INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

K⁺ plays a significant role in plant cells. This role has been best studied in guard cells where transmembrane K⁺ fluxes regulate cell volume, stomatal aperture, and thus gas exchange across the leaf. However, in all cell types, K⁺ plays a major role in osmoregulation. Therefore, plasma membrane K⁺ channels (for review, see Cherel et al., 1996; Maathuis et al., 1997) and their control are of importance in understanding this fundamental process. Inwardly rectifying plant K⁺-channel cDNA clones have been isolated. The first, AKT1 and KAT1, were isolated from Arabidopsis (Anderson et al., 1992; Sentenac et al., 1992) and characterized by functional expression in Xenopus laevies oocytes or yeast (Anderson et al., 1992; Sentenac et al., 1992; Bertl et al., 1994; Schachtman et al., 1994). KAT1 is expressed primarily in guard cells, whereas AKT1 is expressed in roots (Cao et al., 1995). Other cDNA clones encoding inwardly rectifying K⁺ channels have been isolated from Arabidopsis and potato (Cao et al., 1995; Muller-Rober et al., 1995; Ketchum and Slayman, 1996).

Two cDNA clones, KCO1 (Czempinski et al., 1997) and SKOR (Gaymard et al., 1998), encoding voltage activated outwardly rectifying K⁺ channels, have been isolated from Arabidopsis. Properties of KCO1 (Czempinski et al., 1997) were determined by expression studies in baculovirus-infected insect cells (Czempinski et al., 1997). SKOR was characterized by expression in X. laevis oocytes (Gaymard et al., 1998). In contrast to the limited molecular characterization of plant outwardly rectifying K⁺ channels, numerous studies describe the electrophysiological characteristics of outwardly rectifying K⁺ currents (I_Kout) in isolated plant protoplasts. Studies of ion channels in the plasma membranes of fava bean (Vicia faba cv Long Pod) and Arabidopsis guard cells (Blatt, 1988; Schroeder and Hedrich, 1989; Lemtiri-Chlieh, 1996) and mesophyll cells (Li and Assmann, 1993; Romano et al., 1998), tobacco mesophyll cells (Bei and Luan, 1998), Arabidopsis root stelar tissues (Gaymard et al., 1998), and potato leaves (Brandt and Fisahn, 1998) have resulted in the characterization of voltage-activated I_Kout. Stretch-activated K⁺ channels in the plasma membrane of fava bean guard cells have also been described (Cosgrove and Hedrich, 1991).

Effects of abscisic acid (ABA) on K⁺-channel function in guard cells have been studied extensively (Blatt, 1990; MacRobbie, 1993; Assmann et al., 1994; Schwartz et al., 1994). ABA is synthesized and translocated within the plant under stress conditions, and its actions promote physiological responses leading to drought tolerance, osmotic adjustment, and dormancy (Davies and Mansfield, 1983). I_Kout in intact stomatal guard cells is rapidly enhanced by ABA, and ABA inhibits an inward K⁺ current (I_Kin) (Blatt, 1990; Lemtiri-Chlieh, 1996). These results are consistent with the function of that cell type in which ABA is known to inhibit K⁺ uptake and promote K⁺ loss, thus deflating the guard cells and causing stomatal closure, which improves water conservation under stress conditions. Roberts (1998) described the effect of ABA on maize root K⁺ channels. However, in contrast to our knowledge of guard cell mechanisms, the effects of ABA on K⁺ channels in other cell types have not been well studied.

In the present study we performed a patch clamp analysis of ABA regulation of I_Kout in fava bean mesophyll protoplasts and a two-electrode voltage clamp analysis (TEVC) of X. laevis oocytes expressing an I_Kout derived from mesophyll mRNA. ABA inhibited the I_Kout expressed by the oocytes. These results provide evidence for a mesophyll ABA receptor that modulates a mesophyll outwardly rectifying K⁺ channel. We also used the X. laevis oocyte system to compare the ABA signaling pathways in mesophyll and guard cells. Co-injection studies with KAT1 cRNA encoding the guard cell KAT1 channel revealed that the mesophyll ABA signaling pathway that modulates I_Kout is not functionally interchangeable with the guard cell ABA signaling pathway that modulates I_Kin mediated by KAT1.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Mesophyll Protoplast I_Kout Is Modulated by ABA

