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J. Biol. Chem., Vol. 279, Issue 34, 35306-35312, August 20, 2004

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Cyclic Adenosine Monophosphate Regulates Calcium Channels in the Plasma Membrane of Arabidopsis Leaf Guard and Mesophyll Cells^*

Fouad Lemtiri-Chlieh and Gerald A. Berkowitz

From the Agricultural Biotechnology Laboratory, Department of Plant Science, University of Connecticut, Storrs, Connecticut 06269-4067

Received for publication, January 12, 2004 , and in revised form, June 4, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The effect of cAMP on Ca²⁺-permeable channels from Arabidopsis thaliana leaf guard cell and mesophyll cell protoplasts was studied using the patch clamp technique. In the whole cell configuration, dibutyryl cAMP was found to increase a hyperpolarization-activated Ba²⁺ conductance (I_Ba). The increase of I_Ba was blocked by the addition of GdCl₃. In excised outside-out patches, the addition of dibutyryl cAMP consistently activated a channel with particularly fast gating kinetics. Current/voltage analyses indicated a single channel conductance of 13 picosiemens. In patches where we measured some channel activity prior to cAMP application, the data suggest that cAMP enhances channel activity without affecting the single channel conductance. The cAMP activation of these channels was reversible upon washout. The results obtained with excised patches indicate that the cAMP-activated I_Ba seen in the whole cell configuration could be explained by a direct effect of cAMP on the Ca²⁺ channel itself or a close entity to the channel. This work represents the first demonstration using patch clamp analysis of the presence in plant cell membranes of an ion channel directly activated by cAMP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Arabidopsis thaliana genome encodes 20 putative members of the cyclic nucleotide-gated channel (CNGC)¹ family (1–4). Using electrophysiological techniques in combination with heterologous expression systems, prior work from this laboratory (5, 6) demonstrated that the translation products of cloned plant CNGC cDNAs, like their animal counterparts found in rod and olfactory cells, are activated by the binding of cyclic nucleotide monophosphate (cNMP; cAMP and/or cGMP), a defining attribute of this channel family. Animal CNGCs (six genes are present in humans) are nonselective cation (i.e. conducting Ca²⁺, Na⁺, and K⁺) channels whose opening produces membrane depolarization and/or cytosolic Ca²⁺ rise, important components of signal transduction pathways in the animal cell (7).

CNGC proteins are expressed in a number of plant tissues (4); however, the presence and role of the CNGC-activating ligand cAMP in plants is still controversial. Early reviews (8) cite work that questioned the presence in plants of cAMP at physiologically relevant levels. However, more recent studies using a cyclic nucleotide fluorosensor (9) injected into pollen tubes have reported cAMP levels as high as 150 nM. Use of tandem mass spectrometry (10) for cyclic nucleotide quantification has confirmed the presence of cAMP (as well as cGMP) in plant cells. Furthermore, a partial clone encoding a protein with homology to fungal adenylate cyclase has been identified in corn pollen; importantly, its translation product was shown to have adenylate cyclase activity (9). Recent work has identified adenylate cyclase activity in (tobacco) chloroplasts (11), suggesting its ubiquitous presence in photosynthetic tissue. cAMP has been associated with cation uptake and associated cell growth/expansion in pollen tubes (9, 12), as well as cell cycle progression and hormone signaling in plants (see Refs. 4 and 12 for reviews). cGMP may be involved in ion uptake into guard cells and stomatal opening, although the effect of cGMP on ion transport is probably indirect and mediated through a signal cascade (13, 14). Conflicting studies have associated cGMP increases in the guard cell cytosol with stomatal closure and presumably loss of ions from guard cells, however (15, 16).

