Calcium-dependent membrane depolarisation activated by phytochrome in the moss Physcomitrella patens

In caulonemal filaments of Physcomitrella patens which had been preincubated in the dark for 24 h, irradiation with red light (640 nm, fluence rate 85 mol · m^–2 · s^–1) evoked (i) the development of side branch initials and (ii) a rapid, but transient, depolarisation of the plasma membrane by 90 ± 13 mV from a resting potential of -178 ± 13 mV. This was followed by a transient hyperpolarisation to a value 21± 8 mV more negative than the original membrane potential. The refractory period for the transient depolarisation was between 12 and 15 min. The fluence rate of red light required to evoke maximal depolarisation was about 80 mol · m^–2 · s^–1 for a 1-min pulse. At this fluence rate, a depolarising response could be recorded for pulse lengths as small as 7 s. The transient depolarisation was insensitive to 3-(3,4dichlorophenyl)-1,1-dimethyl urea (DCMU) and was unchanged in plants bleached by growth on norflurazon (SAN 9789). Furthermore, the electrical response could be blocked by simultaneous application of far-red light. These results suggest the involvement of the photoreceptor phytochrome in the response. Removing Ca²⁺ from the external medium or replacing Ca²⁺ with Mg²⁺ blocked the depolarisation. The depolarisation could also be blocked by the K⁺ channel-blocker tetraethylammonium (10 mM) and the Cl^– channel-blocker niflumic acid (1 M). Conversely, although calcium channel-antagonists such as nifedipine and lanthanides, applied at a concentration of 100 M, also altered the response, they did not block it. A possible ionic mechanism for the membrane potential transient is advanced, and the physiological significance discussed in the context of early events in the phytochrome signalling pathway.Abbreviations [Ca²⁺]_c cytosolic Ca²⁺ concentration - DCMU 3-(3,4-dichlorophenyt)-1,1-dimethylurea - TEA tetraethylammonium We thank Prof. David Cove (Department of Genetics, University of Leeds) for fruitful discussions, providing plants and advice on culturing methods, Dr. Richard Firn (York) for stimulating discussions, Ian Jennings (York) for technical advice on the electrophysiological apparatus, and Anna Bate (York) for looking after the plant cultures. Financial support was received from the Biotechnology and Biological Sciences Research Council (Grant P87/4043 to D.S. and Grant PDF/14 to E.J.) and The New Phytologist Trust (studentship support to E.E.).     

Phytochrome modulation of calcium fluxes in wheat (Triticum aestivum L.) protoplasts

Employing the metallochromic dye murexide and by monitoring the uptake of radiolabelled calcium, photoreversible calcium fluxes were measured in wheat leaf protoplast suspensions. Results obtained by both methods were identical — red light promoted and subsequent far-red irradiation reversed an influx of Ca⁺⁺ ions into the protoplasts. These findings imply phytochrome regulation of Ca⁺⁺ fluxes across the plasma membrane. The influx of Ca⁺⁺ stimulated by 2 min red irradiation could be maintained in total darkness for the initial 16–18 min after illumination, after which a 6–8 min efflux process was triggered and the basal Ca⁺⁺ level restored. Verapamil, a calcium channel blocker, inhibited the red-promoted influx, whereas the far-red mediated efflux could be checked by the use of the ATPase inhibitor vanadate, and also by the calmodulin antagonist chlorpromazine, thus suggesting a role of ion channels and pumps in phytochrome-controlled Ca⁺⁺ fluxes. The possible involvement of phosphoinositides in phytochrome-modulated calcium fluxes was also investigated.Abbreviations A difference in absorbance - CPZ chlorpromazine - FR far-red (light) - MX murexide - PI phosphatidylinositol - PIP₂ phosphatidylinositol 4, 5-bisphosphate - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - POPOP 1, 4-bis [2-(5-phenyl-1, 3-oxazolyl)]-benzene - PPO 2, 5-diphenyl-1, 3-oxazole - R red (light) - SOV sodium orthovanadate     

Phytochrome and calcium ions are involved in light-induced membrane depolarization in Nitella