To determine whether ABA modulates K⁺ currents in mesophyll cells, the effect of ABA on I_Kout of mesophyll protoplasts was examined by patch clamp analysis. Figure 1A depicts a typical current recording obtained from patch clamp analysis of a mesophyll protoplast. This current was previously characterized and shown to be mediated by a channel with high, although not complete, selectivity for K⁺ (Li and Assmann, 1993). I_Kout is elicited when the protoplast membrane is depolarized from 47 mV to voltages between 15 and +85 mV. The rundown of mesophyll cell K⁺ currents is shown in Figure 1B. There is no significant decay. I_Kout at 15 min is 13.0 ± 2.0 picoampere per picofarad (pA pF¹) and the current at 30 min is 12.9 ± 2.4 pA pF¹ at the +65 mV step (n = 5) (Fig. 1E). The effect of 25 µM ABA on a typical current recording is shown in Figure 1, D versus C. The average effect of 25 µM ABA was to reduce I_Kout by approximately 40% from 11.6 ± 1.8 pA pF¹ to 7.0 ± 1.0 pA pF¹ at +65 mV (n = 11; Fig. 1E).

View larger version (34K): [in this window] [in a new window]   Figure 1.   ABA inhibits outward K+ current (IKout) from fava bean mesophyll protoplasts. A typical whole-cell recording (current versus time) from mesophyll protoplasts at 15 min (A) or 30 min (B) and before (C) and after ABA application (25 µM) (D) is presented. After the whole-cell configuration was obtained, the membrane potential was clamped at 47 mV. Test pulses were from 55 to +85 mV in 20-mV steps. ABA (± cis/trans, Sigma) (25 µM) was added near the protoplast under study 15 min after achieving the whole-cell configuration and recordings obtained 10 to 15 min later. IKout obtained from a mesophyll protoplast before ABA treatment (C) was reduced after application of ABA (D). Mean values of time-activated whole-cell currents were normalized by cell capacitance and plotted as a function of voltage. (x ± SE, n = 5 for control, n = 11 for ABA treatment) (E).

Mesophyll mRNA-Injected Oocytes Express Outward Current

Figure 2, A and B, depicts typical whole-cell recordings in 96 mM Na⁺ solution from mesophyll mRNA and water-injected oocytes, respectively. Oocytes were voltage clamped at a holding potential of 60 mV; test potentials were from 60 to 180 mV in 20-mV steps. An outward current was observed in oocytes injected with mesophyll mRNA that activates with depolarization and does not appear to saturate. The current per voltage curve from mRNA-injected oocytes versus control oocytes shows an approximately 10-fold greater outward current from mRNA-injected oocytes (Fig. 2C).

View larger version (30K): [in this window] [in a new window]   Figure 2.   Fava bean mesophyll mRNA expresses an outward current in X. laevis oocytes. Typical current recording from X. laevis oocytes injected with mRNA (50 ng) (A), water (B). C, Current per voltage relationship of outward current expressed by mRNA-injected oocytes () and water-injected oocytes (). Maximum outward current for each test potential was measured at 800 ms. n, Number of oocytes used for each data point presented. Iout was monitored in 96 mM Na+ solution at a holding potential of 60 mV. Voltage protocol consisted of test potentials stepped from 60 to 180 mV in 20-mV steps.

The Outward Current Is Carried Mainly by K⁺

The reversal potential (E_rev) of the outward current observed in oocytes was determined to identify the major ionic species involved. Tail current analysis (Wollmuth and Hille, 1992) of mesophyll mRNA-injected oocytes in 96 mM K⁺ solution yielded an E_rev of 20 mV. In 2 mM K⁺ (i.e. 96 mM Na⁺ solution) E_rev shifted to 70 mV (Fig. 3A). The voltage protocol consisted of membrane depolarization from 60 to 0 mV for 750 ms followed by 20-mV steps to potentials from 0 to 180 mV . 

View larger version (25K): [in this window] [in a new window]   Figure 3.   The outward current is specific for K+. A, Comparison of Itail versus test potential at 800 ms for mesophyll-mRNA-injected oocytes monitored in 2 mM K+ (96 mM Na+) solution and 96 mM K+ solution. IKout reversed at 70 mV in 2 mM K+ and shifted to 20 mV when monitored in 96 mM K+ solution. The membrane potential was held at 60 mV and then depolarized to 0 mV for 750 ms. The membrane potential was then stepped to more negative potentials in 20-mV intervals. B, Effect of Ba2+ on outward current from water-injected or mRNA-injected oocytes. Maximum IKout at 160 mV, 800 ms, versus concentration of Ba2+ (0, 20, 40 mM) is depicted.

