Light-induced Membrane Potential Changes Research Articles

Investigation of membrane potential changes in Nitellopsis obtusa cells induced by blue and red light stimulation

Light controls multiple physiological processes in plants. Membrane potential is widely used to report the early events as­sociated with changes in ion permeability, thus the effect of monochromatic light on bioelectrical parameters of Nitellopsis ob­tusa cells was examined. In this study blue, red and far-red light-induced membrane potential changes, light-induced photoelectrical reaction and membrane excitability were investigated using K’-anaesthesia method. All investigated lights cause the same bioelectrical events in Nitellopsis obtusa cells. It can be concluded that all photoreception systems exert their effects by first altering the permeability of the plasma membrane to ions and later ion channels perform signal-transducing functions in the complex array of mechanisms that determine plant physiology.

Light-Triggered Modulation of Cellular Electrical Activity by Ruthenium Diimine Nanoswitches

Ruthenium diimine complexes have previously been used to facilitate light-activated electron transfer in the study of redox metalloproteins. Excitation at 488 nm leads to a photoexcited state, in which the complex can either accept or donate an electron, respectively, in the presence of a soluble sacrificial reductant or oxidant. Here, we describe a novel application of these complexes in mediating light-induced changes in cellular electrical activity. We demonstrate that RubpyC17 ([Ru(bpy)(2)(bpy-C17)](2+), where bpy is 2,2'-bipyridine and bpy-C17 is 2,2'-4-heptadecyl-4'-methyl-bipyridine), readily incorporates into the plasma membrane of cells, as evidenced by membrane-confined luminescence. Excitable cells incubated in RubpyC17 and then illuminated at 488 nm in the presence of the reductant ascorbate undergo membrane depolarization leading to firing of action potentials. In contrast, the same experiment performed with the oxidant ferricyanide, instead of ascorbate, leads to hyperpolarization. These experiments suggest that illumination of membrane-associated RubpyC17 in the presence of ascorbate alters the cell membrane potential by increasing the negative charge on the outer face of the cell membrane capacitor, effectively depolarizing the cell membrane. We rule out two alternative explanations for light-induced membrane potential changes, using patch clamp experiments: (1) light-induced direct interaction of RubpyC17 with ion channels and (2) light-induced membrane perforation. We show that incorporation of RubpyC17 into the plasma membrane of neuroendocrine cells enables light-induced secretion as monitored by amperometry. While the present work is focused on ruthenium diimine complexes, the findings point more generally to broader application of other transition metal complexes to mediate light-induced biological changes.

Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping

Microorganisms can use complex photosystems or light-dependent proton pumps to generate membrane potential and/or reduce electron carriers to support growth. The discovery that proteorhodopsin is a light-dependent proton pump that can be expressed readily in recombinant bacteria enables development of new strategies to probe microbial physiology and to engineer microbes with new light-driven properties. Here, we describe functional expression of proteorhodopsin and light-induced changes in membrane potential in the bacterium Shewanella oneidensis strain MR-1. We report that there were significant increases in electrical current generation during illumination of electrochemical chambers containing S. oneidensis expressing proteorhodopsin. We present evidence that an engineered strain is able to consume lactate at an increased rate when it is illuminated, which is consistent with the hypothesis that proteorhodopsin activity enhances lactate uptake by increasing the proton motive force. Our results demonstrate that there is coupling of a light-driven process to electricity generation in a nonphotosynthetic engineered bacterium. Expression of proteorhodopsin also preserved the viability of the bacterium under nutrient-limited conditions, providing evidence that fulfillment of basic energy needs of organisms may explain the widespread distribution of proteorhodopsin in marine environments.

Facilitated permeation of methyl viologen into chloroplasts in situ during electric pulse generation in excitable plant cell membranes

Generation of action potential (AP) in plasma membranes of characean algae has a strong impact on photoreactions occurring in chloroplasts. Under physiological conditions, AP suppresses electron transport in alkaline and acidic regions, although to a different extent; these changes are transient and reversible. In the presence of the artificial electron acceptor, methyl viologen (MV2+), AP-induced changes in electron transport in photosystem II become irreversible. Incubation of Chara corallina internodal cells with MV2+ has no effect on the chlorophyll P700 photooxidation kinetics in photosystem I reaction centers, suggesting that MV2+ is inaccessible for interactions with photosystem I, because its permeation into chloroplasts of a resting cell is hindered by membrane barriers. At the same time, AP generation in the presence of MV2+ is accompanied by irreversible modification of P700 photooxidation kinetics, as can be evidenced from differences in absorption changes at 810 and 870 nm (ΔA810 signals). These findings suggest that MV2+ permeation into chloroplasts in situ is facilitated during or after the AP generation. Similar to the ΔA810 signals, light-induced changes in membrane potential do not depend on the presence of MV2+ in the external medium until the first excitatory stimulus is applied. Electric photoresponses of the cell are irreversibly modified by AP generated in the presence of MV2+ at the expense of non-cyclic photosynthetic electron transport redirected to the MV2+ reduction. It is concluded that AP effects on chloroplast photosynthesis in situ are complex and involve permeability changes for MV2+ in membrane barriers of the “plasmalemma-chloroplast envelope” system.