Isolated internodes of Nitella (N. opaca, N. flexilis) and Nitellopsis spec. were punctured with single microelectrodes and their membrane potentials were recorded continuously during various light treatments. In red light the initial response was always a depolarization. This depolarization began with a lag-time of 0.4-3.5s and reached a steady state within 1–2 min of continuous illumination. Repolarization began within several seconds after turning off the light. The magnitude of the red-light-induced depolarization increased with the Ca²⁺-concentration of the medium. The largest depolarizations were recorded in 5 m mol l^-1 Ca²⁺. Ca²⁺ could not be replaced in this function by Na⁺, Mg²⁺, La³⁺ or mannitol. Far-red light alone had no effect on the resting membrane potential. Far-red light applied immediately after red light accelerated the repolarization of the membrane potential. Far-red light applied simultaneously with red light reduced the amount of depolarization and increased the rate of repolarization. The results indicate that phytochrome and Ca²⁺ are involved in the light-induced depolarization of the membrane. They are consistent with the hypothesis that phytochrome may act by triggering a Ca²⁺-influx at the plasma membrane.Abbreviations APW artificial pond water - Pfr far-red absorbing form of phytochrome - DCMU 3-(3,4-Dichlorphenyl)-1,1-dimethylurea     

Phytochrome-mediated uptake of calcium in Mougeotia cells

Ca²⁺ is proposed to function as a messenger in such phytochrome-mediated responses as localized cell growth, intracellular movements, and control of plasma membrane properties. To test this hypothesis, the uptake of Ca²⁺ in irradiated and non-irradiated regions of individual threads of the green alga Mougeotia was studied with the aid of ⁴⁵Ca²⁺ and low temperature autoradiography: 10–20 cells within 40–60 cell-long threads were irradiated for up to 1 min, transferred to darkness for 3 to 10 min, submersed in a radioactive medium for 1 min, washed in an unlabelled medium for 30 min, and then autoradiographed at-80° C for several days.The autoradiographs show that those cells which had been pre-irradiated with red light did take up 2–10 times more Ca²⁺ than the adjacent non-irradiated cells of the same thread. Cells pre-irradiated with farred light or red light followed by far-red light showed no enhanced uptake of Ca²⁺. These results might be interpreted to indicate, firstly, that phytochrome-Pfr is involved in the enhanced uptake of Ca²⁺ and secondly, that the accumulation of radioactive Ca²⁺ in red light irradiated cells is an expression of an increased intracellular concentration of Ca²⁺. This interpretation is based on the data that (i) the dark interval between irradiation and labelling precluded the involvement of photosynthesis, (ii) the effect of red light was reversible with far-red light, and (iii) the accumulation of Ca²⁺ persisted during the long wash-out period. We speculate, that the red light-enhanced accumulation of Ca²⁺ in Mougeotia cells is caused by a Pfr-mediated increase of the Ca-permeability of the plasma membrane, and perhaps by a Pfr-impeding of an active Ca²⁺-extrusion.Abbreviations APW artificial pond water - EGTA ethylene glycol-bis-(-amino ethyle ether) N,N-tetraacetic acid - R red irradiation - D darkness - FR far-red irradiation - Pfr physiologicallyactive form of phytochrome - Pr physiologically inactive form of phytochrome This paper is part of a Ph. D. Thesis submitted to the University of Erlangen-Nürnberg by E.M. Dreyer     

Role of calcium and other ions in directing root hair tip growth in Limnobium stoloniferum

The magnitude and spatial localization of Ca²⁺, K⁺ and H⁺ fluxes in growing and non-growing Limnobium stoloniferum root hairs was determined using non-invasive, ion-selective vibrating microelectrodes. Both the spatial pattern and magnitude of the ionic flux was dependent on the particular ion in question. Both H⁺ and Ca²⁺ influx was localized almost exclusively to the tips of growing root hairs, suggesting that these fluxes may be involved in directing growth. Influx of K⁺ showed no distinct localization and uptake appeared uniform along the length of the root hair. Competitive inhibition of Ca²⁺ influx using a range of Mg⁺ concentrations indicated that the magnitude of the Ca²⁺ flux entering the root hair tip did not determine growth rate; however, the presence of Ca²⁺ on the external face of the membrane was implicit for root hair integrity. Aluminum proved to be a potent inhibitor of root hair growth. At an exogenous Al concentration of 20 M a complete blockage of Ca²⁺ influx into root hair tips was observed, suggesting that Al blockage of Ca²⁺ influx could be involved in Al toxicity. However, at a lower Al concentration (2 M), Ca²⁺ fluxes were unaffected while inhibition of growth was still observed along with a distinct swelling of the root hair tip. The swelling at the root hair tips was identical in appearance to that seen in the presence of microtubule inhibitors, suggesting that Al could influence a number of different sites at the plasma-membrane surface and within the cell. The possible role(s) of Ca²⁺ and H⁺ fluxes in directing tip growth are discussed.     