Analysis of ion channel blockage aids in identification of the ionic species involved. Figure 3B depicts outward currents measured at 180 mV, 800 ms by TEVC analysis of mesophyll mRNA- and water-injected oocytes in the absence and presence of 20 or 40 mM BaCl₂. Barium had no effect on outward currents from control oocytes. However, the outward current from mesophyll mRNA-injected oocytes was reduced by approximately 50% in 20 mM BaCl₂ and by 75% in 40 mM BaCl₂ solution.

I_Kout from Mesophyll mRNA-Injected Oocytes Is Modulated by ABA

Typical I_Kout recordings were observed from TEVC analysis of oocytes injected with mesophyll mRNA (Fig. 4A) and water (Fig. 4C) in 96 mM Na⁺ solution minus ABA. After the initial analysis in the absence of ABA, 50 µM ABA in 96 mM Na⁺ solution was bath applied, and 5 min later TEVC analysis was repeated on the same oocyte. ABA treatment resulted in a decrease in I_Kout from mesophyll mRNA-injected oocytes (Fig. 4, A and B). Control oocytes showed no significant change of outward current in response to ABA (Fig. 4, C and D).

View larger version (25K): [in this window] [in a new window]   Figure 4.   ABA inhibits IKout in oocytes. IKout traces in 96 mM Na+ solution minus ABA from oocytes injected with mesophyll mRNA (A) or water (C). ABA (50 µM) in 96 mM Na+ solution was applied to the bath and 5 min later IKout was again monitored from the same oocytes injected with mRNA (B) or water (D). Voltage protocol was as described in Figure 2 legend. E, Effect of 10, 25, and 50 µM ABA concentrations on maximum IKout. IKout was measured at 160 mV, 800 ms.

The effect of different concentrations of ABA on I_Kout is represented in Figure 4E. Recovery of I_Kout after ABA inhibition varied. Therefore, different oocytes were used for each ABA exposure. Approximately 50% inhibition of I_Kout was obtained with 10 µM ABA (Fig. 4E). Outward current from control oocytes was not influenced by application of ABA.

The Mesophyll ABA Signal Transduction Pathway Cannot Functionally Substitute for the Guard Cell ABA Signal Transduction Pathway

The finding that ABA modulates K⁺-channel response in mesophyll cells led us to examine whether the ABA signaling pathway encoded by mesophyll mRNA can functionally substitute for the ABA signaling pathway encoded by guard cell mRNA. To ensure that we were examining modulation of the same K⁺ channel and also to limit differences in signaling to the receptor and components upstream of the K⁺ channel, the inwardly rectifying K⁺ channel of guard cells, KAT1, was used for this comparison. Reciprocal experiments with the mesophyll cell outwardly rectifying channel of fava bean were not possible as that channel has not yet been cloned.

I_Kin produced by KAT1 cRNA-injected oocytes in 96 mM K⁺ solution is depicted in Figure 5A. ABA (50 µM) had no effect on I_Kin (761 ± 202 nA [mean ± SE] before ABA, 798 ± 180 nA [mean ± SE] after ABA, n = 6) elicited from oocytes injected with KAT1 cRNA alone (Fig. 5, C and F [lane 1]). Oocytes injected with only guard cell mRNA or only mesophyll mRNA displayed no significant I_Kin (Fig. 5B). Since one would expect guard cell mRNA to include the KAT1 message, the absence of significant levels of I_Kin generated by this mRNA suggests that the KAT1 message may be of low abundance. Oocytes co-injected with KAT1 cRNA and guard cell mRNA express an I_Kin (406 ± 69 nA [mean ± SE], n = 9) that is inhibited by 50 µM ABA (76 ± 38 nA [mean ± SE], n = 8) (Fig. 5, D and F [3]). Replacement of the guard cell mRNA-encoded ABA receptor and possible associated signal transduction components by co-injection of KAT1 cRNA with mesophyll A⁺ RNA produced an I_Kin (482 ± 238 nA [mean ± SE], n = 7) which did not, however, respond to ABA (470 ± 231 nA [mean ± SE], n = 7) (Fig. 5, E and F [2]). Figure 5F depicts average I_Kin measured at 180 mV before and after application of ABA in three situations: (a) oocytes injected with KAT1 cRNA only; (b) oocytes co-injected with KAT1 cRNA and mesophyll mRNA; (c) oocytes co-injected with KAT1 cRNA and guard cell mRNA. A 1.8-fold larger I_Kin is observed for KAT1 cRNA-only injected oocytes compared with the co-injected samples. This could be due to competition with other transcripts for translation of the KAT1 cRNA in co-injected samples or possible inhibition of I_Kin by components of the mRNA preparations or proteins encoded by mesophyll or guard cell mRNA. Nevertheless, it is evident that the mesophyll mRNA cannot substitute for the guard cell mRNA in transducing the inhibitory ABA signal.