Examination of mutagenized plants has identified a role for the Arabidopsis CNGCs AtCNGC2 (17) and AtCNGC4 (18) in plant hypersensitive response to pathogen infection. Interestingly, an early step in plant response to pathogen infection is Ca²⁺ influx into cells (19, 20). The molecular mechanisms facilitating this inward Ca²⁺ current are not known. AtCNGC2 and AtCNGC4 also play a role in normal plant growth and development (i.e. in the absence of pathogen infection); Arabidopsis plants lacking functional copies of these genes display reduced growth as compared with wild type plants (17, 18, 21). AtCNGC2 and AtCNGC1 are probably involved in cation transport in plant cells; translational arrest of these genes affects plant sensitivity to cations in the growth medium (21, 22). In both of these studies, the authors speculated that these plant CNGCs might play a role in Ca²⁺ movement into or within the plant. Support for the hypothesis that CNGCs may be involved in Ca²⁺ fluxes across the plant cell membrane can be found in the work of Volotovsky et al. (23). Using protoplasts isolated from tobacco plants expressing recombinant apoaequorin (a cytosolic Ca²⁺ sensor), they demonstrated that physiological responses such as protoplast swelling and change in cell Ca²⁺ homeostasis occurred in response to exposure of protoplasts to cyclic nucleotides. Having said that, the authors also show that intracellular stores of Ca²⁺ could be activated by cyclic nucleotides, since increase of [Ca²⁺]_cyt can occur in external Ca²⁺-free medium (23).

Prior work from this laboratory has demonstrated that Arabidopsis CNGCs are inward rectified, noninactivating channels that can conduct Ca²⁺ across the cell membrane (5, 6). Cyclic nucleotides are present in plant cells and play critical roles in numerous signal transduction pathways (4, 12). Recent studies (9, 24) have identified proteins with adenylyl (as discussed above) and guanylyl cyclase activities in plants. Thus, the cytosolic machinery necessary for the generation of the activating ligand (cNMP) of CNGCs is present in plant cells. However, no study to date has demonstrated the presence of a cyclic nucleotide-activated inward Ca²⁺-conducting ion channel in plant cell membranes. It was the objective of the work described in this report to apply voltage clamp methods to plant cell protoplasts to probe for the presence of this current in plants.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Protoplast Isolation—A. thaliana (Columbia) seeds were grown on standard potting mix in a controlled environment growth chamber at 18 °C on a 9/15-h light/dark cycle. Guard cell protoplasts and mesophyll cell protoplasts were isolated from 5–6-week-old Arabidopsis plants. Guard cell protoplasts were isolated from abaxial epidermal strips as described previously (25). Briefly, epidermal strips were floated on medium containing 1.8–2.5% (w/v) Cellulase Onozuka RS (Yacult Honsha, Tokyo, Japan), 1.7–2% (w/v) Cellulysin (Calbiochem, Behring Diagnostics, La Jolla, CA), 0.026% (w/v) Pectolyase Y-23 (Yacult Honsha), 0.26% (w/v) bovine serum albumin, and 1 mM CaCl₂ (pH 5.6) with osmolality adjusted to 360 mOsM/kg with mannitol. After 2–3 h of incubation at 28 °C with gentle shaking, released protoplasts were passed through a 25-µm mesh and kept on ice for 2–3 min before centrifugation (100 x g for 4 min at room temperature). For mesophyll cell protoplasts, we used peeled leaves (mesophyll layer exposed to the enzyme solution by removal of the abaxial epidermis), and tissue was incubated for 20 to 30 min prior to centrifugation. The pellet of guard cell or mesophyll cell protoplasts was resuspended and kept on ice in 1–2 ml of fresh medium containing 0.42 M mannitol, 10 mM Mes, 200 µM CaCl₂, 2.5 mM KOH (pH 5.55 and osmolality at 466 mOsM/kg). Unless stated otherwise, all chemicals were from Sigma.

Solutions—Protoplasts were placed in a 0.4-ml recording chamber (model RC-25F from Warner instruments Corp., Hamden, CT), allowed to settle, and then perfused continuously at flow rates of 0.5 ml/min. To record I_Ca, we used two different sets of barium-containing solutions (see "Results"). Set 1 contained 50 mM BaCl₂, 1 mM KCl, and 10 mM Mes (pH 5.5 with KOH) (bath) or 5 mM BaCl₂, 20 mM KCl, and 10 mM Hepes (pH 7.5 with KOH) (pipette). Set 2 contained 100 mM BaCl₂ and 10 mM Mes (pH 5.5 with Tris) (bath) or 100 mM KCl, 1 mM MgCl₂, and 10 mM Hepes (pH 7.5 with KOH) (pipette). All solutions were adjusted to an osmolality of 470 mOsM/kg (bath) or 500 mOsM/kg (pipette) with mannitol. cAMP and the lipophilic cAMP analog Bt₂cAMP were solubilized in deionized water and stored in aliquots of 50–100 µl at a concentration of 0.1 M. A few minutes before the experiment, the cAMP and Bt₂cAMP solutions were diluted to the final desired concentration. The perfusion system in all our experiments was gravity-driven (0.5 ml/min; it takes several minutes for the ligand to reach the recording chamber via the tubing). The flow rate is about one chamber volume/min.

