, and putrescine, strongly inhibited opening and induced closure of stomata. Whole-cell patch-clamp analysis showed that intracellular application of polyamines inhibited the inward K(+) current across the plasma membrane of guard cells. Single-channel recording analysis indicated that polyamine regulation of the K(+) channel requires unknown cytoplasmic factors. In an effort to identify the target channel at the molecular level, we found that spermidine inhibited the inward K(+) current carried by KAT1 channel that was functionally expressed in a plant cell model. These findings suggest that polyamines target KAT1-like inward K(+) channels in guard cells and modulate stomatal movements, providing a link between stress conditions, polyamine levels, and stomatal regulation.     

Expression of a Cs(+)-resistant guard cell K+ channel confers Cs(+)-resistant, light-induced stomatal opening in transgenic arabidopsis.

Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+in channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+in channel, KAT1, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+(in) currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KAT1 functions as a plasma membrane K+ channel in vivo and that K+in channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+in channels in guard cells indicate that components in addition to KAT1 may contribute to the formation of K+in channels in vivo.     

Inhibitory effects of methylglyoxal on light-induced stomatal opening and inward K+ channel activity in Arabidopsis

Methylglyoxal (MG) is a reactive aldehyde derived by glycolysis. In Arabidopsis, MG inhibited light-induced stomatal opening in a dose-dependent manner. It significantly inhibited both inward-rectifying potassium (K(in)) channels in guard-cell protoplasts and an Arabidopsis K(in) channel, KAT1, heterologously expressed in Xenopus oocytes. Thus it appears that MG inhibition of stomatal opening involves MG inhibition of K(+) influx into guard cells.     

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 μM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 μM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

A type of voltage-dependent Ca2+ channel on Vicia faba guard cell plasma membrane outwardly permeates K+

The fine regulation of stomatal aperture is important for both plant photosynthesis and transpiration, while stomatal closing is an essential plant response to biotic and abiotic stresses such as drought, salinity, wounding, and pathogens. Quick stomatal closing is primarily due to rapid solute loss. Cytosolic free calcium ([Ca(2+)](cyt)) is a ubiquitous second messenger, and its elevation or oscillation plays important roles in stomatal movements, which can be triggered by the opening of Ca(2+)-permeable channels on the plasma membrane. For Ca(2+)-permeable channel recordings, Ba(2+) is preferred as a charge-carrying ion because it has higher permeability to Ca(2+) channels and blocks K(+) channel activities to facilitate current recordings; however, it prevents visualization of Ca(2+) channels' K(+) permeability. Here, we employed Ca(2+) instead of Ba(2+) in recording Ca(2+)-permeable channels on Vicia faba guard cell plasma membrane to mimic physiological solute conditions inside guard cells more accurately. Inward Ca(2+) currents could be recorded at the single-channel level, and these currents could be inhibited by micromolar Gd(3+), but their reversal potential is far away from the theoretical equilibrium potential for Ca(2+). Further experiments showed that the discrepancy of the reversal potential of the recorded Ca(2+) currents is influenced by cytosolic K(+). This suggests that voltage-dependent Ca(2+) channels also mediate K(+) efflux at depolarization voltages. In addition, a new kind of high-conductance channels with fivefold to normal Ca(2+) channel and 18-fold to normal outward K(+) conductance was found. Our data presented here suggest that plants have their own saving strategies in their rapid response to stress stimuli, and multiple kinds of hyperpolarization-activated Ca(2+)-permeable channels coexist on plasma membranes.     

Voltage-activation of high-conductance K+ channel in the insulin-secreting cell line RINm5F is dependent on local extracellular Ca2+ concentration

Patch-clamp single-channel current recording experiments have been carried out on intact insulin-secreting RINm5F cells. Voltage-activation of high-conductance K+ channels were studied by selectively depolarizing the electrically isolated patch membrane under conditions with normal Ca2+ concentration in the bath solution but with or without Ca2+ in the patch pipette solution. When Ca2+ was present in the pipette, 40 mV to 120 mV depolarizing pulses (100 ms) from the normal resting potential (-70 mV) regularly evoked tetraethylammonium-sensitive large outward single-channel currents and the average open state probability during the pulses varied from about 0.015 (40 mV pulses) to 0.1 (120 mV pulses). In the absence of Ca2+ in the pipette solution the same protocol resulted in fewer and shorter K+ channel openings and the open-state probability varied from about 0.0015 (40 mV pulses) to about 0.03 (120 mV pulses). It is concluded that Ca2+ entering voltage-gated channels raises [Ca2+]i locally and thereby markedly enhances the open-state probability of tetraethylammonium-sensitive voltage-gated high-conductance K+ channels.     

Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin

Bovine adrenocortical cells express bTREK-1 K(+) (bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca(2+) and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca(2+)-dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca(2+) ionophore ionomycin (1 μM) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5-10 μM) produced a sustained inhibition of bTREK-1 that was reversible upon washing, even in the absence of hydrolyzable [ATP](i). BAPTA was much more effective than EGTA at suppressing bTREK-1 inhibition by ANG II. When intracellular Ca(2+) concentration ([Ca(2+)](i)) was buffered to 20 nM with either 11 mM BAPTA or EGTA, ANG II (10 nM) inhibited bTREK-1 by 12.0 ± 4.5% (n=11) and 59.3 ± 8.4% (n=4), respectively. Inclusion of the water-soluble phosphatidylinositol 4,5-bisphosphate (PIP(2)) analog DiC(8)PI(4,5)P(2) in the pipette failed to increase bTREK-1 expression or reduce its inhibition by ANG II. The open probability (P(o)) of unitary bTREK-1 channels recorded from inside-out patches was reduced by Ca(2+) (10-35 μM) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K(+) channels by a Ca(2+)-dependent mechanism that does not require the depletion of membrane-associated PIP(2). They further indicate that the Ca(2+) source is located in close proximity within a "Ca(2+) nanodomain" of bTREK-1 channels, where [Ca(2+)](i) may reach concentrations of >10 μM. bTREK-1 is the first two-pore K(+) channel shown to be inhibited by Ca(2+) through activation of a G protein-coupled receptor.     

The pore of plant K(+) channels is involved in voltage and pH sensing: domain-swapping between different K(+) channel alpha-subunits

Plant K(+) uptake channel types differ with respect to their voltage, Ca(2)+, and pH dependence. Here, we constructed recombinant chimeric channels between KST1, a member of the inward-rectifying, acid-activated KAT1 family, and AKT3, a member of the weakly voltage-dependent, proton-blocked AKT2/3 family. The homologous pore regions of AKT3 (amino acids 216 to 287) and KST1 (amino acids 217 to 289) have been exchanged to generate the two chimeric channels AKT3/(p)KST1 and KST1/(p)AKT3. In contrast to AKT3 wild-type channels, AKT3/(p)KST1 revealed a strong inward rectification reminiscent of that of KST1. Correspondingly, the substitution of the KST1 by the AKT3 pore led to less pronounced rectification properties of KST1/(p)AKT3 compared with wild-type KST1. Besides the voltage dependence, the interaction between the chimera and extracellular H(+) and Ca(2)+ resembled the properties of the inserted rather than the respective wild-type pore. Whereas AKT3/(p)KST1 was acid activated and Ca(2)+ insensitive, extracellular protons and Ca(2)+ inhibited KST1/(p)AKT3. The regulation of the chimeric channels by cytoplasmic protons followed the respective wild-type backbone of the chimeric channels, indicating that the intracellular pH sensor is located outside the P domain. We thus conclude that essential elements for external pH and Ca(2)+ regulation and for the rectification of voltage-dependent K(+) uptake channels are located within the channel pore.     

Evidences for Regulation of the Inward K+ channels by CDPK in Vicia faba Guard Cells

Regulation of the inward K+ -channels in the guard cell plasma membranes plays impotant roles in regulation of stomatal movement in responses to exogenous and endogenous signals. It is well-known that elevation of cytosolic Ca2+ in guard cells inactivates these inward K + channels, and consequently inhibits stomatal opening or induces stomatal closing, yet the downstream molecular mechanism for the Ca2 + -mediated inhibition of the inward K+ channels remains unknown. The calmodulin-like domain protein kinases (CDPKs) have been identified as an unique group of protein kinases in higher plant cells. As a downstream regulator, CDPK may play roles in mediating Ca2+ regulation on the inward K+ -channels in stomatal guard cells. The authors have applied the patchclamp technique to investigate if CDPK be involved in the regulation of the inward K+ -channels in Vicia faba guard cells by cytosolic Ca2+ . The presence of the 1.5 μmol/L intracellular Ca2 + result-ed in inhibition of the inward K+ channel activity by 60%, while the addition of purified CDPK from the cytoplasmic side resulted in greater inhibition than Ca2+ alone. Histone Ⅲ-S and protamine, which is the substrate and substrate competitive inhibitor of CDPKs respectively, completely reversed the Ca2+ -induced inhibition of the inward K+ channel activities. These results are the first reported evidences for that CDPKs are involved in the Ca2+ -mediated inward K+ -channel regulation in guard cells.     

