two inward k channels in the xylem parenchyma cells a

Abstract. In order to study the mechanism and regula-tion of K+ resorption from the xylem by the cells thatborder the xylem vessels (the xylem parenchyma cells),K+ inward-rectifying channels (KIRCs) in the plasmamembrane of xylem parenchyma cells from Hordeumvulgare L. cv. Apex were studied using the patch-clamptechnique. In the inside-out con®guration, three di�erenttypes of K+ channel and a further K+ conductancecould be identi®ed. Two of these channels, namedKIRC1 and KIRC2, were activated by guanosine5¢-[b,c-imido]triphosphate (Gpp(NH)p; 150 lM), a non-hydrolyzable derivative of GTP, indicating that channelactivity was up-regulated by G-proteins; modulation ofchannel activity occurred via a membrane-delimitedpathway, since the e�ect could be demonstrated in cell-free patches. At 100 mM external K+, KIRC1 had aconductance of 8 pS. There was no e�ect of ATP onchannel activity. Likewise, addition of 150 lM guano-sine 5¢-[b-thio]diphosphate (GDPbS) or adenosine 5¢-[c-thio]triphosphate (ATPcS) failed to activate KIRC1,indicating nucleotide speci®city of the e�ect. A secondK+ channel, activated by Gpp(NH)p (KIRC2) withgating properties clearly di�erent from the ®rst one wasless frequently observed. Four di�erent substates couldbe identi®ed; the main level had a conductance of about2 pS. Gating below the Nernst potential of K+ (EK) wasvoltage-independent. The channel closed at potentialsmore positive than EK. A third, hyperpolarization-activated K+ channel, KIRC3, with a low open prob-ability was encountered in inside-out patches. It had a

conductance of 45 pS in 100 mM K+. Channel activitywas not a�ected by the addition of G-protein modula-tors. Moreover, slowly activating inward currents car-ried by K+ were recorded in several patches that areascribed to a `subpicosiemens conductance'. NeitherGDPbS nor Gpp(NH)p appeared to have an e�ect onthe currents. Whole-cell measurements with these G-pro-tein modulators included in the pipette solution were ingeneral agreement with the results obtained on cell-freepatches. A statistical evaluation revealed that time-dependent inward currents were larger when the G-pro-tein activator Gpp(NH)p was included in the pipettemedium compared to measurements with the inhibitorGDPbS. With the GTP analogue, an additional instan-taneous component was elicited that was ascribed toKIRC2 activity. Data are discussed with respect to theputative role of G-proteins in conveying hormonalsignals. Regulation by G-protein may either serve to®ne-tune K+ uptake by xylem parenchyma cells or toinitiate depolarization, followed by salt-e�ux throughdepolarization-activated cation and anion channels.

Key words: G-protein ± Hordeum (K+ channels) ± K+

inward recti®er ± Patch-clamp ± Root ± Xylemparenchyma

Introduction

In a series of recent studies, new insight was gained intothe biophysical mechanisms of K+ acquisition by theroot and subsequent K+ transport to the shoot. Majoradvances have been made by applying the patch-clamptechnique to protoplasts derived from root cells and bythe cloning and functional reconstitution of K+ trans-porters. The high- and low-a�nity uptake systems forK+ in root hairs and the cortex, previously establishedby ¯ux measurements (Epstein and Hagen 1952), havebeen identi®ed as a H+-K+ symporter and one orseveral inward-rectifying K+ channels, respectively

Planta (1997) 203: 506±516

Two inward K+ channels in the xylem parenchyma cellsof barley roots are regulated by G-protein modulators througha membrane-delimited pathway

Lars H. Wegner*, Albertus H. De Boer

Faculty of Biology, Department of Genetics, Section Plant Physiology Vrije Universiteit Amsterdam, BioCentrum, De Boelelaan 1087,NL-1081HV Amsterdam, The Netherlands

Received 11 October 1996 /Accepted: 21 April 1997

*Present address: Lehrstuhl fuÈ r Biotechnologie, UniversitaÈ t WuÈ rz-burg, Biozentrum am Hubland, D-97074 WuÈ rzburg, Germany

Abbreviations: ABA = abscisic acid; ATPcS = adenosine 5¢-[c-thio]triphosphate; Ek = Nernst potential of K+; GDPbS = gua-nosine 5¢-[b-thio]diphosphate; Gpp(NH)p = guanosine 5¢-[b,c-imido]triphosphate; GTPcS = guanosine 5¢-[c-thio]triphosphate;KIRC = K+ inward-rectifying channel; KORC = K+-selectiveoutward-rectifying conductance

Correspondence to: A.H. de Boer,E-mail: [email protected]; Fax: 31(20)444 7155

(Schachtman et al. 1991; Findlay et al. 1994; Gassmannand Schroeder 1994; Maathuis and Sanders 1994, 1995;Roberts and Tester 1995). A large fraction of K+ that istaken up at the cortex moves radially into the stele via asymplasmic pathway and is subsequently loaded into thedead xylem vessels for transport to the shoot (seeClarkson 1993; Marschner 1995 for recent reviews). Sofar, little is known about the mechanisms involved in ionexchange between the xylem sap and the surroundingtissue, the xylem parenchyma. Recently, however, thesecells have become accessible to electrophysiologicalstudies with the patch-clamp technique (Wegner andRaschke 1994), and a K+-selective outward-rectifyingconductance (KORC), a non-speci®c outward-rectifyingconductance (NORC), a K+ inward recti®er (KIRC)and anion channel activity in the plasma membrane ofthese cells have been characterized (Wegner andRaschke 1994; Wegner et al. 1994). Based on thesestudies, xylem loading of K+ has been suggested tooccur by a passive release via KORC, and xylemperfusion with the speci®c K+-channel blocker tetra-ethylammonium (TEA+) seems to con®rm this hypoth-esis (data not shown). The resorption of K+ from thexylem, on the other hand, is thought to be mediated bythe K+ inward recti®er (Wegner et al. 1994) andenergized by the activity of the proton pump (De Boerand Prins 1985).

