Single Light Flashes Research Articles

Sensitized photoinactivation of minigramicidin channels in bilayer lipid membranes

The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dimer of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double β 5.7-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded β 6.3-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer.

Calcium-Sensitive Downregulation of the Transduction Chain in Rod Photoreceptors of the Rat Retina

In vertebrate rod outer segments phototransduction is suggested to be modulated by intracellular Ca. We aimed at verifying this hypothesis by recording saturated photosignals in the rat retina after single and double flashes of light and determining the time t c to the beginning of the signal recovery. The time course of Ca i after a flash was calculated from a change of the spatial Ca 2+ concentration profile recorded in the space between the rods. After single flashes t c increased linearly with the logarithm of flash intensity, confirming the assumption that t c is determined by deactivation of a single species X* in the phototransduction cascade. The photoresponse was shortened up to 45% if the test flash was preceded by a conditioning preflash. The shortening depended on the reduction of Ca i induced by the preflash. The data suggest that the phototransduction gain determining the amount of activated X* is regulated by a Ca i-dependent mechanism in a short time period (<800 ms) after the test flash. Lowering of Ca i by a preflash reduced the gain up to 20% compared to its value in a dark-adapted rod. The relation between phototransduction gain and Ca i revealed a K 1/2 value close to the dark level of Ca i.

Photoinactivation of Photosystem II by flashing light

Inhibition of Photosystem II (PS II) activity by single turnover visible light flashes was studied in thylakoid membranes isolated form spinach. Flash illumination results in decreased oxygen evolving activity of PS II, which effect is most pronounced when the water-oxidizing complex is in the S2 and S3 states, and increases with increasing time delay between the subsequent flashes. By applying the fluorescent spin-trap DanePy, we detected the production of singlet oxygen, whose amount was increasing with increasing flash spacing. These findings were explained in the framework of a model, which assumes that recombination of the S2QB - and S3QB - states generate the triplet state of the reaction center chlorophyll and lead to the production of singlet oxygen.

Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6

We report three unrelated patients with mutations in the GRM6 gene that normally encodes the glutamate receptor mGluR6. This neurotransmitter receptor has been shown previously to be present only in the synapses of the ON bipolar cell dendrites, and it mediates synaptic transmission from rod and cone photoreceptors to this type of second-order neuron. Despite the synaptic defect, best visual acuities were normal or only moderately reduced (20/15 to 20/40). The patients were night blind from an early age, and when maximally dark-adapted, they could perceive lights only with an intensity equal to or slightly dimmer than that normally detected by the cone system (i.e., 2-3 log units above normal). Electroretinograms (ERGs) in response to single brief flashes of light had clearly detectable a-waves, which are derived from photoreceptors, and greatly reduced b-waves, which are derived from the second-order inner retinal neurons. ERGs in response to sawtooth flickering light indicated a markedly reduced ON response and a nearly normal OFF response. There was no subjective delay in the perception of suddenly appearing white vs. black objects on a gray background. These patients exemplify a previously unrecognized, autosomal recessive form of congenital night blindness associated with a negative ERG waveform.

Evaluation of the role of the retinal G protein-coupled receptor (RGR) in the vertebrate retina in vivo.

The retinal G protein-coupled receptor (RGR) is a protein that structurally resembles visual pigments and other G protein-coupled receptors. RGR may play a role as a photoisomerase in the production of 11-cis-retinal, the chromophore of the visual pigments. As the proposed function of RGR, in a complex with 11-cis-retinol dehydrogenase (RDH5), is to regenerate 11-cis-retinal under light conditions and RDH5 is expected to function in the light-independent part of the retinoid cycle, we speculated that the simultaneous loss of function of both proteins should more severely affect the rhodopsin regeneration capacity. Here, we evaluated the role of RGR using rgr-/- single and rdh5-/-rgr-/- double knockout mice under a number of light conditions. The most striking phenotype of rgr-/- mice after a single flash of light includes light-dependent formation of 9-cis- and 13-cis-retinoid isomers. These isomers are not formed in wild-type mice because either all-trans-retinal is bound to RGR and protected from isomerization to 9-cis- or 13-cis-retinal or because RGR is able to eliminate these isomers directly or indirectly. After intense bleaching, a transient accumulation of all-trans-retinyl esters and an attenuated recovery of 11-cis-retinal were observed. Finally, even under conditions of prolonged light illumination, as investigated in vitro in biochemical assays or in vivo by electroretinogram (ERG) measurements, no evidence of catalytic-like photoisomerization-driven production of 11-cis-retinal could be attained. These and previous results suggest that RGR and RDH5 are likely to function in the retinoid cycle, although their role is not essential and regeneration of visual pigment is only mildly affected by the absence of both proteins in rod-dominated mice.

