Article

Separate Determination of the Electrical Properties of the Tonoplast and the Plasmalemma of the Giant-Celled Alga Valonia utricularis: Vacuolar Perfusion of Turgescent Cells with Nystatin and Other Agents

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Abstract

In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason, a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions (up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport properties of the membrane barrier. Addition of 16 μm of the H+-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to −22 mV and in an increase of the area-specific membrane resistance from 6.8 × 10−2 to 40.6 × 10−2Ωm2. The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol. 29:81–94, 1976). Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water were replaced by a cytoplasma-analogous medium). When 50 μm of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested that the fast and slow relaxations originated exclusively from the electrical properties of the plasmalemma and the tonoplast respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually high (6.21 × 10−2 Fm−2 compared to 0.77 × 10−2 Fm−2 of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of mobile charges of an unknown electrogenic ion carrier system.

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... electrical parameters of plasmalemma as well as tonoplast to describe more precisely the processes following changes in osmotic pressure or turgor pressure. At present, such data are only accessible for V. utricularis and V. ventricosa because the vacuole of these giant algal cells can be perfused under (clamped or variable) turgescent conditions (Wang et al . 1997a;Ryser et al . 1999). Integrated microelectrodes allow measurement of the individual membrane conductances by injection of charge pulses and subsequent measurement of the membrane voltage relaxations. These studies showed (Wang et al . 1997a;Ryser et al . 1999) that the fast voltage relaxation originates from the plasmalemma, whereas the ...
... use the vacuole of these giant algal cells can be perfused under (clamped or variable) turgescent conditions (Wang et al . 1997a;Ryser et al . 1999). Integrated microelectrodes allow measurement of the individual membrane conductances by injection of charge pulses and subsequent measurement of the membrane voltage relaxations. These studies showed (Wang et al . 1997a;Ryser et al . 1999) that the fast voltage relaxation originates from the plasmalemma, whereas the slow one arises from the tonoplast. The analysis showed further that the specific conductance of the tonoplast is low (even lower than the specific conductance of the plasmalemma) and that the vacuolar membrane must be many-folded and/or con ...
... Turgor pressure measurements in combination with chargepulse relaxation studies (Zimmermann, Büchner & Benz 1982;Benz & Zimmermann 1983;Wang, Zimmermann & Benz 1994) were performed by using the perfusion assembly described in detail by Wang et al . (1997a) and Ryser et al . (1999). Even though perfusion experiments were not performed in the work reported herein, the integrated pressure regulator valve of this apparatus could be used to change turgor pressure and clamp it at non-equilibrium values (for more details, see Wang et al . 1997a). This allows the effects of turgor pressure and tr ...
Article
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.
... In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl − conductance of the plasmalemma of the turgescent "mother cells" and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, 1989;Wang et al., 1991Wang et al., , 1997aBisson & Kirst, 1995). However, the interpretation of the electrophysiological (and pressure) data obtained on giant algae is not always straightforward because of the placement of the microelectrodes into the vacuole that occupies more than 97% of the cell volume. ...
... The individual electrical properties of the two membranes could be measured very recently by Wang et al. (1997a) and Ryser et al. (1999) in turgescent giant cells of Valonia utricularis and Ventricaria ventricosa, respectively, by using a vacuolar perfusion technique with integrated microelectrodes for charge pulse relaxation experiments. Separate permeabilization of the tonoplast and the plasmalemma and subsequently performed electrophysiological measurements were achieved by addition of the pore-forming antibiotic nystatin to the vacuolar or external perfusion solutions. ...
... Separation of the electrical properties of the individual membranes in perfused cells by addition of nystatin to the bath or to the perfusion medium (Wang et al., 1997a) showed (C. Ryser and U. Zimmermann, unpublished data) that the potential of the plasmalemma of V. utricularis was in the order of about −25 to −30 mV at a turgor pressure of about 0.1 MPa. ...
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The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant ``mother cells'' (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123–134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl− channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K+ channel (VKC1, Valonia K +Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl− media, VAC1 was most frequently observed and had a single channel conductance of 36 ± 7 pS (n= 4) in the outside-out and 33 ± 5 pS (n= 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 ± 4 mV (n= 4, outside-out) and 9 ± 7 mV (n= 10, cell-attached) close to the Nernst potential of Cl− and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl− media (bath/pipette; E Cl− −20 ± 7 mV (n= 4); Nernst potential −28 mV). Consistent with a selectivity for Cl−, VAC1 was inhibited by 100 μM DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl− conditions yielded a midpoint potential of −12 ± 5 mV (n= 4, outside-out) and −3 ± 6 mV (n= 9, cell-attached) and corresponding apparent minimum gating charges of 15 ± 3 (n= 4) and 18 ± 5 (n= 9). The midpoint potential shifted to more negative values in the presence of a Cl− gradient. VAC2 was activated by voltages more negative than E Cl− and was always observed together with VAC1, but less frequently. It showed a ``flickering'' gating. The single channel conductance was 99 ± 10 pS (n= 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 ± 5 pS (n= 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 ± 27 pS (n= 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl− conductance of the plasmalemma of the turgescent ``mother cells'' and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation.
... Conversely, hyposmotic challenges are overcome by increased passive KCl efflux due to an increase of the volumetric elastic modulus of the cell wall with pressure (Zimmermann & Steudle, 1978). Electrophysiological studies have given evidence (Wang et al., 1997;Ryser et al., 1999) that the tonoplast must be multi-folded, leading to a sponge-like organization of the 5 lm thick cytoplasmic layer situated between the large central vacuole (occupying more than 95% of the cell volume) and the cell wall. ...
... The oil-filled cell pressure probe was also used for measurements on cells of V. utricularis. However, in most experiments, the pressure-controlled perfusion assembly with integrated potentialmeasuring setup was used (for details and a schematic representation of the setup, see Wang et al., 1997). Briefly, the perfusion setup allows independent control of perfusion flow rate and turgor pressure and allows the exchange of perfusion solutions without a pressure shock. ...
... After about 60 min, perfusion was stopped. Previous experiments had shown (Wang et al., 1997) that this perfusion time was sufficient to completely exchange the vacuolar sap (50 to 160 ll) by AVS. Then, the perfused cell was subjected to a perfusion regime outlined below, in order to test the effect of addition of sucrose or of isosmotic replacement of KCl by sucrose. ...
Article
The pressure response of (plant) cells to osmotic challenges depends on the reflection coefficient, sigma, of osmotically active solutes; it is less than predicted by the van't Hoff equation if sigma < 1. In Valonia utricularis, sigma is significantly reduced by internal (and, to a lesser extent, by external) unstirred layers, protecting the cytoplasm against vacuolar osmotic fluctuations. As shown by scanning and transmission electron microscopy, diffusion-restricted spaces are formed by innumerable small vacuoles that are interconnected with each other and with the central vacuole. They are embedded in networks of cytoplasmic strands connecting and encircling the organelles. Unstirred layers are also created in the central vacuole by an extensive network of acid mucopolysaccharide filaments (visualized by alcian blue staining). Mucopolysaccharides apparently also affect steady-state turgor by reducing the water activity. When the effective vacuolar osmotic pressure was adjusted to that of the bath by perfusion with an artificial vacuolar sap (AVS), an "offset turgor pressure" of 17 +/- 5 kPa was recorded. Consistent with the ultrastructural data, sigma values less than unity were calculated from the pressure response upon vacuolar addition of KCl or sucrose by perfusion (sigma(iKCl) = 0.63 +/- 0.13; sigma(isuc) = 0.58 +/- 0.17). Dilution of AVS yielded slightly higher sigma(iKCl) values (0.73 +/- 0.35). External addition to the artificial sea water (ASW) indicated that sigma(e) > sigma(i) for these osmotica. However, even in this case, sigma(esuc) (0.86 +/- 0.09) and sigma(ePEG) (0.58 +/- 0.08) were significantly less than sigma(eNaCl) (0.94 +/- 0.05) and sigma(eKCl) (0.91 +/- 0.13), presumably due to unstirred layers within the 4 micro m thick cell wall. Consistent with the low sigma values, a partial replacement of NaCl by osmotically equivalent amounts of sucrose (ASW(suc)), PEG and dextran, respectively, as well as replacement of Cl(-) by the large anion MES(-) induced an 'anomalous' hyposmotic turgor pressure response followed by the usual backregulation of pressure. After a 2-day preincubation in ASW(suc), significantly lower sigma(e) values were obtained both hyperosmotically (sigma(eNaCl) = 0.78 +/- 0.14; sigma(esuc) = 0.72 +/- 0.15) and hyposmotically (sigma(eNaCl) = 0.70 +/- 0.17; sigma(esuc) = 0.63 +/- 0.09), probably due to long-term effects on membrane structure to be elucidated yet. The freshwater alga Chara corallina lacked these apparently closely related structural and biophysical features of Valonia.
