Electrical properties ofValonia ventricosa

Summary The cytoplasmic electrical potential and membrane resistance of mature cells ofValonia ventricosa have been measured by inserting a microelectrode concentric with another electrode into the vacuole of the cell. The cytoplasmic region was investigated by advancing the microelectrode into the cell wall from the vacuolar side.The results revealed a unique region where the vacuolar electric potential and membrane resistance changed in a simultaneous single step to values close to zero. The measured potential always remained positive immediately after the step.At no time was a highly negative potential region encountered. Further penetration of the microelectrode revealed a low resistance negative potential region of –12.6±1.1 mV associated with the cell wall. Experiments were also carried out on aplanospores ofV. ventricosa to compare mature and immature cells. The chemical composition of the vacuolar and protoplasmic phases of mature cells was determined. The results agreed with previous results except that the Cl^– ion content of the protoplasm was significantly higher at 381±20 mmoles/liter (H₂O). It was concluded that mature cells ofValonia are significantly different from immature cells in that no highly negative potential cytoplasmic region was found in mature cells.It was considered that the measured step change in electric potential and membrane resistance occurred at the plasmalemma and that the tonoplast was a region of very low resistance. The implications of these findings in terms of models of ion transport intoValonia are discussed.     

Longitudinal profiles of the vacuolar pH in internally perfused cells of characean alga

Activities of ion pumps and H⁺-conducting channels in the plasmalemma of illuminated characean algae are distributed inhomogeneously along the internode, which accounts for the shifts of surface pH up to 3.5 units between various cells regions. Spatial variations in cytoplasmic properties provide the basis for uneven distribution of photosynthetic activity along the cell length and might affect the operation of H⁺-transporting systems at the tonoplast. In order to visualize the longitudinal distribution of the vacuolar pH in Chara corallina internodal cells, the pH microelectrode was inserted into the vacuole and the cell sap was gradually displaced along the cell during intracellular perfusion with an artificial medium. Fluorescein was added to the perfusion medium as a fluorescent marker to detect the arrival of the replacing medium into the area of pH and fluorescence measurements. In light-adapted cells, nonuniform longitudinal pH profiles were observed, with pH shifts as large as 2–2.5 units. In dark-adapted cells, the pH shifts in longitudinal profiles did not exceed 0.5 pH units. The occurrence of large pH changes within the vacuole of individual internodes indicates the possibility of nonuniform distribution of the tonoplast H⁺-transporting systems in different regions of the illuminated cell.     

Effect of cyanide on the plasmalemma potential of mnium

By centrifuging Mnium cuspidatum leaf cells, the cytoplasm can be distinguished from the vacuole and a microelectrode tip can be located unambiguously in the cytoplasm. The site of the electrogenic pump is clearly demonstrated to reside in the plasmalemma as shown by depolarization of the cell electropotential induced by CN⁻.     

CELL WALL POTENTIAL IN NITELLA

In the process of inserting a microelectrode into the vacuoleof Nitella three potential levels were recorded. The first onewas at a water phase outside the cell wall, the second one inthe cell wall and the third one across the plasmalemma. Thefirst potential was variable with the distance from the surfaceof the cell wall. When the external solution was 10^–4M KCl, the second potential level was –90 mv and the thirdone –170 mv against an external reference electrode. Thesepotentials were less negative (more negative) with the increase(decrease) of the external KCl concentration and varied to someextent among samples. The vacuolar potential measured againstthe cell wall phase was, therefore, –80 mv inside negativeto outside. A large potential change such as action potentialwas observed only across the plasmalemma. An overshoot of theaction potential of Nitella flexilis was observed very often,when the vacuolar potential was measured against the cell wallphase. This work was supported by a Research Grant from the Ministryof Education of Japan. Part of this work was performed whenR. NAGAI was a Yukawa Research Grant fellow.     