The ®nding that xylem parenchyma cells in roots areequipped with channels for both K+ uptake and releaseprovokes the question of how these conductances arefunctionally coordinated in the root to balance net K+

¯ux to the transpiration stream. One regulating factormay be the K+ concentration in the xylem itself thatstrongly a�ects gating of KORC (data not shown) and,in seminal roots of barley, seems to be coupled to theshoot demand by the amount of K+ that is recirculatedto the root via the phloem (Drew et al. 1990). Further-more, K+ uptake and release by the xylem parenchymais susceptible to hormonal control by auxin and abscisicacid (ABA) indicating that ion transport is stronglyregulated by external signals; K+ resorption from thexylem sap was shown to be stimulated by auxin inhypocotyls of Vigna unguiculata (De Boer et al. 1985),and ABA was reported to have either stimulating orinhibiting e�ects on ion transport to the xylem, depend-ing on the experimental conditions (Pitman et al. 1974;Behl and Jeschke 1979).

Since xylem parenchyma cells should be a primetarget of signalling substances transported by the tran-spiration stream (ABA, auxin, cytokinin, systemin etc.)we decided to investigate the involvement of G-proteinsin the regulation of ion-channel activity in these cells.Elaborate work on animal systems, e.g. the muscarinicK+ channel, has revealed that ion channels are a majortarget of G-protein action (Brown and Birnbaumer1990; Clapham 1994). As for plants, a number of recentreports suggest that this may also be true for the K+

inward recti®er in guard cells (Fairley-Grenot andAssmann 1991; Wu and Assmann 1994; Armstrongand Blatt 1995; Kelly et al. 1995) and the K+ outwardrecti®er in mesophyll cells (Li and Assmann 1993). Using

the patch-clamp technique, Fairley-Grenot and Assm-ann (1991) observed an increase in inward whole-cell K+

currents in guard-cell protoplasts when the G-proteininhibitor guanosine 5¢-[b-thio]diphosphate (GDPbS) wasincluded in the pipette medium, and an inhibition of thiscurrent when, instead they used guanosine 5¢-[c-thio]-triphosphate (GTPcS) which locks G-proteins in theiractive form. Recently, Kelly et al. (1995) have repeatedwhole-cell experiments on guard-cell protoplasts usingG-protein modulators and obtained data that are atvariance with those reported by Fairley-Grenot andAssmann. They observed an inhibition, rather thanactivation, of inward-rectifying K+ channels by GDPbSat low cytoplasmic Ca2+ concentrations and opposingreactions of individual guard-cell protoplasts at highcytoplasmic Ca2+ on treatment with GTPcS, indicatingthat G-protein action may be more complicated, andthat both activating and inactivating G-proteins may bepresent in guard cells.

In this study, we show for the ®rst time that theplasma membrane of xylem parenchyma cells containsfour types of inward-rectifying K+ channel, two ofwhich appear to be activated by G-protein modulators.It should be noted that we interpret our pharmacologicaldata obtained with di�erent G-nucleotides in terms ofregulatory mechanisms on channels exerted by G-pro-teins, in accordance with the literature on both animaland plant cells (Gilman 1987; Fairley-Grenot andAssmann 1991). This leaves open the possibility thatnucleotides act directly on the channel rather than viaactivation or inactivation of G-proteins.

Materials and methods

Plant cultivation and protoplast isolation. Barley (Hordeum vulgareL. cv. Apex; Cebeco Saaten, Celle, Germany) was cultivated asdescribed earlier (Wegner and Raschke 1994). Likewise, protoplastisolation was generally performed as described by Wegner andRaschke (1994). To optimize the selectivity of the procedure forisolation of xylem parenchyma protoplasts, stele segments fromnodal roots 2±3 cm above the root tip were used. Cell walls wereremoved selectively by an enzymatic treatment with an enzymecocktail containing 2% (w/v) cellulase Onozuka R10 (YakultHonsha, Tokyo, Japan), 0.02% (w/v) pectolyase Y-23 (SeishinPharmaceutical, Tokyo, Japan), 2% (w/v) BSA, 10 mM Na-ascorbate, 20 mM glucose, 20 mM sucrose and 1 mM CaCl2. ThepH was adjusted to 5.5. Mannitol was added to an osmolality of500 mosmol á kg-1. After incubation, the suspension was ®lteredthrough a 100-lm mesh and rinsed with a wash medium containing460 mM mannitol, 20 mM sucrose, 20 mM glucose and 1 mMCaCl2. Protoplasts were enriched by two centrifugation steps(10 min at 100 g) and the pellet of the second step was resuspendedin 100 ml wash medium for patch-clamp experiments. Protoplastswere either used immediately or stored overnight at 13 °C in washmedium. No obvious di�erences between ``fresh'' and storedprotoplasts were observed.

Electrical recording and solutions. Conventional patch-clamp ex-periments were performed in the inside-out, cell-attached andwhole-cell con®guration. Pipettes were pulled from borosilicateglass capillaries (Kimax 51; Kimble Products, Vineland, N.J. USA).They had a resistance of about 5 MW for whole-cell experimentsand 20 MW for inside-out experiments in symmetrical 100 mMKCl. For voltage clamp an Axopatch 200A ampli®er (Axon

L.H. Wegner and A.H. De Boer: G-nucleotide-activated K+ channels 507

instruments, Foster city, Calif, USA) was used. Data were storedand pulse protocols were generated on a personal computer (Vectra386; Hewlett-Packard, Palo Alto, Calif., USA) using software fromCambridge Electrical Design (EPC software package, CED,Cambridge, UK), that was connected to the ampli®er via a AD/DA converter, also from CED.

Standard solutions for inside-out recordings were composed asfollows: Pipette: 50 or 100 mM KCl, 10 mM Mes, 2 mM MgCl2,1 mM CaCl2, pH 5.8. Bath: 250 mM K-glutamate, 2 mMMgATP,2 mM EGTA or N-hydroxyethylethylenediaminetriacetic acid(HEDTA), 10 mM Bis-Tris Propane (BTP), pH 7.2 (Mes). Thefree Ca2+ concentration was either adjusted to 150 nM or 1.5 lMwith Ca-gluconate using EGTA or HEDTA as a bu�er, respec-tively. The free Mg2+ concentration was adjusted to 2 mM usingMgCl2. If other solutions were used, their composition is given inthe legend to the ®gures. For whole-cell experiments, the pipettesolution contained 112 mM K-glutamate, 8 mM KOH, 10 mMHepes, 2 mM HEDTA, 2 mM MgATP, 0.03 mM Ca-gluconate(1.5 lM free Ca2+), 1.75 mM MgCl2 (2 mM free Mg2+), pH 7.2(Mes); the bath contained 30 mM KCl, 1 mM CaCl2, 2 mMMgCl2, 10 mM Mes, pH 5.8 (Hepes). Guanosine 5¢-[b,c-imido]tri-phosphate (Gpp(NH)p, tetrasodium or tetralithium salt), adeno-sine 5¢-[c-thio]triphosphate (ATPcS, tetralithium salt) and GDPbS(trilithium salt) were obtained from Fluka (Buchs, Switzerland) anddissolved in 100 mM Tris-Mes pH 7.2. All nucleotides were addedto the patch medium on the day of the experiment. Stock solutionswere stored at )20 °C for maximally one week. The amount ofCa2+ to be added to maintain a given free concentration wascalculated using the program ``Calcium'' (FuÈ hr et al. 1993).Activities of ions were calculated from concentrations accordingto Robinson and Stokes (1968). Liquid junction potentials weredetermined as described previously (Neher 1992) and corrected for,if the value exceeded 2 mV.

