Studies on Electrogenesis under High K+ Concentrations in Internodal Cells of Chara corallina

+ concentration ([K⁺]_o) on the membrane potential (E_m) of Chara corallina was studied. E_m more negative than -100 mV was maintained even at 100 mM [K⁺]_o. Addition of Ca²⁺ to the external medium further increased this tendency. However, E_m responded sensitively to the increase in [K⁺]_o, when the electrogenic proton pump of the plasma membrane was inhibited by treating cells with dicyclohexylcarbodiimide, an inhibitor of proton pump. Analysis using equivalent circuit model of the plasma membrane suggested that the electrogenic proton pump was activated by the increase in [K⁺]_o. In the presence of 100 mM K⁺, action potentials were generated by electric stimuli. The ionic mechanism of generation of action potentials in the presence of K⁺ at high concentration was discussed. Received 3 October 2000/ Accepted in revised form 6 January 2001     

Photosynthetic Fixation of 14Carbon by Internodal Cells of Chara corallina

Maximum fixation rates of 120 and 60 pmol cm^–2 s ^–1wereobtained when exogenous carbon was supplied as ¹CO₂ and H¹⁴CO₃^–respectively. These values are considerably higher than thosepreviously reported for this species. A kinetic analysis wasperformed on this data. Substrate saturation in the concentrationrange 1.0–1.5 mM was observed for both CO₂ and HCO₃^– In the presence of exogenous CO², a linear relationship wasobserved between light intensity and fixation while the HCO₃^–relationship was slightly sigmoidal. Fixation saturated at intensitiesof 15–20 W m^–2 and 13–15 W m^–2 for exogenous¹⁴CO² and H¹⁴CO₃^–respectively. The presence, in this species, of an extremely active HCO₃^–transport system, situated in the plasmalemma, demonstratesthat when alkaline solutions are employed the involvement ofthis ion cannot be ignored during electrical studies on thismembrane. The maximum H¹⁴CO₃^– influxes obtained duringthis study are the largest ionic fluxes measured for any Characeanspecies. It was demonstrated that CO² for fixation can be supplied simultaneouslyby gaseous diffusion and HCO₃^– transport (cf. Raven, 1968).Inhibition of H¹⁴CO₃^– influx was observed in the presenceof Tris, Tricine, and borate buffers, and CO₃^{2 –} alsoappeared to act as a strong inhibitor. The possible mechanism(s)by which this inhibition occurs is discussed.     

N-Ethylmaleimide Blocks the H+ Pump in the Plasma Membrane of Chara corallina Internodal Cells

The sulfhydryl (SH) modifying reagent N-ethylmaleimide (NEM)was applied to the internodal cells of Chara corallina to studythe role of SH residues in the activity of the plasma membraneH⁺ pump. NEM (1 µM) caused a marked depolarizing shiftof the resting potential by 6410mV (n=7) together with depressionof the conductance peak at around —200 mV, indicatinga marked depression of the H⁺ pump activity. This effect ofNEM was partly reversible, the membrane repolarized and theconductance peak was restored after extracellular washing. TheH⁺ pump inhibitor, dicyclohexylcarbodiimide (DCCD), caused noadditive membrane depolarization and/or depression of the H⁺pump conductance, in the presence of NEM. This suggests thatNEM blocks the H⁺ pump and that SH residues play a pivotal rolein maintaining the H⁺ pump activity in Chara corallina. (Received April 10, 1993; Accepted July 29, 1993)     

Cortical Microtubule Organization and Internodal Cell Maturation in Chara corallina

The arrangement of cortical microtubules, as documented by immunofluorescence and immunogold-silver enhancement, changes during the growth and maturation of giant internodal cells of Chara corallina, a process taking approximately 45 days. Transverse microtubules are found throughout growth along with a subset of distinctly non-transverse microtubules. During the second half of the growing period, when relative growth rates are diminishing, these non-transverse microtubules become more abundant but a few days prior to growth cessation, they are mostly absent. At about the time of growth cessation the microtubules, while retaining their locally parallel alignment, begin to show increasing deviation from the transverse axis. Eventually, a mosaic of locally parallel yet variably oriented fields of microtubules forms. Many days after growth stops, microtubules become shorter and less numerous and lose parallel alignment, leading to the formation of a random MT pattern.     