... Mobile and gating charges are a common feature of biological membranes. Mobile charges within the plasma membrane are involved in ion carrier systems ( Benz and Zimmermann, 1983; Wang et al., 1997a) and can play a role in intercellular communication (Turin et al., 1991 ). Their concentration in biological membranes can be markedly increased because of the partition of organic lipid-soluble ions, such as dipicrylamine (DPA ) and derivatives of tetraphenylborate (TPhB ), etc., into the membranes (Benz et al., 1976; Läuger et al., 1981; Benz and Nonner, 1981; Dilger and Benz, 1985). ...
... Although lipophilic anions have been widely used as experimental tools in studies of living cells and cell organelles (Oberhauser and Fernandez, 1995; Klodos et al., 1995; Lu et al., 1995; Bühler et al., 1991; Turin et al., 1991; Lichtenberg et al., 1988; Demura et al., 1985), relatively little is known about the transport of lipophilic ions in biological membranes. This is because the microelectrode methods traditionally used in electrophysiological studies (Zimmermann et al., 1982; Wang et al., 1994b Wang et al., , 1997a) are restricted to cells of sufficiently large size, and to frequencies below 100 kHz because of the high impedance of microelectrodes. An alternative approach to the study of the interaction between lipophilic ions and biological membranes is the single-cell electrorotation technique (Arnold and Zimmermann, 1982). ...
Article
The electrical properties of biological and artificial membranes were studied in the presence of a number of negatively charged tungsten carbonyl complexes, such as [W(CO)5(CN)]−, [W(CO)5(NCS)]−, [W2(CO)10(CN)]−, and [W(CO)5(SCH2C6H5)]−, using the single-cell electrorotation and the charge-pulse relaxation techniques. Most of the negatively charged tungsten complexes were able to introduce mobile charges into the membranes, as judged from electrorotation spectra and relaxation experiments. This means that the tungsten derivatives act as lipophilic anions. They greatly contributed to the polarizability of the membranes and led to a marked dielectric dispersion (frequency dependence of the membrane capacitance and conductance). The increment and characteristic frequency of the dispersion reflect the structure, environment, and mobility of the charged probe molecule in electrorotation experiments with biological membranes. The partition coefficients and the translocation rate constants derived from the electrorotation spectra of cells agreed well with the corresponding data obtained from charge-pulse experiments on artificial lipid bilayers.
... Mobile and gating charges are a common feature of biological membranes. Mobile charges within the plasma membrane are involved in ion carrier systems ( Benz and Zimmermann, 1983; Wang et al., 1997a) and can play a role in intercellular communication (Turin et al., 1991 ). Their concentration in biological membranes can be markedly increased because of the partition of organic lipid-soluble ions, such as dipicrylamine (DPA ) and derivatives of tetraphenylborate (TPhB ), etc., into the membranes (Benz et al., 1976; Läuger et al., 1981; Benz and Nonner, 1981; Dilger and Benz, 1985). ...
... Although lipophilic anions have been widely used as experimental tools in studies of living cells and cell organelles (Oberhauser and Fernandez, 1995; Klodos et al., 1995; Lu et al., 1995; Bühler et al., 1991; Turin et al., 1991; Lichtenberg et al., 1988; Demura et al., 1985), relatively little is known about the transport of lipophilic ions in biological membranes. This is because the microelectrode methods traditionally used in electrophysiological studies (Zimmermann et al., 1982; Wang et al., 1994b Wang et al., , 1997a) are restricted to cells of sufficiently large size, and to frequencies below 100 kHz because of the high impedance of microelectrodes. An alternative approach to the study of the interaction between lipophilic ions and biological membranes is the single-cell electrorotation technique (Arnold and Zimmermann, 1982). ...
... Thus, the data reflect the electrical response of both the tonoplast and the plasmalemma, which are arranged in series. Wang et al. (1997a) and Ryser et al. (1999) were recently able to separate the individual electrical properties of both membranes in the marine giant algae Valonia utricularis and Ventricaria ventricosa. By perfusing the vacuole or the bath with the pore-forming antibiotic nystatin, these authors could selectively short-circuit the tonoplast and the plasmalemma, respectively, thus allowing the measurements of the electrophysiological properties of the individual membranes of turgescent cells. ...
... From the relaxation time of the membrane voltage and the surface area (estimated from the diameter of the spherically shaped protoplasts) a specific resistance of 1.09 ± 0.52 W m 2 (n = 20) and a specific capacity of 0.85 ± 0.21 lF cm À2 (n = 20) were calculated. A similar value for the specific capacity of the plasmalemma of the 'mother cells' was determined by charge pulse-relaxation experiments (0.77 ± 0.16 lF cm À2 ; n = 10; Wang et al., 1997a). The agreement of the values was taken as evidence that the dense lipid vesicle layer did not interfere with clamp experiments in the whole-cell configuration. ...
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The electrical properties of protoplasts of the turgor pressure-regulating giant marine alga Valonia utricularis were investigated by using the patch-clamp technique. In the whole-cell configuration, large inward currents were elicited by negative-going voltage pulses. The time-dependent component was predominantly carried by Cl-, as revealed by 'tail current' analysis. When experiments were performed on protoplasts directly after mechanical release from the 'mother cell', small outward currents were additionally observed at membrane voltages more positive than ECl-. These outward currents disappeared to a large extent after treatment of the protoplasts with a mixture of cell wall-degrading enzymes. Plots of the chord conductance versus the clamped membrane voltage revealed that enzymatic treatment affected the gating properties. By fitting Boltzmann distributions to the data, a midpoint potential of + 5 +/- 5 mV (n = 7) was obtained in symmetrical Cl- solutions for mechanically released protoplasts. In contrast, protoplasts treated additionally with enzymes exhibited a midpoint potential of -13 +/- 5 mV (n = 8). By varying the external and internal Cl- concentration, gating was also shown to depend on the Cl- gradient across the plasmalemma both in enzymatically treated and untreated protoplasts. Plotting of the midpoint potential against the Nernst potential of Cl- rendered a slope less than 1 (0.70 and 0.64, respectively) indicating that gating did not strictly depend on the electrochemical Cl- gradient. The voltage- and Cl--dependence as well as inhibition experiments with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) suggested that the Cl- conductance of the membrane is dominated by the Valonia Anion Channel 1 (VAC1) described by Heidecker, M., Wegner, L.H., Zimmermann, U. 1999: A patch-clamp study of ion channels in proto-plasts prepared from the marine alga Valonia utricularis. J. Membrane Biol. 172:235-247. The relevance of the findings for membrane potential control and turgor regulation in V. utricularis as well as the general implications of the data for electrophysiological work on protoplasts (that are usually obtained by enzymatic digestion of plant tissue) are discussed.
... The vacuole of V. ventricosa forms a complex interface with the alveolate cytoplasm. Theoretical calculations suggest its membrane surface is " multifolded " by a factor of nine (Wang et al. 1997, Ryser et al. 1999). The vacuole is unusual also in that it contains sulphated polysaccharide mucilages. ...
... Vacuolar accumulation of xenobiotics in plants and fungi has recently been attributed to tonoplast-specific ABC transporters (Rea 1999 ). The lack of characteristic vacuolar transporter systems , the accumulation of mucilage, the presence of a highly unusual K pump (Hastings and Gutknecht 1974), and the amplified surface area of the membrane (Wang et al. 1997, Ryser et al. 1999) all make sense if the inner membrane of V. ventricosa is indeed not a true tonoplast. We will argue that it is the inner face of a polarised communal membrane. ...
