Proc. Natl. Acad. Sci. USAVol. 91, pp. 6310-6314, July 1994Cell Biology

A membrane-delimited pathway of G-protein regulation of theguard-cell inward K+ channel

(IOn chne/membrane potnial/somaa/seondm r/Viafba L.)

WEI-HUA WU* AND SARAH M. ASSMANN*tThe Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138

Communicated by Emanuel Epstein, March 28, 1994

ABSTRACT GTP-binding protein (G-protein) regulationof inward ifig K+ chan in the plasma membrane ofVicia (Vida faba L.) guard cells has previously been demon-strated at the whde-cell level. However, whether a cytosolicsignal transduction chain is required for G-protein r tinof Ki ns Vicia ard cells, or in any plant cell type,remains unknown. In the present study, we assayed effects ofseveral G-protein regulators on inward K+ channels isolatedinside-out membrane patches from Vida guard cell protonplasts. Guanosine 5'-[rthloltrIphosphate, a nonhydrolyzableGTP analog that locks G protn into thefr activated state,dereased the open state probability (P.) of single Inward K+

. This decrease aPo,was accomnied by an easein one Of the closed time consats of the K+ channel. Guano-sine 5'-[j-thkltdphosphate, a GDP analog that locks G pro-teins into their Inactivated state, slightly ieased the P. of theinward K+ channel and shortened the closed time consants.Pertussis toxin and cholera tom, which ADP-rlbosylate Gproteins at differentsites, deceed the P. of the Inward K+chae. Our data indi te that G proteins can act via a

membrane-delimited pathway to regulate Inward K+ channelsin the guard-cell plasma membrane.

Stomatal movement, which regulates transpiration and pho-tosynthesis (1), results largely from changes in ion fluxesacross the guard-cell plasma membrane (2). Stomatal aper-tures are in turn regulated by environmental factors such aslight and humidity (1) and by internal factors such as abscisicacid and cytoplasmic Ca2+ concentration (1, 3). Some ofthese signals (4) are known to alter the activity of the plasmamembrane K+ channels (5), which mediate K+ flux into guardcells during stomatal opening, but the biochemical pathwaysthat link signal reception to channel modulation remainlargely unknown.One common mechanism in animal systems for the cou-

pling of membrane-associated receptors and ion channelsinvokes GTP-binding regulatory proteins (G proteins) assecond messengers (6-8). A receptor activated by a stimulusactivates a G protein, which then interacts directly eitherwith the channel or with membrane-bound or cytoplasmiceffectors, resulting in changes in channel activity (8, 9). K+channels are prime targets for the G-protein-mediated regu-latory action of hormones and neurotransmitters (7).There is a growing body of evidence supporting the exis-

tence of G proteins in plants (10-14). There is also someevidence showing the involvement of G proteins in signaltransduction in plant cells (10, 13-16). We have previouslyreported evidence that whole-cell inward K+ currents ofguard cells (17) and whole-cell outward K+ currents ofmesophyll cells (18) are regulated by G proteins. However, itis not known whether G proteins can regulate K+ channels in

plant cells via a cytoplasm-independent pathway as demon-strated in animal systems (6, 8). In the present study, weassayed the effects of guanosine 5'-[ythio]triphosphate(GTP['yS]), guanosine 5'-[3-thio]diphosphate (GDP[PSJ),pertussis toxin (PTX), cholera toxin (CTX), and ATP[-§] oninward K+ channels in isolated membrane patches and ob-tained direct evidence for the existence of a cytoplasm-independent pathway of G-protein regulation in a plant cell.

MATERIALS AND METHODSPreparation of Guard Cdl Protoplam (GCP). Plants of

Vicia faba L. (cv. "Long pod") were grown and guard-cellprotoplasts were prepared as described (17). Before use inpatch-clamp experiments, isolated GCPs were kept in thedark at 0-20C in a solution containing 5 mM Mes (KOH, pH5.5), 0.45 M sorbitol, 1 mM MgCl2, and 1 mM CaCl2.WhoeCel Cl Procedure. Experiments were per-