View larger version (40K): [in this window] [in a new window]   Figure 5.   The mesophyll mRNA encoded-ABA signaling pathway is not functionally equivalent to that encoded by guard cell mRNA. IKin was activated from a holding potential of 60 mV stepped between 180 mV and 0 mV in 20-mV increments. A, IKin from KAT1 cRNA-injected oocytes measured in 96 mM K+ solution. B, Effect of ABA on IKin (presented as absolute values) obtained from control oocytes (water-injected, guard cell mRNA-injected, and mesophyll mRNA-injected). C, Application of ABA (50 µM) did not reduce IKin from KAT1-cRNA-injected oocytes. D, Effect of ABA on IKin produced by oocytes co-injected with guard cell mRNA and KAT1 cRNA. E, Effect of ABA on oocytes co-injected with mesophyll mRNA and KAT1 cRNA. F, Comparison of the maximum negative current (presented as absolute values) at 180 mV obtained from oocytes injected with: (1) KAT1 cRNA (n = 7); (2) KAT1 cRNA and mesophyll mRNA (n = 7); (3) KAT1 cRNA and guard cell mRNA (n = 9).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Our patch clamp results on fava bean mesophyll cells demonstrate that the I_Kout of these cells is inhibited by ABA. Among other roles, ABA is involved in plant acclimation to drought, osmotic, and salt stress (Davis and Mansfield, 1983). All of these stresses would tend to decrease the water potential of plant tissue, and to the extent that ABA inhibition of K⁺ efflux channels counteracts loss of cellular K⁺, it may help to preserve the water status of mesophyll tissue. It is also possible that the K⁺ channels responsible for I_Kout have a limited permeability to Na⁺ influx as described for rye root K⁺ channels (White and Ridout, 1995). If so, ABA-induced reduction of I_Kout-channel activity during salt stress may protect mesophyll cells against harmful Na⁺ uptake.

The exact components of the signaling pathway by which ABA inhibits I_Kout are not yet known. We previously observed that elevated intracellular Ca²⁺ concentrations and G-protein activators also inhibit this current (Li and Assmann, 1993), suggesting possible elements of an ABA signal transduction pathway that could be studied in future experiments. It is notable that I_Kin of guard cells is likewise inhibited by ABA, elevated Ca²⁺, and G-protein activators (Blatt, 1988; Schroeder and Hagiwara, 1989; Fairley-Grenot and Assmann, 1991; Wu and Assmann, 1994; Lemtiri-Chlieh, 1996). These comparisons led us to question whether there was a universal ABA-signaling pathway that modulated K⁺-channel activity in these two cell types. We tested this hypothesis by analyzing ABA regulation of these channels as expressed in X. laevis oocytes.

Mesophyll mRNA apparently encodes an outwardly rectifying channel (Fig. 2) that results in production of outward current under TEVC analysis. The dependency of E_rev on K⁺ concentration and the susceptibility of the outward current to block by Ba²⁺ (Fig. 3), a property also observed for the mesophyll outward K⁺ channel when studied in planta (Li and Assmann, 1993), indicates the outward current is due mainly to K⁺ flux. The I_Kout E_rev of 20 mV in 96 mM K⁺ solution and 70 mV in 2 mM K⁺ solution (96 mM Na⁺ solution); (Fig. 3) are very similar to those described by Li and Assmann (1993) who observed E_rev of 35 to 25 mV in 100 mM K⁺ solution and 75 mV in 1 mM K⁺ solution. These values indicate that the channel is K⁺ permeable but not completely K⁺ selective. We cannot completely exclude the possibility that a translation product of the injected mesophyll mRNA stimulates expression of an oocyte-encoded channel. A delayed rectifier K⁺ current endogenous to oocytes has been described (Lu et al., 1990). However, the oocyte channel does not share the same pharmacological characteristics as those described here. The endogenous K⁺ currents in oocytes are insensitive to 10 mM barium (Lu et al., 1990). The susceptibility of the K⁺ current expressed by mesophyll mRNA-injected oocytes to barium (Fig. 3) suggests that the I_Kout is not due to activation of the endogenous K⁺ channel. In addition, the close similarity of the oocyte and mesophyll protoplast I_Kout currents suggest that the current observed in oocytes indeed results from expression of a plant message.

