Planta (1996)198:279-287 P l a n t ~

�9 Springer-Verlag 1996

Inositol 1,4,5-trisphosphate may mediate closure of K § channels by light and darkness in S a m a n e a s a m a n motor cells

Hak Yong Kim, Gary G. Cot~*, Richard C. Crain

U-125, Department of Molecular and Cell Biology, The University of Connecticut, Storrs, CT 06269-3125, USA

Received: 20 April 1995/Accepted: 14 June 1995

Abstract. Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regu- lated K § fluxes across the plasma membranes of extensor and flexor cells in opposing regions of the leaf-moving organ, the pulvinus. We previously showed that blue light appears to close open K § channels in flexor protoplasts during the dark period (subjective night) (Kim et al., 1992, Plant Physiol 99: 1532-1539). In contrast, transfer to darkness apparently closes open K § channels in extensor protoplasts during the light period (subjective day) (Kim et al., 1993, Science 260: 960-962). We now report that both these channel-closing stimuli increase inositol 1,4,5- trisphosphate [Ins(1,4,5)P3] levels in the appropriate pro- toplasts. If extensor cells are given a pulse of red light followed by transfer to darkness, channels still apparently close (Kim et al. 1993) but changes in Ins(1,4,5)Pa levels are complex with an initial decrease under red light followed by accumulation. Neomycin, an inhibitor of polyphosphoinositide hydrolysis, inhibits both blue-light- induced Ins(1,4,5)P3 production and K+-channel closure in flexor protoplasts and both dark-induced Ins(1,4,5)P3 production and K § channel closure in extensor proto- plasts. The G-protein activator, mastoparan, mimics blue light and darkness in that it both increases Ins(1,4,5)P3 levels and closes K § channels in the appropriate cell type at the appropriate time. These results indicate that phos- pholipase C-catalyzed hydrolysis of phosphoinositides, possibly activated by a G protein, is an early step in the signal-transduction pathway by which blue light and darkness close K § channels in S. saman pulvinar cells.

Key words: Inositol 1,4,5-trisphosphate - K § Channel - Leaf movement - Phototransduction - Samanea (K § channels) - Turgor regulation

*Present address: Department of Biology, Millikin University, Decatur, IL 62522, USA Abbreviations: DiS-C3-(5 ) = 3,3'-dipropylthiadicarbocyanine iod- ide; AF = measure change in Dis-Ca-(5) fluorescence; Fo = initial Dis-Ca-(5 ) fluorescence; Ins(1,4,5)P 3 = inositol 1,4,5-trisphosphate; Ptdlns(4,5)P 2 = phosphatidylinositol 4,5-bisphosphate; rbc = red blood cell Correspondence to: R.C. Crain; FAX: 1 (203) 486 4331; E-mail: crain@uconnvm.uconn.edu

Introduction

Leaflet movements of Samanea saman are produced by changes in the turgor of extensor and flexor cells (collec- tively called motor cells) on the lower and upper sides, respect ively, of the leaf-moving organ, the pulvinus, at the base of the leaf stalk. These turgor changes are caused by fluxes of ions, primarily potassium and chloride, across the plasma membrane (Satter 1979). These ion fluxes are regulated by exogenous signals, such as blue light, red light, or darkness, and by an endogenous circadian clock (Satter et al. 1988). During the dark period, white or blue light signals extensor cells to take up K +, CI- , and water and thus to swell while signaling flexor cells to lose K § C1- and water, and thus to shrink, thereby extending the leaflets towards the light (Lee and Satter 1989; Lowen and Satter 1989). Conversely, during the light period, red light followed by premature darkness triggers the opposite ion fluxes in motor cells, thereby folding the leaflets together.

Inward-rectifying, hyperpolarization-activated K + channels have been described in protoplasts of S. saman motor cells (Moran 1990) and in protoplasts of stomatal guard cells (Schroeder and Fang 1991), which also exhibit turgot-mediated movements. These channels could carry the inward K § fluxes. Using a membrane-potential-sensi- tive dye, we have monitored changes in membrane poten- tial induced by the addition of K § to motor-cell proto- plasts at specific times during a light-dark cycle of 16 h light, 8 h darkness (Kim et al. 1992). Membrane-potential changes consistent with the inward movement of the ad- ded K + occurred in flexor, but not in extensor protoplasts at hours 4 to 8 of the normal dark period, and membrane- potential changes consistent with similar K + movement in extensor but not in flexor protoplasts occurred at hours 10 to 12 of the normal light period. These changes in potential were dependent on K + concentrations, were blocked by tetraethylammonium ion or A13 +, were spe- cifically triggered by K + over Na +, Li § or Ca 2 + and were not affected by the counterion to K +, consistent with the changes being triggered by movement of K § through K § channels. We interpret these results to indicate that inward-rectifying K § channels are open in flexors but not in extensors at hours 4 to 8 of the dark period and are

280

open in extensors bu t no t in flexors at hours 10 to 12 of the light period (Kim et al. 1993). We have also shown that white light or blue light dur ing the dark period abolishes the potent ia l for inward K § movemen t in flexor proto- plasts while act ivat ing the potent ia l for inward K § move- men t in extensor protoplasts . We interpret this to indicate that white light or blue light closes K § channels in flexor protoplas ts while opening them in extensor protoplasts (Kim et al. 1992). Similarly, red light followed by prema- ture darkness dur ing the light period apparent ly closes K § channels in extensor protoplas ts and opens them in flexor protoplas ts (Kim et al. 1993). Premature darkness alone, wi thout the preceding red light t reatment , is also able to inhibi t apparen t inward K § fluxes in extensor cells, bu t it has no apparen t effect on inward K § fluxes in flexor cells (Kim et al. 1993). While these results indicate that light and darkness regulate K § movemen t across the p lasma m e m b r a n e of mo to r cells, the s ignal- t ransduct ion cascade involved in this regulat ion is unknown.

In m a n y an imal systems, extracellular signals such as hormones , neurot ransmi t te rs , and light activate phos- pholipase C, which hydrolyzes phosphat idyl inosi to l 4,5- b isphosphate (Ptdlns(4,5)Pz), p roducing the second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and 1,2-sn-diacylglycerol. Ins(1,4,5)P3 binds to a receptor, trig- gering Ca 2 § mobi l iza t ion from internal stores (Berridge 1993), while 1,2-sn-diacylglycerol, together with calcium in some cases, activates one or more of the isozymes of pro te in kinase C (Kikkawa et al. 1989). Pre l iminary evid- ence indicates that white light triggers phosphoinosi t ide tu rnover in at least some cells of intact S. s a m a n pulvini (Satter et al. 1988).

