Importance of specific hydrogen bonds of archaeal rhodopsins for the binding to the transducer protein

Four rhodopsins, bacteriorhodopsin (bR), halorhodopsin (hR), sensory rhodopsin (sR) and phoborhodopsin (pR) exist in archaeal membranes. bR and hR work as a light-driven ion pump. sR and pR work as a photo-sensor of phototaxis, and form signaling complexes in membranes with their respective cognate transducer proteins HtrI (with sR) and HtrII (with pR), through which light signals are transmitted to the cytoplasm. What is the determining factor(s) of the specific binding to form the complex? Binding of the wild-type or mutated rhodopsins with HtrII was measured by isothermal titration calorimetric analysis (ITC). bR and hR could not bind with HtrII. On the other hand, sR could bind to HtrII, although the dissociation constant (K(D)) was about 100 times larger than that of pR. An X-ray crystallographic structure of the pR/HtrII complex revealed formation of two specific hydrogen bonds whose pairs are Tyr199(pR)/Asn74(HtrII) and Thr189(pR)/Glu43(HtrII)/Ser62(HtrII). To investigate the importance of these hydrogen bonds, the K(D) value for the binding of various mutants of bR, hR, sR and pR with HtrII was estimated by ITC. The K(D) value of T189V(pR)/Y199F(pR), double mutant/HtrII complex, was about 100-fold larger than that of the wild-type pR, whose K(D) value was 0.16 microM. On the other hand, bR and hR double mutants, P200T(bR)/V210Y(bR) and P240T(hR)/F250Y(hR), were able to bind with HtrII. The K(D) value of these complexes was estimated to be 60.1(+/-10.7) microM for bR and to be 29.1(+/-6.1) microM for hR, while the wild-type bR and hR did not bind with HtrII. We concluded that these two specific hydrogen bonds play important roles in the binding between the rhodopsins and transducer protein.     

Phototaxis inEuglena gracilis: Effect of sodium azide and triphenylmethyl phosphonium ion on the photosensory transduction chain

Phototaxis in the flagellateEuglena gracilis was studied by means of a microvideographic analysis, and the light-induced directional movement was determined by computer-based statistical treatments. Lateral white light with an illuminance of 25 lx (0.105 Wm^–2) caused the cells to preferentially swim toward the light source (positive phototaxis), while an illuminance of 1,000 lx (4.2 Wm^–2) induced negative phototaxis. The lipophilic membranepenetrating cation triphenylmethyl phosphonium ion (TPMP⁺) specifically inhibited positive phototaxis, while it hardly affected negative phototaxis. The uncoupler sodium azide, on the other hand, impaired negative phototaxis substantially.     

Photomovement of the red alga Porphyridium cruentum (Ag.) Naegeli

Phototaxis of the unicellular red alga Porphyridium cruentum was studied by staining the slime tracks of individual cells as well as with the aid of a population method. Because of the increased straightness of the movement the mean linear velocity of a unilaterally illuminated population exceeds considerably that of an only photokinetically stimulated one. In white light the phototactic reaction is saturated already at 100 lx. The zero threshold lies at about 1 lx. Spectral sensitivity curves of phototaxis obtained at high photon fluence rates (>=10^–11 mol cm^–2 s^–1) display two main peaks which shift against each other at intermediate irradiances and, finally, form a single maximum in the blue range (443 nm) at low photon fluence rates (10^–12 and 10^–13 mol cm^–2 s^–1). Photon fluence rate-response curves reveal that supraoptimal irradiances decrease the phototactic reaction, especially in the range of the highest sensitivity of the cells. The action spectrum of phototaxis was calculated on the basis of the photon fluence rate-response curves. It shows a maximum at 443 nm and shoulder at 416 nm and between 467 and 477 nm. Wavelengths longer than 540 nm are phototactically inactive even at very high irradiances (25 W m^–2). Thus, this is the first phototactic action spectrum of a biliprotein-containing organism which does not indicate the participation of biliproteins in the absorption of phototactically active light. DCMU and potassium iodide have no specific effects on phototaxis.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Proton excretion and cell expansion in bean leaves