Article
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Ventricaria ventricosa and its relatives have intrigued cell biologists and electrophysiologists for over a hundred years. Historically, electrophysiologists have regarded V. ventricosa as a large single plant cell with unusual characteristics including a small and positive vacuole-to-outside membrane potential difference. However, V. ventricosa has a coenocytic construction, with an alveolate cytoplasm interpenetrated by a complex vacuole containing sulphated polysaccharides. We present a theory relating the coenocytic structure to the unusual electrophysiology of V. ventricosa. The alveolate cytoplasm of V. ventricosa consists of a collective of uninucleate cytoplasmic domains interconnected by fine cytoplasmic strands containing microtubules. The cytoplasm is capable of disassociating into single cytoplasmic domains or aggregations of domains that can regenerate new coenocytes. The cytoplasmic domains are enclosed by outer (apical) and inner (basolateral) faces of a communal membrane with polarised K(+)-transporting functions, stabilised by microtubules and resembling a tissue such as a polarised epithelium. There is evidence for membrane trafficking through endocytosis and exocytosis and so "plasmalemma" and "tonoplast" do not have fixed identities. Intra- and extracellular polysaccharide mucilage has effects on electrophysiology through reducing the activity of water and through ion exchange. The vacuole-to-outside potential difference, at which the cell membrane conductance is maximal, reverses its sign from positive under hypertonic conditions to negative under hypotonic conditions. The marked mirror symmetry of the characteristics of current as a function of voltage and conductance as a function of voltage is interpreted as a feature of the communal membrane with polarised K(+) transport. The complex inhomogeneous structure of the cytoplasm places in doubt previous measurements of cytoplasm-to-outside potential difference.
... It was suggested that the positive inside membrane potential of tonoplasts (+10 to +40 mV) in comparison to the negative potential of plasma membranes of intact plant cells of −120 mV (Higinbotham, Etherton & Foster, 1967) is causal for the resistance of the tonoplast against the AMP (Matic et al., 2005). Furthermore, it was reported that the tonoplast is multiply folded or shows an sponge-like structure, which leads to a 8-fold larger surface area than that of the plasma membrane (Ryser et al., 1999; Wang et al., 1997). The larger surface of the tonoplast could complicate the destruction of the central vacuole because more SN2 molecules are needed to form pores in all the layers of the tonoplast. ...
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Antimicrobial peptides (AMPs) are a diverse group of short, cationic peptides which are naturally occurring molecules in the first-line defense of most living organisms. They represent promising candidates for the treatment of pathogenic microorganisms. Snakin-2 (SN2) from tomato ( Solanum lycopersicum ) is stabilized through six intramolecular disulphide bridges; it shows broad-spectrum antimicrobial activity against bacteria and fungi, and it agglomerates single cells prior to killing. In this study, we further characterized SN2 by providing time-kill curves and corresponding growth inhibition analysis of model organisms, such as E. coli or B. subtilis . SN2 was produced recombinantly in E. coli with thioredoxin as fusion protein, which was removed after affinity purification by proteolytic digestion. Furthermore, the target specificity of SN2 was investigated by means of hemolysis and hemagglutination assays; its effect on plant cell membranes of isolated protoplasts was investigated by microscopy. SN2 shows a non-specific pore-forming effect in all tested membranes. We suggest that SN2 could be useful as a preservative agent to protect food, pharmaceuticals, or cosmetics from decomposition by microbes.
... Steudle & Zimmermann, 1971;Zimmermann & Steudle, 1974;Zimmermann, 1978Zimmermann, , 1989Wendler & Zimmermann, 1982, 1985Ortega et al., 1992;Murphy & Smith, 1998;Tomos & Leigh, 1999). The electrical properties of the tonoplast and the plasmalemma can also be determined separately if the probe is combined with vacuolar microelectrodes and perfusion assemblies (Wang et al., 1997;Ryser et al., 1999;Heidecker et al., 2003a,b;Mimietz et al., 2003). Measurements of the water relations parameters of tissue cells of higher plants were first reported by Hüsken et al. (1978) using a sophisticated modification of the probe. ...
Article
ContentsI. IntroductionII. Can water sustain negative pressures?III. Negative xylem pressures of several megapascals: fact or mystery?IV. The continuity of the xylem water columns: fact or hypothesis?V. The ‘Multi-Force’ or ‘Watergate’ TheoryVI. Conclusions AcknowledgementsReferencesAppendix 1Appendix 2SummaryThe Cohesion Theory considers plant xylem as a ‘vulnerable pipeline’ isolated from the osmotically connected tissue cells, phloem and mycorrhizas living in symbiosis with plant roots. It is believed that water is pulled exclusively by transpiration-induced negative pressure gradients of several megapascals through continuous water columns from the roots to the foliage. Water under such negative pressures is extremely unstable, particularly given the hydrophobicity of the inner xylem walls and sap composition (lipids, proteins, mucopolysaccharides, etc.) that prevents the development of stable negative pressures larger than about −1 MPa. However, many plant physiologists still view the Cohesion Theory as the absolute and universal truth because clever wording from the proponents of this theory has concealed the recent breakdown of the Scholander pressure bomb (and other indirect methods) as qualified tools for measuring negative pressures in transpiring plants. Here we show that the arguments of the proponents of the Cohesion Theory are completely misleading. We further present an enormous bulk of evidence supporting the view that – depending on the species and ecophysiological context – many other forces, additional to low tensions, can be involved in water ascent and that water can be lifted by a series of watergates (like ships in staircase locks).
... Turgor pressure was measured by impalement of the cells with a turgor pressure probe as described in detail elsewhere ( Zimmermann et al. 1969, Zimmermann 1978. For the determination of structural changes in response to osmotic pressure gradients under the light microscope, the vacuolar perfusion assembly described recently was used ( Wang et al. 1997a;Ryser et al. 1999;Heidecker et al. 2003a, b). Osmotic pressure gradients were established by manipulation of the osmotic pressure of the external medium and/or of the vacuolar sap. ...
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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.
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Corticomuscular coherence (CMC) is an index utilized to indicate coherence between brain motor cortex and associated body muscles, conventionally. As an index of functional connections between the cortex and muscles, CMC research is the focus of neurophysiology in recent years. Although CMC has been extensively studied in healthy subjects and sports disorders, the purpose of its applications is still ambiguous, and the magnitude of CMC varies among individuals. Here, we aim to investigate factors that modulate the variation of CMC amplitude and compare significant CMC between these factors to find a well-developed research prospect. In the present review, we discuss the mechanism of CMC and propose a general definition of CMC. Factors affecting CMC are also summarized as follows: experimental design, band frequencies and force levels, age correlation, and difference between healthy controls and patients. In addition, we provide a detailed overview of the current CMC applications for various motor disorders. Further recognition of the factors affecting CMC amplitude can clarify the physiological mechanism and is beneficial to the implementation of CMC clinical methods.
Article
Various studies on mangroves and other tall trees rooting in high-salinity water have given compelling evidence that tension is not the only factor in water lifting as thought by plant physiologists. A characteristic feature of these trees is that the tissue cells, the apoplastic space and, in particular, the lumen and the inner walls of many xylem vessels of the roots, the trunk and the branches (up to the apex) contain mucilage. Data on single marine giant algal cells are presented that show that mucilage reduces the chemical activity of water. Longitudinal gradients in the chemical activity of water and interfacial forces are presumably the dominant forces for water lifting. In order to save water on its tortuous pathway to the uppermost foliage trees apparently use different strategies (as revealed by 1H-NMR imaging), e.g. reduction of the conducting xylem area in the branches at intermediate height by mucilage or interruption of the xylem water columns by gas-filled segments and water lifting through mucilage networks and surface films. Pressure bomb experiments over the entire height of the trees revealed clearly that balancing pressure values cannot be taken as a measure for xylem tension. Such values can be used generally for an estimation of the chemical potential of water in the xylem of leafy twigs under atmospheric pressure, µw,h =0, provided that a species-specific "threshold pressure" (depending on wood density, elastic forces of the tissue, hydraulic coupling between xylem and tissue cells, intercellular spaces, cellular osmotic pressure etc.) is subtracted from the balancing pressure values. Transpiration increases the "threshold pressure" considerably and in an unpredictable way. Thus, as shown here, predawn balancing pressure data taken at various heights can yield information about the height dependence of µw (measured at h=0) under field conditions, particularly when the water content of the xylem is simultaneously determined in a reliable manner (e.g. by the compression/decompression method in combination with centrifugation).