Characterization of two phases of chlorophyll formation during greening of etiolated barley leaves.

The esterification kinetics of chlorophyllide, obtained by a single flash of light, were investigated in etiolated barley ( Hordeum vulgare L.) and oat ( Avena sativa L.) leaves. A rapid phase, leading to esterification of 15% of total chlorophyllide within 15-30 s, was followed by a lag-phase of nearly 2 min and a subsequent main phase, leading to esterification of 85% of total chlorophyllide within 30-60 min. The presence of additional protochlorophyllide, produced in the leaves by incubation with 5-aminolevulinate, did not change the esterification kinetics. The rapid phase was identical after partial (11-15%) and full (>80%) photoconversion of protochlorophyllide; the ability for a second rapid esterification phase was restored in a dark period of at least 10 min. Cooling the leaves to 0 degrees C abolished the esterification of the main phase while the rapid phase remained unchanged. The prolamellar bodies were already in part transformed into prothylakoid-like structures within 2-5 min after a full flash but not after a weak flash (11% photoconversion); in the latter case, the corresponding transformation required a dark period of about 45 min. The existence of subcomplexes of prolamellar bodies containing NADPH:protochlorophyllide oxidoreductase and chlorophyll synthase in the ratio 7:1 is discussed.

Effect of heterogeneity in the distribution of ligands and proteins among disconnected particles: the binding of ubiquinone to the bacterial reaction center

The decay from the photo-induced charge separated state of a photosynthetic reaction center (RC) depends strongly on the occupancy of the binding site of the secondary quinone, QB. In the case of RCs from the purple bacterium Rhodobacter sphaeroides, the kinetics of charge recombination following a single turnover flash of light were found to be markedly different in detergent micelles and phospholipid vesicles at the same quinone to RC ratio. We ascribed this behavior to a statistical heterogeneity in the number of RC and quinone molecules distributed among the micelle and vesicle population. A general description of this heterogeneity in terms of the distribution functions of RC and quinone among the host particle ensemble was developed and the kinetics of charge recombination were calculated accordingly. The results obtained by assuming that the distribution functions of RC and quinone are gamma-distributions agree with the measured kinetics and show deviations from the behaviour of a homogeneous system. The deviations are particularly strong at a low mean number of proteins and ligands per host particle. The model developed has a general relevance to the study of quinone–membrane protein interactions in both artificial and native vesicular systems.

Visual illusion induced by sound

We present the first cross-modal modification of visual perception which involves a phenomenological change in the quality—as opposed to a small, gradual, or quantitative change—of the percept of a non-ambiguous visual stimulus. We report a visual illusion which is induced by sound: when a single flash of light is accompanied by multiple auditory beeps, the single flash is perceived as multiple flashes. We present two experiments as well as several observations which establish that this alteration of the visual percept is due to cross-modal perceptual interactions as opposed to cognitive, attentional, or other origins. The results of the second experiment also reveal that the temporal window of these audio–visual interactions is approximately 100 ms.

EPR studies of the oxygen-evolving complex reveal a light-adaption process in Photosystem II