formed using conventional whole-cell recording techniques(17, 34). GCP were placed in bath solution containing (in mM)100 potassium glutamate, 1 MgCl2, 1 CaCl2, 10 Hepes (titrat-ed with KOH to pH 7.20), and mannitol to give a finalosmolality of 460 mmol/kg. The final K+ concentration ofthis bath solution was 103 mM. Glass pipettes (Kimax-51capillaries; VWR, Bridgeport, NJ) had resistances around 20Mfl when filled with solution containing (in mM) 10 Hepes(KOH, pH 7.2), 80 potassium glutamate, 20 KCI, 2 MgCl2, 2EGTA, 2 MgATP, and mannitol to give an osmolality of 500mmol/kg. The final Ki concentration for this pipette solutionwas 107 mM. Whole-cell clamping was performed at roomtemperature (20 ± 20C) under green light (no. 874 filter;Roscolene, Woburn, MA). Seal resistances were between 1and S Gil. Cell capacitance was measured for each cell usingthe capacity compensation device of the amplifier and wasbetween 5.5 and 7.5 pF.

Whole-cell currents were measured using an Axopatch-1Damplifier (Axon Instruments, Foster City, CA) interfaced(TL-1 DMA Interface; Axon Instruments) with a computer(486/33c; Gateway 2000, North Sioux City, SD). PcLMP5.5.1 (Axon Instruments) software was used to acquire andanalyze whole-cell currents. After obtaining the whole-cellconfiguration, membrane potential (V.') was clamped to -52mV. Voltage pulse protocols as shown in Fig. lC wereapplied to the clamped cell during data acquisition. Whole-cell current was filtered at 1 KHz with an eight-pole Besselfilter using the Axopatch-1D amplifier before acquisition (2ms per sample) to computer disk.

Abbreviations: Vm, membrane potential; P., open state probability;PTX, pertussis toxin; CTX, cholera toxin; G protein, GTP-bindingregulatory protein; GTP[yS], guanosine 5'-[y-thio]triphosphate;GDP[ISI, guanosine 5'-[P-thio]diphosphate; GCP, guard cell proto-plasts..*Present address: Department of Biology, Pennsylvania State Uni-versity, 208 Mueller Laboratory, University Park, PA 16802.tTo whom reprint requests should be addressed.

6310

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Nadl. Acad. Sci. USA 91 (1994) 6311

Whole-cell currents were leak-subtracted before generat-ing whole-cell current-voltage relations. Leak currents foreach cell were determined from the first one to three datapoints obtained after Vm was stepped from holding to testvoltage. The mean values of whole-cell currents were deter-mined from samples obtained between 3.1 and 3.3 s afterimposition of the test voltage, when current amplitude hadplateaued. After subtraction of leak currents, the final time-activated whole-cell currents were expressed per unit capac-itance (pF) to account for variation in cell surface area.Single-Chane Recording Procedure. GCP were placed in a

chamber filled with solution containing (in mM) 80 potassiumglutamate, 20 KCI, 10 CaCl2, 2 MgCl2, 10 Hepes (pH 7.2), andmannitol (osmolality adjusted to 480 ± 10 mmol/kg). Whenplaced in this solution, glass pipettes filled with the bathsolution used for whole-cell experiments had resistances be-tween 30 and 40 Ml. Seal resistances between 5 and 10 Gilformed in %20%o of attempts. Inside-out membrane patcheswere obtained after sealformation by retracting the pipette andexposing the pipette tip briefly to the air. After the formationof a stable inside-out patch, the bath solution was changed tothe pipette solution used for whole-cell experiments as de-scribed above, which had an estimated final Ca2+ concentra-tion of2 nM (17). Data acquisition was conducted 10 min afterthis solution change for those patches that maintained stableseal resistances and channel signals, and again 10 min afterimposition of a given treatment. Single-channel currents weremeasured and analyzed using the same equipment and soft-ware package as for the whole-cell experiments. Data werefiltered with an eight-pole Bessel filter at 1 kHz, digitized at 4kHz, and stored on computer disk. Liquid junction potentialswere both measured and calculated (19), and Vm valuesreported have been appropriately corrected.Open state probabilities (PO) were determined as the ratio of

total open time to the total recording time. Current amplitudefor the open state at a given voltage was obtained fromGaussian fitting of open state amplitude histograms. Closedand open time constants (T) were determined from exponentialfittings of closed and open time histograms. The goodness offit was determined by evaluating standard errors of timeconstants (lOo of r), PCLAM software defined "goodness offit" (-2.0), and fit area as percent of data area (295%).