Effects of Lanthanum on Calcium and Magnesium Contents and Cytoplasmic Streaming of Internodal Cells of Chara corallina

Biological and environmental effects of lanthanide series of elements have received much attention recently due to their wide applications. In this study, effects of La³⁺ treatments on calcium and magnesium concentrations as well as cytoplasmic streaming of internodal cells of Chara corallina were investigated. At all treatment concentrations (10, 100, and 1,000 μM), La³⁺ significantly decreased calcium concentrations in the cell-wall fractions after 5-h treatments. Calcium concentrations in the cell contents and magnesium concentrations in the cell-wall fractions were reduced by 100 and 1,000 μM La³⁺ treatments. However, cytoplasmic streaming as an indicator of [Ca²⁺]_cyt was only inhibited at the highest La³⁺ concentration (1,000 μM). The results suggest that La³⁺ may affect cellular calcium homeostasis by actions other than as a simple Ca²⁺ antagonist. La³⁺ could partially compensate for calcium deficiency at certain concentrations.     

Cooperative behavior of K+ channels in the tonoplast of Chara corallina.

Spontaneous cooperatively of K+ channels is studied in excised patches of Chara corallina tonoplasts. Bar histograms (dwell time versus number of open channels) are constructed from the time series of current by means of the higher-order Hinkley detector (R. Schultze and S. Draber. 1993. J. Membr. Biol. 132:41-52). A statistical test, based on these bar histograms, shows that the channels are not independent. Further analysis reveals that the channels are cooperatively changing their open probability, which leads to the idea of cooperative mode shifting.     

Entry of Methylammonium and Ammonium Ions into Chara Internodal Cells

Low concentrations of ammonia and methylamine greatly affectmembrane transport by, and the electrical properties of, cellsof Chara corallina (=C. australis). In the presence of theseamines, influx of Cl^– and efflux of K⁺ increase and alarge depolarizing current flows through the cell membrane. Measurements with [¹⁴C]methylamine show that methylamine isabsorbed rapidly over a wide pH range, and that the absorptionisotherm is complex. Methylamine influx is not affected by presenceor absence of Cl^–, K⁺, or Na⁺, but is decreased by additionof . The depolarizing current is associated with a small increase in membrane conductance, except at highpH, and both these effects are reversible. The current showssaturation with increasing amine concentration; when methylamineis 10–12 times more concentrated than ammonia, it producesa current of the same magnitude. The results show that the amines enter the cells as cations( or CH₃) except where external pH is high, and that a specific, selective electrogenicporter is involved. There is no need to invoke active transport,as there is no evidence that internal amine concentrations exceedthe equilibrium (Nernst) concentrations.     

Conduction Velocity and Blockage of Action Potential in Chara Internodal Cells

Conduction velocity of the action potential in Chara brauniiinternodal cells was 0.21 ?0.05 cms in moist air and 1.5?0.9cms in artificial pond water (APW). The action potential waspropagated at an almost constant velocity along the cell inmoist air except within 0.3 cm from an end of the cell, whereasin APW, the velocity increased to 5.7 ? 2.3 cms within 1.8 cmfrom the end of the cell. When part of the cell was put in moistair and the other part was immersed in APW, conduction of theaction potential in moist air decreased in velocity and sometimesstopped in the vicinity of the boundary between moist air andAPW. Some cells from the plants collected in late autumn towinter generated an action potential which could not propagatein moist air. In these cells, an increase in the threshold andpartial cessation of protoplasmic streaming were observed. ¹Present address: Department of Physiology, Tohoku UniversitySchool of Dentistry, Seiryo-machi, Sendai 980 ²Present address: Biology Laboratory, Kyoritsu Women's University,Hachioji, Tokyo 193, Japan. (Received March 10, 1987; Accepted July 6, 1987)     