Photosystem II utilizes solar energy to drive electrons from the Mn cluster at the lumenal side to the quinone at the stromal side of the thylakoid membrane. The source of electrons is H2O, which is split to oxygen by the OEC. The water-splitting process involves cycling of the Mn-cluster through four semi-stable oxidation states, termed the S0, S1, S2, and S3 states. The OEC can be trapped in the different S-states, and studied by EPR techniques. The S2 state is often obtained by exposing a dark-adapted (S1 state) sample to continuous illumination at low temperature, or to a single flash of light at room temperature followed by freezing. Both procedures capture the S2 state, as reached by a single oxidation of the OEC following a period of dark-adaptation. With a laser flash procedure, the S2 state can be obtained a second time after 5 flashes, when the S-cycle has been completed once. We have discovered differences between the S2 state formed after one flash and the S2 state formed after five flashes, in terms of the relaxation behavior of the S2 multiline EPR signal. These data indicate a change within the Mn cluster that builds up during the first turnovers after dark-adaptation. Pulsed field-swept spectra of samples given 0-5 flashes of light show a similar trend: the S1 state obtained by zero and four flashes differ significantly from one another. Based on the data presented and similar reports in the literature, we propose that the OEC undergoes a light-adaptation process during the first two turnovers after dark-adaptation, adjusting the system for efficient continuous water-splitting. We tentatively suggest that light-adaptation involves rearrangements of the proton network in the OEC, possibly as a means of setting up proton channels. (Less)

Formation of long-wavelength chlorophyllide (Chlide695) is required for the assembly of Photosystem II in etiolated barley leaves

Chlorophyll(ide) spectroscopic properties and Photosystem II assembly, monitored by 77 K variable fluorescence, were studied in etiolated barley leaves as a function of the extent of protochlorophyllide photoreduction by a single millisecond light flash of different intensities. Variable fluorescence, measured 2 hours after the flash, was only detected when the extent of phototransformation was higher than a threshold value of 0.4. Its development paralleled the formation of a chlorophyll emission component at 685 nm, which itself derived from long-wavelength chlorophyllide with an emission maximum at 695 nm. At low flash intensities, short-wavelength chlorophyllide forms preferentially accumulated and no Photosystem II fluorescence was detected after 2 hours. Chlorophyllide esterification was independent of the extent of phototransformation. These results suggested that the formation of long-wavelength chlorophyllide was essential for further assembly of Photosystem II. This interpretation was strengthened by the observed inhibition of both long-wavelength chlorophyllide formation and of variable fluorescence development in leaves treated with δ-aminolevulinic acid or in untreated leaves subjected to repeated flashes of low intensity.

Light Flash Stimulation Alters the Nightingale's Singing Style: Implications for Song Control Mechanisms

To investigate decision processes during song performances in a species with a large repertoire and a versatile singing style we studied the characteristics of brief song interruptions in male nightingales Luscinia megarhynchos. Interruptions were induced experimentally by exposure to single light flashes. (1) The temporal positioning of stimuli within songs had no clear effect on the likelihood of a song interruption. (2) The delay between stimulus presentation and song interruption showed considerable variation. (3) Song types exposed to the stimulus were repeated significantly more often than in normal singing bouts. (4) In day time singing, durations of silent intersong intervals were not affected by stimulus presentations. During nocturnal singing however silent intersong intervals increased in duration after stimulus presentations unless a song type repetition was following subsequently. The results suggest a mechanism facilitating song type repetitions for songs that were disturbed during singing performance. Whether or not temporal control is affected seems to depend on both time of day and on whether or not a song is repeated after a stimulus presentation.

Photodynamic inactivation of gramicidin channels: a flash-photolysis study

Photosensitized inactivation of ionic channels formed by gramicidin in the planar bilayer lipid membrane (BLM) has been studied upon exposure of the BLM to single flashes of visible light in the presence of tetrasulphonated aluminium phthalocyanine. The gramicidin photoinactivation is inhibited by the addition of unsaturated phospholipids to the membrane-forming solution as well as by the addition of azide to the bathing solution, consistent with involvement of singlet oxygen. The characteristic time of the photoinactivation (T) does not change markedly under these conditions. Moreover, T remains nearly constant upon alteration of the flash energy and the photosensitizer concentration. The value of T appears to be sensitive to the gramicidin concentration and to the factors affecting the open time of the gramicidin channels, namely the temperature and the solvent used in the membrane-forming solution. The photoinactivation is not observed with covalent gramicidin dimers. The equations derived from the model of Bamberg and Laeuger (J. Membrane Biol. (1973) 11, 177–194), describing the relaxation of the gramicidin-induced conductance after a sudden distortion of the dimer-monomer equilibrium, are shown to explain consistently the time course of the photoinactivation provided that the damage of the gramicidin molecules leads to deviation from the equilibrium.

EPR and ENDOR studies of the water oxidizing complex of Photosystem II

A comparative study of X-band EPR and ENDOR of the S2 state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S2 state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S2 state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. (1)H-ENDOR experiments performed at two different field positions of the S2 state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382-2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D2O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S2.