Impact of hypoosmotic challenges on spongy architecture of the cytoplasm of the giant marine alga Valonia utricularis

Summary. The ultrastructure of the several micrometers thick cytoplasmic layer of the giant marine alga Valonia utricularis displays characteristics which are apparently linked with the capability of this alga to regulate turgor pressure. Transmission and scanning electron microscopy of cells prefixed in different ways, including a protocol that allows prefixation of the alga in a turgescent state, revealed a highly dendritic network of cytoplasmic strands connecting and enveloping the chloroplasts and the nuclei. Innumerable vacuolar entities are embedded in the network, giving the cytoplasm a spongy appearance. Vacuolar perfusion of turgor-pressure-clamped cells with prefixation solution containing tannic acid presented evidence that these vacuolar entities together with the huge central vacuole form a large unstirred continuum. In contrast to the tonoplast, the plasmalemma followed smoothly the lining of the cell wall, even at the numerous cell wall ingrowths. Sucrose, but not polyethylene glycol 6000, induced chloroplast clustering. Acute hypoosmotic treatment (established by reduction of external NaCl or by replacement of part of the external NaCl by equivalent osmotic concentrations of sucrose or polyethylene glycol 6000) resulted in a local relocation of the chloroplasts and cytoplasm towards the central vacuole. This effect did not occur when the relatively low reflection coefficients of these two osmolytes were taken into account. The increase in spacing between the spongy cytoplasm and the plasmalemma by chloroplast relocation (viewed by confocal laser scanning microscopy) was associated with a speckled appearance of the affected surface area under the light microscope. As indicated by electron microscopy, hypoosmotically induced chloroplast relocation resulted from disproportionate swelling of the vacuolar entities located close to the plasmalemma. The cytoskeleton in the cytoplasm and the mucopolysaccharide network in the central vacuole apparently resisted swelling of these compartments. This finding has the important consequence that relevant hydrostatic pressure gradients can be built up throughout the entire multifolded vacuolar space. This gradient could represent the trigger for turgor pressure regulation which is manifested electrically first in the tonoplast.Correspondence and reprints: Lehrstuhl für Biotechnologie, Biozentrum, Am Hubland, 97074 Würzburg, Federal Republic of Germany.     

Tonoplast action potential inNitella in relation to vacuolar chloride concentration

Summary The action potential ofNitella internode was studied in relation to K⁺ and Cl^– concentrations in the vacuole. When the vacuole ofNitella pulchella was filled with an artificial solution with extremely low Cl^– concentration, a diphasic action potential (DAP) was observed. The first phase consists of a rapid depolarization followed by a relatively rapid repolarization, and the second one consists of a strong hyperpolarization followed by a gradual return to the resting potential.When the cell was stimulated immediately after the generation ofDAP, a monophasic action potential which resembles an action potential of the natural cell was observed, indicating that theDAP consists of two components with different refractory periods. The refractory period of the component responsible for the depolarizing phase is shorter than that of a component responsible for the hyperpolarizing phase. Measuring the plasmalemma potential and vacuolar potential separately, it was demonstrated that the hyperpolarizing component ofDAP originates from the tonoplast.The action potential of the tonoplast, in contrast with that of the plasmalemma, could be generated independently of concentration of K⁺ in the vacuole. Since the maximum amplitude of hyperpolarization decreased significantly by increasing Cl^– concentration of the vacuole, it is concluded that the tonoplast is very sensitive to Cl^– during excitation.     

Turgor pressure changes trigger characteristic changes in the electrical conductance of the tonoplast and the plasmalemma of the marine alga Valonia utricularis