Analysis of the ABA effect on I_Kout in oocytes (Fig. 4) also agreed with that observed in planta (Fig. 1). The dose-dependent inhibition of I_Kout by ABA (Fig. 4) suggests the involvement of an ABA receptor. These results indicate that mesophyll mRNA not only encodes an outwardly rectifying K⁺ channel but also all or part of an ABA-signaling pathway that modulates the channel. The strong correlation between the results obtained from mesophyll mRNA-injected oocytes with those obtained from direct patch clamp analysis of mesophyll protoplasts provided confidence for use of the oocyte system to address questions regarding modulation of mesophyll K⁺ channels by ABA.

KAT1 cRNA expression in oocytes has been characterized (Schachtman et al., 1994; Very et al., 1995), and our results of I_Kin mediated by KAT1 are similar to those of other researchers. The lack of ABA inhibition of I_Kin from oocytes injected only with KAT1 cRNA shown in Figure 5 provides evidence for the absence of endogenous ABA receptors or signaling components in oocytes that can couple to the KAT1 channel. ABA inhibition of I_Kin obtained from co-expression of guard cell mRNA and KAT1 cRNA in oocytes (Fig. 5) shows clearly that guard cell mRNA encodes all or part of an ABA-signaling pathway that functions to inhibit I_Kin, just as observed in the in planta experiments on this channel (Blatt, 1990; MacRobbie, 1993; Assmann and Wu, 1994; Schwartz et al., 1994).

These data strongly indicate the presence of an ABA signaling pathway encoded by guard cell mRNA that modulates I_Kin and a mesophyll cell mRNA-encoded ABA signaling pathway that modulates I_Kout. To determine whether these two ABA pathways are the same or similar enough to function interchangeably, we co-injected KAT1 cRNA with mesophyll mRNA. The I_Kin current obtained from KAT1 cRNA and mesophyll mRNA co-injected oocytes is due to KAT1 expression, since oocytes injected with only mesophyll mRNA do not express detectable I_Kin. The inability of the mesophyll mRNA-encoded ABA signaling pathway to modulate KAT1 leads us to conclude that this ABA signaling pathway cannot function interchangeably with the guard cell mRNA-encoded ABA signaling pathway.

If we assume that the oocytes are providing the second messenger components (membrane-associated and/or cytoplasmic) then we can conclude that two different ABA receptor types are involved in modulation of I_Kin and I_Kout in guard cells versus mesophyll cells. Alternatively, it is possible that identical ABA receptors are present in the two cell types, but they are coupled to distinct downstream signaling elements in guard cells versus mesophyll cells.

To date there has been one other study using the oocyte system to dissect hormonal signal transduction pathways of plants. Leyman et al. (1999) previously observed an ABA-stimulated Ca²⁺-dependent Cl current in oocytes injected with mRNA from drought-stressed tobacco leaves. A syntaxin-like protein of tobacco, Nt-SYR1 was implicated in ABA signal transduction by these experiments (Leyman et al., 1999). They also showed that application of clostridium botulinum type C, known to disrupt syntaxin function in animal systems, prevented ABA inhibition of I_Kin in guard cells as well as altering ABA regulation of I_Kout and anion channels in this cell type. Additional studies can now be pursued to further distinguish the two tissue-specific ABA signal transduction pathways described in the present report. Expression cloning strategies (Hollmann et al., 1989) may now be used to identify key components of the unique ABA signal transduction pathway of mesophyll cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Material

Fava bean (Vicia faba cv Long Pod) plants were grown for 3 weeks in plant growth chambers with a 10-h light (100 µmol m² s¹ white light, 21°C) and 14-h dark (18°C) regime. Bifoliate leaves with 80% to 90% expansion were used to isolate protoplasts.