Current-Voltage Recording and Analysis—Patch pipettes (5–10 µm) were pulled from Kimax-51 glass capillaries (Kimble 34500; Kimble, Owens, IL) using a Flaming/Brown micropipette puller (Sutter P-87; Sutter Instrument Company, Novato, CA). Experiments were performed at room temperature (20–22 °C) using standard whole cell patch clamp techniques, with an Axopatch 200B integrating patch clamp amplifier (Axon Instruments, Inc., Union City, CA). Voltage commands and simultaneous signal recordings and analyses were assessed by a microcomputer connected to the amplifier via a multipurpose input/output device (Digidata 1320A) using pClamp 9.0 software (Axon Instruments, Inc.). After gigaohm seals were formed (cell-attached configuration), the whole cell configuration was achieved by gentle suction, and the membrane was immediately clamped to a holding potential of –30 mV. Excised outside-out patches were achieved by gentle tapping at the amplifier head stage if and when cells were no longer attached to the bottom of the chamber. Inside-out patches were pulled from cells well attached to the bottom of the chamber. In all configurations, protoplasts (or membrane patches) were perfused for 3–5 min before starting any current measurements. Current was measured prior to and after (times noted in figure legends) the addition of activating ligand (0.75–2 mM cyclic nucleotide, as noted in the figure legends) to the perfusion bath. The lipophilic cAMP analog Bt₂cAMP was used to activate currents in the whole cell and outside-out patch configurations; cAMP was added to the perfusion bath for studies with inside-out patches. It should be noted that the concentration of cAMP (or Bt₂cAMP) used in the studies reported here is substantially higher than the ambient level thought to be present in plant cells, although both temporal and spatial spikes in plant cell [cAMP] can occur (8, 9, 10, 26). However, the cAMP level we used is of a similar magnitude as the level of cyclic nucleotide ligand used to activate CNGCs in many patch clamp studies. For example, 4 mM cGMP was used to activate an animal CNGC in native membrane preparations and also when the channel was expressed in HEK cells (27); 2 mM was used in the electrophysiological analysis of another animal CNGC (28). Even in the patch configuration, approximately 1 mM cyclic nucleotide have been used to obtain maximal currents in studies of animal (e.g. see Refs. 29–31) and plant (18) CNGCs. Prior work from this laboratory involving whole cell voltage clamp electrophysiological analysis of cloned plant CNGCs used 0.1 to 5 mM activating ligand (6). It is also noteworthy that unusually high concentrations of cNMP phosphodiesterases are found in plant cells (see Ref. 8). CNGCs (plant and animal) are known to bind calmodulins (32, 33); calmodulin binding blocks cyclic nucleotide activation of CNGCs (7, 34). Exposure of (animal) CNGCs to high concentrations of cyclic nucleotide facilitates dissociation of calmodulin from these channels (35). The dissociation rate constant (8 min) for removal of calmodulin from CNGCs is much longer that the rate constant for binding. If the channel responding to our exogenous application of cAMP is, in fact, a CNGC (see "Discussion"), then endogenous calmodulin present in the plant cell could require long exposure to high levels of exogenous cAMP in order to dissociate from the channel and allow for cAMP binding.