The giant marine alga Valonia utricularis is capable of regulating its turgor pressure in response to changes in the osmotic pressure of the sea water. The turgor pressure response comprises two phases, a fast, exponential phase arising exclusively from water shifting between the vacuole and the external medium (time constant about 10 min) and a second very slow, almost exponential phase adjusting (but not always) the turgor pressure near to the original value by release or uptake of KCl (time constant about 5 h). The changes in the vacuolar membrane potential as well as in the individual conductances of the tonoplast and plasmalemma accompanying turgor pressure regulation were measured by using the vacuolar perfusion assembly (with integrated microelectrodes, pressure transducers and pressure‐regulating valves) as described by Wang et al. (J. Membrane Biology 157, 311–321, 1997). Measurements on pressure‐clamped cells gave strong evidence that the turgor pressure, but not effects related to water flow (i.e. electro‐osmosis or streaming potential) or changes in the internal osmotic pressure and in the osmotic gradients, triggers the cascade of osmotic and electrical events recorded after disturbance of the osmotic equilibrium. The findings definitely exclude the existence of osmosensors as postulated for other plant cells and bacteria. There was also evidence that turgor pressure signals were primarily sensed by ion transporters in the vacuolar membrane because conductance changes were first recorded in the many‐folded tonoplast and then significantly delayed in the plasmalemma independent of the direction of the osmotic challenge. Consistently, turgor pressure up‐regulation (but not down‐regulation) could be inhibited reversibly by external addition of the K⁺ transport inhibitor Ba²⁺ and/or by the Cl^– transport inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). Extensive studies under iso‐, hyper‐ and hypo‐osmotic conditions revealed that K⁺ and Cl^– contribute predominantly to the plasmalemma conductance. Addition of 0.3 mm NaCN showed further that part of the K⁺ and Cl^– transporters depended on ATP. These transporters are apparently up‐regulated upon hyper‐osmotic, but not hypo‐osmotic challenge. These findings explain the strong increase of the K⁺ influx upon lowering turgor pressure and the less pronounced pressure‐dependence of the Cl^– influx of V. utricularis reported in the literature. The data derived from the blockage experiments under hypo‐osmotic conditions were also equally consistent with the experimental findings that the K⁺ efflux is solely passive and progressively increases with increasing turgor pressure due to an increase of the volumetric elastic modulus of the cell wall. However, despite unravelling some of the sequences and other components involved in turgor pressure regulation of V. utricularis the co‐ordination between the ion transporters in the tonoplast and plasmalemma remains unresolved because of the failure to block the tonoplast transporters by addition of Ba²⁺ and DIDS from the vacuolar side.     

Studies on the tonoplast action potential ofNitella flexilis

Summary The origins of the two peaks of the action potential inNitella flexilis were analyzed by inserting two microelectrodes. one into the vacuole and the other into the cytoplasm. It was unequivocally demonstrated that the rapid first peak was generated at the plasmalemma and the slow second peak at the tonoplast. MnCl₂ applied in the external medium abolished the second, tonoplast, peak but not the first, plasmalemma, peak, MnCl₂ also inhibited the cessation of the cytoplasmic streaming accompanying the action potential. CaCl₂ added in MnCl₂-containing medium recovered generation of the tonoplast action potential and the streaming cessation. Since it has been established that the cessation of cytoplasmic streaming on membrane excitation is caused by an increase in cytoplasmic free Ca^2– (Williamson, R.E., Ashley, C.C., 1982.Nature (London) 296:647–651: Tominaga, Y., Shimmen, T., Tazawa, M., 1983,Protoplasma 116:75–77), it is suggested that the tonoplast action potential is also induced by an increase in cytoplasmic Ca²⁺ resulting from the plasmalemma excitation. When vacuolar Cl was replaced with SO ₄ ² by vacuolar perfusion, the polarity of the second, slow peak was reversed from vacuolar positive to vacuolar negative with respect to the cytoplasm, supporting the previous report that the tonoplast action potential is caused by increase in Cl permeability (Kikuyama, M., Tazawa, M., 1976.J. Membrane Biol.29:95–110).     