Article
Die kombinierte Mikroperfusions-/Ladungspulstechnik an einzelligen marinen Riesenalgen ermöglicht die getrennte Darstellung der elektrischen Eigenschaften von Plasmalemma und Tonoplast durch gezielte Manipulation des vakuolären und externen Mediums. Dabei kann der für die Physiologie der Zellen wichtige hydrostatische Innendruck (Turgor) ständig kontrolliert werden. Die Applikation eines Ladungspulses resultierte bei Valonia utricularis und Ventricaria ventricosa in einer biphasischen Relaxation des Gesamt-membranpotentials, die durch die Summe zweier Exponentialfunktionen beschrieben werden konnte. Die Zeitkonstanten dieser beiden Relaxationen lagen dabei im Bereich von 0.1 ms und 1 ms (V. utricularis), bzw. 0.1 ms und 10 ms (V. ventricosa). Addition von Nystatin (einem membranimpermeablen und porenbildenden Antibiotikum) zu der vakuolären Perfusionslösung führte bei beiden Spezies zu einem Verschwinden der langsamen Relaxation des Spektrums, während über das Badmedium (extern) dotiertes Nystatin die Zeitkonstante der schnellen Komponente dramatisch verringerte. Die jeweils andere Relaxation blieb dabei unbeeinflusst. Folglich muss die schnelle Relaxation den RC-Eigenschaften des Plasmalemmas und die langsame den Eigenschaften des Tonoplasten zugeordnet werden. Dies ist ein klarer Beweis für das sog. "Zwei-Membranen Modell". In Übereinstimmung damit beeinflussten externe Ionentauschexperimente sowie externe Zugabe von Kanal/Carrier-Inhibitoren wie TEA (Tetraethylammonium), Ba2+ und DIDS (4,4'-Diisothiocyanatostilben-2,2'-Disulfonsäure), nur die schnelle Relaxation des Ladungspulsspektrums, nicht aber die langsame. Dagegen hatte die Zugabe dieser Inhibitoren zu der vakuolären Perfusionslösung keinen signifikanten Einfluss auf das Relaxationspektrum der marinen Algen. Bei der Berechnung der passiven Membranparameter fiel eine ungewöhnlich hohe flächenspezifische Kapazität des Tonoplasten auf, die nach elektronenmikroskopischen Untersuchungen mit einer etwa 9-fachen Oberflächenvergrößerung erklärt werden konnte. Diese resultierte aus tubulären Ausstülpungen des Tonoplasten, die in das Zytosol hineinreichen. Es konnte darüber hinaus gezeigt werden, dass - entgegen der Lehrmeinung - der Widerstand des Tonoplasten mariner Algen hoch ist (0.3 bis 1.1 Ohm m²) und somit Werte aufweist, die mit Messungen an Vakuolen höherer Pflanzen vergleichbar sind. Darüber hinaus ergaben Nystatin-Experimente, dass das Zytoplasma von V. ventricosa stark negativ geladen ist (bis zu -70 mV). Neben vielen Gemeinsamkeiten existieren auch klare Unterschiede in der Physiologie dieser Algen. Während bei V. ventricosa die Plasmalemmaleitfähigkeit durch Kalium dominiert wurde, war das Plasmalemma von V. utricularis deutlich permeabler für Chlorid als für Kalium. Variation des externen pH-Wertes wirkte sich nur bei V. utricularis, nicht aber bei V. ventricosa, im Relaxationsspektrum in einer drastischen Erhöhung der schnellen Zeitkonstante aus. Für die Analyse turgorgesteuerter Membrantransportprozesse an marinen Algen bedeuten diese Arbeiten einen methodischen Durchbruch, so dass die vollständige Aufklärung der biophysikalischen Prozesse, die mit der Turgorregulation assoziiert werden, unmittelbar bevorsteht. The intergrated perfusion/charge-pulse technique allows the separated determination of the electrical properties of the tonoplast and the plasmalemma of giant-celled marine algae with exact manipulation of the vacuolar and external solution. The hydrostatic pressure in the cells (the so-called turgor-pressure), which is one of the most important parameters for the physiology of the cell, can easily be regulated during the experiments. The charge-pulse relaxation spectrum of Valonia utricularis and Ventricaria ventricosa showed a biphasic shape that could be described by the sum of two exponentials decays. The time constants of the two relaxations were calculated to be around 0.1 ms and 1 ms (V. utricularis), and 0.1 ms and 10 ms (V. ventricosa), respectively. Addition of nystatin (a membrane-impermeable and pore-forming antibiotic) to the the vacuolar perfusion-solution of both species resulted in the disappearance of the slow exponential of the spectrum, wheras the presence of nystatin in the bath decreased dramatically the time constant of the fast component. The other relaxation remained unaffected. Consequently, the fast relaxation process must be assigned to the RC-properties of the plasmalemma and the slow one to those of the tonoplast. This is a clear proof for the so-called "two-membrane model". Consistent with this, external variation of the major ions as well as external addition of channel/carrier inhibitors like TEA (tetraethyammonium), Ba2+ and DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) affected only the fast relaxation, but not the slow one. In contrast, addition of these blockers to the vacuolar perfusion solution showed no measurable effect on the charge-pulse relaxation spectrum of the marine algae. Calculating the individual passive electrical parameters of the membranes, an unusually high area-specific capacitance of the tonoplast was detected. According to electronmicroscopic studies this could be explained by a approximately 9-fold enlargement of the tonoplast surface showing tubular "fingers" invading the cytoplasm. Above that it could be demonstrated that the area-specific resistance of the tonoplast of V. utricularis and V. ventricosa is high (0.3 to 1.1 ohm m²) and thus comparable with data observed on vacuoles of higher plants. Furthermore, the nystatin-studies indicated that the cytoplasm is negatively charged (up to -70 mV). Although they have a lot in common, the two species exhibit some clear differences in their physiology. With V. ventricosa, the conductance of the plasmalemma was dominated by K+, whereas the plasmalemma of V. utricularis was more permeable to Cl- than to K+. Variation of the external pH had a significant effect on the charge-pulse relaxation spectrum only of V. utricularis but not of V. ventricosa. The presented studies can be seen as a technical breakthrough in the analysis of turgor-regulated transport processes of marine algae. A complete elucidation of the biophysical properties which contribute to the turgorregulation can now be achieved.