Quantification of the electrochemical proton gradient and activation of ATP synthase in leaves

We have developed a new method to quantify the transmembrane electrochemical proton gradient present in chloroplasts of dark-adapted leaves. When a leaf is illuminated by a short pulse of intense light, we observed that the light-induced membrane potential changes, measured by the difference of absorption (520 nm–546 nm), reach a maximum value (~190 mV) determined by ion leaks that occur above a threshold level of the electrochemical proton gradient. After the light-pulse, the decay of the membrane potential follows a multiphasic kinetics. A marked slowdown of the rate of membrane potential decay occurs ~100 ms after the light-pulse, which has been previously interpreted as reflecting the switch from an activated to an inactivated state of the ATP synthase (Junge, W., Rumberg, B. and Schröder, H., Eur. J. Biochem. 14 (1970) 575–581). This transition occurs at ~110 mV, thereby providing a second reference level. On this basis, we have estimated the Δμ˜H+ level that pre-exists in the dark. Depending upon the physiological state of the leaf, this level varies from 40 to 70 mV. In the dark, the Δμ˜H+ collapses upon addition of inhibitors of the respiratory chain, thus showing that it results from the hydrolysis of ATP of mitochondrial origin. Illumination of the leaf for a period longer than several seconds induces a long-lived Δμ˜H+ increase (up to ~150 mV) that reflects the light-induced increase in ATP concentration. Following the illumination, Δμ˜H+ relaxes to its dark-adapted value according a multiphasic kinetics that is completed in more than 1 h. In mature leaf, the deactivation of the Benson–Calvin cycle follows similar kinetics as Δμ˜H+ decay, showing that its state of activation is mainly controlled by ATP concentration.

CO2 provides an intermediate link in the red light response of guard cells.

Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K+-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO2. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO2-free air, depolarized after a switch to air with 700 micro l l(-1) CO2, in a reversible manner. As a result, K+-fluxes across the plasma membrane changed direction, to favour K+ extrusion and stomatal closure in the presence of CO2. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO2 concentration by about 250 micro l l(-1), as measured with a miniature CO2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO2 concentration.

The Role of Light-Induced Membrane Potential Changes in Guttation in Gametophytes of Asplenium trichomanes

Gametophytes of the fern Asplenium trichomanes exhibit guttation when illuminated. Membrane potential changes evoked by light were measured in the presence of ion channel and proton pump inhibitors to elucidate the nature of the response and a possible link to guttation. Light-induced depolarization was suppressed by the anion channel inhibitors: anthracene-9-carboxylic acid and niflumic acid. Potassium channel blockers: TEA and Ba2+ caused an increase of the amplitude of light-induced membrane potential changes. Calcium channel inhibitors, La3*, Gd3+, diltiazem, nifedipine and verapamil had no significant effect on the membrane potential changes. Similarly, proton pump inhibitors, diethylstilbestrol and vanadate, had only minor effects on the response. A possible role of Cl~ and K+ fluxes in light-induced guttation is discussed.

Light-induced cytosolic calcium transients in green plant cells. ll. The effect on a K + channel as studied by a kinetic analysis in Chara corallina

The fluorescent dye chlorotetracycline was used to study the relationship between the light-induced decrease in cytosolic free calcium concentration, [Ca2+]c, and its effect on ion transport at the plasma membrane in the giant cells of Chara corallina Klein ex Willd. A kinetic analysis of the simultaneously measured light-induced changes in membrane potential and in [Ca2+]c led to the same time constant of about 40 s. The reversal potential of the light effect on membrane potential was in agreement with the dominant role of a K+ channel in the plasma membrane. Thus, the experiments reported here provide evidence for the following light-driven signal transduction chain from the chloroplasts to K+ transport of the plasma membrane: (i) light causes an uptake of Ca2+ into the chloroplasts, (ii) this causes a decrease in cytosolic [Ca2+]c, (iii) this leads to a decrease in the activity of a K+ channel. The results also initiated a re-analysis of previously published data of the light effect on the velocity of cytosolic streaming and supported the hypothesis that Ca2+ fluxes coming out of the chloroplasts upon darkening cause a Ca2+-induced phosphorylation of myosin, which slows down cytoplasmic streaming.

Light-induced membrane potential changes of epidermal and mesophyll cells in growing leaves of Pisum sativum

Light transiently depolarizes the membrane of growing leaf cells. The ionic basis for changes in cell membrane electrical potentials in response to light has been determined separately for growing epidermal and mesophyll cells of the argenteum mutant of pea (Pisum sativum L.). In mesophyll cells light induces a large, transient depolarization that depends on the external Cl− concentration, is unaffected by changes in the external Ca2+ or K+ concentration, is stimulated by K+-channel blockers tetraethylammonium (TEA+) and Ba2+, and is inhibited by 3-(3′-4′-dichlorophenyl)-1,1-dimethylurea (DCMU). In isolated epidermal tissue, light induces a small, transient depolarization followed by a hyperpolarization of the membrane potential. The depolarization is enhanced by increasing the external Ca2+ concentration and by addition of Ba2+, and is not sensitive to DCMU. Epidermal cells in contact with mesophyll display a depolarization resembling the response of the underlying mesophyll cells. The light-induced depolarization in mesophyll cells seems to be mediated by an increased efflux of Cl− while the membrane-potential changes in epidermal strips reflect changes in the fluxes of Ca2+ and in the activity of the proton-pumping ATPase.