Data analysis. For analysis of pulsed whole-cell and patch data,software from CED was employed. Continuous recordings in theinside-out con®guration were evaluated with the program TRAN-SIT, provided by A.M.G. van Dongen Medical Center, Depart-ment of Pharmacology, Duke University Durham, N.H., USA;Van Dongen 1996). Open probabilities of channels (po) andchannel activity (n*po) were determined from the dwell time perlevel according to n*po �

Pn � an � 0 n*t with n = number of channels

that were open simultaneously; tn = fraction of time in which nchannels are open simultaneously; a = maximal number of chan-nels observed simultaneously. The amplitude of unitary currentswas obtained from amplitude histograms, either using all datapoints or sorted by the channel detection procedure of TRANSIT.

Sign convention. Membrane potentials are de®ned as the voltageon the cytoplasmic side of the membrane with respect to thephysiological outside. Inward currents in the physiological sense,irrespective of the technical direction, are shown as downwardde¯ections.

Results

Two inwardly rectifying K+ channels, KIRC1 andKIRC2, that are activated by the pharmacologicalG-protein activator Gpp(NH)p.

Activation of KIRC1 by Gpp(NH)p. In the majority ofinside-out patches, channel activity at hyperpolarizationwas very low (po < 0.01%) in the absence of Gpp(NH)p(Fig. 1; exceptions are mentioned below). We screenedfor channel activity by imposing hyperpolarizing pulseprotocols from a holding potential of 0 mV (usuallylasting 4 s) and by a prolonged clamp of the membraneto various potentials (usually 2±4 min per voltage).

When the pharmacological agent was applied to thepatch at a ®nal concentration of 150 lM, either by bathperfusion or by adding a small aliquot of a stocksolution to the bath, K+-channel activity was elicited.The more frequently observed channel (Fig. 1) isdenoted as K+ Inward-Rectifying Channel 1 (KIRC1).This channel had a conductance of 6.2 pS at 30 mMexternal K+, 7.3 pS at 50 mM external K+ and 8.3 pSat 100 mM external K+ (Fig. 2a). Channel activity wasobserved within 2 min when Gpp(NH)p was added bybath perfusion (in order to disperse the agonist evenly).In 14 out of 20 inside-out patches, KIRC1 channelscould be elicited by adding Gpp(NH)p to the bath(70%). Channel activation appeared to be reversible;removing the agonist from the bath by prolongedwashing resulted in a loss of channel activity (Fig. 3).Potassium selectivity was established by rapid voltagescans of the open channel (Fig. 2b). The voltage atwhich the direction of single-channel currents reversedwas close to the Nernst potential of potassium(Erev = )14 mV versus EK = )20 mV), indicating that

Fig. 1. Activation of barley root KIRC1 by Gpp(NH)p in an inside-out patch. Representative current traces before and after addition of150 lM Gpp(NH)p to the bath at three di�erent potentials each.Standard solutions were used with 100 mM KCl in the pipette. FreeCa2+ in the bath was bu�ered to 150 nM with 2 mM EGTA.Continuously recorded currents were ®ltered at 500 Hz and sampledat 2 kHz. Note that channel activity increases with a hyperpolariza-tion of the patch.Dashed lines indicate the current level correspondingto the closed state and up to three channels being open simultaneous-ly. An arrowhead at the right of the traces marks the closed level

508 L.H. Wegner and A.H. De Boer: G-nucleotide-activated K+ channels

currents were mostly carried by this ion. Note thatsolutions were designed in such a way that the equilib-rium potentials of Cl) and Ca2+ were far negative andpositive from EK, respectively. The position of thereversal potential indicates that the permeability of thechannel for these ions is very low. In Fig. 2c, the voltagedependence of channel activity in one inside-out patch isshown. The open probability was extrapolated from thefraction of time that the channel resided in the openstate. It strongly increased with a hyperpolarization ofthe patch below about )100 mV. In other patches, ananalysis of gating properties of the channel was ham-pered by the fact that a slow rundown was superimposedon the response of KIRC1 to subsequently applied

voltages. Activation kinetics of the channel were inves-tigated by repeatedly applying hyperpolarizing voltagesteps (from )50 to )180 mV). Channel activity elicitedby this protocol was averaged for 47 traces (Fig. 2d).The averaged current response re¯ected the mean timecourse of activation. A time constant of 123 ms wasobtained by ®tting the trace with a single exponentialfunction.

The channel KIRC1 is not activated by ATPcS andGDPbS. For a G-protein-activated channel, the e�ect ofGTP and GTP derivatives should be speci®c and notreplaceable by other nucleotides. In the experimentsreported above, 2 mM ATP was present in the control,

Fig. 2a±d. Properties of KIRC1. a Unitary currents from KIRC1 as a function of the patch potential. Data from six inside-out patches with theG-protein activator added to the bath were pooled. Experiments were performed with standard solutions (100 mM K+ in the pipette, 150 nMfree Ca2+). The mean conductance was determined to be 8.3 pS (bold line). It ranged from 6.3 to 11.4 pS (thin lines denoting the upper and lowerlimit). b Voltage scan performed on an inside-out patch after application of Gpp(NH)p. At least three channels are simultaneously active in thepatch (see smooth lines). The scan over the voltage range shown lasted 300 ms and was applied from a holding potential of 4 mV. All linesintersect at the reversal potential of the channel that was determined to be)14 mV for this scan. The Nernst potential of K+ was )20 mV, thoseof the other ions were )87 mV (Cl)), and +180 mV (Ca2+), indicating a high selectivity for K+. Further experimental conditions were asdescribed in Fig. 1. c Voltage dependence of channel activity (n * po) in an inside-out patch in the presence of Gpp(NH)p. Pipette solution:30 mM K-glutamate, 1 mM CaCl2, 2 mM MgCl2, 10 mM Hepes. The osmolality was adjusted to 500 mosmol á kg)1 with mannitol, pH � 5:8(Mes). Bath solution: 120 mM KCl, 1.5 lM free Ca2+ (31 lM total Ca2+, calculated according to FuÈ hr et al. 1993), 2 mM free Mg2+, 150 lMGpp(NH)p, 2 mMHEDTA, 2 mMMg-ATP, pH � 7:2. Mannitol was added to an osmolality of 500 mosmol á kg)1. d Activation of KIRC1 byhyperpolarizing voltage steps from)50 to )180 mV (four examples and average of 47 traces at the bottom). The averaged trace was ®tted with anequation of the form I � Iaverage*�1ÿ exp�ÿt=s��, with s � 123 ms (smooth line). Quality of the ®t was R � 0:87. The ®t was not improved by theintroduction of a second time constant. For experimental conditions see c. Data were ®ltered at 500 Hz and sampled at 2 kHz. Dashed linesindicate the closed state (see arrowhead at the right of the trace) and the open state