Characterization of the Ca2+-Dependent Cl- Efflux in Perfused Chara Cells

The mechanism of the Ca²⁺-dependent Cl^– efflux was studiedin tonoplast-free cells, in which the intracellular chemicalcomposition can be freely controlled. Tonoplast-free cells wereprepared by perfusing the cell interior of internodal cellsof Chara corallina with a medium that contained EGTA. The Ca²⁺-inducedCl^– efflux was measured together with the membrane potentialduring continuous intracellular perfusion. The dependenciesof Cl^– efflux and the membrane potential on the intracellularCa²⁺ or Cl^– concentrations were analyzed. When perfusionwas started with medium that contained Ca²⁺ ions, Cl^–efflux and membrane depolarization were induced. The amountof Cl^– efflux varied considerably among individual cells.The rate of efflux decreased exponentially but a residual effluxremained detectable. The Cl^– efflux was induced at concentrationsof Ca²⁺ ions above 1 µM and reached a maximum at 1 mM.By contrast, the membrane depolarization reached a maximum atabout 10 µM Ca²⁺. The rate of Cl^– efflux increasedlinearly with logarithmic increases in the intracellular Cl^–concentrations. These findings suggest that more than two kindsof Ca²⁺-dependent Cl^– channel might be present in theplasma membrane. Addition of ATP or its removal from the perfusion medium didnot affect the Ca²⁺-dependent Cl^– efflux. Calmodulin antagonistsslightly inhibited the Ca²⁺-dependent Cl^– efflux. ¹Present address: Biological Laboratory, Hitotsubashi University,Naka 2-1, Kunitachi, Tokyo, 186 Japan.     

Studies on alkaline band formation in Chara corallina: ameliorating effect of Ca2+ on inhibition induced by osmotic shock

Although the decrease in cell turgor by application of sorbitol to the external medium did not inhibit the alkaline band formation in Chara corallina, recovery of normal turgor severely inhibited it. Alkaline-loading analysis suggested that the inhibition of alkaline band formation was caused by inhibition of HCO(3)(-) influx but not that of OH(-) efflux. In the presence of 10 mM CaCl(2), the capacity of alkaline band formation was maintained during osmotic treatment. Cells could not form alkaline bands, when plasmolysis was induced by application of sorbitol at a higher concentration. Addition of 10 mM CaCl(2) could ameliorate the inhibition caused by plasmolyis.     

Effect of Divalent Cations on Na+ Permeability of Chara corallina and Freshwater Grown Chara buckellii

The effect of elevated Na⁺ concentration on Na⁺ permeability(P_Na) and Na⁺ influx in the presence of two levels of externaldivalent cations was determined in Chara corallina and freshwater-culturedChara buckellii. When Na⁺ in the medium was increased from 1.0to 70 mol m^–3, Na⁺ influx increased in both species ifCa²⁺ was low (0.1 mol m–3). If Ca²⁺ was increased to 7.0mol m^–3 when Na⁺ was increased, Na⁺ influx remained atthe low control level in C. corallina, and showed only a temporaryincrease in C. buckellii. Mg²⁺ was a better substitute for Ca²⁺in C. buckellii than in C. corallina. Na⁺ permeability data suggest that when the external Ca²⁺ concentrationis low, P_Na does not increase in the presence of elevated NaCl;the increase in Na⁺ influx appears to be due to the increasein external Na⁺ concentration alone. Ca^{2 +} supplementation appearsto decrease P_Na whereas supplemental Mg²⁺ has no effect. Na⁺ effluxes were computed from previously determined net fluxesand the influxes. It was found that for both species, fluxesin both directions were stimulated in response to all experimentaltreatments, but Na⁺ influx always exceeded efflux. This resultedin net Na⁺ accumulation in the vacuoles of both species. The results are discussed with reference to net flux and electrophysiologicaldata obtained previously under identical conditions, as wellas the comparative salinity tolerance of both species and theNa⁺/divalent cation ratio. Key words: Na⁺ influx, Na⁺ tolerance, membrane potential, permeability, Chara     