Functional MR imaging of visual and motor cortex stimulation at high temporal resolution using a FLASH technique on a standard 1.5 Tesla scanner.

Functional magnetic resonance imaging (fMRI) was performed on a conventional 1.5 T scanner by means of a modified FLASH-technique at temporal resolutions of 80 and 320 ms. The method's stability was assessed by phantom measurements and by investigation of three volunteers resulting in a low amplitude (3%) periodic (4 s) signal modulation for the in vivo measurements, which was not observable in the phantom experiments. fMRI activation studies of motor and visual cortices of four adjacent slices were carried out on 12 healthy right-handed volunteers. Stimulation was performed by a triggered single white light flash or single finger-to-thumb opposition movement, respectively. Event-related response of visual and motor activation was traced over 10.24 s with a temporal resolution of 320 ms for the four slice measurements. Brain activation maps were calculated by correlation of measured signal time course with a time-shifted boxcar function. Activation was quantified by calculation of percentual signal change in relation to the baseline. Observed signal magnitudes were about 5-7% in visual and about 8-12% in primary motor cortex. While photic response was delayed by about 2 s, motor stimulation showed an instantaneous increase of the MR signal. MR signal responses for both stimuli had decayed completely after about 5 s. Our results show that event-related fMRI enables mapping of brain function at sufficient spatial resolution with a temporal resolution of up to 80 ms on a conventional scanner.

A patch clamp method for determining single turnover charge separations in the chloroplast membrane

This report describes the application of the chloroplast patching technique to study the primary electrogenic events in the thylakoid membrane. It is shown that by applying single turnover saturating light flashes the actual number of functional charge transferring reaction center channels in the thylakoid membrane and the voltage generated by this charge transfer can be determined in a single individual chloroplast. Moreover, low conducting barriers between the thylakoid membrane and the adjacent lumenal and stromal phases, respectively, are likely to exist.

Oscillations of Reaction Center II-D1 Protein Degradation in Vivo Induced by Repetitive Light Flashes: CORRELATION BETWEEN THE LEVEL OF RCII-QB- AND PROTEIN DEGRADATION IN LOW LIGHT

The D1 protein subunit of the photochemical reaction center II (RCII) turns over rapidly in oxygenic photosynthetic organisms exposed to the light. At high photon flux densities (PFD), photoinactivation of RCII precedes the degradation of the D1 protein. We found that the apparent quantum yield for the D1 protein degradation in Chlamydomonas cells is severalfold higher at low PFDs (10-100 mumol m-2 s-1) as compared to that observed at PFDs which induce photoinactivation of RCII (1.5-3 x 10(3) mumol m-2 s-1). Relative high levels of reduced RCII secondary plastoquinone acceptor, QB-, are induced in cells exposed to low PFDs as determined by thermoluminescence measurements. The probability of generating elevated levels of QB- which may recombine with the S2,3 oxidized states of the oxygen evolving complex decreases with increase in the light intensities at which consecutive double reduction of QB and exchange with the plastoquinone pool prevail. We have used light flashes to test if a correlation exists between the degradation of D1 protein and the relative level of QB-. D1 protein degradation could be induced in dark-incubated cells exposed to a series of 1.4 x 10(3) single light flashes given at intervals compatible with generation of elevated levels of QB- and its decay by charge recombination. Oscillations of the QB- level in cells exposed to 960-1440 series of 1 to several flashes correlated with oscillations of the D1 protein degradation in Chlamydomonas cells and in the Scenedesmus wild type but not in the LF-1 mutant lacking photosystem II donor side activity. In this mutant the "S state cycle" and QB- oscillations are abolished. We propose that the process of recombination of long lived RCII-QB- with the S2,3 states may involve damaging events related to the D1 protein degradation induced by light flashes or continuous low light in vivo.