All current traces shown were low pass-filtered at 2 kHz before analog-to-digital conversion and were uncorrected for leakage current or capacitive transients. Membrane potentials were corrected for liquid junction potential as described (36). Nernst potentials were calculated without correction for ionic activities. Current-voltage relationships for I_Ba were plotted as steady-state currents versus test potentials.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
cAMP Modulates an Inward Rectifying Ca²⁺ Current (I_Ca) across the Plasma Membrane of Guard Cell Protoplasts—Arabidopsis guard cell protoplasts were patch-clamped (whole cell configuration) in order to assess the effect of cAMP on the hyperpolarization-activated Ba²⁺ current (I)_Ba. Ba²⁺ was preferred as the charge carrier instead of Ca²⁺, since the use of Ba²⁺ has the advantages of (a) blocking inward and outward K⁺ currents through K⁺-selective channels and therefore unmasking other less dominant conductances, and (b) Ba²⁺ permeates Ca²⁺ channels much better than Ca²⁺ itself (37).

In the whole cell configuration, the addition of Bt₂cAMP to the perfusion bath resulted in an enhancement of I_Ba in seven of nine guard cell protoplasts assayed. In the presence of Bt₂cAMP, I_Ba measured at –140 mV increased by an average of 3.4 ± 0.28-fold (n = 7). A typical experiment demonstrating such an effect (i.e. increase in whole cell current upon the addition of activating ligand) is shown in Fig. 1. In this case, the I-V relationships of I_Ba measured from one Arabidopsis guard cell prior to and 8 min following the application of 1 mM Bt₂cAMP are shown. In the absence of Bt₂cAMP, only a small background conductance is noticeable (–13 pA at –110 mV), but in the presence of Bt₂cAMP, a much larger conductance (–47 pA at –110 mV) was measured, a 3.5-fold increase (Fig. 1A). The Bt₂cAMP-induced I_Ba current, obtained by subtracting the control I-V curve (–db-cAMP) from the test I-V curve (+db-cAMP), is plotted in Fig. 1B. This analysis shows an inward rectification of this conductance in the whole cell configuration, with a voltage threshold for activation approaching –30 mV. Results presented in Fig. 1C show the whole range of the Bt₂cAMP-activated I_Ba-V plot, as we extended the range of the hyperpolarizing voltage ramp to values near –190 mV. At these negative voltages, approximately –195 pA current could be measured. The addition of GdCl₃ at 50 µM, a concentration at which only Ca²⁺ channels are affected (see Ref. 38), and keeping the same concentration of Bt₂cAMP in the bath, led to a dramatic and swift block of the Bt₂cAMP-activated I_Ba (Fig. 1C). Perfusing guard (or mesophyll) cells with control solutions (no ligand) for up to 35 min did not affect the background current (n = 6; data not shown). Several groups (13, 14) have previously identified a possible role of cGMP in promoting stomatal opening and, further, have associated this effect with changes in cytosolic Ca²⁺ in the guard cell. In these prior studies, this association of cGMP with increased cytosolic Ca²⁺ level in guard cells did not include any measurements of plasma membrane ion fluxes. In addition, the putative effect of cGMP on promoting increased guard cell cytosolic Ca²⁺ was attributed to an indirect effect of cGMP on ion transport (i.e. cGMP was not postulated to act directly on ion channels) (13). Prior studies of cloned plant CNGCs indicate that cGMP as well as cAMP could activate currents (5). We therefore investigated whether the activation of a Ba²⁺ current by cAMP observed here with native membranes could be reproduced using cGMP. In a preliminary analysis (studies done on four cells), however, we found no effect of cGMP on I_Ba (data not shown). Thus, any effect of cyclic nucleotide in activating inward currents in the work reported here is limited to the ligand cAMP.

View larger version (21K): [in this window] [in a new window]   FIG. 1. The hyperpolarization-activated IBa is up-regulated by cAMP and blocked by Gd3+. Whole cell currents from one A. thaliana guard cell protoplast (bath and pipette media set 1) are shown. A, current to voltage (I-V) curves taken before () and 8 min after () extracellular perfusion of 1 mM Bt2cAMP. IBa currents were induced by voltage ramps going from +88 to –112 mV in 2 s. B, I-V curve of the Bt2cAMP-induced IBa current obtained by subtracting the control curve () from the test curve () shown in A. C, I-V curves in response to voltage ramps from 0 to –192 mV (1.4 s long) before () and 5 min after () the addition of 50 µM GdCl3.1 mM Bt2cAMP was present in the bath for both recordings.