Membrane deletion during plasmolysis in hardened and non-hardened plant cells

Abstract Video recordings of interference phase contrast microscopy were used to study plasmalemma deletion during plasmolysis in hardened and non-hardened suspension cultured cells of Brassica napus, alfalfa, and cells isolated from rye seedlings. Although different hardening regimes and different cells were used, the responses to plasmolysis were consistent. Hardened cells uncoupled the volume to surface area ratio during plasmolysis both by forming a large number of strands between the cell wall and protoplast and by leaving rivulet-like networks of membranes on the cell wall surface. Tonoplast membrane was deleted as sac-like intrusions into the vacuole. Non-hardened cells produced few strands during plasmolysis. They also deleted plasmalemma and tonoplast into the vacuole as endocytotic vesicles. During deplasmolysis of hardened cells both the individual membrane strands and the rivulets of membrane material vesiculated into strings of vesicles. The vesicles were osmotically active and were re-incorporated into the expanding protoplast. Conversely, deplasmolysis in non-hardened cells resulted in few osmotically active vesicles and many broken strands. The vacuolar sac-like intrusions in hardened cells were re-incorporated into the vacuole whereas the endocytotic vesicles in non-hardened cells were not re-incorporated. Therefore, the non-hardened cells underwent expansion-induced lysis.     

Voltage Noise in Acer pseudoplatanus Cells : Basic Cellular Noise and Gramicidin a Induced Noise

The present contribution is devoted to studying the electrical noise of Acer pseudoplatanus cells in culture suspensions. Spontaneous voltage noise of the cells was recorded by means of a microelectrode inserted in the vacuole. The small signal impedance of the cell was measured so that it was possible to study the intensity spectra of the noise. We recorded intensity spectra with cells incubated in 10⁻³ molar gramicidin A. Difference spectra showed characteristics of a channel noise. By using the calculated conductance of gramicidin A in an artificial membrane, and by simplifying assumptions for the ionic transports through plasmalemma and tonoplast, we were able to estimate the electrochemical potential difference for K⁺ ions across the plasmalemma (3.2 ± 1 millivolt).     

Movement of Abscisic Acid Across the Plasmalemma and the Tonoplast of Guard Cells of Valerianella locusta

Uptake experiments and efflux compartmental analyses of abscisic acid (ABA) with acid treated epidermal peels of Valerianella locusta were performed to elucidate the mechanisms of transport of ABA across the plasmalemma and tonoplast of guard cells. ABA uptake across the plasmalemma is linearly correlated with external ABA concentration in the incubation medium. Under alkaline conditions ABA-uptake was not significantly above background, indicating that ABA uptake occurs mainly by diffusion of undissociated ABAH as the most permeable species, which is trapped afterwards in the alkaline cytosol as impermeable ABA^?. Efflux analysis of ABA revealed a saturable component of ABA transfer across the tonoplast. A Woolf-Augustinsson-Hofstee analysis suggested the existence of two transport systems for ABA at the tonoplast. The high affinity transport system had a K_M of 0.21 mol m^?3 and a V_max 85.8 amol ABA cell^?1 h^?1. Using the data of the uptake and efflux experiments we calculated the permeability coefficients of ABA for the plasmalemma and the tonoplast of guard cells, which are 2.46 10^?7 m s–¹ and 1.26 10^?8m s^?1, respectively. The distribution of the pH-probe (¹⁴C)-DMO between medium, cytosol and vacuole was investigated and used to calculate cytosolic and vacuolar pH. The vacuolar pH is too low to explain the high vacuolar ABA concentration by trapping of ABA^?, whereas the cytosol is sufficiently alkaline to act as an efficient anion trap. Therefore we conclude that ABA transport across the guard cell tonoplast is catalyzed by a saturable uptake component.     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

Electrical Properties of the Plasmalemma and Tonoplast in Valonia ventricosa

Studies were made on the electric potentials of the plasmalemma (E_co) and tonoplast (E_vc) in small cells (1-3 mm diameter) of Valonia ventricosa. To measure E_co, microelectrodes with long tapers were inserted into the vacuole with the path of electrode entry off-center. The microelectrode then was pushed across the vacuole and into the cytoplasm on the opposite side of the cell. A reference electrode was placed in the artificial seawater bathing the cell. A similar method was used to measure E_vc except that the reference electrode was placed in the vacuole.     