Visual and somatosensory evoked potentials are mediated by excitatory amino acid receptors in the thalamus

In pentobarbital-anaesthetized rats early somatosensory evoked potentials (SEPs) were recorded from the sensory cortex in response to electrical stimulation of the contralateral forepaw and visual evoked potentials (VEPs) from the primary visual cortex in response to single light flashes. Microapplication of the specific non-NMDA antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) into the ventro-basal thalamus (VB) resulted in a pronounced decrease in amplitude and an increase in latency of SEPs, whereas injection of DNQX into the dorsal lateral geniculate nucleus (DGL) induced a pronounced decrease in amplitude and an increase in latency of VEPs. These changes were: (1) dose-dependent (DNQX 0.01–1.0 nmol), (2) receptor-specific, and (3) site-specific. In contrast, the specific NMDA antagonist 2-amino-7-phosphonoheptanoate (AP7; 0.5–5 nmol) did not affect SEPs after microapplication into the VB and less potently reduced the amplitude and increased the latency of VEPs after microapplication into the DGL. The present findings are consistent with the assumption that an excitatory amino acid serves as transmitter at synapses in the rat thalamus mediating the nervous impulses responsible for the generation of SEPs and of VEPs. In addition the results suggest that this transmitter preferentially interacts with non-NMDA receptors.

Calibration and time resolution of lumenal pH-transients in chromatophores of Rhodobacter capsulatus following a single turnover flash of light: Proton release by the cytochrome bc1-complex is strongly electrogenic

The flash-induced proton release into the lumen of chromatophores from Rhodobacter capsulatus was studied with Neutral red as pH-indicator. Calibration of the acidification jump after a single flash yielded a much larger figure, at least 0.8 units, than previously thought. A slow kinetic phase of proton release (85–90% of total) was sensitive to inhibitors of the cytochrome bc 1-complex. Its half-rise time, about 10 ms, was the same as the rise time of the electrogenic reaction in the cytochrome bc 1-complex that was recorded by electrochromism of carotenoids. The oxidoreduction of the two b-hemes was significantly faster ( t 1 2 ≅ 3 ms). Thus the major electrogenic event in the cytochome bc 1-complex is proton and not electron transfer.

Oxidation-reduction potential dependence of photosystem II carbonic anhydrase in maize thylakoids.

In characterizing the carbonic anhydrase (CA) found in maize thylakoid membranes, it was observed that the enzyme's activity was inhibited somewhat when the Hill oxidant, ferricyanide, was given in the dark [Stemler, A. (1986) Biochim. Biophys. Acta 850, 97-107]. In the present work, a redox titration of this effect shows that the CA activity is mediated by a component that has a midpoint potential (Em) of about 485 mV at pH 6.5 and a pH dependence of 60 mV/pH. These redox titration characteristics are identical to those of the redox mediator "D480", which modulates formate and bicarbonate binding affinity to photosystem II (PS II). Bicarbonate binds to PS II more readily, and CA activity is higher, when D480 is reduced, whereas both bicarbonate binding and thylakoid-bound CA activity are low when D480 is oxidized in the dark by ferricyanide. Both the low bicarbonate binding affinity and the low CA activity induced by the presence of ferricyanide are reversed by a single saturating flash of light. In contrast, the activity of soluble CA, which is extracted from maize mesophyll cytosol, does not exhibit any redox dependence in the range 400-550 mV. Furthermore, thylakoid-bound CA activity is inhibited by 5 mM ZnCl2 by as much as 75%, whereas the activity of soluble CA shows no significant decrease induced by ZnCl2. Also, at a medium potential of 400 mV, ferricyanide (1 mM) inhibits soluble CA activity by 88% and thylakoid-bound CA activity by only 18%. It is concluded from these results that CA activity observed in thylakoids arises from CA inherent to PS II and is not some form of contamination by soluble CA. Possible roles of CA in PS II reaction mechanisms are discussed.

Effect of flash photoreactivation on Escherichia coli recA induction by ultraviolet light.

Excision-deficient Escherichia coli, carrying the gene for the photolyase on a multicopy plasmid, were irradiated with ultraviolet (UV) light then photoreactivated by illumination delivered from a camera flash unit. Such instantaneous illumination monomerizes only cyclobutane pyrimidine dimers already bound by the photolyase. Whereas the lethal effect of UV light and the number of C-to-T transition-type mutations induced by UV irradiation were both significantly reduced by subsequent irradiation with a single flash of light, single-flash photoreactivation did not reverse the induction of the recA gene by UV light. The results indicate, therefore, that non-photoreactivable DNA lesions play a role in recA induction.