but failed to activate the channel. This indicates thatchannel activation by Gpp(NH)p was not due to anunspeci®c requirement for nucleotides. To test thisassessment more rigorously, ATPcS, a non-hydrolyzablederivative of ATP, was applied. Inside-out patches wereestablished in a bath solution containing 150 lMATPcS. As shown in Fig. 4, no channel activity wasrecorded under these conditions. As a control for thepresence of KIRC1 in the patch, Gpp(NH)p wassubsequently substituted for ATPcS. In two out of fourpatches, KIRC1 activity could be elicited by bathperfusion with the GTP derivative (Fig. 4). In twoinside-out patches, large-hyperpolarization-activated in-ward currents, reminiscent of whole-cell currents wereobserved after the G-protein modulator had been added(data not shown). This may indicate that channelscluster under certain conditions.

Using the same approach, GDPbS was likewiseshown to be ine�ective in stimulating channel activity.Binding of GDPbS locks G-proteins in their inactivestate. The presence of KIRC1 channels in the patchesexposed to GDPbS was demonstrated by substitutingGpp(NH)p for GDPbS by bath perfusion; channelactivity was evoked in two out of four experiments(data not shown).

Activation of KIRC2 by Gpp(NH)p. In several inside-out patches, a second type of channel (KIRC2) wasactivated by Gpp(NH)p; KIRC2 clearly di�ered fromKIRC1 with respect to its conductance and gatingproperties. Representative recordings are shown inFig. 5. Due to its low conductance, we could identifyKIRC2 activity only in recordings with a low noise level

(high seal resistances � 10 GW) and with no otherchannel activity (e.g. KIRC1) superimposed. We there-fore cannot determine the frequency of channel activa-tion by Gpp(NH)p in inside-out patches. Four distinctconductance levels could be distinguished (Fig. 6a±c).Transitions between these conductance levels were notrandom, indicating that they represented substates of thesame channel: from the closed state the channel switchedmore frequently to one of the two larger conductancestates, but then moved rapidly to a long-lived smallerstate (Fig. 6d,e; 1.4 pS with 50 mM K+ and 2 pS with120 mM K+ in the pipette). This e�ect leads to a``spiky'' appearance of channel openings. Virtually nochannel openings were observed at potentials positive ofEK (Fig. 5), indicating that gating of KIRC2 wasstrongly inward rectifying. By voltage scans of the openchannel, a reversal potential close to EK was obtained(Fig. 6f, central panel), indicating a high selectivity forK+. Note that scanning of open channels renders morereliable information on the selectivity than extrapolationfrom current amplitudes in continuous recordings (com-pare Fig. 6d and f ). Only in a few scans, (about 20%, seecentral panel, Fig. 6f ), was the channel observed to pass

Fig. 3. Activation of KIRC1 in inside-out patches is reversible whenthe Gpp(NH)p is washed away. Current traces result from ahyperpolarizing voltage step from )50 to )150 mV before and afteraddition of Gpp(NH)p (®nal concentration 150 lM) to the bath andafter washing the agonist away (6 min bath perfusion). Solutions asindicated in the legend to Fig. 2c (apart from Gpp(NH)p being leftout in the bath for control and wash). Data were ®ltered at 500 Hzand sampled at 2 kHz. The closed state is marked by the arrowheadplus dashed line, whereas the open state is marked by a dashed linealone

Fig. 4. Another non-hydrolysable nucleotide, ATPcS, does notactivate KIRC1. Current traces from recordings on an inside-outpatch with 150 lM ATPcS in the bath and after substituting it withGpp(NH)p at the same concentration. Standard bath and pipettesolution with 100 mMKCl in the pipette and 1.5 lM free Ca2+ in thebath. The ®lter was set to 500 Hz with a sample frequency of 2 kHz.Dashed lines, closed state and open states, respectively. Arrowheads,closed states


an outward current. In the majority of the scans, single-channel currents elicited by voltage ramps were stronglyinward rectifying (Fig. 6f, bottom panel); probably, thiswas no property of the channel pore itself but resultedfrom an immediate transition to the closed state upon

depolarization beyond EK and instantaneous re-openingfollowing repolarization (see bottom panel of Fig. 6f,arrowhead). Gating was voltage independent at poten-tials negative to EK (Fig. 6e). Again, ATP and its non-hydrolysable analogue, ATPcS, were ine�ective inactivating the channel (data not shown), as should beexpected for a channel regulated by G-proteins.

Inwardly rectifying K+ conductances that do not requireG-protein modulators for activation. A third type ofinward-rectifying K+ channel (KIRC3) was observed ininside-out patches (Fig. 7). In most recordings, only veryfew openings were observed both in the presence andabsence of Gpp(NH)p (po < 0.01; the traces shown inFig. 7 are not representative in that respect, but werechosen to give an impression of the kinetics and voltagedependence of the channel), suggesting that it was notsubjected to regulation by G-proteins. It could easily beidenti®ed since it gave rise to relatively large unitarycurrents. As shown in Fig. 7, channel activity increasedwith hyperpolarization. Two smaller, infrequent sub-states were observed (see arrowheads Fig. 7a). Theconductance was 45 pS with 100 mM K+ in the pipette(i.e. the external face of the membrane) and 250 mM inthe bath (Fig. 7). This channel may be identical to a30 pS K+ channel at 30/120 mM K+ previously identi-®ed in outside-out patches from the same membrane(Wegner and Raschke 1994). The generally low openprobability precluded a more-detailed analysis ofKIRC3.