Preactivatlon of PIX and CTX. PTX (100 pg/ml) waspreactivated by incubation in medium containing 50 mMHepes (pH 7.5), 20 mM dithiothreitol (DTT), 1 mg of bovineserum albumin per ml, and 0.1% (wt/vol) SDS at 30TC for 30

min (cf. ref. 20). The PTX stock was then diluted with thebath solution for single-channel recording experiments togive a PTX stock solution with a concentration of 20 pg/ml.PTX stock solution was added to bath solution to give a finalPTX concentration of 300 ng/ml. The final SDS concentra-tion was 0.0002% (wt/vol), and this low concentration ofSDSwill not affect the integrity of biomembranes (21).CTX (1 mg/ml) was preactivated by incubation in a me-

dium containing 10mM Hepes (pH 7.3), 100mM NaCl, 5 mMDTT, and 0.5% (wt/vol) SDS at 300C for 30 min (cf. ref. 20).The stock was subsequently diluted with bath solution andwas used at a final concentration of 300 ng/ml. For PTX andCTX treatments, 5 juM NAD+ was added as the substrate forADP-ribosylation of G proteins.Chemical Reagents. GTP[yS], GDP[(3S], and ATP[-v] were

obtained as lithium salts from Calbiochem. PTX and CTXwere from List Biological Laboratories (Campbell, CA). Allother chemicals were from Sigma.

RESULTSEffects of Guani Nucleotides on Whole-Cell K+ Current.

The whole-cell currents observed in our experiments resultmainly from K+ flux; as shown in Fig. 1A, the reversalpotential of the whole cell currents is near 0 mV, which isclose to the K+ reversal potential predicted from the exper-imental conditions.

In our previous report (17), experimental conditions underwhich G-protein effects on K+ currents were assayed wouldnot be conducive to stomatal opening. In the present study,the high external K+ concentrations employed will promotestomatal opening in Vicia epidermal peels, even in theabsence of illumination (22). Given the hypothesis (23) thatenvironmental conditions might influence whether activatingor inhibiting G proteins are available for regulation byGTP['yS] and GDP[.S], it was of interest to ascertain theeffects of G-protein regulators under these high K+ condi-tions. Effects on K+ currents were essentially the same asthose shown previously with low external K+ concentra-tions. GTP[-y], which activates G proteins, inhibits whole-cell inward K+ currents (Fig. 1 C and E), whereas GDP[,BS],which inactivates G proteins, enhances whole-cell inward K+currents (Fig. 1 D and E).

Identification of Single K+ Channels. Single-channel re-cordings shown in Figs. 2A, 3, and 5 were identified as K+channel currents on the basis of their reversal potential,

FIG. 1. G-protein regulation of whole-cell K+ current in Vicia guard-cell protoplasts. (A) Reversal potential of whole-cell current. Measuredreversal potential of whole-cell current was near 0 mV (arrow), near the predicted K+ reversal potential (Kin/Kout = 107/103). (B) Whole-cellcurrent of a guard-cell protoplast at different membrane potentials. The voltage protocols and time and current amplitude scales shown in Calso refer to B and D. (C) Inhibition of whole-cell inward K+ current by 500 pAM cytoplasmic GTP[ 61]. (D) Enhancement of whole-cell inwardK+ current by 500 ,uM cytoplasmic GDP[(3S] . (E) Average (n = 6, -+SE) whole-cell current (IO) as a function ofvoltage in the absence ofG-proteinregulator (o) and in the presence of 500 pM GTP[yS] (o) or 500 ILM GDP[PS] (v).

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6312 Cell Biology: Wu and Assmann

A mV Vm(mv) k B P0 C., . _ - - - --e O -1120 -810 -40 ,'C- -.....-38

_ -8-58 -2

-7 -18 3- Irl-~.-88 *7 01- r~~r~~\wrt~~rr--88 -_r,-98 / -~~~~~~~~~~~~~~~~-4*j

-

_I-108 Is - _5 * sThr~~~4VVW~~f'~~~~rr198~-50-118_ -120 - -80 -40

lob~s IK(PA4 Vm(mV)