Temperature Sensitivity of the Tonoplast Ca2+-Activated K+ Channel in Chara: The Influence of Reversing the Sign of Membrane Potential

A detailed temperature dependence study of a well-defined plant ion channel, the Ca²⁺-activated K⁺ channel of Chara corallina, was performed over the temperature range of their habitats, 5–36°C, at 1°C resolution. The temperature dependence of the channel unitary conductance at 50 mV shows discontinuities at 15 and 30°C. These temperatures limit the range within which ion diffusion is characterized by the lowest activation energy (E _a = 8.0 ± 1.6 kJ/mol) as compared to the regions below 15°C and above 30°C. Upon reversing membrane voltage polarity from 50 to −50 mV the pattern of temperature dependence switched from discontinuous to linear with E _a = 13.6 ± 0.5 kJ/mol. The temperature dependence of the effective number of open channels at 50 mV showed a decrease with increasing temperature, with a local minimum at 28°C. The mean open time exhibited a similar behavior. Changing the sign of membrane potential from 50 to −50 mV abolished the minima in both temperature dependencies. These data are discussed in the light of higher order phase transitions of the Characean membrane lipids and corresponding change in the lipid-protein interaction, and their modulation by transmembrane voltage. Received: 14 June 2000/Revised: 20 September 2000     

Analysis of Rapid Light-Induced Potential Change in Cells of Chara corallina

Perfused Chara cells were used to measure the rapid light-inducedpotential change (rapid LPC) caused by activation of a K⁺ channelin the plasma membrane through photosynthesis in the presenceof various photosynthetic inhibitors. The rapid LPC was inhibitedby DCMU but recovered on addition of phenazinemethosulfate (PMS)in the presence of DCMU. Carbonylcyanide m-chlorophenylhydrazone(CCCP) stimulated the rapid LPC. DCCD partially inhibited therapid LPC with a partial inhibition of oxygen evolution. Itis concluded that both cyclic and noncyclic electron flows arecoupled with the rapid LPC. To understand the mechanism of K⁺ channel activation by photosyntheticelectron flow, the rapid LPC was measured under continuous internalperfusion. It was suggested that a diffusible substance wasnot released from chloroplasts, since vigorous continuous perfusiondid not inhibit the rapid LPC. The suggestion that the rapid LPC is caused by changes in surfacecharge density of chloroplasts was supported by the fact thatthe rapid LPC was inhibited by increasing the ionic strengthof the perfusion medium. (Received February 28, 1986; Accepted April 30, 1986)     

HCO(3) Influx across the Plasmalemma of Chara corallina: Physiological and Biophysical Influence of 10 mm K

The effect of 10 mm K⁺ on the HCO₃⁻ influx in Chara corallina has been used to distinguish a Ca²⁺-dependent membrane integrity site from the HCO₃⁻ transport site which is also Ca²⁺-dependent (Lucas and Dainty, Plant Physiology 1977 60: 862-867).     

Passive Ca2+ Transport and Ca2+-Dependent K+ Transport in Plasmodium falciparum-Infected Red Cells

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca²⁺ permeability. The magnitude of the increase is greater than that normally required to activate the Ca²⁺-dependent K⁺ channel (K _Ca channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K _Ca channel and the Ca²⁺ permeability properties of pRBC. For pRBC suspended in media containing Ca²⁺, K _Ca channel activation was elicited by treatment with the Ca²⁺ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca²⁺ into pRBC in which the Ca²⁺ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (10¹³ cells · hr)⁻¹), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca²⁺ influx increased to over 1 mmol (10¹³ cells · hr)⁻¹, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca²⁺ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni²⁺. Possible roles for this pathway in pRBC are considered. Received: 12 May 1999/Revised: 8 July 1999     

Molecular cloning and sequencing of the cDNA for vacuolar H+-pyrophosphatase from Chara corallina1

We have cloned a cDNA for vacuolar proton-translocating pyrophosphatase of Chara corallina that is one of the closest green algae to the land plants. The deduced protein consists of 793 amino acid residues. Its sequence is 71% identical to the H+-pyrophosphatases of land plants, and is less than 46% identical to those of marine alga and phototrophic bacterium.     