Light-induced expansion of Phaseolus vulgaris L. leaf cells is accompanied by increased cell-wall plasticity. The possibility that leaf-cell walls are loosened by excreted protons has been investigated. First, light causes acidification, detected at the leaf surface, within 5–15 min. Growth starts 10–20 min after exposure to light. Second, exogenous acid induces loosening of isolated leaf cell walls. Third, infiltration of the tissue with a neutral buffer inhibits light-induced growth. Fourth, fusicoccin stimulates growth of as well as H⁺ excretion by bean leaf cells, without light. These findings show that the acid-growth theory is applicable to light-induced growth of leaf cells, and indicate that light-induced proton excretion initiates cell enlargement in leaves.Abbreviations FC fusicoccin - RL red light - WEx wall extensibility - WL white light     

Responses of strawberry plantlets cultured in vitro under superbright red and blue light-emitting diodes (LEDs)

Unrooted strawberry cv. `Akihime' shoots with three leaves obtained from standard mixotrophic cultures were cultured in the ``Culture Pack'-rockwool system with sugar-free MS medium under CO<sub>2</sub>-enriched condition. To examine the effect of superbright red and blue light-emitting diodes (LEDs) on in vitro growth of plantlets, these cultures were placed in an incubator, ``LED PACK', with either red LEDs, red LEDs1blue LEDs or blue LEDs light source. To clarify the optimum blue and red LED ratio, cultures were placed in ``LED PACK 3' under LED light source with either 100, 90, 80, or 70% red + 0, 10, 20, 30% blue, respectively, and also under standard heterotrophic conditions. To determine the effects of irradiation level, cultures were grown under 90% red LEDs + 10% blue LEDs at 45, 60 or 75 mol m^–2 s^–1 . Plantlet growth was best at 70% red + 30% blue LEDs. The optimal light intensity was 60 mol m^–2 s^–1. Growth after transfer to soil was also best after in vitro culture with plantlets produced were 70% red LEDs + 30% blue LEDs.     

Chloride ion binding to bacteriorhodopsin at low pH: an infrared spectroscopic study

Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-induced ion pumps in the cell membrane of Halobacterium salinarium. Under normal conditions bR is an outward proton transporter, whereas hR is an inward Cl- transporter. There is strong evidence that at very low pH and in the presence of Cl-, bR transports Cl- ions into the cell, similarly to hR. The chloride pumping activity of bR is connected to the so-called acid purple state. To account for the observed effects in bR a tentative complex counterion was suggested for the protonated Schiff base of the retinal chromophore. It would consist of three charged residues: Asp-85, Asp-212, and Arg-82. This quadruplet (including the Schiff base) would also serve as a Cl- binding site at low pH. We used Fourier transform infrared difference spectroscopy to study the structural changes during the transitions between the normal, acid blue, and acid purple states. Asp-85 and Asp-212 were shown to participate in the transitions. During the normal-to-acid blue transition, Asp-85 protonates. When the pH is further lowered in the presence of Cl-, Cl- binds and Asp-212 also protonates. The binding of Cl- and the protonation of Asp-212 occur simultaneously, but take place only when Asp-85 is already protonated. It is suggested that HCl is taken up in undissociated form in exchange for a neutral water molecule.     

Light-induced fluorescence changes in<Emphasis Type="Italic"> Phycomyces</Emphasis>: evidence for blue light-receptor associated flavo-semiquinones

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH^·), and the flavo-hydroquinone (FlH₂) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH₂, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.Abbreviations FAD flavin adenine dinucleotide - Fl oxidized flavin - FlH flavo-semiquinone radical - FlH₂ flavo-hydroquinone - LIAC light-induced absorbance change - LIFC light-induced fluorescence change - Pt oxidized pterin - PtH₂ dihydro-pterin - PtH₄ tetrahydro-pterin     

Effects of L-alanine and L-alanine peptides on the chemotaxis of tetrahymena: Evolutionary conclusions

L-alanine and its peptides (L-Ala-2–6) do not attract or repulse Tetrahymena in a 10^–8M concentration. In 10^–10M concentration there is a consistent repellent effect. Twenty four hours after L-alanine or L-alanine-peptides' pretreatment (imprinting) the progeny generation of the cells react differently to the same materials. L-Alanine, L-alanine penta- and hexapeptide in both concentrations are chemoattractant, while L-alanine tetrapeptide is repellent. L-Alanine dipeptide is inert in 10^–10M and repellent at 10^–8M concentrations, while L-alanine tripeptide is strongly repellent at 10^–10M and attractant at 10^–8M concentrations. This means, that the first encounter (imprinting) with an exogeneous amino acid or peptide is decisive to the later reaction of the protozoan cell. The chain length is important in the imprinting, however the reaction is not consistent. The experiments call the attention to the significance of imprinting in the receptor and hormone evolution.     