Mesophyll Protoplasts

Isolation of mesophyll cell protoplasts was performed as described by Li and Assmann (1993) with some modification. Leaves were cut into slices after removing the abaxial epidermis and midrib, and the slices were put into an enzyme digestion solution containing 0.6 M mannitol, 1 mM CaCl₂, 0.4% (w/v) macerozyme (Yakult R-10, Karlan Research Products, Santa Rosa, CA), 1% (w/v) cellulase (Onozuka R-10), 0.1% (w/v) polyvinypyrrolidone 40, and 0.2% (w/v) bovine serum albumin, pH 5.5. The mixture was vacuum infiltrated for 2 min followed by a 5-min digestion at 24°C with fast shaking. The debris and disintegrated cells were discarded by decantation. New enzyme solution was added, and leaf slices were digested for 1 h at 24°C with gentle shaking. The digestion mixture was filtered through 60-µm mesh. The filtrate was centrifuged twice at 200g for 3 min, and each time the supernatant was decanted and the pellet containing the protoplasts resuspended in 5 mL of rinsing medium (0.6 M manitol/1 mM CaCl₂). Protoplasts for electrophysiological analysis were incubated in darkness on ice for at least 1 h before starting patch clamp experiments. Protoplasts for RNA isolation were quick frozen in liquid N₂.

Guard Cell Protoplasts

Guard cell protoplasts were prepared as described by Miedema and Assmann (1996). Epidermal peels, obtained by blending leaf sections followed by filtration through cotton mesh, were treated with an enzyme solution containing 0.7% Cellulysin (Trichoderma viride, Calbiochem, La Jolla, CA). The digest was shaken in the dark at 27°C for 30 min, diluted with basic medium (0.45 M mannitol, 0.5 mM CaCl₂, 0.5 mM MgCl_2, 0.5 mM ascorbic acid, 10 µM KH₂PO₄, and 5 mM MES pH 5.5) and shaken for a further 5 min. Peels were collected on 220-µm nylon mesh and transferred to a second enzyme solution containing 1.5% (w/v) Onozuka RS cellulase (Yakult Honsha, Tokyo), 0.02% (w/v) Pectolyase Y-23 (Seishin, Tokyo). This digest was continued with shaking in the dark at 19°C for 20 min. The speed was reduced and the digest continued for an additional 25 min. Resulting protoplasts were filtered through 30-µm nylon mesh and collected by centrifugation at 200g for 4 min. Pellets were rinsed in a medium consisting of 0.35 M mannitol and 1 mM CaCl₂, and then quick frozen in liquid N₂.

RNA Isolation

Frozen protoplasts were thawed and homogenized in 4 M guanidinium thiocyanate solution. The homogenate was centrifuged at 8,000g for 10 min at 4°C. The resulting supernatant was layered over a 5.7 M CsCl cushion. Ultracentrifugation was performed as per the procedure of Chirgwin et al. (1979). Poly(A⁺) RNA was obtained by chromatography on oligo(dT) columns (Qiagen USA, Valencia, CA). The mRNA for injection into oocytes was resuspended in water. All water used for RNA resuspension or oocyte injection was autoclaved twice after treatment with diethyl pyrocarbonate (Sigma, St. Louis).

In Vitro Transcription

KAT1 cDNA was subcloned into the pCITE-4c vector (Novagen, Madison, WI; Li et al., 1998). Since pCITE-4c contains a T7 terminator, uncut plasmid containing KAT1 cDNA (1 µg) was used in the in vitro transcription reaction. Standard in vitro transcription was performed at 37°C for 1 h with T7 RNA polymerase as per mMessage kit (Ambion, Austin, TX). Resulting transcripts were precipitated with LiCl (3 M), washed with 70% (v/v) ethanol, and resuspended in water.

Oocyte Preparation and Injection

Oocytes were obtained from Xenopus laevis. Follicular membranes were removed at room temperature by digestion with collagenase at 2 mg/mL, (type 1A, Sigma) dissolved in Ca²⁺-free saline solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 mM HEPES [4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid]/NaOH, pH 7.4). Oocytes were injected with 50 nL of (1 ng/nL) RNA or 50 nL of water (control). Following injection, oocytes were incubated for 3 to 7 d in "96 mM Na⁺ solution" (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 5 mM HEPES/NaOH, pH 7.5) supplemented with 25 mM sodium pyruvate, 50 mM gentamycin, 10 µg/mL penicillin/streptomycin, and 0.5 mM theophylline.

Recordings

Patch Clamp Measurements

1

1

TEVC Measurements

2