D E

~ sec a Isec

inhibition by high cytoplasmic Ca2+, voltage dependence,and time dependence. (i) As shown in Fig. 2B, the predictedK+ reversal potential (VK) of 1 mV (K+Ot/K+ij = 103/107)is met well by our single-channel data. (ii) We infrequentlyobserved single-channel events before the original bath so-lution, containing millimolar Ca2+, was changed to the lowCa2+ solution described in Materials and Methods. P. was0.012 ± 0.004 (n = 8) in the high Ca2+ solution and 0.21 +0.028 (n = 8) in the low Ca2+ solution. It is known that theinward K+ channel in the guard-cell plasma membrane isstrongly inhibited by high cytoplasmic Ca2+ levels (24),whereas anion channels of guard cells are activated byelevated cytoplasmic Ca2+ (25). (iii) The nonlinear voltage-dependent PO distribution of the single channels (Fig. 2C) isexactly as would be predicted from the nonlinear current/voltage relation of whole cell K+ currents (Fig. 1E). (iv)Inspection of single-channel traces (e.g., Fig. 2D) and en-semble summing of single-channel cells (Fig. 2E) reveal thatsingle-channel and whole-cell currents (Fig. 1 A-D, -120 mVtraces) exhibit similar, time-dependent activation and pro-vides additional confirmation ofthe identity ofthese channelsas K+ channels.

Effects of GTP[yS] and GDP[I3SJ on Single K+ ChannelCurrents. Studies of G-protein-mediated ion channel regula-

FIG. 2. Properties of single K+ channels. (A)Recordings of inward K+ channel current froman inside-out membrane patch derived from aguard cell protoplast. The membrane potentialof each recording is shown on the right. Arrowsto the left ofeach trace indicate the closed level.(B) Open channel current as a function of volt-age. The arrow indicates the predicted K+ re-versal potential (Vy), which is very close to theextrapolated reversal potential ofthe recordings(intersection ofthe abscissa and the dotted line).The conductance of the inward K+ channelshown is 39 pS, as calculated from the slope ofthe current/voltage regression. (C) Voltage de-pendence of open state probability (P.). (D)Time dependence of PO in an isolated patchshowing activity of two channels. Vm was -118mV. (E) Sum of 24 isolated patch recordings at-118 mV, showing the time dependence ofchannel activation (compare Fig. 1, whole-cellcurrent at -120 mV).

tion in animal systems typically employ 100 IAM to 1 mMconcentrations of nonhydrolyzable guanine nucleotide ana-logs (26-28). In our experiments, the addition of 500 A&MGTP[IySI on the cytoplasmic side of isolated inside-out mem-brane patches significantly decreased PO of the inward K+channel (Fig. 3, Table 1). This effect, and the inhibitoryeffects ofPTX and CTX (see below), cannot be attributed tochannel "run-down" because decreases in PO were notobserved in control patches monitored over an even longertime period (Table 1) nor in patches treated with ATP[yS] orGDP[3S] (Table 1). The absence of an effect of ATP[IS] onPO confirms the nucleotide specificity of the GTP[yS] effect.Open and closed time distributions of all our data were

fitted well by double exponentials, which indicates the exis-tence of more than one closed (or open) state (see Discus-sion). GTP[yS] increased one of the closed time constants ofthe K+ channel and did not significantly affect the open timeconstants (Fig. 4, Table 2). GDP[j3S] slightly increased P.(Table 1, Fig. 5 A and B) and shortened the closed timeconstants (Table 2) ofthe K+ channel. The effect ofGDP[,fS]is not as great as that of GTP[yS], perhaps because underthese isolated patch conditions most of the G protein wasalready inactivated, but the result is consistent with thewhole-cell data shown in Fig. 1.

Control A

C,-P0.-

Ca

-is

2LIC)

ON\ | Po=0.26

a - Xz ICURRENT

GTPAYS CCaI_-.

'-1- T F

FIG. 3. Effect of GTP[yS] on inwardK+ channel activity recorded at -118mV. (A) Single K+ channel recordings ofthe control. The closed (c) and open (o)levels are indicated by arrows. (B) All-point amplitude histogram from the patchshown in A. (C) Single K+ channel re-cordings in the presence of 500 IAM cy-toplasmic GTP[yS]. (D) All-point ampli-tude histogram from the patch shown inC. Open state probability decreased from0.26 to 0.09 after application ofGTP[-S].

j1'~{' 't'2 I'"I I"11'moALS

Proc. Natl. Acad. Sci. USA 91 (1994)

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Table 1. Effects of various G-protein regulators on PO of theinward K+ channel