Article
The charge-pulse relaxation spectrum of nonperfused and perfused (turgescent) cells of the giant marine alga Ventricaria ventricosa showed two main exponential decays with time constants of approximately 0.1 msec and 10 msec, respectively, when the cells were bathed in artificial sea water (pH 8). Variation of the external pH did not change the relaxation pattern (in contrast to other giant marine algae). Addition of nystatin (a membrane-impermeable and pore-forming antibiotic) to the vacuolar perfusion solution resulted in the disappearance of the slow exponential, whereas external nystatin decreased dramatically the time constant of the fast one. This indicated (by analogy to corresponding experiments with Valonia utricularis, J. Wang, I. Spiess, C. Ryser, U. Zimmermann, J. Membrane Biol. 157: 311-321, 1997) that the fast relaxation must be assigned to the RC-properties of the plasmalemma and the slow one to those of the tonoplast. Consistent with this, external variation of [K+]o or of [Cl-]o as well as external addition of K+- or Cl--channel/carrier inhibitors (TEA, Ba2+, DIDS) affected only the fast relaxation, but not the slow one. In contrast, addition of these inhibitors to the vacuolar perfusion solution had no measurable effect on the charge-pulse relaxation spectrum. The analysis of the data in terms of the "two membrane model" showed that K+- and (to a smaller extent) Cl--conducting elements dominated the plasmalemma conductance. The analysis of the charge-pulse relaxation spectra also yielded the following area-specific data for the capacitance and the conductance for the plasmalemma and tonoplast (by assuming that both membranes have a planar surface): (plasmalemma) Cp = 0.82 * 10(-2) F m-2, Rp = 1.69 * 10(-2) Omega m2, Gp = 5.9 * 10(4) mS m-2, (tonoplast) Ct = 7. 1 * 10(-2) F m-2, Rt = 14.9 * 10(-2) Omega m2 and Gt = 0.67 * 10(4) mS m-2. The electrical data for the tonoplast show that (in contrast to the literature) the area-specific membrane resistance of the tonoplast of these marine giant algal cells is apparently very high as reported already for V. utricularis. The exceptionally high value of the area-specific capacitance could be explained - among other interpretations - by assuming a 9-fold enlargement of the tonoplast surface. The hypothesis of a multifolded tonoplast was supported by transmission electronmicroscopy of cells fixed under maintenance of turgor pressure and of the electrical parameters of the membranes. This finding indicates that the tonoplast of this species exhibited a sponge-like appearance. Taking this result into account, it can be easily shown that the tonoplast exhibits a high-resistance (1.1 Omega m2). Vacuolar membrane potential measurements (performed in parallel with charge-pulse relaxation studies) showed that the potential difference across the plasmalemma was mainly controlled by the external K+-concentration which suggested that the resting membrane potential of the plasmalemma is largely a K+-diffusion potential. After permeabilization of the tonoplast with nystatin the potential of the intact membrane barrier dropped from about slightly negative or positive (-5.1 to +18 mV, n = 13) to negative values (-15 up to -68 mV; n = 8). This indicated that the cytoplasm of V. ventricosa was apparently negatively charged relative to the external medium. Permeabilization of the plasmalemma by addition of external nystatin resulted generally in an increase in the potential to slightly more positive values (-0.8 to +4.3 mV; n = 5), indicating that the vacuole is positively charged relative to the cytoplasm. These findings apparently end the long-term debate about the electrical properties of V. ventricosa. The results presented here support the findings of Davis (Plant Physiol. 67: 825-831, 1981), but are contrary to the results of Lainson and Field (J. Membrane Biol. 29: 81-94, 1976).
Article
Exposure of the giant marine alga Valonia utricularis to acute hypo-osmotic shocks induces a transient increase in turgor pressure and subsequent back-regulation. Separate recording of the electrical properties of tonoplast and plasmalemma together with turgor pressure was performed by using a vacuolar perfusion assembly. Hypo-osmotic turgor pressure regulation was inhibited by external addition of 300 μM of the membrane-permeable ion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). In the presence of 100 μM NPPB, regulation could only be inhibited by simultaneous external addition of 200 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), a membrane-impermeable inhibitor of Cl− transport. At concentrations of about 100 μM, NPPB seems to selectively inhibit Cl− transporters in the tonoplast and K+ transporters in the plasmalemma, whereas 300 μM NPPB inhibits K+ and Cl− transporters in both membranes. Evidence was achieved by measuring the tonoplast and plasmalemma conductances (G t and G p) in low-Cl− and K+-free artificial seawater. Inhibition of turgor pressure regulation by 300 μM NPPB was accompanied by about 85% reduction of G t and G p. Vacuolar addition of sulfate, an inhibitor of tonoplast Cl− transporters, together with external addition of DIDS and Ba2+ (an inhibitor of K+ transporters) also strongly reduced G p and G t but did not affect hypo-osmotic turgor pressure regulation. These and many other findings suggest that KCl efflux partly occurs via electrically silent transport systems. Candidates are vacuolar entities that are disconnected from the huge and many-folded central vacuole or that become disconnected upon disproportionate swelling of originally interconnected vacuolar entities upon acute hypo-osmotic challenge.
Article
Full-text available
Charge pulse relaxation studies were performed on cells of the giant marine alga Valonia utricularis. Two exponential voltage relaxations were recorded as found previously (Benz, R., and U. Zimmermann. 1983. Biophys. J. 43:13-26.). The parameters of the two exponential voltage decays were studied as a function of the chloride concentration in the artificial sea water. Replacement of external chloride by 2(N-morpholino)ethanesulfonate (Mes(-)) had a dramatic influence on the four relaxation parameters. This chloride dependence could not be satisfactorily explained by the simplified model used earlier. Accordingly, additional reaction steps had to be included in the model. Only two relaxation processes could be resolved under all experimental conditions. This means that the heterogeneous complexation reactions, k(R) (association), and k(D) (dissociation) were too fast to be resolved. Therefore a carrier model with equilibrium heterogeneous surface reactions was used to fit the experimental results. From the charge pulse data at different chloride concentrations the translocation rate constants of the free and complexed carriers, k(S) and k(AS), through the membrane, as well as the total surface concentration of carrier systems, N(0), could be evaluated. The results described here indicate that the cell membrane of Valonia utricularis contains an electrogenic transport system for chloride.
Article
Full-text available
The electrical membrane resistance ρ0 of the marine alga Valonia utricularis shows a marked maximum in dependence on the turgor pressure. The critical pressure, Pc, at which the maximum occurs, as well as its absolute value, ρ0max, are strongly volume-dependent. Both Pc and ρ0max, increase with decreasing cell volume. It seems likely, that these relationships reflect the elastic properties of the cell wall, because the volumetric elastic modulus, ε, is also volume-dependent, increasing hyperbolically with cell volume. Both Pc and ρ0max can be affected by external application of indole-3-acetic acid at concentrations of 2·10−7m to 2 ·10−5m. The critical pressure is shifted by 1.2 to 6 bars toward higher pressures and the maximum membrane resistance increased up to 5.6-fold. During the course of the experiments (up to 4 hours), however, IAA had no effect on the volumetric elastic modulus, ε.The maximum in membrane resistance is discussed in terms of a pressure-dependent change of potassium fluxes. The volume dependence of Pc and ρ0max suggests that not only turgor pressure but also ε must be considered as a regulating parameter during turgor pressure regulation. On this basis a hypothesis is presented for the transformation of both, a pressure signal and of changes in the elastic properties of the cell wall into alterations of ion fluxes. It is assumed that the combined effects of tension and compression of the membranes as well as the interaction between membrane and cell wall opposingly change the number of transport sites for K+ providing a turgor-sensing mechanism that regulates ion fluxes. The IAA effects demonstrated are consistent with this view, suggesting that the basic mechanisms for turgor pressure regulation and growth regulation are similar.Any relation connecting growth rate with turgor pressure should be governed by two parameters, i.e. by a yielding pressure, at which cell growth starts, and by the critical pressure, at which it ceases again.
Chapter
The giant celled marine alga, Valonia macrophysa, maintains a 1.5 atm turgor pressure by holding the osmolarity of its intracellular fluid about 65 mosM above the osmolarity of the external sea water. This difference is maintained approximately constant over a wide range of sea water salinities. The osmotically active species in the vacuolar sap are the inorganic ions K+, Na+, and Cl- whose concentrations are, in mM: 480, 150, and 630, respectively. Our earlier observations on internally perfused cells of both V. ventricosa and V. macrophysa showed that inward K+ transport is stimulated by abolishing the normal turgor pressure of 1.5 atm (GUTKNECHT, 1968, and unpublished observation). The observations suggest that a negative feedback loop is used to regulate the turgor pressure. An increase in the turgor pressure is sensed by the cell and this leads to a reduction of K+ transport. An important part of the feed back loop is the pressure sensitive transducer, which links the turgor pressure (a physical parameter) to the K+ pump (a biochemical process). In this investigation we further studied the pressure transducer.
Article
It is the aim of this Chapter to set out and to classify the equations that seem most useful to those who work on transport across biological membranes. So that they may be the more confidently used—or rejected—the equations will be accompanied by details of the assumptions upon which they are based. The intention is to be of service; the licence is claimed, which is allowed to some servants, to pass occasional moral judgements.
Article
Simultaneous measurements of the separate plasmalemma and tonoplast capacitance and conductance (as a function of frequency) are described. For frequencies > 1 Hz, the capacitance of both membranes increased with decreasing frequency. Below 1 Hz, two distinctly different types of behaviour were observed: either the membrane capacitances continued to increase, reaching values of 0.5 F/m² (50 µF/cm²) at 0.07 Hz, or the membranes became inductive at very low frequencies. At frequencies > 10 Hz, the tonoplast capacitance was 1.5-3 times higher than that of the plasmalemma and increased more rapidly with decreasing frequency. Notwithstanding this, in each case the frequency dependence of the capacitance determined from vacuolar measurements (i.e. tonoplast, cytoplasm and plasmalemma in series) was nearly the same as that of the plasmalemma alone. The conductance of the tonoplast at all frequencies was 4-10 times higher than that of the plasmalemma.