Characterization of calcium fluxes across the envelope of intact spinach chloroplasts

Calcium fluxes across the envelope of intact spinach chloroplasts (Spinacia oleracea L.) in the light and in the dark were investigated using the metallochromic indicator arsenazo III. Light induces Ca²⁺ influx into chloroplasts. The action spectrum of light-induced Ca²⁺ influx and the inhibitory effect of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) indicate an involement of photosynthetic electron transport in this process. The driving force for light-induced Ca²⁺ influx is most likely a change in the membrane potential component of the proton motive force. This was demonstrated by the use of agents modifying the membrane potential (lipophilic cations, ionophores, different KCl concentrations). The activation energy of the observed Ca²⁺ influx is about 92 kJ mol^-1. Verapamil and nifedipine, two Ca²⁺-channel blockers, have no inhibitory effect on light-induced Ca²⁺ influx, but enhance ferricyanide-dependent oxygen evolution. Inhibition of Ca²⁺ influx by ruthenium red reduces the light-dependent decrease in stromal NAD⁺ level.Abbreviations and symbols Chl chlorophyll - DCMU 3-(3',4'-dichlorophenyl)-1,1-dimethylurea - FCCP earbonyl cyanide p-trifluoromethoxyphenylhydrazone - PGA 3-phosphoglyceric acid - ABA⁺ tetrabutylammonium chloride - TPP⁺ tetraphenylphosphonium chloride - E membrane potential     

Calcium influx at the plasmalemma of Chara corallina

Influx of ⁴⁵Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La³⁺-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5–15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca²⁺ externally was 0.25–0.5 pmol·cm^-2·s^-1, but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K⁺. In cells in which the control flux was less than about 0.5 pmol·cm^-2·s^-1 there was no effect of 50 M nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm^-2·s^-1 (whether by natural variability, pretreatment, or by depolarisation in 20 mM K⁺), the flux was reduced by 50 M nifedipine to a value in the range 0.25–0.59 pmol·cm^-2·s^-1. It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 M BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm^-2 with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca²⁺ the measured influx is a reasonable estimate of the influx at the plasmalemma.Abbreviations 0.4K-APW6 artificial pondwater, pH 6, containing 0.4 mM KCl - 20 K-APW6 artificial pondwater, pH 6, containing 20 mM KCl - Ca_o external Ca²⁺     

Use of an extracellular,ion-selective,vibrating microelectrode system for the quantification of K+, H+, and Ca2+ fluxes in maize roots and maize suspension cells