Comparison of a lipophilic cation and microelectrodes to measure membrane potentials of the giant-celled algae,Chara australis (Charophyta) andGriffithsia monilis (Rhodophyta)

Summary The vacuolar equilibrium potential of the lipophilic cation TPMP⁺ (triphenyl methyl phosphonium) in the giant algaeChara australis andGriffithsia monilis was directly measured. The TPMP⁺ equilibrium potential was approximately 100mV less negative than the measured vacuolar electrical potential. Thus TPMP⁺ does not act as a probe of the vacuolar electrical potential and appears to be extruded against an electrochemical gradient. Measurement of the plasmalemma equilibrium potential of TPMP⁺ showed that extrusion of TPMP⁺ apparently occurred at both the tonoplast and plasmalemma inChara and at the plasmalemma inGriffithsia. It is concluded that TPMP⁺ cannot be used as a membrane potential probe inChara orGriffithsia.     

Light-Induced H+ Accumulation in the Vacuole of Nitellopsis obtusa

The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H⁺-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.1–0.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pH_o). At pH_o 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pH_o valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.3–0.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pH_o 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pH_o 9.0 to pH_o 7.5results in a cell hyperpolarization by 60–80 mV and adecrease of the vacuolar pH by 0.4–0.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H⁺ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H⁺ in the cytoplasm. The H⁺ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H⁺ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H⁺ conductance and on the electrochemical gradientfor H⁺ at the plasmalemma.     

Turgor regulation inValonia macrophysa following acute osmotic shock

Summary The marine algaValonia macrophysa an inhabitant of shallow subtropical waters, is subjected to sudden dilutions of external seawater during rain showers. This study describes the mechanisms involved in turgor pressure regulation following acute hyposmotic shock. Turgor regulation is 88% effective and complete within 4 hr following hyposmotic shocks of up to –10 bar. Loss of vacuolar K⁺, Na⁺ and Cl^– accounts for the decrease in vacuolar osmotic pressure associated with turgor regulation. A novel mechanism of turgor regulation is exhibited byValonia macrophysa given hyposmotic shocks greater than about –4 bar. Such an osmotic shock causes cell wall tension to increase above a critical value of about 6×10⁵ dyne/cm, whereupon the protoplasm ruptures and the cell wall stretches irreversibly at a localized site. The protoplasm rupture is suggested by (1) a large abrupt increase in K⁺ efflux (as measured by⁸⁶Rb⁺), (2) a rapid decrease in turgor pressure as measured with a pressure probe, and (3) sudden depolarization of the vacuole potential. Evidence for an increase in cell wall permeability includes efflux from the vacuole of dextran (mol wt 70,000), which normally has a very low cell wall permeability, and scanning electron micrographs which show a trabeculated scar area in the cell wall. This mechanism of turgor regulation is physiologically important because 98% of the cells regained normal growth rate and turgor following acute osmotic shock.     

Electrical coupling between cells of higher plants: A direct demonstration of intercellular communication

Summary Electrical coupling between adjacent cells of Elodea canadensis has been demonstrated using a microelectrode technique in which the membrane potentials were recorded during the passage of a current pulse from the vacuole of one cell to the external solution. The changes in membrane potential resulting from the passage of the current may be simulated by an equivalent circuit in which the tonoplast:plasmalemma:plasmodesmata resistances are in the ratio 1.0:5.6:2.2. On this basis, the specific resistances are 3.1 k cm² for the plasmalemma, 1.0 k cm² for the tonoplast and 0.051 k cm² for the junction between the cells. Although the plasmodesmata permit the passage of current, it is estimated that they have a resistance about 60 times higher than would be the case if they were completely open channels. Electrical coupling has also been demonstrated between parenchymal cells in oat coleoptiles and between cortical cells in maize roots. The significance of these findings is discussed in relation to the symplastic transport of ions and other small molecules and in relation to the quantitative measurement of membrane resistance in multicellular tissue.     