Article
The capacitance and conductance of the plasmalemma and tonoplast of Chara were measured simultaneously in space-clamped cells. At a frequency of 5 Hz the capacitance and conductance of the tonoplast were 60 ± 5 mF m−2 (i. e. 6.0 μ F cm−2) and 6.5 ± 0.6 S m−2 respectively. These values were respectively 2.9 ± 0.3 and 3.7 ± 0.4 times greater than those of the plasmalemma. It is shown that any leakage of current around the cytoplasmic electrode will not drastically affect the calculated area-specific values of the tonoplast parameters under the experimental conditions used, providing that the cytoplasm possesses a reasonable longitudinal conductivity. An examination of the relative measured impedances of the plasmalemma and tonoplast supports this conclusion.
Article
This chapter discusses the electrophysiology of giant algal cells. The giant algae provide a unique system suitable for the study of a single plasma membrane in vivo. The methods described in this chapter are biased toward freshwater giant algae. Most of these techniques can be extended to giant-celled marine algae. The presence and origin of the vesicles are interesting in themselves, and the system might provide some data on vacuole formation. Despite the fact that the structure of the fragments is more complex than originally expected, they still constitute a convenient system for the study of the plasmalemma. The study of giant algae spans several disciplines, which makes it a more interesting but also a more demanding field of research. Knowledge of computing and electronics will be a great advantage. Some of the techniques defy detailed scientific description, such as making the setup noise-free or performing cytoplasmic perfusion successfully.
Article
Taking advantage of vacuolar perfusion, concentrations of K+, Cl–, and H+ in the vacuole ofNitella pulchella were changed in a wide range. Both the potential difference (E vo ) and specific resistance (R vo ) between the vacuole and the external medium were scarcely affected by K+ in the vacuole, while they responded sensitively to K+ in the external medium. E vo also responded to Cl– in both internal (vacuolar) and external medium. However, the sign of the response was opposite to that expected from the constant field assumption.R vo was almost independent of Cl–-concentrations of both internal and external medium.The response ofE vo to internal pH was similar to that of external pH. Between pH's 4 and 8,E vo changed by about 10 mV for one unit change of both external and internal pH.E vo responded very sensitively to internal pH in the strongly acid region (30–60 mV at pH 3–4) irrespective of the concentration of KCl in the vacuole. In the alkaline region, however,E vo responded to vacuolar pH only when the KCl concentration in the vacuole was low (0.1 mM).R vo increased significantly when the vacuolar pH was lowered to 4 or 3.Increase in tonicity of the vacuolar medium to twice normal caused no significant change in bothE vo andR vo , while it raised the threshold for excitation.Even when the chemical potential gradient between the internal and external medium was made zero by replacing the cell sap for the same solution used for the external medium, a significant amount ofE vo was observed. The short-circuit current which was first outward decreased to zero or changed its direction with time. Light did not affect the current. These facts show that the possibility for the contribution of an ion pump toE vo can be excluded.The results were discussed under the assumption that responses ofE vo andR vo to either internal or external ions reflect the passive property of either tonoplast or plasmalemma.
Article
Charge-pulse experiments were performed on giant algal cells ofValonia utricularis. If the tonoplast and plasmalemma in series are charged to voltages of the order of 10mV, the decay of the initial voltage with time can be described by the sum of two or three exponential relaxations. It is not possible to explain the exponential decay of the voltage by twoRC-circuits in series (e.g. tonoplast and plasmalemma), because this would lead to unreasonable values for the specific capacities of the two membranes. The exponential relaxations might be attributable to the transport of mobile negative charges present in both membranes, possibly as a part of a transport system. From an analysis of the experimental results in terms of the proposed model, the translocation rate constantk and the total surface densityN t of the mobile charges in one membrane could be evaluated. On averagek is of the order of 600 sec–1 andNt is about 510–12 mol cm–2 (average turgor pressure 1.6 bar). The transport properties of the mobile charges within the tonoplast and plasmalemma were studied as a function of different parameters such as external pH, glutardialdehyde, electrical breakdown and turgor pressure. When the pH is lowered from 8.2 to 4 or 5 the mobile charges disappear completely, presumably as the result of protonation of the anionic groups. This pH effect was found to be completely reversible. Electrical breakdown causes a reversible disappearance of the relaxation with the longer half-time due to the decrease in membrane resistance. The value of the electrical breakdown voltage determined by injection of charge pulses of 300-sec duration into the cell is pH-independent and therefore is consistent with the mobile charge model and with results previously reported (U. Zimmermann & R. Benz.J. Membrane Biol 53:33–43, 1980). Addition of glutardialdehyde leads also to a disappearance of the mobile charges probably due to cross-linkage. Increase of the turgor pressure from 0.05 bar to 2 bar results in an increase ink by a factor of 2 and inNt by about 30%. The increase ink is in reasonable agreement with that expected on the basis of the assumed compressibility of the membranes. The elastic compressive modulus perpendicular to the membrane plane calculated from the pressure dependence of the translocation rate constantk is in very good agreement with that derived from electrical breakdown experiments (14 and 13 bar, respectively). The presence of charges within the membranes as well as the compressibility of the membranes are discussed in terms of a possible turgor-pressure-sensing mechanism.
Article
Ionic composition and turgor pressure in the giant celled marine alga,Valonia macrophysa, were measured at environmental salinities ranging from 15 to 60 (11–44 atm). The steady-state turgor pressure, which is normally about 1.5 atm, changes only 2.5 atm in response to a 25 atm change in seawater osmotic pressure. Thus, turgor regulation is 90% effective. The salts important in turgor regulation are KCl and NaCl. During turgor regulation changes in intracellular KCl concentration account for 85% of the change in sap osmolality, and changes in NaCl account for the remaining 15%. Potassium is actively transported into the vacuole, whereas chloride appears to be passively transported as the counter ion. Thus, potassium transport, which we have shown previously to be sensitive to the turgor pressure, accounts for most of the turgor regulation at all salinities.
Article
Turgor pressure relaxation curves for individual plant cells represent an important source of information for the plant physiologist. However, the accurate interpretation of these curves is strongly dependent on the model chosen to describe the plant cell. If the compartmentation of the cell into vacuole and cytoplasm is taken into account, a theoretical analysis shows that pressure relaxation curves can be represented by the sum of two exponential functions. Givena priori assumptions about the exchange area of the tonoplast and its reflection coefficient, the hydraulic conductivities of the plasmalemma and tonoplast can be determined and the proportion of the total cell volume occupied by the cytoplasm is also obtained. Numerical solutions to the flow equations have shown that the biphasic nature of pressure relaxations is maintained even when a permeable tonoplast is assumed. Depending on the magnitude of the reflection coefficient and the permeability of the vacuolar membrane, large errors can arise in the determination of the hydraulic conductivity of the tonoplast. However, under certain conditions, even a highly permeable tonoplast may behave like a nonpermeable membrane during pressure relaxation.
Article
By injuring cells ofValonia ventricosa, one of two survival strategies — wound-healing and protoplast formation — is induced. The present study revealed that turgor pressure, as well as Ca2+ in bathing medium, is involved in the choice between these survival strategies. On the process of wound-healing, turgor pressure is recovered in the presence of both the wound plug, which closes the wound immediately after an injury, and the aggregation of protoplasm around the wound, which serves to protect the inflow of outer medium into the protoplasm layer and also to strengthen the wound plug. When the size of the wound is more than 150 m in diameter, the protoplasmic aggregate strengthen the wound plug incompletely and, as a result, wound-healing is unsuccessful. In this case, the ejection of vacuolar sap is repeated, due to partial restoration of turgor pressure. In each ejection, the wound plug is blown off, together with the aggregated protoplasm and, after several ejections are repeated, the cell is unable to heal the wound because of a lack of protoplasm around the wound. Continuous depression of turgor pressure, during the repeat of the unsuccessful wound-healing, induces disorganization of the protoplasm layer. Under these conditions, the centrifugal propagation of protoplasmic aggregation, followed by the protoplasts formation, takes place easily. Effects of turgor pressure and Ca2+ in the bathing medium upon the wound healing is discussed and the cytoplasmic behavior for survival of wounded cells is presented schematically.