Delayed fluorescence study on P*QA→ P+QA − charge separation energetics linked to protons and salt in reaction centers from Rhodobacter sphaeroides

The energetics of the first stable charge separated state, P⁺Q_A– relative to that of P–Q_A was examined in isolated RC from Rhodobacter sphaeroides by delayed fluorescence. The temperature dependence of the delayed fluorescence indicates that the charge separation is a highly enthalpy-driven process (H = – 818 ± 20 meV at pH 8) and the free energy gap between P–Q_A and P⁺Q_A– drops with increasing pH (40 ± 4 meV between pH 6 and 10). The pH-dependence of the free energy change of the P⁺Q_A– state runs parallel to the (integrated) net proton uptake due to the PQ_A/P⁺Q_A– redox change in a wide pH range and under different ionic conditions. Elevation of the ionic strength increases the delayed fluorescence intensity and decreases the (dark and light) pK_a values as well as the light-induced pK_a changes of the protonatable groups of the protein. The observed dependence of the energetics of P⁺Q_A– on the concentration and composition of mobile ions is discussed in terms of binding and screening of protonatable groups and surface charges as dominant modes of electrostatic interaction between RC and salt.     

Storage of light-driven transthylakoid proton motive force as an electric field (Δψ) under steady-state conditions in intact cells of <Emphasis Type="Italic">Chlamydomonasreinhardtii</Emphasis>

Proton motive force (pmf) is physiologically stored as either a ΔpH or a membrane potential (Δψ) across bacterial and mitochondrial energetic membranes. In the case of chloroplasts, previous work (Cruz et al. 2001, Biochemistry 40: 1226–1237) indicates that Δψ is a significant fraction of pmf, in vivo, and in vitro as long as the activities of counterions are relatively low. Kinetic analysis of light-induced changes in the electrochromic shift (ECS) in intact leaves was consistent with these observations. In this work, we took advantage of the spectroscopic properties of the green alga, Chlamydomonas reinhardtii, to demonstrate that light-driven Δψ was stored in vivo over the hours time scale. Analysis of the light-induced ECS kinetics suggested that the steady-state Δψ in 400 μmol photons m⁻² s⁻¹ red light was between 20 and 90 mV and that this represented about 60% of the light-induced increase in pmf. By extrapolation, it was surmised that about half of total (basal and light-induced) pmf is held as Δψ. It is hypothesized that Δψ is stabilized either by maintaining low chloroplast ionic strength or by active membrane ion transporters. In addition to the strong implications for regulation of photosynthesis by the xanthophyll cycle, these results imply that pmf partitioning is important across a wide range of species.     

Fast Fluorescence Quenching from Isolated Guard Cell Chloroplasts of Vicia faba Is Induced by Blue Light and Not by Red Light

Chlorophyll a fluorescence transients from isolated Vicia faba guard cell chloroplasts were used to probe the response of these organelles to light quality. Guard cell chloroplasts were isolated from protoplasts by passing them through a 10-μm nylon net. Intact chloroplasts were purified on a Percoll gradient. Chlorophyll a fluorescence transients induced by actinic red or blue light were measured with a fluorometer equipped with a measuring beam. Actinic red light induced a monophasic quenching, and transients induced by blue light showed biphasic kinetics having a slow and a fast component. The difference between the red and blue light-induced transients could be observed over a range of fluence rates tested (200-800 μmol m⁻² s⁻¹). The threshold fluence rate of blue light for the induction of the fast component of quenching was 200 μmol m⁻² s⁻¹, but in the presence of saturating red light, fluence rates as low as 25 μmol m⁻² s⁻¹ induced the fast quenching. These results indicate that guard cell chloroplasts have a specific response to blue light.     

Effect of external factors on phototaxis of Chlamydomonas reinhardtii

The effects of several cations on phototaxis of Chlamydomonas reinhardtii have been studied with the aid of an automated phototaxis monitoring device, coupled with a continuous culture. Sodium, potassium and magnesium ions, if added to the complete nutrient medium, have only slight effects on phototaxis at lower concentrations (10^-3 mol), but inhibit at higher concentrations (10^-2 mol). This inhibitory effect is not specific because motility is also impaired. Addition of 10^-3 mol calcium enhances the phototactic reaction for some hours, but then the stimulation decreases gradually. Addition of 10^-2 mol calcium causes strong inhibition. However, the reactivity recovers gradually during the following hours. If 10^-3 mol potassium which does not influence phototaxis if added alone is applied simultaneously with calcium, the stimulation by calcium is enhanced. By the addition of 5·10^-4—2·10^-3 mol Ca²⁺ or Ca²⁺+K⁺ cicadian rhythms with an average period length of 24 h are initiated which damp out after 1–2 weeks. If the cells are grown in a calcium deficient medium or if calcium is removed, phototactic activity decreases to very low reaction values or to zero, but is drastically increased immediately after the addition of calcium. The stimulatory effect of Ca²⁺ ions is specific. Ca²⁺ cannot be fully substituted by Ba²⁺ or Sr²⁺, and phototaxis is reversibly inhibited by lanthanum which is known to inhibit the calcium pump.     