PO

Membrane patch

Treatment 1 2 3 4 Mean ± SDControl A* 0.21 0.18 0.23 0.17 0.20 ± 0.024Control B* 0.23 0.20 0.21 0.18 0.21 ± 0.018

Control 0.26 0.24 0.16 0.22 0.22 ± 0.036GTP[rS] 0.09 0.08 0.06 0.10 0.082 ± 0.01Control 0.18 0.24 0.15 0.18 0.19 ± 0.033GDP[(S] 0.21 0.28 0.16 0.23 0.22 ± 0.043Control 0.21 0.25 0.18 0.19 0.21 ± 0.027PTX 0.05 0.02 0.01 0.01 0.02 ± 0.018

Control 0.19 0.21 0.08 0.16 0.16 ± 0.048CTX 0.15 0.15 0.09 0.09 0.12 ± 0.030

Control 0.27 0.23 0.20 0.16 0.22 ± 0.040ATP[-yS] 0.25 0.24 0.22 0.19 0.23 ± 0.023The concentrations of GTP[yS] and GDPIIS] were 500 AM, and

the concentrations of PTX and CTX were 300 ng/ml. The concen-tration of ATPb['S] was 1 mM. Each pair of data for control andparticular treatment was obtained from the same membrane patchrecorded at -118 mV.*Control B data were recorded 20 min after control A data, from thesame membrane patch. Control A data, and all other controls, wererecorded 10 min after imposition of the low Ca2+ bath solution (seetext).

Effects of PTX and CTX. When applied to the cytoplasmicside of membrane patches, PTX and CTX inhibited openingof inward K+ channels. Table 1 and Fig. 5 C and D clearlyshow the strong inhibitory action of PTX on the opening ofthe inward K+ channel. Addition of 300 ng ofPTX per ml leftso few channel events (Fig. 5 CandD) that it was not possibleto carry out a valid analysis for open and closed time kinetics.CTX was less inhibitory than was PTX (Tables 1 and 2, Fig.5), but the results shown here with PTX and CTX areconsistent with effects observed previously in the whole-cellconfiguration (17).

cna) Control A GTPSYS Cz I

> ~~~~~~T,0.63 ZLU.. T 503Z

m A iA,~~~~~~~~cA

z flj -z -0 5 10 15 20 ms 0 5 10 15 20 ms

OPEN TIME OPEN TIME

u) Control B | GTP'YS Dz z> T, = 0TO97T10=2.26LL- T2=9.57 U.~X 0 o T2=45.0

z z -

0 l] 22 33 44ms0 40 80 120 160 msCLOSED TIME CLOSED TIME

Table 2. Effects of G-protein regulators on the open and closedtime constants (x) of the inward K+ channel

1T

Open time, ms Closed time, msTreatment T1 T2 T1 T2Control 0.6 ± 0.28 8.5 ± 3.9 1.4 ± 0.41 14 ± 3.2GTP[yS] 0.6 ± 0.03 10 ± 2.6 1.5 ± 0.59 30 ± 14Control 0.7 ± 0.14 7.3 ± 2.2 1.7 ± 0.44 15 ± 3.2GDP[3SI 0.7 ± 0.23 7.8 ± 2.6 1.5 ± 0.25 11 ± 2.2Control 0.6 ± 0.12 5.1 ± 0.78 1.8 ± 0.36 12 ± 1.1CTX 0.5 ± 0.22 5.3 ± 0.73 2.3 ± 0.75 26 ± 5.6The concentrations of GTP[(yS] and GDP(3S] were 500 PM and

CTX concentration was 300 ng/ml. Each number represents mean ±SD (n = 4). Each pair of original data for the control and theparticular treatment was obtained from one membrane patch re-corded at -118 mV.

DISCUSSIONG proteins, a family of heterotrimeric (a, A, 'y) proteins, act inanimal systems on membrane-associated targets such as ionchannels (7, 8). G proteins are membrane messengers andsecond messengers for signal transduction pathways thatbegin with G-protein-coupled receptors and may include cy-toplasmic ormembrane-bound third, fourth, etc., messengers.

