Excitable Active Electrogenic Ion-Transport Units in the Plasmalemma of Nitellopsis obtusa

With the use of voltage clamp and current clamp techniques thesupposition was proved that during the hyperpolarizing response(HR) N. obtusa cells generate active electromotive force (emf)at the expense of metabolic energy. Threshold inward currentsent through the plasmalemma of the cell which was depolarizedwith 100 mol m^–3 KG resulted in the HR with the transferof the membrane's excitable units from the high-conductive stateto the low-conductive state. During the HR the membrane potentialV_m increased from –135±10 mV to –290±15mV, the membrane resistance increased from 3.3±1.5 kOhmcm² to 5.8±1.2 kOhm cm² and the membrane emf E_m increasedfrom –20±4 mV to –93± 15 mV. Changesin the external concentration of K^–, Na⁺, Cl^– andH^– did not affect the patterns of HR. Cells which weredepolarized by light also generated HR (in normal medium) whichwas accompanied with the increase of V_m, R_m and E_m. The highvalue of Em generated during the HR can be explained only withthe involvement of active electrogenic charge transfer acrossthe membrane. 0.05 mol m^–3 DCCD added to the externalmedium inhibited the HR in both cases. Key words: Active ion transport, Hyperpolarizing response, Nitellopsis obtusa     

H+ Transport across the Plasmalemma and Tonoplast of Nitellopsis obtusa during Excitation

Measurements of intra- and extracellular pH during the excitationof Nitellopsis obtusa were carried out with antimony microelectrodesat conditions of dark adaptation and of continuous illumination.The pH of the vacuolar sap of both dark-adapted and illuminatedcells increased during cell excitation by 0·1–0·15units. H⁺ ions which had entered the cytoplasm during excitationin dark-adapted cells were extruded back into the vacuole acrossthe tonoplast. After cell excitation in the light H⁺ ions wereextruded from the cytoplasm also into the external medium probablyacross the light-stimulated active H⁺-channels. Protoplasmicstreaming ceased during excitation in the dark for 1–3min, and during excitation in the light—for 5–20s.     

Rhythmic Excitation in Nitella at Conditions of Voltage Clamp

Rhythmic excitation of Nitella cells initiated in 100 mM NaCloccurred when the voltage across the plasmalemma was fixed notonly at the resting level, but also at different hyperpolarizedlevels. The results presented indicate that the activation ofthe excitable units is not potential-dependent but is insteadchemical, reflecting changes in the ionic status within themembrane. Key words: Nitella, Plasmalemma potential difference, Rhythmic excitation     

Chlorolemma-A Continuous Membrane Layer between the Plasmalemma and the Streaming Cytoplasm in Nitellopsis obtusa and Nitella translucens, which takes part in the Generation of the Resting Potential, in its Light-Induced Changes and in the Generation of the Action Potential

At slow stepwise insertion of the glass micro-electrode intothe cell of Nitellopsis obtusa andNitella translucens four potentialjumps were recorded which corresponded to the penetration ofthe micro-electrode across the cell wall, the plasmalemma, thesupposed chlorolemma and the tonoplast, respectively. The photo-electricdepolarization appeared after the third jump only, which wassupposed to be the penetration of a membrane layer between theplasmalemma and the streaming part of the cytoplasm. The vibrationof this chlorolemma, caused probably by the protoplasmic streaming,resulted in large (up to 100 mV) hyperpolarizing and depolarizingpotential fluctuations when the micro-electrode tip was nearto this membrane. During cell excitation the potential difference(p.d.) had changed also across the chlorolemma- It was shownthat the potential changes across the chlorolemma, caused bylocal illumination on the isolated part of the cell, were measurablealso at a considerable distance (more than 1.4 cm) from theilluminated and isolated sequence. The light- and electron microphotographsobtained showed that the chloroplasts might really form a verycompact layer with tight connections between the neighbouringchloroplasts and that there existed at least one continuousmembrane layer which separated the immobile chloroplasts fromthe streaming cytoplasm. It was concluded that the p.d. measuredbetween the streaming cytoplasm and the external medium wasthe sum of the p.d.s across the cell wall, the plasmalemma andthe chlorolemma, respectively. Key words: Resting potential, Photo-electric responses, Characean algae, Chlorolemma     

Light-Activated H+ Transport into the Vacuole of Valonia ventricosa

Using glass capillary microelectrodes for the measurement ofpotential differences (PD) and antimony microelectrodes forthe measurement of pH, we investigated the light-induced changesof PD between the central vacuole and the external medium, ofpH in the vacuole (pH_v), as well as of pH in the external medium(pH_o) of the green marine alga Valonia ventricosa. PD in thedark was about +30 to +40 mV (vacuole positive), pH_v 6.3, andthe resistance of the protoplast (cell wall-plasmalemma-tonoplast)17.8 kOhm cm². Illumination caused an increase of the positivePD (after a few oscillations) up to +80 to +100 mV, acidificationof the vacuolar sap, alkalinization of the external medium,and a decrease in the resistance of the protoplast to 7.6 kOhmcm². The kinetics of the changes of PD, pH_v, and pH_o were similarto each other. It is concluded that a light-stimulated activeH⁺ flow occurs from the external medium into the central vacuoleof Valonia ventricosa as a result of the onset of photosyntheticactivity.     

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.     

Voltage Clamp Studies on the Slow Inward Current during the Excitation of Nitellopsis obtusa

The ionic currents across the plasmalemma of Nitellopsis obtusawere measured in voltage clamp experiments. Depolarization ofthe cell by 30–100 mV from the level of the resting potentialresulted in (1) a rapid inward current, (2) a subsequent slowinward current, and (3) a stationary outward current. The firstcurrent component changed sign at –20 to –30 mV.The second component decreased to a minimum at this clampedlevel. With increasing depolarizing steps some slow transientcurrent component reappeared without changing sign. This transientinward current occurred also when the potential was clampedeither at large depolarizing (+80 mV) or at large hyperpolarizing(–300 mV) potentials. In cases when the slow inward currentcomponent was evident cessation of protoplasmic streaming wasobserved. The ATPase inhibitor dicyclohexylcarbodiimide (DCCD)at a concentration of 2 x 10^–5 M in the external mediuminhibited the slow transient inward current without affectingthe first rapid current component. It is suggested that theirreversible slow transient current component reflects the onsetof some active ion-transport system in the plasmalemma duringcell excitation.     

Excitable Channels in Nitellopsis obtusa as Part of an Active Ion-Transport System

The present paper is a study on the rapid and the slow excitablechannels of Nitellopsis obtusa. The working hypothesis is thatduring the excitation of these cells a Ca²⁺-dependent activeion-transport system in the plasmalemma is activated (Gyenesand Bulychev, 1979; Gyenes, Bulychev, and Kurella, 1980) whichmay interact with a light-dependent active transport systemalso present in the plasmalemma. It is found that under conditionsof maximal light-induced current changes, registered in voltageclamp experiments, the amplitudes of both action current componentsare relatively small (10–15 µA cm²) and they increaseup to 100–150 µA cm^–2 during 15–30 minin the dark. Cells may also be excited chemically under conditionsof unchanged voltage across the plasmalemma. It is suggestedthat in the excitation process of Nitellopsis obtusa two typesof ion channels take part/emdash electrically excitable passiveand chemically excitable active channels-both incorporated inone proteolipid complex of a Ca²⁺-dependent ATPase of the plasmalemma.