We here report that phosphoinos i t ide turnover is in- duced by blue light in extensor protoplasts and by darkness in flexor protoplasts , and that Ins(1,4,5)P3 pro- duc t ion correlates with apparen t K§ closure, suggesting that Ins(1,4,5)P3 elevation mediates closure. We also report that phosphol ipase C can be activated by the G-pro te in act ivator m a s t o p a r a n and that this activa- t ion also correlates with apparen t K+-channe l closure. These results suppor t a hypothesis that light signals which close inward K § channels and reduce cell turgor are t ransduced by G-pro te in -media ted produc t ion of the sec- ond messenger Ins(1,4,5)P3.

M a t e r i a l s and m e t h o d s

Chemicals. Reagents for the preparation of protoplasts were de- scribed earlier (Kim et al. 1'992, 1993). 3,3'-Dipropylthiadicar- bocyanine iodide [DiS-Ca-(5)] was purchased from Molecular Probes, Inc. (Junction City, Ore., USA), and dissolved in dimethyl- sulfoxide (DMSO) at 1 mg'm1-1. Stock solutions of 1 mM mas- toparan and 1 mM neomycin (both from Sigma Chemical Co., St. Louis, Mo., USA) were prepared in water. A stock solution of 40 ~tM Ins(1,4,5)P 3 (LC Laboratories, Woburn, Mass., USA) was prepared in water and stored in 100-btl aliquots at - 20~ Before use, it was diluted 1:10 with water. D-myo-[3H]Inositol 1,4,5-trisphosphate (New England Nuclear/DuPont, Boston, Mass., USA) was diluted 1 : 100 with distilled water just before use.

Plant material. Samanea saman (Jacq.) Merr. trees were grown at 26 + 1.5 ~ on a 16-h light: 8-h dark cycle (cool-white fluorescent light, 200 ~tmol photons.m-Z-s-I). Secondary pulvini were

H.Y. Kim et al.: Ins(1,4,5)P 3 Regulation of K + Channels in S. saman

harvested from the second-youngest fully mature leaf at hours 9 to 10 of the light period, at which time the leaves were fully extended. Protoplasts were isolated under room lights from separated extensor and flexor regions of the harvested pulvini as previously described (Kim et al. 1992, 1993) and were maintained at 25~ under the same light:dark cycle as in the growth chamber (Kim et al. 1993).

Measurement o f relative membrane potential. Changes in the plasma- membrane potential of protoplasts were detected by changes in the intensity of fluorescence emission of the potential-sensitive dye DiS- C3-(5 ) (Kim et al. 1992, 1993). The assay medium consisted of 20 mM 2-(N-morpholino)ethanesulfonic acid (Mes) (pH5.5), 400mM sorbitol, 20mM KCI, lmM CaC12, and 1 x 105 protoplasts per milliliter. Because of the lengthy incubations of protoplasts used for experiments in the light period, 90 mM sucrose was added as a car- bon source and 0.2 mM dithiothreitol (DTT) was added to protect the dye from oxidation. The dye DiS-C3-(5 ) was added in DMSO to a final concentration of 2/.tM 1 h before experiments in the dark period, or 5 min before experiments in the light period since the dye was labile in light. The final concentration of DMSO was 0.0125% (v/v). The dye was excited with 620 nm light and emission was measured at 668 nm. The measured change in fluorescence (AF) was divided by the initial fluorescence (Fo) to normalize for variation between experiments.

Measurement o f lns(1,4,5)P 3 concentration. Protoplasts were incu- bated in the same medium as described above for measurement of relative membrane potential, except that DiS-C3-(5 ) in DMSO was not added. At specific time points following treatment with light, darkness, or mastoparan, 0.5-ml aliquots of the protoplast suspen- sions were mixed with 1/20 vol. of ice-cold 100% (w/v) tri- chloroacetic acid and the precipitate was removed by centrifugation at 650-g for 10 min at 4~ Trichloroacetic acid was removed by extracting four times with 5 vol. of water-saturated ether and the extract was adjusted to pH 7.5-8.0 with 16% (w/v) sodium carbon- ate. Samples were concentrated fourfold by lyophilization in a vac- uum centrifuge and reconstitution in distilled H20. Concentrations of Ins(1,4,5)P 3 were determined by an isotope-dilution assay using bovine adrenal Ins(1,4,5)P 3 receptors following the protocol of Van Haastert (1989) as previously described (Quarmby et al. 1992). Re- covery of Ins(1,4,5)P 3 in the extract was 85-90%, based on recovery of 3H-Ins(1,4,5)P3 added to protoplast suspensions just before ex- traction, in agreement with results of Van Haastart (1989). The assay can detect 0.15 pmoi of Ins(1,4,5)P 3 in a 100-btl sample.

Validation of lns(1,4,5)P 3 radio-receptor assay. To determine whether S. saman contains phosphate metabolites which bind to the bovine Ins(1,4,5)P 3 receptor, extensor and flexor tissues from iso- lated pulvini were finely minced and incubated with 32pi for 4 h. Trichloroacetic acid (TCA) was added to a final concentration of 10% (w/v); precipitated macromolecules and lipids were removed by centrifugation at 650"g for 10 min; TCA in the supernatant was removed by ether extraction; and the resulting aqueous extract was adjusted to pH 8.0 with sodium carbonate (Quarmby et al. 1992). The extract was concentrated fourfold by lyophifization in a vacuum centrifuge and reconstituted in distilled water. A 0.2-ml sample of extract was allowed to react with 4mg bovine adrenal Ins(1,4,5)P 3 receptor under the same conditions as used in the Ins(1,4,5)P 3 assay, in the presence or absence of 1 ~tM Ins(1,4,5)P 3 (LC Laboratories Inc., Woburn, Mass., USA). The receptor protein was sedimented and bound metabolites were extracted by treatment with 5% TCA (w/v) as described above. The bound labeled metabolites were re- solved and identified by HPLC on a Partisphere SAX column as previously described (Cot6 et al. 1987).