An ion-selective vibrating-microelectrode system, which was originally used to measure extracellular Ca²⁺ gradients generated by Ca²⁺ currents, was used to study K⁺, H⁺ and Ca²⁺ transport in intact maize (Zea mays L.) roots and individual maize suspension cells. Comparisons were made between the vibrating ion-selective microelectrode, and a technique using stationary ion-selective microelectrodes to measure ionic gradients in the unstirred layer at the surface of plant roots. The vibrating-microelectrode system was shown to be a major improvement over stationary ion-selective microelectrodes, in terms of sensitivity and temporal resolution. With the vibrating ion microelectrode, it was easy to monitor K⁺ influxes into maize roots in a background K⁺ concentration of 10 mM or more, while stationary K⁺ electrodes were limited to measurements in a background K⁺ concentration of 0.3 mM or less. Also, with this system it was possible to conduct a detailed study of root Ca²⁺ transport, which was previously not possible because of the small fluxes involved. For example, we were able to investigate the effect of the excision of maize roots on Ca²⁺ influx. When an intact maize root was excised from the seedling at a position 3 cm from the site of measurement of Ca²⁺ transport, a rapid fourfold stimulation of Ca²⁺ influx was observed followed by dramatic oscillations in Ca²⁺ flux, oscillating between Ca²⁺ influx and efflux. These results clearly demonstrate that wound or perturbation responses of plant organs involve transient alterations in Ca²⁺ transport, which had previously been inferred by demonstrations of touch-induced changes in cytoplasmic calcium. The sensitivity of this system allows for the measurement of ion fluxes in individual plant cells. Using vibrating K⁺ and H⁺electrodes, it was possible to measure H⁺efflux and both K⁺ influx and efflux in individual maize suspension cells under different conditions. The availability of this technique will greatly improve our ability to study ion transport at the cellular level, in intact plant tissues and organs, and in specialized cells, such as root hairs or guard cells.Symbol X amplitude of vibration The authors would like to thank Richard Sanger for his invaluable work on the design and improvement of the ion-selective vibratingmicroelectrode system. The research presented here was supported in part by U.S. Department of Agriculture Competitive Grant No. 90-37261-5411 to Leon Kochian and William Lucas.     

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.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - CCCP carbonylcyanide m-chlorophenylhydrazone - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - LID _e light-induced depolarization in epidermal cells - LID _m light-induced depolarization in mesophyll cells - LIH light-induced hyperpolarization - TEA⁺ tetraethylammonium Ecotrans paper #43. This research was supported by National Science Foundation grants DCB-8903744 and MCB-9220110 to E.V.     

Calcium and tip growth inNeurospora crassa

Summary We examined the ionic regulation of tip growth inNeurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from –200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K⁺) or change dramatically (e.g., Ca²⁺ influx) but had no effect on hyphal growth rates. Therefore, ionic fluxes (including Ca²⁺ influx) may not be required for tip growth. However, intracellular Ca²⁺ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca²⁺ directly by ionophoresis. Elevated Ca²⁺ induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca²⁺ gradient within 10 m of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca²⁺ chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca²⁺ and dissipation of the tip-high gradient. We conclude that Ca²⁺ plays a regulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca²⁺ gradient is generated from intracellular Ca²⁺ stores in the ascomyceteN. crassa.Abbreviations BAPTA 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate - [Ca²⁺]_i intracellular Ca²⁺ concentration - fluo-3 2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4-methyl-2,2-(ethylenedioxy)dianiline-N,N,N,N-tetraacetic acid     

Calcium influx at the plasmalemma of isolated guard cells of Commelina communis

The influx of ⁴⁵Ca into isolated guard cells of Commelina communis L. has been measured, using short uptake times, and washing in ice-cold La³⁺-containing solutions to remove extracellular tracer after the loading period. Over 0.5–4 min the uptake was linear with time, through the origin. Over 20–200M external Ca²⁺ the influx measured with 10–20 mM external KCl was in the range 0.3–2.3 pmol·cm^-2·s^-1 (on the basis of estimated guard-cell area); with only 1 mM KCl externally the ⁴⁵Ca influx was significantly reduced, in the range 0.3–1.1 pmol·cm^-2·s^-1 for external Ca²⁺ of 50–100 M. The results indicate that the Ca-channel is voltage-sensitive, opening with depolarisation. No consistent effect of the addition of abscisic acid could be found. In different experiments, on the addition of 0.1 mM abscisic acid the Ca²⁺ influx was sometimes stimulated by 28–79%, was sometimes unaffected, and was sometimes inhibited by 16–29%. The results rule out a long-lasting stimulation of ⁴⁵Ca influx by ABA, but they do not rule out a transient stimulation followed by inhibition, perphaps as a consequence of down-regulation of Ca²⁺ influx by increasing cytoplasmic Ca²⁺. The hypothesis that ABA may act via an action on Ca²⁺ influx, increasing cytoplasmic Ca²⁺, with consequent effects on voltage-dependent and Ca²⁺-dependent ion channels in both plasmalemma and tonoplast, is neither proved nor disproved by these results.Abbreviations ABA abscisic acid - Ca_o, K_o external Ca and K concentrations     