In excised outside-out patches (as well as inside-out), the cytosolic machinery of the cell is believed to be lacking (43); hence, these techniques constitute powerful electrophysiological tools that help discriminate between direct and indirect effects of ligand application on a target channel. To test for the possibility of a direct effect of cAMP-induced Ca²⁺ channel activation, we performed outside-out patch clamp experiments on membrane patches pulled from guard cell protoplasts. Current recordings were made on a total of six membrane patches pulled from six different guard cell protoplasts; in all six tests, application of cyclic nucleotide either promoted the activation of Ba²⁺-conducting channels that were silent in the absence of added ligand or dramatically potentiated channels that were opening even before the addition of exogenous Bt₂cAMP. A representative set of data is shown in Fig. 2 from one Arabidopsis guard cell plasma membrane patch. Note the virtual absence of channel activity before Bt₂cAMP application in control current traces (at all but one voltage; –132 mV) and compare it with the strong activation that followed the application of Bt₂cAMP (Fig. 2A). Plotting the I-V relationship of this current prior to and after Bt₂cAMP application (Fig. 2B) reveals that both curves change sign virtually at the same reversal potential (+35 mV), just slightly positive of E_Ba (+29 mV) but far away from E_Cl (–31 mV) and/or E_K (–75 mV). In this particular example, it can be seen from the current traces shown in Fig. 2A (right panel) that the probability of opening of Ca²⁺ (i.e. Ba²⁺) channels depends not only on the presence of the ligand Bt₂cAMP, but also on the membrane voltage. In Fig. 2C, the Bt₂cAMP-induced I_Ba (obtained by subtracting the control background current curve from the +Bt₂cAMP curve) is shown. Although it is difficult to distinguish channel open and closed states when channel gating is as fast as is the case here with these cAMP-activated I_Ba channels, it can be estimated that the I_Ba curve plotted in Fig. 2C shows up to five open states. Each of the dashed lines was fitted to a linear equation with a basic slope of 13 pS for one channel being open: O₁ (26 pS for O₂, 39pSfor O₃, etc.). Also, notice the reversal potential just slightly positive of the calculated E_Ba pointing to a predominant Ba²⁺ conductance in the presence of Bt₂cAMP.

View larger version (31K): [in this window] [in a new window]   FIG. 2. Bt2cAMP (db-cAMP) increases single channel activity of IBa in excised outside-out patches. Current traces and I-V relationships from one A. thaliana guard cell protoplast (bath and pipette media 1) are shown. A, six current traces (membrane voltages in mV are indicated to the right) of single channel activity present in the membrane patch prior to and 10 min after perfusing with bath solution containing 2 mM Bt2cAMP. B, I-V relationship before () and after () the addition of 2 mM Bt2cAMP to the perfusion bath. Currents were induced by voltage ramps going from +48 to –160 mV in 2 s. C, I-V relationship of the cAMP-induced single channel current activity obtained by subtracting the control curve () from the test curve () shown in B. Respective positions of the calculated values of EBa, ECl, and EK are indicated in B and C by arrows along the voltage axis. Five open channel levels are shown and are indicated by O1, O2, etc.; each of the five corresponding dashed lines is a fit of a linear equation (see "Results" for details). The conductance values used to generate the lines corresponding to these five open states were obtained from the analysis (Fig. 3C) of the membrane patch shown in Fig. 3.