Chemotactic behavior of mutants of the nematodeCaenorhabditis elegans that are defective in osmotic avoidance

Summary Wild-typeC. elegans and seven derivative strains previously selected for absence of the wild-type tendency to avoid highly concentrated solutions (Culotti and Russell, 1978) were tested for responsiveness in 11 assays of chemotaxis, including 6 attractant and 3 repellent stimuli. The two strains altered in the geneosm-1 (P808 and P816) did not respond in any test except, possibly, one or two weak responses. Strain P801 responded to one attractant and two repellents. Strain P802 made moderately strong responses to most stimuli and avoided CO₂ in phosphate buffer as strongly as the wild-type. Of particular interest, this strain avoided OH^– which is attractive to wild-type. Strain P821 avoided CO₂ in phosphate buffer weakly, if at all, but did respond to the attractants Na⁺ and Cl^–. Conversely, strains P813 and P811 made little if any response to any attractant but did respond to the two strong repellents. Taken together with other results, these findings suggest that the osmotic response has more gene requirements in common with both attractive and repellent chemical stimuli than with thermal or mechanical stimuli. In addition, they indicate that the known chemical stimuli and the osmotic stimulus are probably mediated by at least 9 different receptors.This research was supported by the National Science Foundation and the National Institutes of Health. I would also like to thank Ms. Georgann Hardin, Mr. Kirt Rusenko, and Ms. Deborah Higgins for their assistance in carrying out the experiments. Drs. J.G. Culotti and R.L. Russell generously provided the mutant strains they had isolated.     

Photoresponses of late instar <Emphasis Type="Italic">Chaoborus punctipennis</Emphasis> larvae to UVR

With a few clear exceptions (e.g., Daphnia) it is uncertain if most aquatic invertebrates can detect and respond to ultraviolet radiation (UVR). It is known that many aquatic invertebrates are vulnerable to UVR and that anthropogenically-induced increases in surface UVR have occurred in recent decades. We examined the photoresponses of late larval instars of Chaoborus punctipennis to different combinations of UVA (320–400 nm), UVB (300–320 nm) and visible light (400–700 nm) to determine whether the larvae can detect and/or avoid UVR. To accomplish this, we exposed late instar C. punctipennis larvae to a directional light source of UVR only (peak wavelength at 360 nm), visible light only or visible plus various wavebands of UVR. We examined negative phototaxis for 10 min at a quantum flux of 2.62 x 10¹³ quanta s^–1 cm^–2 (S.D. = 3.63 x 10¹² quanta s^–1 cm^–2). In the dark, larvae stayed close to the surface of the experimental vessels. Under all treatments containing visible light the larvae exhibited negative phototaxis and occupied the bottom of the vessels. Under UVR only, the larvae occupied the middle of the water column. Our results suggest that late instar C. punctipennis larvae are unable to detect and avoid UVB and short UVA wavelengths but they can detect long UVA wavelengths.     

Light quality affects photosynthesis and leaf anatomy of birch plantlets in vitro

Cultures in vitro of Betula pendula Roth were subjected to light of different spectral qualities. Photosynthetic capacity was highest when the plantlets were exposed to blue light (max recorded photosynthesis, 82 mol CO₂ dm^–2 h^–1) and lowest when irradiated with light high in red and/or far-red wave lengths (max recorded photosynthesis, 40 mol CO₂ dm^–2 h^–1). Highest chlorophyll content (2.2 mg dm^–2 leaf area) was found in cultures irradiated with blue light, which also enhanced the leaf area. Morphometric analysis of light micrographs showed that the epidermal cell areas were largest in plantlets subjected to blue light and smallest in those subjected to red light. Morphometric analysis of electron micrographs of palisade cells, showed that the functional chloroplast area was largest in chloroplasts of leaves subjected to blue light and smallest in those exposed to red light. We suggest that light quality affects photosynthesis both through effects on the composition of the photosynthetic apparatus and on translocation of carbohydrates from chloroplasts.     