To further test the identity of receptor:binding activity in S. saman tissue, extracts were incubated with red-blood-cell (rbc) mem- branes essentially as described by Irvine et al. (1989). The rbc ghosts were prepared as described previously (Cotret al. 1987) and stored at - 2 0 ~ until used. The rbc membranes' (0.7 mg protein) were incubated with S. saman pulvinar extracts in the presence or absence of 7.4kBq of [3H]Ins(l,4,5)P3 (New England Nuclear, Boston,

H.Y. Kim et al.: Ins(1,4,5)P 3 Regulation of K § Channels in S. saman

Mass., USA) in 300 ~tl of a buffer containing 10 mM Hepes (pH 7.4) 5000 - and 2.5mM MgC12 (Downes and Michell 1981). To prevent activa- tion of the endogenous rbc phospholipase C by contaminating Ca 2 + 4000 in the pulvinar extract EGTA was added to a final concentration of 0.5 mM (Cot6 et al. 1987; Downes and Michel11981). The incubation was stopped at various times by mixing an aliquot of the reaction "~ 3000 mixture with TCA (final concentration 10% w/v) and chilling on ice. ~ B The TCA was removed, the pH adjusted to 8.0 and the sample ~ 2000 concentrated twofold as described above. Receptor binding activity was assayed by the usual method, while hydrolysis of added aH- Ins(1,4,5)P3 was monitored by scintillation counting following 1000 chromatographic separation of [3H]Ins(1,4,5)P3 from [3I-I-Jlns(1,4)P2 on Amiprep trimethylaminopropyl SAX minicolumns (Amersham, 0 Arlington Heights, II1., USA) as described by Huang et al. 20 24 (1995).

Experimental treatments. Protoplasts were irradiated with blue light between hours 4 and 8 of the dark period or with red light between hours 10 and 12 of the light period. Blue light and red light were from a fiber-optic source (Reichert Scientific Instruments, Vienna, Austria) equipped with heat-absorbing filters and interference filters as previously described (Kim et al. 1992, 1993). The photon fluence rate was 200 ~tmol-m-2-s - 1 for blue pulses, 80 ~tmol-m-2-s - 1 for high-intensity red pulses, and 3-4 lamol-m-2-s - 1 for low-intensity red pulses. White light was provided with Sylvania (Danvers, Mass., USA) GTE fluorescent bulbs (32 W, 4100 K) at a photon fluence rate of 40 ~tmol-m-2.s - 1. Neomycin was added 30 s before treatment with light, darkness, or mastoparan. Protoplasts were incubated for 150 s with mastoparan or for 180 s after a change in lighting before 200 mM K + was added to monitor the state of the K + channels.

Results

Validation o f Ins(1,4,5)P3 radio-receptor assay. Figure 1 shows tha t a me tabo l i t e which comig ra t ed with au th- entic Ins(1,4,5)P3 on H P L C , b o u n d to the bovine ad rena l Ins(1,4,5)P3 receptor , and tha t this b ind ing was speci- fically e l imina ted by compe t i t i on with unlabe led Ins(1,4,5)P3. This me tabo l i t e was unde tec tab le in a 5% a l iquot of the to ta l ex t rac t (not shown), and was great ly concen t r a t ed by recep tor binding. In contras t , a p p r o x i m - ately 10% of the to ta l label in each of the o ther 32p_ labeled S. saman metabo l i t e s assoc ia ted with the recep tor prote in , but the b ind ing of these metabo l i t e s was non- specific since it was no t at all inh ib i ted by unlabe led Ins(1,4,5)P3.

F igure 2 shows tha t r ecep to r -b ind ing act ivi ty f rom S. saman t issues was hydro lyzed by the rbc inosi to l t r i sphos- pha t e 5 ' - m o n o p h o s p h a t a s e (Downes et al. 1982) with the same kinet ics as bo th au thent ic [3H]Ins(1,4,5)P3, and the endogenous Ins(1,4,5)P3 present in the rbc ghosts .

Blue light during the dark period alters Ins(1,4,5)P3 levels. Basal levels of Ins(1,4,5)P3 in f lexor p ro top l a s t s averaged 1.9 _+ 0.3 pmol/105 cells (mean _+ SD) and in- creased 2.1 _+ 0.5-fold wi thin 15 s of blue l ight t rea tment , a l t hough the levels decl ined somewha t dur ing con t inued i r r ad i a t i on (Fig. 3). Conversely , basa l levels in ex tensor p ro top la s t s ave raged 4 . 3 _ 0.1 pmol/105 cells and de- creased to 36 _+ 13% of the basa l level wi th in 30 s of b lue l ight i r r ad i a t i on (Fig. 3). The increased Ins(1,4,5)P3 levels in flexor p ro top la s t s c o r r e l a t e d with a p p a r e n t K + - c h a n - nel c losing observed after 3 min of blue l ight while decreased Ins(1,4,5)P3 levels in ex tensor p ro top la s t s cor- re la ted with a p p a r e n t K + - c h a n n e l open ing s imilar ly

281

---O-- -IP 3

+IP3 I - - ~ 3H_lP 3 i

2500

�9 2000

1500

500

0

28 32

a7

elution time (min)

Fig. 1. Separation by HPLC of 32p-metabolites bound to the Ins(1,4,5)P3 receptor of Samanea saman motor cells in the presence or absence of excess unlabeled Ins(1,4,5)P3. Minced pulvinar tissue was labeled with 32Pi and extracted as described in Materials and methods. The a 2p.labele d extracts were incubated with a preparation of bovine adrenal Ins(1,4,5)P3 receptor in the absence of unlabeled Ins(1,4,5)P3 (open circles) or in the presence of 1.0 ~tM Ins(1,4,5)P3 (open squares) and extracted as described in Materials and methods. The samples were separated by HPLC as previously described (Cot6 et al. 1987). An authentic standard of [3H]Ins(1,4,5)P3 was applied to the same column in a separate experiment, its elution is indicated by closed circles and a dashed line

9o ~

75i ~ 6 0 -

~" 4 5

30.

15

~'~'.!.

E i i

10 20 30 40 50

time

Fig. 2. Time course of degradation of Ins(1,4,5)P3 by rbc inositol trisphosphate 5' phosphatase. Extracts from S. saman pulvinar tis- sue, in the presence or absence of tracer [3H]Ins(1,4,5)P3, were incubated with rbc membranes at 37 ~ as described in Materials and methods. Control incubations without pulvinar extracts were performed to monitor endogenous rbc Ins(1,4,5)P3. Reactions were terminated with 10% trichloroacetic acid and Ins(1,4,5)P3 levels were determined as described in Materials and methods. Degrada- tion of [3H]Ins(1,4,5)P3 was assessed following chromatographic separation of inositol phosphates and is expressed as percent of label in Ins(1,4,5)P3 at time 0 (closed circles). Degradation of endogenous rbc Ins(1,4,5)P3 (closed triangles) and degradation of pulvinar plus rbc Ins(1,4,5)P3 (closed squares) were determined by radio-receptor assay and are expressed as percent of the levels at time 0. The rbc Ins(1,4,5)pa was approximately 30% of the total in incubations with pulvinar extract.

observed after 3 min of blue l ight (Kim et al. 1992 and Table 1).