View larger version (55K): [in this window] [in a new window]   FIG. 3. Bt2cAMP (db-cAMP) increases single channel activity of IBa in excised outside-out patches without affecting the single channel conductance. Current traces, single channel I-V relationships, and plots of the dwell time versus single channel amplitude are shown for one Arabidopsis guard cell protoplast (bath and pipette media 1). A, four current traces (membrane voltages in mV are indicated to the left) of single channel activity recorded prior to (left panel) and 10 min after the addition of 1 mM Bt2cAMP to the perfusion bath (right panel). Note that the time scale for recordings at –11.8 mV is different from the time scale for recordings obtained at all the other command voltages. The arrows (left panel) indicate fast "open-shut transitions" that were seen in single channel traces even before the application of Bt2cAMP. The box delineated with a solid line (right panel) highlights an unusual and relatively long period of silence occurring even in the presence of Bt2cAMP. B, analysis of the dwell times of channel opening/closing events occurring during the current recordings shown in A. At each command voltage (as shown to the left in A), the dwell time at which current was maintained at a given value (i.e. the time between channel opening and/or closing events as evidenced by shifts in current amplitude) is plotted against the current amplitude maintained over that time period during the current trace. Results of this analysis are shown for the membrane patch in the absence (left) and presence (right) of Bt2cAMP. For each command voltage, data recorded over an equal length of time either in the presence or absence of Bt2cAMP were used for this analysis. Note the varying scale for dwell time in the different panels. C, I-V relationship used to determine the unitary channel conductance obtained from the well resolved and slow single channel traces occurring before application of Bt2cAMP () and from the not so well resolved and fast flickering single channel traces obtained after application of Bt2cAMP () presented in A. The data points were fitted to a linear equation with a slope of 13.2 pS. Respective positions of the calculated values of EBa, ECl, and EK are indicated by arrows along the voltage axis (EK =–75 mV). Data points were gathered from the segments shown and other segments not shown. Each data point is an average measurement of at least six opening events ± S.E. D, "flickery" channel opening events occurring in the presence of Bt2cAMP are shown with an expanded time scale. A segment (as highlighted by the box delineated with the broken line in the right panel of A) of the data recorded at –31.8 mV command potential from the membrane patch in the presence of Bt2cAMP is shown.

The I_Ba-V relationship of this membrane patch is shown in Fig. 3C. The data points were fitted to a linear relation with a slope factor of 13.2 pS. In this experiment, the apparent reversal potential (Fig. 3C) of +20 mV is slightly less positive than the calculated E_Ba but is still far removed from both E_Cl and/or E_K; this result is in good agreement with the primarily Ba²⁺ nature of this conductance. A portion (i.e. the region within the box bordered by broken lines) of the recording made at –31.8 mV command potential from this membrane patch in the presence of Bt₂cAMP as shown in Fig. 3A (right panel) is presented in Fig. 4D with an expanded time scale. In this manner, discreet channel opening/closing events can be more easily discerned.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Bt2cAMP reversibly increases single channel activity of IBa in excised membrane patches from mesophyll cells. I-V relationships were recorded from three A. thaliana mesophyll cell protoplasts. A, I-V relationship before () and 8 min after () the addition of 1mM Bt2cAMP to the perfusion bath. Currents were induced by voltage ramps going from +50 to –100 mV in 2 s. B, I-V relationship before () and 5 min after washout of 1 mM Bt2cAMP (). Currents were induced by voltage ramps going from +50 to –190 mV in 2 s. For A and B, the membrane patches were in the outside-out configuration, and bath and pipette media 2 were used; respective positions of the calculated values of EBa, ECl, and EK are indicated by the arrows along the voltage axis. C, I-V relationship of an inside-out membrane patch before () and 8 min after () the addition of 1 mM cAMP to the perfusion bath. Currents were induced by voltage ramps going from +50 to –100 mV in 2 s, and bath and pipette media 1 were used; the position of the calculated EBa value is indicated by an arrow along the voltage axis. D, I-V curve of the cAMP-induced IBa current obtained by subtracting the control curve () from the test curve () shown in C. Respective positions of the calculated values of EBa, ECl, and EK are indicated in C and D.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Data presented in this report show that cAMP, a well established cytosolic second messenger in animals, activates a Ca²⁺ (i.e. Ba²⁺)-conducting channel located in the plasma membrane of plant cells. In whole cell experiments designed to measure I_Ca activated by hyperpolarization (e.g. 37, 45–48), Arabidopsis guard cell protoplasts increased their I_Ca in response to a lipophilic analog of cAMP (Bt₂cAMP) by an average of 3–4-fold (Fig. 1A). The cAMP-induced activation of I_Ca, measured in guard cell protoplasts in the whole cell configuration, showed a marked inward rectification with no apparent current flow in the outward direction at voltages up to +100 mV (Fig. 1B). It should be noted that prior studies have identified a hyperpolarization-activated I_Ca current in the guard cell plasma membrane that is modulated by abscisic acid and hydrogen peroxide (47, 48). The fast gating kinetics and short dwell time (i.e. "flickery" nature) of the cAMP-activated inward current we observe in our studies is similar to that of the guard cell hyperpolarization-activated Ca²⁺ current reported by Hamilton et al. (47). We also note with interest that the single channel conductance reported by Hamilton et al. (47) (13 pS) is similar to that reported here for the guard cell cAMP-activated Ca²⁺ channel; documentation that the same channel is responding to all three of these messenger molecules (i.e. cAMP, hydrogen peroxide, and abscisic acid) awaits further study, however.