Additionally, we noticed slow inward currents devel-oping in some patches when hyperpolarizing voltagesteps were imposed from a holding potential of 0 mV(data not shown). Tail currents could be recorded thatturned close to EK, suggesting that the currents weremainly carried by K+ and did not result from deterio-rating seal resistance. These currents are denoted here asa ``subpicosiemens conductance'' in the sense ofVogelzang and Prins (1995), either re¯ecting low-con-ductance channel activity for which unitary current stepscould not be resolved, or activity of a carrier. Theactivation potential of these currents was around )50mV. This subpicosiemens conductance was only ob-served in four inside-out patches but seemed to be morefrequent in cell-attached recordings (approx. 10%),suggesting that a cytosolic factor required for activationmay be lost by patch excision. This current appeared notto be a�ected by modulators of G-protein activity.

Whole-cell currents. Channel activation by Gpp(NH)pas observed in inside-out patches should also bedetectable in whole-cell recordings when G-proteinmodulators are included in the pipette solution. There-fore, whole-cell currents were recorded with 150 lMGpp(NH)p and 400 lM GDPbS, respectively, added tothe internal medium, and a statistical approach was usedto analyze the e�ects of the G-protein modulators.Previously, time-dependent inward K+ currents hadbeen observed in a fraction of protoplasts with no GTPanalogue added to the pipette (about 30% at low accessresistance compared with about 70% at high access

Fig. 5. A second K+ channel, KIRC2, is activated by the G-proteinactivator. Traces recorded before and after application of Gpp(NH)pto an inside-out patch are depicted, showing characteristic, slow gatingof the channel. Data were ®ltered at 200 Hz. Standard solutions wereapplied with 50 mMKCl in the pipette. Ca2+ in the bath was adjustedto 150 nM. Distinct states of the channel are indicated by dashed lines,the closed state is additionally marked by arrowheads


Fig. 6a±f. Properties of KIRC2. a All-point histogram obtained from the experiment shown in Fig. 5 ()172 mV). One open state can be clearlydistinguished (denoted as o) from the closed state (c). The asymmetric ¯anks of the open-state distribution indicate the presence of several openstates that cannot be resolved properly from this plot.b Idealization of a trace segment with the `TRANSIT' algorithm for multiple-state channels(Van Dongen 1996). The smooth line represents the idealized trace. c Distribution of current amplitudes obtained after idealization of the trace(42 s). Note that every level resulting from more than two data points contributed one observation, irrespective of its duration. Closed states wereomitted. The superimposed curve represents the probability density function (pdf) ®tted to the histogram. The pdf forms the sum of four Gaussiandistributions with peaks at 0.12, 0.23, 0.33 and 0.43 pA, respectively. d Current voltage curves were constructed by plotting current amplitudes(obtained from pdf ®ts as shown in c) as a function of the membrane potential. Four di�erent substates could be distinguished (di�erent symbols).By linear regression, conductances were determined to be 0.14 (s), 1.4 (d), 2.12 (r) and 2.72 ()) pS, respectively (solid lines). e Occupancyprobabilities of the di�erent substates (see d for the symbols, same patch), that were voltage-independent below)50 mV as indicated by the lines.The continuous line corresponds to the 1.4 pS subconductance; the dashed line corresponds to the 2.12 pS subconductance. f Two scans of KIRC2single-channel currents obtained by voltage ramps (same patch as in a; see top panel for the protocol). Data were corrected for leak currents bysubtracting averaged ``empty'' scans without channel activity. In most scans (80%), the channel appeared to be strongly inward rectifying (lowertrace). In some cases, however, KIRC2 carried an outward current as well (upper trace), indicating that ``recti®cation'' was not intrinsic but camefrom a rapid closure of the channel at depolarization and an immediate opening on repolarization (seearrowheads at the traces). From scans likethe upper one, the reversal potential of KIRC2 was determined by linear regression (smooth line). Note that the reversal potential was close to EK,as indicated by the vertical line


resistance, data not shown). Here, only measurements atlow access resistance (up to 20 MW) were selected for acomparison of inward K+ currents in protoplaststreated with the G-protein activator and inhibitor,respectively, since the chance of ®nding inward currentswithout adding a GTP analogue to the pipette was lowerunder these conditions. Moreover, a low access resis-tance allows a rapid exchange between cytoplasm andpipette solution. Current amplitudes were recordedabout 2 min after the whole-cell con®guration had beenestablished; repeated hyperpolarizations in several ex-periments indicated that currents had reached a stablelevel by that time. Figure 8a shows representative whole-cell current traces upon hyperpolarizing voltage steps inthe presence of the G-protein activator and inhibitor,respectively. With Gpp(NH)p, large inwardly rectifying

currents were recorded. Note that these currents hadboth an instantaneous and a slowly activating compo-nent. Double-pulse experiments indicated K+ selectivityof delayed currents (not shown). When GDPbS wasadded to the pipette, time-dependent inward currentstypically were small or absent and no inward-rectifyingcomponent was observed. Results are summarized inFig. 8b,c. Notably, in all of the cells treated withGpp(NH)p, both instantaneous and time-dependentinward currents were recorded (n = 10, protoplastsfrom four di�erent batches). Current density variedconsiderably among these protoplasts [Fig. 8b (see alsothe inset) and 8c). Inward currents were signi®cantlysmaller in seven out of nine protoplasts dialyzed withGDPbS (protoplasts from two di�erent batches). In twoprotoplasts, however, large time-dependent inward

Fig. 7a±b. Characterization ofKIRC3. aKIRC3 activity in an inside-out patch evoked by hyperpolarizingvoltage steps (see inset). Downwardde¯ections represent channel openings.Data were ®ltered at 1 kHz (samplerate 3 kHz). Standard solutions with100 mM KCl in the pipette were used(1.5 lM free Ca2+ in the bath). bCurrent voltage relation obtained fromthe traces shown in a. The conductancewas 45 pS, as determined by linearregression (solid line). Arrowheadsmark infrequent substates. The Nernstpotential of K+was )20 mV and thoseof Cl) and Ca2+ were )87 mV and+180 mV, respectively

Fig. 8a±c. Whole-cell currents withG-protein modulators in the pipette. aInward currents elicited by a voltageprotocol ranging from )50 to)210 mV (holding potential )30 mV)with 150 lM Gpp(NH)p or 400 lMGDPbS in the pipette medium. Datawere ®ltered at 500 Hz (sample rate2 kHz). For solutions, see Materialsand methods. b Time-dependent in-ward currents in the presence ofGpp(NH)p (solid symbols, mean ofeight experiments) and GDPbS (opensymbols, mean of seven experiments).Error bars represent SD. Inset, sametype of plot, but di�erent scaling.Data from two cells treated withGDPbS and one cell treated withGpp(NH)p. The currents in these cellswere several times larger than thosefound in the other protoplasts. Sincethese currents were considered tore¯ect a separate situation, they werenot included in the statistics for themain ®gure. c Instantaneous inwardcurrents with GDPbS (open symbols)and Gpp(NH)p, (solid symbols), res-pectively


currents were recorded (Fig. 8b, inset) with a currentdensity similar to that recorded with Gpp(NH)p in othercells. There was no di�erence in activation kineticsbetween currents recorded in the presence of Gpp(NH)pand GDPbS (data not shown).