Red light and darkness during the light period alter Ins(1,4,5)P3 levels in extensor, but not in f l exor proto- plasts. Levels of Ins(1,4,5)P3 in ex tensor p r o t o p l a s t s de- creased a b o u t 55% dur ing 30 s of red l ight a t hours 10 to 12 of the l ight pe r iod and then increased to near basa l

282 H.Y. Kim et al.: Ins(1,4,5)P 3 Regulation of K + Channels in S. saman

--• 4

%

, , , i , , , i J , , i , , , i , , , [ , , ,

-30 3 0 9 0 1 5 0 210 270 330

T i m e ( s )

Fig. 3. Time course of changes in Ins(1,4,5)P3 levels in response to blue light. Levels Ins(1,4,5)P3 were assayed in extensor protoplasts (closed circles) or flexor protoplasts (open circles) from S. saman as described in Materials and methods at varying times during illumina- tion with 2001amol-m-a-s -1 blue light at hours 4 to 8 of dark period. The black bar at the bottom of the figure indicates darkness and the double-hatched bar indicates blue light. Values represent mean + SD of three to four separate experiments

levels in subsequent da rkness (Fig. 4A). If red l ight illu- m i n a t i o n was con t inued for 5 min, decreased Ins(1,4,5)P3 levels were sus ta ined dur ing tha t t ime (da ta no t shown). The Ins(1,4,5)P3 levels increased a b o u t 1.7-fold in extensor p ro top l a s t s 30 s after t ransfer to darkness wi thout red l ight i l lumina t ion (Fig. 4B). I r r a d i a t i o n with 80 rtmol ' m - Z . s - 1 red l ight for 30 s fol lowed by transfer to low- intensi ty red l ight (3-4 lamol' m - Z.s- 1), ins tead of to da rk - ness, had the same effect on Ins(1,4,5)P3 levels in extensor p ro top l a s t s as red l ight fol lowed by darkness; Ins(1,4,5)P3 levels decreased a b o u t 62% dur ing i l lumina t ion with h igh- in tens i ty red l ight and increased to near basa l levels dur ing subsequent low- in tens i ty red l ight (data not shown). All of these t r ea tments with red l ight and da rk - ness cause a p p a r e n t c losure of K + channels in extensor p ro top l a s t s measu red after 3 min of l ight t r ea tmen t (Kim et al. 1993 and Tab le 2). N o n e of these red l ight and da rk t r ea tmen t s affected Ins(1,4,5)P3 levels in flexor p ro top las t s (Fig. 4A, B and results no t shown). Since some of these t r ea tmen t s (a l though no t all) open K § channels in these p r o t o p l a s t s (Kim et al. 1993 and Table 2), these results suggest tha t Ins(1,4,5)P3 levels do not p l ay a role in regula t ing K § open ing in flexor cells dur ing the l ight per iod. Blue l ight had no effect on Ins(1,4,5)P3 levels

. 6

"~ 4

f -~ 2 g~

o

~ 6

f ~ 2

0

A

Flexor

E x t e n s o r

-30 3 0 9 0 1 5 0

Time ( s )

210 270 3 3 0

Fig. 4A, B. Time course of changes in Ins(1,4,5)P3 levels in response to darkness or red light followed by darkness. Levels of Ins(1,4,5)P3 were assayed in extensor protoplasts (closed circles) or flexor proto- plasts (open circles) of S. saman as described in Materials and methods at varying times after transfer from white light to darkness with (A) or without (B) an intervening 30-s irradiation with 80 lamol.m- 2.s- t red light. Transfers were performed at hours 10 to 12 of the light period. The white bars at the bottom of the graphs indicates white light illumination, the hatched bar indicates red light illumination, and the black bars indicate darkness. Values represent mean _+ SD of three to four separate experiments

in ei ther extensor or flexor p ro top las t s in the l ight pe r iod (da ta no t shown), consis tent wi th the fact that it does not affect K § channels in ei ther extensor or flexor p ro top las t s dur ing the l ight per iod (Kim et al. 1993). Basal levels of Ins(1,4,5)P3 were abou t 4 pmol in bo th cell types at this t ime po in t (Fig. 4), abou t the same as basa l levels in ex tensor p ro top las t s in the da rk period.

Neomycin blocks Ins(1,4,5)P3 production and subsequent changes in the state o f K § channels. In o rde r to de te rmine whether the changes in Ins(1,4,5)P3 levels are due to the ac t iva t ion of phospho l ipase C, we tested the effect of neomycin , an inh ib i tor of hydrolys is of Ptdlns(4,5)P2 by

Table 1. The effects of blue light and drug treatments on the state of K + channels in extensor and flexor protoplasts Samanea saman at hours 4 to 8 of the dark period. Protoplasts were isolated during the light period as described in Materials and methods and transferred to darkness at the time of normal lights off in the growth chamber. Blue light treatment was for 3 min and mastoparan incubation was for 150 s before 200 mM K § was added to test the state of the K § channels. Neomycin, when used, was added, in darkness, 30 s before blue light treatment. An increase in AF/Fo indicates that K - can enter the cell and change the membrane potential; the channels are presumed to be open (Kim et al. 1992, 1993). No change in AF/Fo indicates that K § cannot enter the cell; the channels are presumed to be closed (Kim et al. 1992). Values given are the mean + SD of three to four independent measurements. Values for dark and blue light conditions are from (Kim et al. 1992). Abbreviations: D, darkness; BL, blue light

Extensor protoplasts Flexor protoplasts Treatment

light AF/Fo(%) K + channel A F/Fo(%) K + channel

Dark 0 + 0 closed 5.95 _ 1.77 open D ~ BL 3.15 + 0.15 open 0 ___ 0 closed D + mastoparan 4.38 + 0.22 open 0.45 + 0.04 ..closed D ~ BL (with neomycin) 3.06 -I- 0.36 open 4.05 _+ 1.32 open

H.Y. Kim et al.: Ins(1,4,5)P 3 Regulation of K + Channels in S. saman 283

Table 2. The effects of red light, darkness, and drug treatments on the state of K + channels in extensor and flexor protoplasts of S. saman at hours 10 to 12 of the light period. Protoplasts were prepared and maintained as described in the legend to Table 1 and were transferred to white light at the usual time of lights-on in the growth chamber. Dark treatments were for 3 min and incubation with mastoparan was for 150 s before 200 mM K + was added to test the state of the K § channels. Red light pulses were for 30 s and neomycin, when used, was added under white light 30 s before red light or darkness was initiated. Values given are the mean + SD of three to four independent measurements. Values for conditions without neomycin or mastoparan are from (Kim et al. 1993). ND, not done; RL, red light; WL, white light; other abbreviations as in Table 1