Modulation of HERG channel inactivation by external cations

We investigated the effect of external cations on the permeability characteristics and gating kinetics of the human ether-à-go-go-related gene (HERG) current using the whole-cell patch-clamp technique. Inward HERG currents were recorded on hyperpolarization in 140 mM external Cs⁺ and Rb⁺, as well as K⁺. The permeability ratios of Rb⁺ and Cs⁺ relative to K⁺ were 1.25 and 0.56, respectively. Biphasic outward currents were recorded on depolarization in 140 mM Cs⁺ and in Rb⁺ with much smaller amplitude. The voltage dependence of inactivation was affected by external cations, such that the half-inactivation voltage shifted from –69.4±3.7 mV in K⁺ to –30.7±1.6 mV in Cs⁺ and to –35.8±1.9 mV in Rb⁺ (n=5). The time constants of inactivation were also changed significantly by external cations; of inactivation at +40 mV was 16.4±2.2 ms in 140 mM K⁺, 181±20.3 ms in Cs⁺, and 94.1±7.6 ms in Rb⁺ (n=5). Voltage dependence of activation was not altered significantly. The inhibition of the rapid inactivation mechanism by large cations may suggest that the foot-in-the-door model of gating is involved in HERG channel inactivation.     

Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini : Involvement of K Channels and the H -ATPase

Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide. White and blue light induced transient membrane hyperpolarization of both extensor and flexor protoplasts; red light had no effect. Following white or blue light-induced hyperpolarization, the addition of 200 millimolar K⁺ resulted in a rapid depolarization of extensor, but not of flexor protoplasts. In contrast, addition of K⁺ following red light or in darkness resulted in a rapid depolarization of flexor, but not of extensor protoplasts. In both flexor and extensor protoplasts, depolarization was completely inhibited by tetraethylammonium, implicating channel-mediated movement of K⁺ ions. These results suggest that K⁺ channels are closed in extensor plasma membranes and open in flexor plasma membranes in darkness and that white and blue light, but not red light, close the channels in flexor plasma membranes and open them in extensor plasma membranes. Vanadate treatment inhibited hyperpolarization in response to blue or white light, but did not affect K⁺ -induced depolarization. This suggests that white or blue light-induced hyperpolarization results from activation of the H⁺ -ATPase, but this hyperpolarization is not the sole factor controlling the opening of K⁺ channels.     

Elevating intracellular free Mg2+ preserves sensitivity of Na+−K+ pump to ATP at reduced temperatures in guinea pig red blood cells

Red cells of hibernating species have a higher relative rate of Na⁺–K⁺ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na⁺–K⁺ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37°C by measuring ouabain-sensitive K⁺ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg²⁺ to 2 mmol·l^-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20°C, rather than to decrease, as occurs in cells not loaded with Mg²⁺. In ground squirrel cells raising intracellular free Mg²⁺ had little effect on apparent affinity of the pump for ATP at 20°C. ATP affinity rose slightly with cooling both in Mg²⁺-enriched and in control ground squirrel cells. Increased intracellular free Mg²⁺ in guinea pig cells stimulated Na⁺–K⁺ pump activity so that at 20°C the pump rate was the same in the Mg²⁺-enriched guinea pig and control ground squirrel cells. Pump activity in Mg²⁺-enriched guinea pig cells at 5°C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na⁺–K⁺ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg²⁺. Conversely, in ground squirrel red cells natural rise of free Mg²⁺ may in part account for the preservation of the ATP affinity of their Na⁺–K⁺ pump with cooling.Abbreviations K _m Michaelis-Menten constant for apparent affinity - MOPS 3-(N-morpholino)-propanesulphonic acid - [Mg²⁺]_i intracellular concentration of free Mg²⁺ - OD optical density - RBC red blood cell(s) - T _b body temperature     

Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells

Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regulated 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 protoplasts. 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)P₃ 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 protoplasts. 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 phospholipase 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.Abbreviations DiS-C₃-(5) 3,3-dipropylthiadicarbocyanine iodide - F measure change in Dis-C₃-(5) fluorescence - F_o initial Dis-C₃-(5) fluorescence - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PtdIns(4,5)P₂ phosphatidylinositol 4,5-bisphosphate - rbc red blood cell 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 Connecticut Biotechnology Center for the use of a fluorescent spectrophotometer.