Discussion

Regulation of inward-rectifying K+ conductances: whole-cell and single-channel experiments. This study, togetherwith a previous publication (Wegner and Raschke 1994),demonstrates the existence of at least three di�erentinward-rectifying K+ channels (KIRC1±3) and ofanother hyperpolarization-activated K+ conductancein the plasma membrane of xylem parenchyma cellsfrom barley roots. For two of the channels (KIRC1 andKIRC2), pharmacological evidence for activation byG-protein modulators has been obtained. Note that thepresence of heterotrimeric G-proteins in the plasmamembrane of root cells of a monocotyledonous planthas previously been shown using a�nity chromatogra-phy with biotinylated GTP (De Boer et al. 1994).G-protein-mediated activation of inward-rectifying K+

channels is a well studied phenomenon in several typesof excitable animal cells, especially atrial cells. Unlikethose cells (Breitwieser and Szabo 1985), KIRC1 activitywas lost in inside-out patches from xylem parenchymacells after washing away the agonist by prolonged bathperfusion (Fig. 3). Since channel activity had been stablein this patch for 40 min in the presence of Gpp(NH)p, itseems unlikely that the loss of channel activity after theremoval of the agonist was due to a sudden, unspeci®crundown. Apart from KIRC1 and KIRC2, two furtherconductances (KIRC3 and the subpicosiemens conduc-tance) were observed that appear not to be regulated byG-proteins, at least not by a membrane-delimitedpathway.

Our ®ndings indicate that four separately regulatedpathways for K+ uptake coexist in the plasma mem-brane of xylem parenchyma cells, contributing to inwardK+ currents. This may provide a mechanism to ®ne-tunethe magnitude of whole-cell currents, adjusting them tophysiological requirements. For example, the activity ofKIRC3 and the subpicosiemens conductance may con-tribute to a G-protein-independent component of in-ward K+ current. Note that some inward current isretained when G-proteins are inhibited by 400 lMGDPbS in about half of the protoplasts; in two cells,these ``residual'' currents were large. In general, anenhancement of inward currents by Gpp(NH)p, aspredicted from single-channel experiments, could beobserved. Interpretation of the data is complicated bythe fact that whole-cell inward-rectifying K+ currents inthe absence of GTP analogues were even more variablein the experimental conditions chosen here. At lowaccess resistance in the whole-cell con®guration, inwardcurrents were absent in the majority of protoplasts(about 70%). In the remaining fraction, K+ inwardcurrents as large as those recorded in the presence ofGpp(NH)p could occur. This variability precluded a

statistical approach to the data. At present, we cannotdecide if G-protein-independent conductances were un-regulated in protoplasts that showed inward K+ cur-rents in the absence of Gpp(NH)p, or if G-proteins werekept in an active state by residual, cellular GTP. Kellyet al. (1995) reported similar problems with analyzingK+ currents in guard cells (but see also Fairley-Grenotand Assmann 1991). However, one important predictionthat could be made from single-channel studies onexcised patches from xylem parenchyma cells for whole-cell experiments, is that inward currents should alwaysbe present in whole-cell recordings when Gpp(NH)p isincluded in the pipette medium, even in conditions likelyto inactivate the K+ inward recti®er such as whole-cellclamps at a low access resistance. Indeed, in recordingswith access resistances ranging from 0 to 20 MW, inwardK+ currents occurred in 100% of the cells treated withGpp(NH)p but only in about 50% of the cells treatedwith GDPbS. Another important prediction is that thecontribution of KIRC1 and KIRC2 to G-protein-activated inward currents can be separated on the basisof their kinetics and voltage dependence. Inward cur-rents mediated by KIRC2 are expected to activateinstantaneously with negative-going pulses and to in-crease linearly with the applied voltage (Fig. 6e,f).Indeed, such a component was observed in whole-cellrecordings when Gpp(NH)p was added to the pipettemedium (Fig. 8c). In contrast, KIRC1 gives rise tostrongly voltage-dependent, slowly activating currents(compare Fig. 2c,d and Fig. 8c). It may be concluded,that whole-cell data were generally in agreement with®ndings on the single-channel level. Whole-cell data donot allow any conclusion as to whether G-proteins alsoact on inward-rectifying K+ channels via other regula-tory pathways that are not membrane-delimited. Theexistence of such routes can, however, not be ruled out.

G-protein regulation of ion channels in guard cells versusxylem parenchyma cells. Thus far, all studies except one(Li and Assmann 1993) on G-protein regulation of ionchannels in plants have been performed on guard cells(Fairley-Grenot and Assmann 1991; Wu and Assmann1994; Armstrong and Blatt 1995; Kelly et al. 1995).G-protein-mediated signalling in xylem parenchymacells, as in guard cells, is directed to inward-rectifyingK+ channels, whereas the outward recti®er remainsuna�ected (data not shown; this is in contrast tomesophyll cells, as reported by Li and Assmann 1993).Notably, G-proteins seem to have opposing e�ects inguard cells and xylem parenchyma cells on thosechannels: in guard cells, inward K+ currents (and theactivity of the corresponding K+ channel) are reducedby exposing the cytosolic side of the membrane to thenon-hydrolyzable G-protein derivative GTPcS (Fairley-Grenot and Assmann 1991; Wu and Assmann 1994),whereas in xylem parenchyma cells, channel activity isincreased by Gpp(NH)p. This indicates that a G-proteinis involved in channel inactivation in guard cells andchannel activation in xylem parenchyma cells. We noticethough that for guard cells, controversial data have beenreported suggesting that the situation may be more