Effect of action potential on photosynthesis and spatially distributed H+ fluxes in cells and Chloroplasts of Chara corallina

When exposed to light, the cells of characean algae produce intermittent regions of H⁺ extrusion and H⁺ absorption, featuring different photosynthetic activities. Methods for local measurements of outer pH, O₂ content, and photochemical activity of photosystem II (PSII) were applied to examine microscopic regions of Chara coralline Klein ex Willd. internodes. The results show that the functional spatial heterogeneity of these excitable cells is controlled not only by light but also by electric excitation of the plasma membrane. Generation of a single action potential (AP) induced a reversible transition to the state with homogenous pH distribution and had different effects on photosynthesis in cell regions producing alkaline and acid zones. The effective quantum yield of PSII primary processes and the maximal chlorophyll fluorescence decreased after AP in the alkaline cell regions but were almost unaffected in the acidic cell regions. The suppression of photosynthesis after AP was also evident in the decrease of photosynthetic O₂ evolution. The results provide evidence that electric signals arising at the plasmalemma are transmitted to the level of thylakoid membranes. The effects of electric excitation on fluorescence and the quantum yield of PSII photochemistry were best pronounced at low light intensities and low level of nonphotochemical quenching. The sensitivity of chlorophyll fluorescence in resting and excited cells to light intensity and protonophores indicates that the AP-induced fluorescence changes derive from the increase in pH gradient at the thylakoid membrane. The temporal elimination of alkaline zones and inhibition of photosynthesis apparently arise from parallel operational sequences that have a common initial stage. A possible role of cytosolic Ca²⁺ rise in the mechanism of photosynthesis suppression after electric excitation of the plasma membrane is discussed.     

Regulation of the H+ pump activity in the plasma membrane of internally perfused Chara corallina

The role of cytoplasm for the maintenance of the H+ pump activity in Chara corallina internodal cells was examined by the intracellular perfusion technique. Cytoplasm-rich and -poor states were obtained by changing the perfusion time, short-term (less than 2 min) and long-term (more than 5 min), respectively. A large portion of cytoplasm was left by short-term perfusion but most of the cytoplasm was removed by long-term perfusion. The activities of the H+ pump of these two different conditions were examined by measuring current-voltage relation (I-V curve) and conductance-voltage relation (G-V curve) under voltage clamp conditions. The H+ pump conductance decreased to 37%, 9% and zero by short-term, long-term and hexokinase perfusion, respectively, whereas the passive channel conductance decreased to 71%, 39% and 73% by short-term, long-term and hexokinase perfusion, respectively. On the other hand, the electromotive-force of the H+ pump (approximately -260 mV) and the passive channel (approximately -130 mV) were not affected by either short- or long-term perfusion. It is indicated that the cytoplasm plays an essential role to regulate the activity of both the H+ pump and the passive channel together with ATP.     

The Effects of pH on the Ionic and Electrical Properties of the Internodal Cells of Chara australis

The effects of pH on the resting and action potentials and onthe fluxes of potassium, sodium, and chloride across the membranesof internodal cells of Chara australis have been investigated. Experiments were carried out in an artificial pond water (A.P.W.)of standard composition: CaCl₂, 01 mM; KCl, 0.1 mM; NaCl, 1.0mM. The resting potential decreased as the pH was lowered from6.5, being depolarized by about 75 mV at pH 4.5. Measurementsof the ion fluxes as a function of pH suggested that this depolarizationwas caused by an increase in the permeability to sodium anda decrease in permeability to potassium at pH 4.5. Action potentialsof constant peak value can be elicited for some time at pH 4.5,but after 20 min or so the cell becomes refractory. All theseeffects on resting and action potentials are fully reversible.We briefly speculate about the mechanism of these pH effects.