Our results indicate that Arabidopsis mesophyll cells also contain an inward rectified Ca²⁺ channel directly activated by cAMP. In other studies (data not shown), we have also identified a cAMP-activated inward rectified Ca²⁺ channel in the plasma membrane of guard cell protoplasts isolated from Vicia faba. The cyclic nucleotide activated channel in Vicia appears to have similar properties as that of the Arabidopsis guard cell channel we have characterized in the work presented here.

The Ca²⁺ channel blocker Gd³⁺ (38, 47, 49) completely blocked the cAMP-activated current. These results provide the first electrophysiological evidence supporting previous speculations that increase of cytosolic [Ca²⁺] triggered by cNMP occurs mainly through a plasma membrane-localized Ca²⁺-permeable channel (23, 50).

The effect of Bt₂cAMP in the whole cell configuration could be interpreted as either indirect (i.e. mediated through cAMP-activated protein kinases (which could in turn activate I_Ca by phosphorylation; see Ref. 51)) or a direct effect of the ligand on the I_Ca channel itself. Our results with excised patches indicate that cAMP was able to stimulate channel activation directly. Thus, cAMP facilitated the opening of single I_Ca channels upon hyperpolarization in excised patch modes. In other cases, single channel activity could be resolved in cAMP-free patches containing only a few channels. The cAMP-induced I_Ca current activation occurred without apparent modification of the single channel conductance (Fig. 3C). The reversal potential obtained from the single channel I-V relationship (+20 mV) indicated that these channels were preferentially carrying Ba²⁺ despite the presence of other ions (K⁺ and Cl^–) in the recording medium. Prior work from this laboratory (5, 6) has shown that cloned plant CNGCs expressed in heterologous systems conduct Ca²⁺ (as well as K⁺) and, further, that exogenous Ca²⁺ partially blocks K⁺ conductance by these channels. We note that in some experiments, cAMP-activated current shows a reversal potential very close to E_Ba (Figs. 2B and 4D), whereas in others (Figs. 3C and 4B) the reversal potential is near (relative to E_K and E_Cl) but not at E_Ba. This result could be explained by the cAMP-activated channel predominantly conducting Ca²⁺ (Ba²⁺) but also allowing, to some extent, K⁺ to permeate the channel. These results are consistent with the conductance properties of cloned plant CNGCs as reported in earlier work from this laboratory (5, 6).

As discussed above, the use of the excised patch configurations allowed us to identify cAMP as a direct activator of Ca²⁺ channels. To our knowledge, this is the first report showing a direct stimulatory action of cAMP on any ion channel present in a native plant membrane. We conclude that the work presented in this report showing cAMP-dependent activation of Ca²⁺ permeation in these plant cell membranes is consistent with the presence of functional CNGCs in these cells.

It should be noted that in all cases reported to date, CNGCs have been found to be heterotetramers composed of subunits encoded by different CNGC genes (7). The translation products of at least 12 of the 20 different CNGC genes in Arabidopsis are expressed in leaves (4). Thus, we cannot know if the cyclic nucleotide-activated current we recorded from the leaf cell membrane in our experiments can be attributed to one or several different CNGC channel protein complexes or if the presumed CNGC channel protein(s) facilitating the currents we recorded is composed of one or several CNGC gene translation products.

	FOOTNOTES

 
^* This work was supported by National Science Foundation Grant 0344141. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Agricultural Biotechnology Laboratory, Dept. of Plant Science, University of Connecticut, U-4067, Storrs Rd., Storrs, CT 06269-4067. Tel.: 860-486-1945; Fax: 860-486-0682; E-mail: gerald.berkowitz{at}uconn.edu.

¹ The abbreviations used are: CNGC, cyclic nucleotide-gated channel; Mes, 4-morpholineethanesulfonic acid; pS, picosiemens.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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