complicated and that several G-proteins with an oppos-ing e�ect on K+-channel activity may exist (Fairley-Grenot and Assmann 1991; Kelly et al. 1995). Interest-ingly, G-proteins seem to act via a membrane-delimitedsignalling pathway in both guard cells and xylemparenchyma cells since GTP derivatives modulatedchannel activity in the inside-out con®guration whenadded to the bath (Wu and Assmann 1994). In guardcells, however, cytosolic Ca2+ may also be involved(Fairley-Grenot and Assmann 1991; Kelly et al. 1995)although this is still a matter of discussion (Armstrongand Blatt 1995). We have looked for an e�ect ofcytosolic Ca2+ on the channel activity described here;however, no evidence for a Ca2+-dependent regulationhas been found. This ®nding is in line with ourconclusion that the e�ect of G-protein modulators ismembrane delimited. In xylem parenchyma cells, theputative G-protein e�ect at the single-channel level ismore pronounced than the opposite e�ect occurring inguard cells. Here, considerable K+-channel activity isretained in the presence of a G-protein activator (theopen probability is reduced from 0.26 to 0.09 withaddition of GTPcS, Wu and Assmann 1994) whereas inxylem parenchyma cells, channel activity is induced fromalmost complete inhibition. In xylem parenchyma cells,however, other conductances exist besides those activat-ed by G-proteins, ``bu�ering'' the e�ect of G-proteins onwhole-cell K+ currents. Hence, in both xylem paren-chyma cells and guard cells, G-proteins have a limited,variable e�ect on K+currents. However, the mode ofaction di�ers and the result is disparate.

The regulation of inward K+ currents by G-proteins:physiological implications. A physiological interpretat-ion of the ®ndings reported here will be preliminary,since the signal conveyed by G-proteins is unknown, andwe are still far from understanding how ion transport toand resorption from the xylem are balanced to controlthe composition of the xylem sap. Intuitively, one wouldassociate an increase in inwardly rectifying K+-channelactivity with an increase in K+resorption. This, in turn,could reduce net K+transport to the xylem. However, amoderate change in inward K+-current density, byitself, tends to be compensated by a depolarization of theplasma membrane; for an increase in K+ uptake, protonexcretion by xylem parenchyma cells would have to beaccelerated likewise in order to maintain charge balance(see also discussion in Kelly et al. 1995, referring toguard cells). Depending on physiological conditions,however, K+ uptake may be blocked completely (com-pare Roberts and Tester 1995) unless K+ channels areactivated by a G-protein-mediated signal. Hence, onefunction of G-protein activation of the inward recti®ermay be to provide su�cient capacity to take up K+

under physiological conditions that entail G-proteinactivation in xylem parenchyma cells. Another functionmay be (counter-intuitively) to initiate salt e�ux bykeeping the membrane su�ciently depolarized so thatdepolarization-activated anion currents (KoÈ hler andRaschke 1995), and, in turn, the K+ outward recti®ercan come into action (Wegner and Raschke 1994).

In animal cells, (heterotrimeric) G-proteins conveyexternal signals that are mediated by a class of seven-helix receptors. A well-studied example is the activationof the muscarinic K+ channel in atrial cells, followingbinding of acetylcholine to the muscarinic acetylcholinereceptor (Yatani et al. 1987; Brown and Birnbaumer1990). Evidence is accumulating that G-proteins have asimilar function in plant cells. Recently, Armstrong andBlatt (1995) addressed this question using mastoparanand mas 7 as probes for interaction of a seven-helixreceptor with the inward recti®er in intact guard cells.They observed a current reduction that is in line with thepreviously published patch-clamp data for G-proteinmodulation of these currents. Possible in-vivo candi-dates for receptor-mediated signalling are the planthormones ABA and auxin, that also have a function inregulating ion ¯ux to and out of the xylem (seeIntroduction). Interestingly, short-term e�ects of ABAare opposite in guard cells and xylem parenchyma cells,leading to salt e�ux from guard cells but salt retentionin the xylem parenchyma. This is reminiscent of theopposing e�ects of G-proteins on K+channels. Studieson ion-channel regulation in root cells will also help usto understand how radial net ion ¯ux from the soilsolution to the xylem is maintained even when plants arenot transpiring. Recent work by Roberts and Tester(1995) on maize roots suggests that most cortical cellsare only equipped with inward-rectifying K+channels tomediate K+uptake, whereas 80% of the stelar cellsexclusively show outward-rectifying K+ currents, rem-iniscent of KORC, for K+ release to the xylem.However, depolarization-activated K+ channels havebeen recorded more frequently in cortical cells orrhizodermal cells from other species (Schachtman et al.1991; Vogelzang and Prins 1994, 1995; White andLemtiri-Clieh 1995), and this study on xylem parenchy-ma cells shows that several channels for K+ uptake existin these cells. Taken together, these observations provideevidence that cortical and stelar plasma membranes areboth capable of K+ uptake and release, but regulationof these processes is opposite in the two cells types.Transport of K+ is balanced towards K+ uptake in thecortex and K+ release in the xylem parenchyma.Recently, single-channel studies on cortical cells fromArabidopsis roots were undertaken by Maathuis andSanders (1995). They observed a 5-pS and a 40-pSconductance very reminiscent of KIRC1 and KIRC3,respectively. Interestingly, the 5-pS conductance wasrecorded in inside-out patches in the absence of aG-protein modulator, indicating that G-protein activa-tion was not required. Future work should be under-taken on cortical cells from barley to investigate whethera channel corresponding to KIRC1 is present in thesecells and if so, whether this channel is G-proteinregulated. Comparing regulatory mechanisms on ionchannels in cortical cells and xylem parenchyma cellswill help us to understand how net ion ¯ux in the intactroot is controlled.

The diversity of inward-rectifying K+ channels andconductances demonstrated by Maathuis and Sanders(1995) and in this study may also be taken as a warning


of several pitfalls inherent to a genetic approach tostudying the physiology of K+ uptake in plant cells.Undoubtedly, analysis of K+-channel properties byfunctional reconstitution (e.g. Schroeder 1994) is apowerful tool in transport physiology. So far, severalgenes encoding for inward recti®ers (e.g. KAT1 andAKT1) have been cloned and studied that way (e.g.Hoshi 1995). However, channel properties may notre¯ect K+-uptake properties of the cell if other K+

conductances that may not be structurally related to thecloned channel and may be subjected to di�erent modesof regulation contribute to it.

Thanks are due to the Studienstiftung des Deutschen Volkes for®nancial support to LHW.