Diacidic motif is required for efficient transport of the K+ channel KAT1 to the plasma membrane

For a number of mammalian ion channels, trafficking to the plasma membrane was found to be controlled by intrinsic sequence motifs. Among these sequences are diacidic motifs that function as endoplasmic reticulum (ER) export signals. So far it is unclear if similar motifs also exist in plant ion channels. In this study we analyzed the function of four diacidic DXE/DXD motifs of the plant K(+) channel KAT1. Mutation of the first diacidic DXE motif resulted in a strong reduction of the KAT1 conductance in both guard cell protoplasts and HEK293 cells (human embryonic kidney cells). Confocal fluorescence microscopy of guard cells expressing the mutated KAT1 fused to green fluorescent protein revealed localization of the mutated channel only in intracellular structures around the nucleus. These structures could be identified as part of the ER via coexpression of KAT1 fused to yellow fluorescent protein with an ER-retained protein (HDEL) fused to cyan fluorescent protein. Block of vesicle formation from the ER by overexpression of the small GTP-binding protein Sar1 fixed in its GDP-bound form led to retention of wild-type KAT1 in similar parts of the ER. Mutation of the three other diacidic motifs had no effect. Together, the results demonstrate that one diacidic motif of KAT1 is essential for ER export of the functional channel in both guard cell protoplasts and HEK293 cells. This suggests that trafficking of plant plasma membrane ion channels is controlled via a conserved mechanism.     

Outer pore residues control the H(+) and K(+) sensitivity of the Arabidopsis potassium channel AKT3

The Arabidopsis phloem channel AKT3 is the founder of a subfamily of shaker-like plant potassium channels characterized by weak rectification, Ca(2+) block, proton inhibition, and, as shown in this study, K(+) sensitivity. In contrast to inward-rectifying, acid-activated K(+) channels of the KAT1 family, extracellular acidification decreases AKT3 currents at the macroscopic and single-channel levels. Here, we show that two distinct sites within the outer mouth of the K(+)-conducting pore provide the molecular basis for the pH sensitivity of this phloem channel. After generation of mutant channels and functional expression in Xenopus oocytes, we identified the His residue His-228, which is proximal to the K(+) selectivity filter (GYGD) and the distal Ser residue Ser-271, to be involved in proton susceptibility. Mutations of these sites, H228D and S271E, drastically reduced the H(+) and K(+) sensitivity of AKT3. Although in K(+)-free bath solutions outward K(+) currents were abolished completely in wild-type AKT3, S271E as well as the AKT3-HDSE double mutant still mediated K(+) efflux. We conclude that the pH- and K(+)-dependent properties of the AKT3 channel involve residues in the outer mouth of the pore. Both properties, H(+) and K(+) sensitivity, allow the fine-tuning of the phloem channel and thus seem to represent important elements in the control of membrane potential and sugar loading.     

Blue light inhibits guard cell plasma membrane anion channels in a phototropin-dependent manner

Guard cells respond to light through two independent signalling pathways. The first pathway is initiated by photosynthetically active radiation and has been associated with changes in the intercellular CO(2) concentration, leading to inhibition of plasma membrane anion channels. The second response is blue-light-specific and so far has been restricted to the activation of plasma membrane H(+)-ATPases. In a search for interactions of both signalling pathways, guard cells of Vicia faba and Arabidopsis thaliana were studied in intact plants. Vicia faba guard cells recorded in CO(2)-free air responded to blue light with a transient outward plasma membrane current that had an average peak value of 17 pA. In line with previous reports, changes in the current-voltage relation of the plasma membrane indicate that this outward current is based on the activation of H(+)-ATPases. However, when V. faba guard cells were blue-light-stimulated in air with 700 microl l(-1) CO(2), the outward current increased to 56 pA. The increase in current was linked to inhibition of S-type anion channels. Blue light also inhibited plasma membrane anion channels in A. thaliana guard cells, but not in the phot1 phot2 double mutant. These results show that blue light inhibits plasma membrane anion channels through a pathway involving phototropins, in addition to the stimulation of guard cell plasma membrane H(+)-ATPases.     

ABA regulation of K(+)-permeable channels in maize subsidiary cells

An antiparallel-directed potassium transport between subsidiary cells and guard cells which form the graminean stomatal complex has been proposed to drive stomatal movements in maize. To gain insights into the coordinated shuttling of K(+) ions between these cell types during stomatal closure, the effect of ABA on the time-dependent K(+) uptake and K(+) release channels as well as on the instantaneously activating non-selective cation channels (MgC) was examined in subsidiary cells. Patch-clamp studies revealed that ABA did not affect the MgC channels but differentially regulated the time-dependent K(+) channels. ABA caused a pronounced rise in time-dependent outward-rectifying K(+) currents (K(out)) at alkaline pH and decreased inward-rectifying K(+) currents (K(in)) in a Ca(2+)-dependent manner. Our results show that the ABA-induced changes in time-dependent K(in) and K(out) currents from subsidiary cells are very similar to those previously described for guard cells. Thus, the direction of K(+) transport in subsidiary cells and guard cells during ABA-induced closure does not seem to be grounded solely on the cell type-specific ABA regulation of K(+) channels.     

Laser microsurgery of higher plant cell walls permits patch-clamp access.

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.