Extensor protoplasts Flexor protoplasts Treatment

light AF/Fo(%) K + channel AF/Fo (%) K + channel

WL 2.40 + 0.20 open 0 ___ 0 closed WL ~ D 0.03 ___ 0.03 closed 0 + 0 closed WL ~ RL ~ D 0 + 0 closed 2.17 + 0.11 open WL + mastoparan 0.40 + 0.14 closed 2.57 + 0.26 open WL ~ D (with neomycin) 2.30 + 0 open 0.17 + 0.05 closed WL --* RL --* D (with neomycin) ND 2.60 + 0.09 open

phospho l ipa se C, on the b l u e l ight- and da rk - i nduc e d changes in Ins(1,4,5)P3 levels and on the closing of K + channels . W h e n a d d e d 30 s before blue l ight i l lumina- t ion at hours 4 to 8 of the da rk per iod, 10 laM neomyc in inhib i ted b lue- l igh t - induced Ins(1,4,5)P3 p r o d u c t i o n (Fig. 5A) and b locked b lue- l igh t - induced K + - c h a n n e l c losure (Table 1) in flexor p ro top las t s . W h e n a d d e d 30 s before t ransfer to darkness at hours 10 to 12 of the l ight per iod, neomyc in b locked the increase in Ins(1,4,5)P3 levels (Fig. 5B) and da rk - i nduced K + channel c losure (Table 2) in ex tensor p ro top las t s . I t had no effect on the K + - c h a n n e l open ing induced by red l ight fol lowed by darkness in flexor p ro top la s t s (Table 2) or on the blue- l igh t - induced open ing of K + channels in ex tensor p ro to - plasts (Table 1). These d a t a suppor t the hypothes is tha t the Ins(1,4,5)P3 accumula t i on which corre la tes with K +- channel c losure on blue l ight t r ea tmen t of flexor p ro to - plasts in the da rk per iod and da rk t r ea tmen t of ex tensor p ro top la s t s in the l ight pe r iod is p r o d u c e d by phos- pho l ipase C act ivat ion.

The effects o f mastoparan on Ins(1,4,5)P3 levels in the dark and in the light. To test whether G pro te ins might media te the blue- l ight- and da rk - i nduced changes in Ins(1,4,5)P3 levels in ex tensor and flexor p ro top l a s t s (Figs. 3, 4), we examined the effects of m a s t o p a r a n , a wasp venom tet- r adecapep t ide , which is a po ten t ac t iva to r of G pro te ins (Higash i j ima et al. 1990). At hours 4 to 8 of the d a r k per iod, 10 g M m a s t o p a r a n mimicked blue l ight i l lumina- t ion, increas ing Ins(1,4,5)P3 levels 2.7-fold in flexor p ro to - plasts and decreas ing Ins(1,4,5)P3 levels 1.6-fold in exten- sor p ro top la s t s (Fig. 6A). Changes in Ins(1,4,5)P3 levels induced by m a s t o p a r a n were s lower than s imilar changes induced by blue light, p r e sumab ly because m a s t o p a r a n mus t cross the p l a s m a m e m b r a n e and diffuse to its tar - get(s). The effect of m a s t o p a r a n was dose dependen t in bo th types of p r o t o p l a s t (da ta no t shown). At hours 10 to 12 of the l ight per iod, 10 g M m a s t o p a r a n mimicked da rk - ness in its effects on ex tensor p ro top las t s ; Ins(1,4,5)P3 levels increased a b o u t twofold (Fig. 6B). N e o m y c i n also b locked m a s t o p a r a n - i n d u c e d Ins(1,4,5)P3 accumula t ion in bo th cell types at the t imes when m a s t o p a r a n was effective (da ta not shown).

3-

f t

- 1

3 ~ O

fl o

- 1

F l e x o r

B

' ' ' r ' ' ' I ' ' ' I ' ' ' I ' ' ' I ' ' '

-30 30 90 150 210 270 330

Time ( s )

Fig. 5A, B. Neomycin blocks blue-light- and dark-induced Ins(1,4,5)P3 accumulation in S. saman protoplasts. (A) At hours 4 to 8 of the dark period, protoplasts were incubated with 10 gM neomycin for 30 s in darkness and were then illuminated with blue light. Changes in Ins(1,4,5)P3 levels in flexor protoplasts were meas- ured and plotted. Compare with Fig. 3. (B) At hours 10 to 12 of the light period, extensor protoplasts were incubated with 10 gM neomycin for 30s before the white light was turned off, and Ins(1,4,5)P3 levels in extensor protoplasts were measured and plot- ted. Compare with Fig. 4. The bars at the bottom of the figures indicate the light conditions: white bar, white light; black bar, dark- ness; double-hatched bar, blue light. Values represent mean + SD of four separate experiments

To test whether m a s t o p a r a n - i n d u c e d increases in Ins(1,4,5)P3 levels cor re la te with changes in the state of K § channels , we m o n i t o r e d the effects of m a s t o p a r a n on K § changes in the m e m b r a n e po ten t i a l of exten- sor and flexor p ro top las t s . At hours 4 to 8 of the d a r k per iod, m a s t o p a r a n mimicked the effect of b lue l ight on the state of K § channels , c losing open K § channels in flexor p ro top las t s and open ing closed K § channels in ex tensor p ro top las t s (Table 1). W h e n admin i s t e red at hours 10 to 12 of the l ight per iod, m a s t o p a r a n mimicked the effect of red l ight fol lowed by darkness , c losing open K § channels in ex tensor p ro top la s t s and open ing closed

284

12

10

6

4

2

0

6

2

A

E x D s o r

Extensor

mas

-30 30 90 150 210 270 330

T i m e (s)

Fig. 6A, B. The effect of mastoparan on Ins(1,4,5)P3 levels. The Ins(1,4,5)P3 levels were monitored following the addition, at time 0, of 10 gM mastoparan at hours 4 to 8 of the dark period (A) and at hours 10 to 12 of the light period (B). Levels in extensor protoplasts are represented by closed circles and levels in flexor protoplasts by open circles. The black bar at the bottom in A indicates darkness while the white bar in B indicates white light. Values represent mean + SD of three to four separate experiments. The arrow labeled

"mas" indicates the time of addition of mastoparan

3

2 A

1

0 I I I I

0 2 4 6 8 10

[Mastoparan] (~M)

Fig. 7. Concentration dependence of the effect ofmastoparan on the K +-induced changes in membrane potential of S. saman protoplasts in the light period. Extensor (closed circles) and flexor (open circles) protoplasts were incubated with 2 laM DiS-C3-(5) for 5 rain under white light. Mastoparan was added 150 s before addition of 200 mM K +. The AF measured 30s after addition of the external K + was normalized by dividing by Fo and plotted against mastoparan concen- tration. Values represent mean _+ SD of three separate experiments

K + channels in flexor protoplasts (Table 2), an effect that was concent ra t ion-dependent (Fig. 7).