References

Armstrong F, Blatt RM (1995) Evidence for K+ channel control inVicia faba guard cells coupled by G-proteins to a 7TMSreceptor mimetic. Plant J 8: 187±198

Behl R, Jeschke WD (1979) On the action of abscisic acid ontransport, accumulation, and uptake of K+ and Na+ in excisedbarley roots; e�ects of accompanying ions. Z P¯anzenphysiol95: 335±354

Breitwieser GE, Szabo G (1985) Uncoupling of cardiac muscarinicand b-adrenergic receptors from ion channels by a guaninenucleotide analogue. Nature 317: 538±540

Brown AM, Birnbaumer L (1990) Ionic channels and theirregulation by G-protein subunits. Annu Rev Physiol 52: 197±213

Clapham DE (1994) Direct G-protein activation of ion channels?Annu Rev Neurosci 17: 441±464

Clarkson DT (1993) Roots and the delivery of solutes to the xylem.Phil Trans R Soc London Ser B 341: 5±17

De Boer AH, Prins HBA (1985) Xylem perfusion of tap rootsegments of Plantago maritima: the physiological signi®cance ofelectrogenic pumps. Plant Cell Environ 8: 587±594

De Boer AH, Katou K, Mizuno A, Kojima H, Okamoto H (1985)The role of electrogenic xylem pumps in K+ absorption fromthe xylem of Vigna unguiculata: the e�ects of auxin andfusicoccin. Plant Cell Environ 8: 579±585

De Boer AH, van Hunnik E, Korthout HAAJ, Sedee NJA, WangM (1994) A�nity puri®cation of GTPase protein from oat rootplasma membranes using biotinylated GTP. FEBS Lett 337:281±284

Drew MC, Webb J, Saker LR (1990) Regulation of uptake andtransport to the xylem in barley roots; K+ distributiondetermined by electron probe x-ray microanalysis of frozenhydrated cells. J Exp Bot 41: 815±825

Epstein E, Hagen CE (1952) A kinetic study of the absorption ofalkali cations into barley roots. Plant Physiol 27: 457±474

Fairley-Grenot K, Assmann SM (1991) Evidence for G-proteinregulation of the guard cell inward K+ channel. Plant Cell 3:1037±1044

Findlay GP, Tyerman SD, Garril A, Skerett M (1994) Pump andK+ inward recti®ers in the plasmalemma of wheat rootprotoplasts. J Membr Biol 137: 99±107

FuÈ hr KJ, Warchol W, Gratzl M (1993) Calculation and control offree divalent cations in solutions used for membrane fusionstudies. Methods Enzymol 6: 70±81

Gassmann W, Schroeder JI (1994) Inward-rectifying K+ channelsin root hairs of wheat. Plant Physiol 105: 1399±1408

Gilman AG (1987) G-proteins: transducers of receptor-generatedsignals. Annu Rev Biochem 56: 615±649

Hoshi T (1995) Regulation of voltage dependence of the KAT1channel by intracellular factors. J Gen Physiol 105: 309±328

Kelly WB, Esser JE, Schroeder JI (1995) E�ects of cytosolic Ca2+

and limited, possible dual, e�ects of G-protein modulators onguard cell inward potassium channel. Plant J 8: 479±489

KoÈ hler B, Raschke K (1995) Xylem loading: activity of anionchannels in xylem parenchyma protoplasts from roots of barley.10th International Workshop on Plant Membrane Biology,Regensburg, Abstr. No. 64

Li W, Assmann SM (1993) Characterization of a G-protein-regulated outward K+ current in mesophyll cells of Vicia fabaL. Proc Natl Acad Sci USA 90: 262±266

Maathuis FJM, Sanders D (1994) Mechanism of high a�nitypotassium uptake in roots of Arabidopsis thaliana. Proc NatlAcad Sci USA 91: 9272±9276

Maathuis FJM, Sanders D (1995) Contrasting roles in iontransport of two K+ channel types in root cells of Arabidopsisthaliana. Planta 197: 456±464

Marschner H (1995) Mineral nutrition of higher plants, 2nd edn.Academic Press, London

Neher E (1992) Correction for liquid junction potentials in patch-clamp experiments. Methods Enzymol 207: 123±130

Pitman MG, LuÈ ttge U, LaÈ uchli A, Ball E (1974) Action of abscisicacid on ion transport as a�ected by root temperature andnutrient status. J Exp Bot 25: 147±155

Roberts SK, Tester M (1995) Inward and outward K+ selectivecurrents in the plasma membrane of protoplasts from maizeroot cortex and stele. Plant J 8: 811±825

Robinson RA, Stokes RM (1968) Electrolyte solutions. Butter-worth, London

Schachtman DP, Tyerman SD, Terry BR (1991) The K+/Na+

selectivity of a cation channel in the plasma membrane of rootcells does not di�er in salt-tolerant and salt-sensitive wheatspecies. Plant Physiol 97: 598±605

Schroeder JI (1994) Heterologous expression and functionalanalysis of higher plant transport proteins in Xenopus oocytes.Methods: A companion to Methods Enzymol 6: 70±81

Van Dongen AMJ (1996) A new algorithm for idealizing single ionchannel data containing multiple unknown conductance levels.Biophys J 70: 1303±1315

Vogelzang SA, Prins HBA (1994) Patch-clamp analysis of thedominant plasma membrane K+ channel in root cell proto-plasts of Plantago media L. Its signi®cance for the P and Kstate. J Membr Biol 141: 113±122

Vogelzang SA, Prins HBA (1995) Kinetic analysis of two simul-taneously activated K+ currents in root cell protoplasts ofPlantago media L. J Membr Biol 146: 59±71

Wegner LH, Raschke K (1994) Ion channels in the xylemparenchyma of barley roots. Plant Physiol 105: 799±813

Wegner LH, De Boer AH, Raschke K (1994) Properties of the K+

inward recti®er in the plasma membrane of xylem parenchymacells from barley roots: e�ects of TEA+, Ca2+ Ba2+ and La3+.J Membr Biol 142: 363±379

White PJ, Lemtiri-Clieh F (1995) Potassium currents across theplasma membrane of protoplasts derived from rye roots: Apatch-clamp study. J Exp Bot 286: 497±511

Wu WH, Assmann SM (1994) A membrane delimited pathway ofG-protein regulation of the guard cell inward K+ channel. ProcNatl Acad Sci USA 91: 6310±6314

Yatani A, Codina J, Brown AM, Birnbaumer L (1987) Directactivation of mammalian atrial muscarinic potassium channelsby GTP regulatory protein GK. Science 235: 207±211


two inward k channels in the xylem parenchyma cells a

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