It was previously thought that G-protein regulation of ionchannels was mediated mainly by cytoplasmic effectors, butit is now clear that G proteins can also interact with ionchannels within the plane of the membrane in a so-calledmembrane-delimited manner (8). The G-protein regulation ofK+ channels in plant cells previously observed at the whole-cell level (17) could therefore have resulted from the actionof either cytosolic or membrane-associated messengers.Whether G proteins could in fact regulate K+ channels via amembrane-delimited pathway in plant cells was not known.The data presented here clearly indicate the existence ofsucha membrane-delimited (or cytoplasm-independent) pathwayfor G-protein regulation of plant K+ channels. This obser-vation does not rule out the possibility ofadditional G-proteinregulation via a cytoplasm-dependent pathway.

FIG. 4. Open and closed time analy-sis of the inward K+ channel. Data usedfor analysis are from the same patch as inFig. 3. All open and closed time histo-grams were fitted well by double expo-nentials. (A and B) Open and closed timehistograms of the control. (C and D)Open and closed time histograms in thepresence of GTP[FyS].

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6314 Cell Biology: Wu and Assmann

A Control B +GDPBS

jw~~i~w~A"Trl11 ~lOOms

C Control D +PTX

E Control F ±CTX_ P Ffl AJ T U,' '' UPft nrrrr~m

FIG. 5. Effects of GDP[.8S], PTX, and CTX on K+ channelactivity recorded at -118 mV. Arrows indicate the closed level ofthechannel. The time and amplitude scales shown in A refer to allrecordings. Each data pair (A and B, C andD, Eand F) was obtainedfrom one membrane patch.

Changes in whole-cell current may reflect changes inchannel conductance or permeation (35) or changes in chan-nel PO (32). In this study we used single-channel recording todistinguish between these possibilities. The data presented inFigs. 3 and 5 and Table 1 indicate that the effects ofG-proteinregulators on whole-cell K+ currents presented in Fig. 1 canbe attributed to changes in P. and not to changes in channelconductance.PTX and CTX covalently modify the a-subunits ofmany G

proteins by catalyzing the transfer of ADP-ribose fromNAD+ to specific amino acid residues (29, 37). In animalsystems, PTX typically inhibits G-protein activation (30),while CTX activates G proteins (9, 30). However, we foundhere that both PTX and CTX inhibited inward K+ currents,the inhibitory effect of PTX being greater than that of CTX.These results are consistent with those ofour previous report(17). Our results may suggest that (i) unlike the situation inanimals, both PTX and CTX activate an inhibitory G proteinin plant cells or (ii)PTX may inhibit a secondG protein whoseactivated form enhances inward K+ currents in Vicia guardcells.

Previous analysis of whole-cell K+ currents (31) had indi-cated that inward K+ current could B'e fitted by a singleexponential model. However, as show"i in Fig. 4 and Table2, the closed and open time distributiobs of single-channelrecordings in all of our experiments were fitted well by twoexponentials. The faster component P(efer to the shorter timeconstants) revealed by the single-cha nel analysis (Table 2)was presumably not resolvable by tie analysis procedureused for the whole-cell currents.A double exponential fit suggests hat, at a minimum, the

inward K+ channel must reside in Pro closed or two openstates. In theory, several different models could explain suchdata. For example, in one model there would be one open andtwo closed states: closed1 -open = closed2. One of theclosed states could be an inactivated (or inhibited) state. Onthe basis ofthis possible explanation, there would be two rateconstants for the opening direction, which refer to the two

closed time constants, and two rate constants for the closingdirection, which refer to the two open time constants. Theincrease of one closed time constant caused particularly byGTP[ -S] and PIX means the decrease of one rate constant inthe opening direction. In other words, activation or inacti-vation of G proteins in guard cells may regulate inward K+currents by changing the opening kinetics of K+ channels.

Results presented here provide evidence for a membrane-delimited pathway of G-protein regulation in plant cells, butthe detailed coupling mechanism(s) between G protein(s) andK+ channels is not yet known. G proteins might act uponchannel proteins either directly without requiring any othermembrane component (8, 32, 36) or indirectly via interme-diate membrane-associated effectors (6). Further elucidationof the coupling mechanism between G proteins and K+channels in guard cells will require single-channel studiesemploying regulators of typical intermediary proteins, suchas kinases and phosphatases (33), and, ultimately, functionalreconstitution of the interacting proteins in an artificial mem-brane system.

This research was supported by U.S. Department of AgricultureGrant 91-37304-6578 to S.M.A.

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