Discussion

The uptake and release of K + by plant cells is central to their ability to regulate cell volume and turgor (Satter

H.Y. Kim et al.: lns(1,4,5)P 3 Regulation of K + Channels in S. saman

1979) and therefore central to both turgor-mediated movements and plant responses to osmotic stress (Cot6 and Crain 1994). Regulation of K + t ransport has been studied in leaflet-moving organs of nyctinastic legumes because such movements depend, at least in part, on the redistribution of K + between cells on opposite sides of the pulvinus (Lowen and Satter 1989). Calcium has been im- plicated in light-induced pulvinar movements (Roblin et al. 1989) which led us to postulate that phosphoinosi t ide turnover and the resulting product ion of Ins(1,4,5)P3 (Berridge 1993) might regulate K + channels in pulvinar cells. We previously reported that light stimulates phos- phoinositide turnover in intact pulvini of the tropical legume S. s a m a n (Satter et al. 1988). However, the intact pulvinus is a poor system for the study of cellular re- sponses to light because extensor and flexor cells behave oppositely to the same signal (Fleurat-Lessard 1990). We have therefore isolated protoplasts of both extensor and flexor cells from the pulvini of S. s a m a n (Kim et al. 1992) and have shown indirectly, using fluorescent dyes sensitive to membrane potential, that the ability of the cells to sustain inward K + fluxes, presumably through inward- rectifying channels, is controlled by light and darkness (Kim et al. 1992, 1993).

We now report that the apparent closure of K + chan- nels correlates with Ins(1,4,5)P3 accumulat ion in flexor cells. Blue light during the dark period prevents inward K + fluxes (Kim et al. 1992) and increases Ins(1,4,5)P3 levels in these protoplasts. The effects of blue light on Ins(1,4,5)P3 levels are not nonspecific since levels are not increased in extensor protoplasts where the same light treatments apparently open the channels. That these changes are physiologically relevant in leaf movements is supported by the fact that they correlate with measured redistribution of K + in the intact pulvinus; blue light in the dark period increases extracellular K + on the flexor side of the pulvinus (Lowen and Satter 1989). Apparent closure of K + channels may also correlate with Ins(1,4,5)P3 accumulat ion in extensor cells, but the situ- at ion is more complex in these cells. Premature darkness during the light period both prevents inward K + fluxes (Kim et al. 1993) and increases lns(1,4,5)P3 levels. This correlates with the dark induced redistribution of K + from extensor cells to flexor cells (Lowen and Satter, 1989). The increase in Ins(1,4,5)P3 levels is not nonspecific as it does not occur in flexor cells where darkness has no effect on the channels.

However, t reatment of extensor cells with red light followed by darkness or by dim red light, like transfer to darkness, appears to close the channels. Levels of Ins(1,4,5)P3 decrease in the red light and then increase in subsequent darkness or dim red light. These three treat- ments which appear to close the channels in extensor protoplasts have in c o m m o n an increase in Ins(1,4,5)P3 levels at the time of transfer to darkness or dim red light, consistent with a dark-induced increase in Ins(1,4,5)P3 levels contr ibuting to the closure of K + channels in exten- sor cells at this time. However, the absolute concentra- tions of Ins(1,4,5)P3 cannot be the only variable regulating K + channels in these cells at this time since Ins(1,4,5)P3 levels are as high in white light, when channels are open, as in subsequent darkness following red light treatment,

H.Y. Kim et al.: Ins(1,4,5)P 3 Regulation of K + Channels in S. saman

when channels are closed. This may reflect compartmen- talization of Ins(1,4,5)P3 so that some of the Ins(1,4,5)P3 detected may not be active in signal transduction. Com- partmentalization of some portion of cellular Ins(1,4,5)P3 has been suggested previously (reviewed by Balla and Catt 1994).

In these experiments, Ins(1,4,5)P3 levels were meas- ured by a radio-receptor assay in which Ins(1,4,5)P3 in the plant extract competes with added [3H]Ins(1,4,5)P3 for binding to Ins(1,4,5)P3 receptor prepared from bovine adrenal glands. The bovine adrenal receptor is specific for Ins(1,4,5)P3; only Ins(1,3,4,5)P4 and synthetic Ins(2,4,5)P3 compete significantly, with 20-fold and 19-fold lower affin- ity, respectively (Palmer and Wakelam 1990). All other compounds tested competed with affinity more than 1000- fold lower.

The validity of this assay for quantification of Ins 1,4,5-P3 in the alga C h l a m y d o m o n a s re inhard t i i was pre- viously demonstrated by parallel HPLC analysis of 3Zp labeled cell extracts (Quarmby et al. 1992). We here demonstrate that the assay is valid with extracts of S. s a m a n pulvinar tissue. First, we showed that S. s a m a n tissues contain no metabolite incorporating phosphate which specifically binds to the receptor, except Ins(1,4,5)P3 (Fig. 1). In particular, we saw no evidence for the binding of Ins(1,3,4,5)P4. The approximately 10% nonspecific binding of other metabolites is consistent with observation of approximately 10% nonspecific binding previously reported with this assay (Palmer and Wakelam 1990). Second, we showed that the receptor binding activ- ity in pulvinar extracts is hydrolyzed by the rbc inositol trisphosphate 5'-monophosphatase with the same kinetics as both commercial authentic Ins(1,4,5)P3 and rbc-ghost Ins(1,4,5)P3 (Fig. 2). This phosphatase has been shown to be specific for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (Downes et al. 1982). The receptor binding activity is unlikely to be Ins(1,3,4,5)P4 since we would not expect it to be hy- drolyzed with the same kinetics as Ins(1,4,5)P3, and since we saw no evidence of Ins(1,3,4,5)P4 binding to the recep- tor (see Fig. 1). Taken together, these results verify that the receptor binding activity we see in S. s a m a n extracts is Ins(1,4,5)P3. While we cannot rule out that S. s a m a n might, under some circumstances, produce another, un- known, metabolite which competes with Ins(1,4,5)P3 for binding to the receptor, the results here described suggest that this is unlikely.