Article
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.
Article
Nystatin and amphotericin B induce a cation-selective conductance when added to one side of a lipid bilayer membrane and an anion-selective conductance when added to both sides. The concentrations of antibiotic required for the one-sided action are comparable to those employed on plasma membranes and are considerably larger than those required for the two-sided action. We propose that the two-sided effect results from the formation of aqueous pores formed by the hydrogen bonding in the middle of the bilayer of two "half pores," whereas the one-sided effect results from the half pores alone. We discuss, in terms of the flexibility of bilayer structure and its thickness, how it is possible to have conducting half pores and "complete pores" in the same membrane. The role of sterol (cholesterol and ergosterol) in pore formation is also examined.
Article
A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or alteration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and Cl currents, lasts for approximately 5 min. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above approximately 1 microM ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].
Article
Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: (1) transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; (2) when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; (3) this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; (4) stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; (5) inhibition of proton secretion using acetazolamide in CO2 and HCO 3−-free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; (6) stimulation of proton transport with CO2 and HCO 3−-containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.
Article
Internally perfused cells of the marine alga Valonia actively transport potassium from external seawater into the cell vacuole. This active uptake of potassium is reduced by restoring the normal turgor pressure of 1 atmosphere by means of a mercury manometer attached to the internal perfusion system. This inhibition of salt uptake by a small hydrostatic pressure suggests that Valonia and other walled cells may regulate their turgor pressures by adjusting their rates of salt uptake.
Article
Ion transport in the giant celled marine alga, Valonia ventricosa, was studied during internal perfusion and short-circuiting of the vacuole potential. The perfusing and bathing solutions were similar to natural Valonia sap and contained the following concentrations of major ions: Na 51, K 618, and Cl 652 mM. The average short-circuit current (I(sc)) was 97 pEq/cm(2) sec (inward positive current), and the average open-circuit potential difference (PD) was 74 mv (vacuole positive to external solution). Perfused and short-circuited cells showed a small net influx of Na (2.0 pEq/cm(2) sec) and large net influxes of K (80 pEq/cm(2) sec) and Cl (50 pEq/cm(2) sec). Unidirectional K influx was proportional to I(sc), but more than one-half of the I(sc) remained unaccounted for. Both the I(sc) and PD were partly light-dependent, declining rapidly during the first 1-2 min of darkness. Ouabain (5 x 10(-4)M) had little effect on the influx of Na or K and had no effect on I(inf) or PD. Fluid was absorbed at a rate of about 93 pliter/cm(2) sec. Reversing the direction of fluid movement by adding mannitol to the outside solution had little effect on ion movements. The ionic and electrical properties of normal and perfused cells of Valonia are compared.
Article
Relaxation techniques have been widely used in kinetic studies of chemical reactions in homogeneous solution (Eigen & DeMayer, 1963). The principle of this method is well known: an external variable such as temperature or pressure is suddenly changed and the time course of a state parameter of the system such as concentration is recorded as it approaches a new steady value. Relaxation techniques can also be used for studying the rate of elementary processes in membranes. This method has proved particularly useful for the investigation of ion transport systems (ion carriers, channels, pumps) in artificial planar bilayer membranes. In this review we describe different relaxation techniques which have been developed for this purpose during the last years, as well as applications to a number of ion transport systems.
Article
The impedance of Necturus gallbladder epithelium was measured with transepithelial and intracellular microelectrodes in different transport states. The data are analysed with five electrical equivalent circuits, which differ with respect to the configuration of the paracellular shunt path (lumped vs distributed model of the lateral space), and of the apical cell membrane (non-ideal capacitance or surface amplification by micro-tubular infoldings). Least square fits indicate: 1. that the lumped model cannot represent the epithelium properly, even under control conditions; 2. that the distributed model, which considers the lateral intracellular space separately as a cable-like structure, describes the data well, both under control conditions and during collapse of the lateral spaces; and 3. that the above indicated variations of the apical membrane configuration improve the fits, but have little effect on the magnitude of the calculated circuit parameters. Quantitatively the analysis of 214 measurements on 25 gallbladders under control conditions yields the following results: The resistances of the tight junctions, of the lateral intercellular space, and of the apical and basal cell membrane are Rj = 123, Rlis = 35.5, Ra approximately 3,500, and Rb = 225 (all in omega cm2), and the capacitances of the cell membranes are Ca = 4.95 and Cbl = 26.5 (mu F/cm2). In oxygen deficiency and after cessation of chamber perfusion transport decreased, the spaces collapsed, and Rlis increased to approximately 130 omega cm2. Although the accuracy of the estimated Ra values is still limited, the analysis shows that it is possible to determine Rj and Rlis separately and to follow their changes in response to experimental maneuvers.
Article
A method has been developed to determine rapidly and simultaneously the resistance of the tight junctions, the resistance of the lateral intercellular space, and the resistances and capacitances of the apical and basolateral cell membrane in leaky epithelia by alternating current spectroscopy. The present paper describes the experimental procedures. Multi-frequency sine-wave currents are simultaneously applied across the tissue and the transepithelial and intracellular voltage responses are recorded with shielded microelectrodes, digitized, and stored in a computer. Using Fast Fourier Transform techniques the frequency-dependent transepithelial impedance and an apparent basal cell membrane impedance are then calculated from the voltage and current waveform. By fitting appropriate model circuits to the data the above listed individual resistances can be deduced. The model calculations and the fit results are described in the subsequent paper [14]. In the present design the analysis covers the frequency range between 2.5 Hz and 12.5 kHz. The minimal measuring time is in the order of 1-2s.
Article
Charge-pulse relaxation studies were performed on cells of the giant marine alga Valonia utricularis with microelectrodes inserted into the vacuole. If the cell was charged by short pulses of 200 ns duration, the decay of the initial membrane voltage could be described by two relaxation processes at normal pH (8.2). The fast exponential relaxation had a time constant of approximately 100 microseconds whereas the the time constant of the slow relaxation ranged between 2 and 15 ms. The ratio of the two amplitudes varied between 10 and 20 and was found to be independent of the initial voltage, up to 400 mV. In contrast to the time constants, the amplitude ratio was a function of the duration of the charge pulse. As the pulse length was increased to 10 ms, the fast relaxation disappeared. A change in pH of the natural sea water from 8.2 to 4 resulted in the disappearance of both exponential processes and the appearance of one single exponential with a 1-ms time constant over the whole pulse-length range. The analysis of the data in terms of a two-membrane model leads to unusual values and a pH-dependence of the specific capacitances (0.6 and 6 microF cm-2) of the two membranes, which can be treated as two serial circuits of a capacitor and a resistor in parallel. The charge-pulse and the current-clamp data are consistent with the assumption that the cell membrane of V. utricularis contains mobile charges with a total surface concentration of approximately 4 pmol cm-2. These charges cross the membrane barrier with a translocation rate constant around 500 s-1 and become neutralized at low pH. From our experimental results it cannot be completely excluded that the tonoplast has also a high specific resistance. But in this case it has to be assumed that the tonoplast and plasmalemma have very similar electrical properties and contain both mobile charges, so that the two membranes appear as a single membrane. Experiments on artificial lipid bilayer membranes in the presence of the lipophilic ion dipicrylamine, support our mobile charge concept for the cell membrane of V. utricularis.