Permeation of Ca2+ through K+ channels in the plasma membrane of Vicia faba guard cells

Summary The whole-cell patch-clamp method has been used to measure Ca²⁺ influx through otherwise K⁺-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K⁺ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K⁺ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K⁺ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca²⁺ is more negative than or equal to the K⁺ equilibrium potential (–47 mV), indicating that the current is due to Ca²⁺ influx through the K⁺ channels prior to their closure. Decreasing internal [Ca²⁺] (Ca _i ) from 200 to 2 nm or increasing the external [Ca²⁺] (Ca _o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca²⁺ influx also decrease the amplitude of time-activated current, indicating that Ca²⁺ permeation is slower than K⁺ permeation, and so causes a partial block. Increasing Ca _o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca^2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K⁺ channels in Zea mays guard cells also appear to have a Ca _i -, and Ca _o -dependent ability to mediate Ca²⁺ influx. We suggest that the inwardly rectiying K⁺ channels are part of a regulatory mechanism for Ca _i . Changes in Ca _o and (associated) changes in Ca _i regulate a variety of intracellular processes and ion fluxes, including the K⁺ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.     

Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H⁺-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca²⁺-channel blockers (verapamil, La³⁺) or by reducing extracellular free Ca²⁺ by treatment with ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca²⁺ is involved. No effects of the K⁺-channel blocker tetraethylammonium (TEA⁺) were detected. Vanadate and KCN, used to inhibit the H⁺-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11–26% after 1–2 min of BL. Input resistance of trichome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H⁺-ATPase with subsequent transient activation of one or more types of ion channels.Abbreviations and Symbols BL blue light - CI current injection - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - TEA⁺ tetraethylammonium - V_m membrane voltage We wish to thank Drs. Adam Bertl and Clifford L. Slayman, Yale School of Medicine, New Haven, Conn., USA, for helpful discussions. This work was supported by a Natural Sciences and Engineering Research Council of Canada Scholarship (E.P.S.) and National Science Foundation Grant DMB-8351030 (D.J.C.).     

Light sensitivity of the ciliate Tetrahymena vorax induced by the fluorescent dye acridine orange

The ciliate Tetrahymena vorax is normally insensitive to light. However, after uptake of acridine orange, blue light evokes instant backward swimming. The dye accumulates mainly in posterior vacuoles, with half-maximal uptake after 1 min. Illumination for 10 s induced a depolarisation of approximately 15 mV lasting less than 2 s, followed by a sustained hyperpolarisation of approximately 20 mV. Deciliated cells displayed a similar response. The hyperpolarisation was linked to reduced membrane resistance, showed a reversal potential of approximately −55 mV and was blocked by 1 mmol l⁻¹ TEA. The rate of rise of electrically evoked Ca²⁺-spikes was reduced during the hyperpolarisation, which is compatible with elevated cytosolic Ca²⁺ concentration. This suggests that the hyperpolarisation may be caused by activation of Ca²⁺-sensitive K⁺ channels. The depolarisation was abolished in Ca²⁺-free medium, whereas the hyperpolarisation was unaffected. Illumination for 2 s, or prolonged stimulation restricted to the anterior part of the cell, induced depolarisation only. Illumination of the posterior part caused delayed hyperpolarisation with no preceding depolarisation. We conclude that the induced backward swimming is associated with Ca²⁺ influx through anterior channels, while Ca²⁺ released from intracellular stores activates K⁺ channels responsible for the delayed hyperpolarisation.     

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.     

Effect of sodium on potassium fluxes at the cell membrane and vacuole membrane of red beet

Slices of red beet (Beta vulgaris) washed for 5 to 6 days are known to accumulate Na⁺ in preference to K⁺ from solutions containing both ions. The present work, using ion concentrations of 1.0 mm or less, with Ca²⁺ added in some cases, shows that Na⁺ strongly inhibits K⁺ influx at the cell membrane (plasmalemma) while K⁺ efflux is increased to a lesser extent. This result from compartmental analysis is confirmed by short (15-minute) influx experiments, which indicate an immediate inhibitory effect of Na⁺ on K⁺ influx at the cell membrane. It is concluded that cation selectivity, even when Na⁺ is favored for uptake, is primarily determined at the cell membrane. Nevertheless, a high level of K⁺ in the cytoplasm is maintained during Na⁺ influx, by an inhibition of K⁺ transfer to the vacuole.     