The increased levels of Ins(1,4,5)P3 apparently result from increased hydrolysis of PtdIns(4,5)P2 by phos- pholipase C since the increases are blocked in both cell types by the phospholipase C inhibitor neomycin. Neo- mycin binds PtdIns(4,5)P/ and inhibits phospholipase C at micromolar concentrations (reviewed in McDonald and Mamrack 1995). At very low molar ratios of neomycin to PtdIns(4,5)P2 (less than 1:4) activation of phospholipase C has been observed in vitro (McDonald and Mamrack 1995). At millimolar concentrations, neomycin inhibits Ins(1,4,5)P3-mediated C a 2+ release (Prentki et al. 1986) and activates G proteins (Aridor and Sagi-Eisenberg 1990). Our results are most consistent with neomycin acting through inhibition of phospholipase C since we have used neomycin only at micromolar levels and have shown that neomycin inhibition of the apparent

285

closure of the channels correlates with neomycin inhibi- tion of increases in Ins(1,4,5)P3 levels. We note that basal levels of Ins(1,4,5)P3 decreased after the 30-s pretreatment with neomycin (compare Fig. 5 with Figs. 3 and 4), which may indicate that basal levels of phospholipase C activity are responsible, at least in part, for the basal levels of Ins(1,4,5)P3. We have previously reported similar reduc- tions in basal levels of Ins(1,4,5)P3 in C. re inhard t i i cells (Quarmby et al. 1992) and in soybean cells (Legendre et al. 1993) incubated for 15 30 s with 5-10 laM neomycin.

The increased activity of phospholipase C may be triggered by activation of a G-protein since the G-protein activator mastoparan increases Ins(1,4,5)P3 levels and closes K + channels in both cell types at the appropriate times. Mastoparan has been shown to activate hetero- trimeric G proteins, including Gi and Go (Higashijima et al. 1990) and small G proteins such as rho (Koch et al. 1991). However, it may have other effects as well; it inter- acts with purified calmodulin (Ohki et al. 1991) and antag- onizes calmodulin effects at micromolar levels (Busse and Miilsch 1994). At higher concentrations, it may directly activate phospholipase C (Wallace and Carter 1989) and it may disrupt cell membranes. Our results are most consis- tent with mastoparan acting through a G protein which activates phospholipase C since we have used mastoparan only at micromolar levels and have shown that masto- paran-induced closure of the channels correlates with mastoparan-induced increases in Ins(1,4,5)P3 levels.

Mastoparan was also effective in opening K + channels in flexor protoplasts in the light and in extensor proto- plasts in the dark. Channel opening does not appear to involve phosphoinositide hydrolysis since Ins(1,4,5)P3 levels were not increased by these channel-opening stimuli or by mastoparan. In fact, Ins(1,4,5)P3 levels decreased in extensor protoplasts treated with blue light or mas- toparan during the dark period. This suggests that G pro- teins also regulate K +-channel opening, but either directly or through another signaling pathway which does not involve phospholipase C and phosphoinositide hydroly- sis. There is evidence for direct regulation of K + channels by G proteins in both animals (Birnbaumer et al. 1990) and plants (Fairley-Grenot and Assmann 1991; Assmann 1993). Another signaling pathway that might be involved in K +-channel opening is the phospholipase-Az-mediated signal-transduction pathway (Liscovitch 1992). Products of phospholipase Az activity, including arachidonic and linolenic acid, and lysophospholipids, activate K + chan- nels in animals (Ordway et al. 1991) and in plants (Lee et al. 1994).

We hypothesize that Ins(1,4,5)P3, generated by phos- pholipase-C-catalyzed hydrolysis of PtdIns(4,5)-P2 mobil- izes intracellular Ca z+ and that this Ca z+ inhibits K + influx and activates C1- efflux, leading to cell shrink- ing and leaflet movement of S. saman. Phospholipase C is activated by an activated blue-light receptor in flexor cells and by some signal of darkness, perhaps the absence of blue light, in extensor cells. In each case a G protein may mediate the activation of phospholipase C by the ac- tivated receptor. Although Ins(1,4,5)P3 does not appear to be involved in K +-channel opening in extensor cells, it could play a role in keeping channels closed in these cells in darkness since lns(1,4,5)P3 levels drop when blue light

286

t r iggers open ing of K + channels in f lexor cells. However , Ins(1,4,5)P3 levels do no t change in flexor cells when K + channels are opened by red l ight fol lowed by da rk - hess.

Like leaflet movemen t , open ing and closing of leaf s t o m a t a also depends on changes in turgor , in this case in the s toma ta l gua rd cells. M i c r o m o l a r Ca 2 § levels inhibi t the inward ly -d i rec ted K § channels of guard cells (Schroeder and H a g i w a r a 1989) and s t imula te an ion channels (Hedr ich et al. 1990) which could car ry C I - etttux. The Ins(1,4,5)P3, re leased f rom a "caged" der ivat ive prev ious ly micro in jec ted into gua rd cells, t r iggers a rap id and t rans ient increase in cytosol ic Ca z +, fol lowed by loss of t u r g o t and closure of the s t o m a t a (Gi l roy et al. 1990). T rea tmen t with the p h y t o h o r m o n e abscisic acid induces cell shr inking, p r e sumab ly in pa r t by inhibi t ing po tass ium influx, and was observed to increase Ins(1,4,5)P3 levels in guard cells (Cot6 and Cra in 1994). These da ta , which suggest tha t Ins(1,4,5)P3 is the second messenger which closes inward ly -d i rec ted K + channels in s tomata l guard cells, are consis tent with our results ind ica t ing an ident ical role for Ins(1,4,5)P3 in S. s a m a n m o t o r cells.

F u t u r e s tudies on signal t r ansduc t ion in S. s a m a n

leaflet m o v e m e n t will focus on events downs t r eam of Ins(1,4,5)P3 accumula t ion , tha t is, on the roles of Ca 2+ and p ro t e in kinases in regula t ing K + channels. We will a lso seek to identify the G protein(s) involved in bo th K +-channel open ing and K +-channel c losure and we will seek to e luc ida te the signal t r ansduc t ion pathway(s) tha t open inward-rec t i fy ing K + channels and cause cells to swell.

Supported by grants from NSF (IBN 9206179 and MCB 9305154) and U.S.-Israel Binational Agricultural Research and Development Fund (IS-1670-90RC) to R.C.C. We thank the University of Con- necticut Biotechnology Center for the use of a fluorescent spectro- photometer.