Photosensory transduction in ciliates. I. An analysis of light-induced electrical and motile responses in Stentor coeruleus.

Light-induced membrane potential changes and motile responses have been studied in Stentor cells with intracellular microelectrodes and video microscopy, respectively. Intracellular microelectrode recordings showed that step-up increase in light intensity induced an electrical membrane response which consisted of an initial membrane depolarization (photoreceptor potential) followed by an action potential and maintaining phase of depolarization (afterdepolarization). The amplitude of the receptor potential is dependent on the intensity of light stimulus and the action potential appears with a lag period (latency) after the onset of light stimulus. The extent of the membrane afterdepolarization is dependent on the intensity and duration of stimulus used. A close time correlation has been established between the latency for the action potential and the onset of ciliary reversal (stop response). A time correlation was also observed between the duration of the membrane afterdepolarization and the duration of backward swimming. The action spectrum for the photoreceptor potential amplitude of Stentor resembled the action spectra for the latency of ciliary reversal and the photoresponsiveness, indicating that the photomovement response and membrane potential changes are coupled through the same photosensor system. A hypothesis on the photosensory transduction chain in Stentor is discussed according to which the photoreceptors and the ciliary apparatus is mediated by the membrane potential changes.

Lanthanum mimicks the trp photoreceptor mutant of Drosophila in the blowfly Calliphora

The effect of lanthanum on the light response of blowfly (Calliphora erythrocephala) photoreceptors was studied. The electrophysiological behaviour of the photoreceptors in the presence of La can be summarized as follows: 1. Upon long stimulation the photoreceptors responded with a 'transient receptor potential', i.e. the cells depolarized at the onset of the stimulus and then repolarized to (or below) the resting potential. This effect was dependent on stimulus intensity and occurred only at high intensities. During illumination membrane noise was reduced. 2. The light-induced changes in membrane potential were paralleled by changes in membrane resistance. 3. The time course of the receptor response was slowed down. 4. Light adaptation led to an increase in response latency. 5. The recovery of the receptor response after light adaptation was slowed down. 6. The sensitivity of the receptor cells measured by the response to short light stimuli was reduced. In summary, the electrophysiological behaviour of Calliphora photoreceptors in the presence of La was very similar to that of the photoreceptors of the trp (transient receptor potential) mutant of Drosophila melanogaster. This result suggests that La and trp mutation affect the same cellular processes in the photoreceptors.

Light induced membrane potential changes in Halobacterium halobium observed with high time resolution by resonance Raman spectroscopy

A τ=80 μs value was found for the build-up of the membrane potential in intact Halobacterium halobium cells. This time constant was determined from carotenoid resonance Raman intensity changes connected to the red shift of the carotenoid absorption band upon the increase of the membrane potential.

Side effects of chlorinated hydrocarbon insecticides on membranes of plant cells: I. The influence of lindane on the membrane potential of Elodea densa leaf cells

Side effects of the insecticide lindane on plant cells are not well understood. To get more information about this problem, effects of sublethal doses of lindane on the membrane potential of the submerged macrophytic fresh-water plant Elodea densa were studied. Using standard intracellular microelectrode techniques, membrane potential levels and time-courses in intact and lindane-treated leaves were investigated in light and in darkness. Lindane produced a time-dependent inhibition of light-induced membrane potential changes. More than 10 hr application of 10 ppm lindane (effective concentration) resulted in a complete insensitivity of the membrane potential to illumination. The basis of this effect is not quite clear, but several possibilities are discussed. On the other hand effects of lindane on the diffusion membrane potential were studied. Lindane decreased the level of the diffusion potential. Additionally, the voltage responses of the diffusion potential to rapid changes in the potassium/sodium relation of the outer solution were found to be lowered drastically by lindane. These results suggest a direct interaction of lindane with the plasmalemma, resulting in a loss of the potassium/sodium selectivity. The findings indicate that biomembranes appear to be a favored target of lindane action in living plant cells.

Light-induced membrane potential changes and rectification in Acetabularia.

Light-induced membrane potential changes and rectification in Acetabularia.

Light-induced changes in membrane potential of algal cells associated with photosynthetic electron transport

The negative membrane potential of Nitella translucens and Nitellopsis obtusa increases reversibly upon illumination. Relative efficiencies of 430-, 676-, 690- and 705-nm light quanta indicate that the changes are caused by light ever conversion in the photosynthetic apparatus. DCMU (1 μM) and CCCP (2 μM) inhibit the light effect. Lower concentrations of CCCP (⩾ 0.2 μM) cause an increase in the rate of the dark recovery of the membrane potential after illumination. The results are discussed in terms of a proposed model, in which changes in H + concentration in the cytoplasmic chloroplast layer, which are coupled with photosynthetic electron transport, are considered.