Calcium and potassium currents in the Fucus egg

The electrical properties of unfertilized eggs of Fucus serratus L. were characterized using voltage clamp and current clamp with single electrodes. The plasma membrane of the unfertilized egg is excitable. Depolarizing the egg in current clamp induced a transient depolarizing voltage response, the amplitude of which was dependent on the presence of external Ca²⁺ or Ba²⁺ and was blocked by La³⁺. The repolarizing phase was blocked by tetraethylammonium ions. Repeated stimulation at frequencies greater than 0.5 Hz caused a transient loss of excitability. Voltage-clamp experiments revealed that an inward current with an activation threshold of -35 mV underlies the depolarizing phase of the voltage response. This current showed rapid activation and slow inactivation. The current was blocked by La³⁺ and could be carried by Ca²⁺ and Ba²⁺ but not by Sr²⁺ or Na⁺. Further depolarization to values more positive than-5 mV induced a slowly activating outward K⁺ current in addition to the inward current, which corresponded to the repolarizing phase of the voltage response. This K⁺ current showed little or no inactivation during stimulation and slow deactivation on return to the resting potential. Hyperpolarizing the egg elicited an inward current. On fertilization, the Fucus egg generates a depolarizing fertilization potential. Voltage-clamp experiments revealed an inward fertilization current underlying the fertilization potential. Within 15 min of fertilization a dramatic, irreversible increase in resting K⁺ permeability developed. The roles of the plasma-membrane channels in generation of the fertilization potential and egg activation are discussed.Abbreviations and Symbols ASW artificial seawater - SECC single-electrode current clamp - SEVC single-electrode voltage clamp - TEA tetraethylammonium - Vm membrane potential This work was supported by The Marine Biological Association U.K., Science and Engineering Research Council U.K. and The Royal Society of London.     

K+ Channels and the Intracellular Calcium Signal in Human Melanoma Cell Proliferation

K⁺ channels, membrane voltage, and intracellular free Ca²⁺ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K⁺ channel and a calcium-activated K⁺ channel. The inwardly rectifying K⁺ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K⁺ channel blockers quinidine, tetraethylammonium chloride and Ba²⁺ and elevated extracellular K⁺ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca²⁺ followed by a long-lasting Ca²⁺ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca²⁺, illustrating a transmembrane flux of Ca²⁺ following its electrochemical gradient. We conclude that K⁺ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca²⁺, a messenger in the mitogenic signal cascade of human melanoma cells. Received: 9 May 1995/Revised: 30 January 1996     

Effects of membrane potential on calcium fluxes of Pelvetia eggs

⁴⁵Ca²⁺ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K⁺]) increases, and depolarization (with high [K⁺]) decreases the influx of Ca²⁺ over the range of [K⁺] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of ⁴⁵Ca²⁺ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of ⁴⁵Ca²⁺ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca²⁺].     

The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts

Protoplasts from dark-grown wheat (Triticum aestivum L.) maintained at a constant osmotic potential at 22°C, were found to swell upon red irradiation (R) and the effect was negated by subsequent far-red light (FR), indicating phytochrome involvement. Swelling only occurred when Ca²⁺ ions were present in the surrounding medium, or were added within 10 min after R. Furthermore, Mg²⁺, Ba²⁺ or K⁺ could not replace this requirement for Ca²⁺. The presence of K⁺ did not enhance the Ca²⁺-dependent swelling response. When the Ca²⁺-ionophore A 23187 was added to the medium, protoplasts swelled in the dark to the same extent as after R. Both the Ca²⁺-channelblocker Verapamil and La³⁺ inhibited R-induced swelling. It is proposed that R causes the opening of Ca²⁺-channels in the plasma membrane. Boyle-van't Hoff analyses of protoplast volume after R and FR are consistent with the conclusion that R irradiation causes changes in membrane properties.Abbreviations EDTA ethylenediaminetetraacetic acid - FR far-red light - nov non-osmotic-volume - Pfr FR-absorbing form of phytochrome - Pr R-absorbing form of phytochrome - R red light