References

Aridor M, Sagi-Eisenberg R (1990) Neomycin is a potent secreta- gogue of mast cells that directly activates a GTP-binding protein involved in exocytosis. J Cell Biol 111:2885 2891

Assmann SM (1993) Signal transduction in guard cells. Annu Rev Cell Biol 9:345 375

Balla T, Catt KJ (1994) Phosphoinositides and calcium signaling. New aspects and diverse functions in cell regulation. Trends Endocrinol Metab 5:250-255

Berridge MJ (1993) lnositol trisphosphate and calcium signaling. Nature 361:315-325

Birnbaumer L, Abramowitz J, Brown AM (1990) Receptor- effector coupling by G proteins. Biochim Biophys Acta 1031: 163 224

Busse R, Mfilsch A (1994) Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett 265: 133 136

Cot6 GG, Crain RC (1994) Why do plants have phosphoinositides? BioEssays 16:39-46

Cot6 GG, Morse MJ, Crain RC, Satter RL (1987) Isolation of soluble metabolites of the phosphatidylinositol cycle from Samanea saman. Plant Cell Rep 6:352-355

Downes CP, Michell RH (1981) The polyphosphoinositide phosphodiesterase of erythrocyte membranes. Biochem J 198: 133-140

H.Y. Kim et al.: Ins(1,4,5)P 3 Regulation of K + Channels in S. saman

Downes CP, Mussat MC, Michell RH (1982) The inositol trisphos- phate phosphomonoesterase of the human erythrocyte mem- brane. Biochem J 203:169-177

Fairley-Grenot K, Assmann SM (1991) Evidence for G-protein regulation of inward K + channel current in guard cells of lava bean. Plant Cell 3:1037-1044

Fleurat-Lessard P (1990) Structure and ultrastructure of the pul- vinus in nyctinastic legumes. In: Satter RL, Gorton HL, Vogel- mann TC (eds) The pulvinus: motor organ for leaf movement. Am Soc Plant Physiol, Rockville, pp 101 129

Gilroy S, Read ND, Trewavas AJ (1990) Elevation of cytoplasmic calcium by caged calcium or caged inositol trisphosphate initi- ates stomatal closure. Nature 346:769 771

Hedrich R, Busch H, Raschke K (1990) Ca 2+ and nucleotide dependent regulation of voltage dependent anion channels in the plasma membrane of guard cells. EMBO J 9: 3889-3892

Higashijima T, Burnier J, Ross EM (1990) Regulation of G i and G o by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity. J Biol Chem 265:14176-14186

Huang C-H, Tate BF, Crain RC, Cot6 GG (1995) Multiple phos- phoinositide-specific phospholipases C in oat roots. Plant J 8:257 267

Irvine RF, Letcher AJ, Lander D J, Drobak BK, Dawson AP, Musgrave A (1989) Phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)phosphate in plant tissues. Plant Physiol 89:888-892

Kikkawa U, Kishimoto A, Nishizuka Y (1989) The protein kinase C family: Heterogeneity and its implications. Annu Rev Biochem 58:31M4

Kim HY, Cot6 GG, Crain RC (1992) Effects of light on the mem- brane potential of protoplasts from Samanea saman pulvini: Involvement of K + channels and the H +-ATPase. Plant Physiol 99:1532-1539

Kim HY, Cot6 GG, Crain RC (1993) Potassium channels in Samanea saman protoplasts controlled by phytochrome and the biological clock. Science 260:960-962

Koch G, Haberman B, Mohr C, Just I, Aktories K (1991) Interaction of mastoparan with the low molecular mass GTP-binding pro- teins rho/rac. FEBS Lett 291:336-340

Lee Y, Satter RL (1989) Effects of white, blue, red light and darkness on pH of the apoplast in the Samanea pulvinus. Planta 178: 31M0

Lee Y, Lee HJ, Crain RC, Lee A, Korn SJ (1994) Polyunsaturated fatty acids modulate stomatal aperture and two distinct K + channel currents in guard cells. Cell. Signaling 14:96-100

Legendre L, Yueh YG, Crain R, Haddock N, Heinstein PF, Low PS (1993) Phospholipase C activation during elicitation of the oxidative burst in cultured plant cells. J Biol Chem 268: 24559-24563

Liscovitch M (1992) Cross talk among multiple signal-activated phospholipases. Trends Biochem Sci 17:393-399

Lowen CZ, Satter RL (1989) Light-promoted changes in apoplastic K + activity in the Samanea saman pulvinus, monitored with liquid membrane microelectrodes. Planta 179:421-427

McDonald LJ, Mamrack MD (1995) Phosphoinositide hydrolysis by phospholipase C modulated by multivalent cations La 3+, AI 3+, neomycin, polyamines, and melittin. J Lipid Mediators Cell Signaling 11:81-91

Moran N (1990) The role of ion channels in osmotic volume changes in Samanea motor cells analysed by patch-clamp methods. In: Satter RL, Gorton HL, Vogelmann TC (eds) The pulvinus: motor organ for leaf movement. Am Soc Plant Physiol, Rockville, pp 142-159

Ohki S, Yazawa M, Yagi K, Hikichi K (1991) Mastoparan binding induces Ca 2+-transfer between two globular domains of calmodulin: A 1H NMR study. J. Biochem. (Tokyo) 110: 737-742

Ordway RW, Singer J J, Walsh JV, Jr (1991) Direct regulation of ion channels by fatty acids. Trends Neurosci 14:96 100

H.Y. Kim et al.: Ins(1,4,5)P 3 Regulation of K + Channels in S. saman

Palmer S, Wakelam MJO (1990) Mass measurement of inositol 1,4,5-trisphosphate using a specific binding assay. In: Irvine RF (ed) Methods in inositide research, Raven Press, New York, pp 127-134

Prentki M, Deeney JT, Matschinsky FM, Joseph SK (1986) Neomycin: a specific drug to study the inositol-phospholipid signalling system? FEBS Lett 197:285-288

Quarmby LM, Yueh YG, Cheshire JL, Keller LR, Snell W J, Crain RC (1992) Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii. J Cell Biol 116: 737-744

Roblin G, Fleurat-Lessard P, Bonmort J (1989) Effects of com- pounds affecting calcium channels on phytochrome- and blue pigment-mediated pulvinar movements of Cassia fasciculata. Plant Physiol 90:697-701

Satter RL (1979) Leaf movements and tendril curling. In: Haupt W, Feinleib ME (eds) Encyclopedia of plant physiology, N S, Vol. 7: Physiology of movements, Springer-Verlag, Berlin, pp 442~484

287

Satter RL, Morse M J, Lee Y, Crain RC, Cot6 GG, Moran N (1988) Light- and clock-controlled leaflet movements in Samanea saman. A physiological, biophysical, and biochemical analysis. Bot Acta 101:205-213

Schroeder JI, Fang HH (1991) Inward-rectifying K + channels in guard cells provide a mechanism for low-affinity K + uptake. Proc Natl Acad Sci USA 88:11583-11587

Schroeder JI, Hagiwara S (1989) Cytosolic calcium regulates ion channels in the plasma membrane of Vicia faba guard cells. Nature 338:427~430

Van Haastert PJM (1989) Determination of inositol 1,4,5-trisphos- phate levels in Dietyostelium by isotope dilution assay. Anal Biochem 177:115-119

Wallace MA, Carter HR (1989) Effects of wasp venom peptide, mastoparan on phosphoinositide-specific phospholipase C puri- fied from rabbit brain membranes. Biochim Biophys Acta 1006: 311-316