Article
The cell membrane of Valonia utricularis contains an electrogenic carrier system for chloride (Wang et al., Biophys J. 59:235-248 (1991)). The electrical impedance of V. utricularis was measured in the frequency range between 1 Hz and 50 kHz. The analysis of the impedance spectra from V. utricularis and its comparison with equivalent circuit models showed that the transport system created a characteristic contribution to the impedance in the frequency range between 10 Hz and 5 kHz. The fit of the impedance spectra with the formalism derived from the theory of carrier-mediated transport allowed the determination of the kinetic parameters of chloride transport through the cell membrane of V. utricularis, and its passive electrical properties. Simultaneous measurements of the kinetic parameters with the charge pulse method demonstrated the equivalence of both experimental approaches with respect to the evaluation of the translocation rate constants of the free and the charged carriers and the total density of carriers within the membrane. Moreover, the impedance spectra of the protonophor-mediated proton transport by FCCP (carbonylcyanide p-trifluoromethoxyphenyl-hydrazone) were measured in model membranes. The carrier system made a substantial contribution to the impedance of the artificial membranes. The analysis of the spectra in terms of a simple carrier system (Benz and McLaughlin, 1983, Biophys. J. 41:381-398) allowed the evaluation of the kinetic and equilibrium parameters of the FCCP-mediated proton transport. The possible application of the measurement of impedance spectra for the study of biological transport systems is discussed.
Article
The effect of metabolic inhibitors, such as cyanide, antimycin A and azide was studied on the chloride transport system of the giant marine alga Valonia utricularis by using the charge pulse relaxation method. Two clearly defined voltage relaxations were resolved. The addition of 10-30 microM cyanide to the artificial sea water (ASW) bathing the algal cells increased the time constants of the slow voltage relaxation, tau 2, significantly when the algal cells were kept in the dark. The cyanide-effect reached a plateau value at 100-300 microM and was fully reversible when cyanide was removed from the ASW. Analysis of the charge pulse data in terms of the Läuger-model demonstrated that the translocation rates of the free, kS, and the charged carrier, kAS, decreased. The decrease of kS was more pronounced than that of kAS. 10 microM antimycin A and 3 mM azide had similar effects on the rate constants when the light was switched off. Upon illumination the cyanide- and antimycin A-, but not the azide-mediated effects disappeared. At concentrations higher than 1 mM cyanide caused a further, dramatic decrease of kS and kAS, while the surface concentration of the carrier molecules, N0, was not affected. This cyanide-effect was also reversible, but not light-dependent. Measurements of the ATP level showed that 3 mM cyanide reduced the ATP level by about 70% both under light and dark conditions. In the presence of 30 microM cyanide (or 10 microM antimycin A) the ATP level decreased by about 50%, but only in the dark. These results suggest two different effects of cyanide on the Cl(-)-carrier system: in the micromolar concentration range cyanide (and antimycin A) reduced predominantly the translocation of the free carrier by inhibition of ATP synthesis by oxidative phosphorylation, whereas in the millimolar concentration range cyanide apparently inhibits the translocation rates of both the free and charged carriers by its binding to the carrier. The results provide some evidence that the chloride transport of V. utricularis could be coupled to metabolic energy but it is an open question whether it is a pump or not.
Article
Cation conductance and efflux induced by polyene antibiotics amphotericin B (AMB), amphotericin B methyl ester (AME), nystatin, mycoheptin, and levorin on frog isolated skeletal muscle fibers and whole sartorius muscles were investigated. Conductance was measured under current-clamp conditions using a double sucrose-gap technique. Cation efflux was studied using flame emission photometry. Some new data were obtained concerning the effects of levorin and mycoheptin on biological membranes. The power dependence of polyene-induced cation transport on antibiotic concentration in muscle membrane was lower than that in bilayers. The decline in the equilibrium conductance caused by polyene removal (except for levorin) was very fast. There was reverse temperature dependence of AMB- and nystatin-induced conductances. Both induced conductance and efflux values demonstrated a correlation with the order of antifungal activities: levorin > AMB, mycoheptin > AME > nystatin, except for AME, which was more potent on yeastlike cells. These effects were interpreted in terms of possible differences in the kinetics of channel formation in biological and model membranes and in light of the role of nonconducting antibiotic forms in biological membranes.
Article
The effect of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the Cl(-)-transport system located in the plasmalemma of cells of the giant marine alga Valonia utricularis was studied by using the charge pulse relaxation technique. Analysis of the biphasic relaxation patterns in terms of the kinetic model published previously (Wang, J., Wehner, G., Benz, R. and Zimmermann, U. (1991) Biophys. J. 59, 235-248) demonstrated that extracellular DIDS dramatically reduced the translocation rate, KAS, of the Cl(-)-carrier complex (maximal inhibition 79%). The translocation rate of the free carrier molecules, KS, as well as the total surface concentration of the carrier, No, were not affected. A Hill-plot of DIDS inhibition on KAS yielded an half-maximal inhibition concentration (IC50) of 3.9 x 10(-5) M and Hill-coefficient of 1.61, suggesting a co-operative binding of the inhibitors to the Cl(-)-carrier. The maximal inhibition of DIDS was dependent on the extracellular Cl(-)-concentration. This inhibition was not competitive to chloride, since it increased and did not decrease with increasing chloride concentration. The DIDS effect decreased with increasing pH-value (investigated pH range between 6.5 and 10). Intravascular DIDS or SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) had no effect on the biphasic voltage relaxation pattern. These results showed that the binding sites of DIDS must be located on the outer surface of the plasmalemma of V. utricularis and, in turn, supported previous conclusions that the Cl(-)-carrier (which is assumed to be part of the turgor-pressure-sensing mechanism) is only located in the outer membrane.
Article
The active transports of sodium and chloride ions, between the vacuole and environmental solutions, were measured in the giant coenocyte of the marine alga Halicystis ovalis. Ion fluxes, determined isotopically, of individual single cells were measured by the short-circuit technique of Ussing and Zerahn (1951). Concentric pipettes were used to replace the vacuole sap with sep water and to short-circuit the vacuole potential difference to zero. The mean net efflux of sodium represented 39.2%, S. D. = 5.4, and the mean net influx of chloride 57.6%, S. D. = 5.3, of the current flowing through a short-circuited cell. Therefore a summation of the current carried by the two net active fluxes can account for the total short-circuit current. Micro-electrode penetration of the protoplasm of the cell indicated that the potential difference of the protoplasm was identical with the vacuole potential; that the total potential difference develops at the outer membrane. A theory is presented which evaluates the effect of these transport systems on the total potential difference and on the osmotic stability of the cell relative to its environment.
Article
The three envelope systems of frog and newt oocytes, the nucleus membrane, the cell membrane, and the epithelial envelope, were examined with intracellular microelectrodes. The two latter were found to be strong barriers for ion movements, the former to be a much more permeable structure: 1.1. The epithelial envelope has a high electrical resistance and capacitance, and sustains a resting potential of 6 mV, with its inner face negative.2.2. The cell membrane has the high resistance and capacitance of other cell structures, and sustains a resting potential (cytoplasm negative). The potential and resistance depend on the developmental stage of the cell. The potential, ranging from 5 to 35 mV, increases with the diameter of the cell. The specific resistance, ranging from 20 to 15,000 Ω cm2, increases roughly logarithmically with the cell diameter. The cell membrane capacitance of 5 μF/cm2 is independent of cell size.3.3. The nucleus membrane of oocytes has no appreciable resistance or potential. This contrasts with the nucleus membrane of another type of cell [21, 23]. A possible structural basis of the high conductance of the nucleus membrane of oocytes is discussed.
Article
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.Both E(co) and E(vc) were influenced by light. In the light, E(co) was -70 millivolts and it changed to -60 millivolts in the dark (cytoplasm-negative to outside). For E(vc), the potentials were +86 millivolts in the light and +69 millivolts in the dark (vacuole-positive to cytoplasm). The vacuole potential (E(vo)) was demonstrated to be the algebraic sum of E(co) and E(vc). For example, in the light, the sum of the means (+/-se) for E(co) (= -70 +/- 1) and E(vc) (= +86 +/- 5) is +16 millivolts, which is comparable to the measured E(vo) of +17 +/- 2 millivolts. In the dark, the sum of E(co) (= -60 +/- 3) and E(vc) (+69 +/- 6) is +9 millivolts and the measured value of E(vo) is +9 +/- 4 millivolts.The external K(+) concentration had a controlling effect on both E(co) and the direct current resistance of the plasmalemma, which suggests that E(co) is largely a K(+) diffusion potential. The tonoplast electrical properties were affected only slightly by external K(+).The data presented are indicative of a K(+) electrogenic influx pump in the tonoplast. It is also considered possible that H(+) might be electrogenically pumped from the cytoplasm both into the vacuole and to the cell exterior.
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