The Role of the Contractile Vacuole in the Osmoregulation of Tetrahymena pyriformis*

The relationship of cell size and contractile vacuole efflux to osmotic stress was studied in Tetrahymena pyriformis strain W, after transfer into fresh solutions iso- or hypoosmotic to the growth medium. Microscopic measurements of the cell and contractile vacuole dimensions, made with an image-sharing ocular at 27 C, allowed the calculation of the cell size and shape and the vacuolar efflux rate which provide a measure of osmoregulation. The contractile vacuole cycles have no homeostatic oscillations. In 0.03–0.10 osmolar solutions, the cell size and shape are constant while the vacuolar efflux rate has an inverse linear dependence upon extracellular osmolarity. Regression analyses indicate that for cells with systole faster than 0.1 sec (the major part of the population), it is only the final diastolic volume of the contractile vacuole that is related to osmotic stress while the frequency of systole is independent of osmotic stress and has a constant period of 7.7 ± 0.2 sec. Therefore, osmotic stress upon Tetrahymena is regulated by a corresponding change in the filling rate of its contractile vacuole to allow an unaltered cell size and shape. Kinetic measurements of vacuoles during diastole fit the model (dV/dt = K_1-K₂A), where (dV/dt) is the vacuolar filling rate and (A) is the vacuolar surface area. This dependence of vacuolar volume upon its surface area may be ascribed either to elastic components of the vacuolar membrane or to an increasing leakiness of this membrane during diastole. Mitochondrial inhibitors were used to observe the energy requirements of vacuolar operation and of intracellular secretion of water.     

Studies on the perfused plasmalemma ofChara corallina: I. Current-voltage curves: ATP and potassium dependence

Summary The electrical properties of theChara cell membrane have been studied using a perfusion method based on that of Williamson, R.E. 1975.J. Cell Sci. 17655. The vacuole, tonoplast, and inner cytoplasm are removed by a brief rapid perfusion. Electrical properties of the plasmalemma indicate that it remains intact after this perfusion.The membrane potential difference after perfusion and with no ATP was close to the potassium equilibrium potential; the current-voltage characteristic had a slope that was time- and voltage-dependent, indicating that the steady-state potassium conductance increased with depolarization. At –125 mV the membrane conductance of the plasmalemma depended on [K⁺]₀. This dependence was inhibited by perfusing with 2.0mm ATP or by clamping at a more negative membrane potential. The addition of ATP to the perfusion medium of unclamped cells caused a hyperpolarization ofca. 50 mV, presumably by activating the proton pump. In clamped cells, perfusion with ATP caused currents ofca. 20 mA m^–2, whose magnitude depended on pH₀. ATP induced membrane conductance changes which were variable. 2.0mm ADP inhibited the proton pump. The intersection points of current-voltage characteristics can set limits on the stalling potential; the resulting stoichiometry of the proton pump appears to be 1.5–2.0 H⁺ per ATP.     

Inactivation of plasmalemma conductance in alkaline zones of <Emphasis Type="Italic">Chara corallina</Emphasis> after generation of action potential

A microelectrode study with Chara corallina cells has shown that post-excitation changes of membrane potential and plasmalemma resistance, induced by the action potential (AP) generation, differ substantially for cell areas producing zones of high and low external pH. In cell regions producing alkaline zones, the AP generation was followed by post-excitation hyperpolarization by about 50 mV, concomitant with four- to eightfold increase in plasmalemma resistance and a considerable drop of pericellular pH. In the acidic areas the post-excitation hyperpolarization was weak or absent, and the membrane resistance showed no significant increase within 1–2 min after AP. The membrane excitation in the acidic zones was accompanied by a noticeable pH increase near the cell surface, indicative of the inhibition of plasma membrane H⁺ pump. The results suggest that the high local conductance of the plasmalemma is closely related to alkaline zone formation and the depolarized state of illuminated cell under resting conditions. Excitation-induced changes of membrane potential and pH in the cell vicinity were fully reversible, with the recovery period of ∼15 min at a photon flux density of ∼100 μE/(m² s). At shorter intervals between excitatory stimuli, differential membrane properties of nonuniform regions turned smoothed and could be overlooked. It is concluded that the origin of alkaline zones in illuminated Chara cells cannot be ascribed to hypothetical operation of H⁺/HCO₃⁻ symport or OH⁻/HCO₃⁻ antiport.