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     

Cable correction of membrane currents recorded from root hairs of Arabidopsis thaliana L.

Membrane currents were recorded under voltage clamp from roothairs of Arabidopsis thaliana L. using the two-electrode method.Concurrent measurements of membrane voltage distal to the pointof current injection were also carried out to assess the extentof current dissipation along the root hair axis. Estimates ofthe characteristic cable length,     

Action of Mono- and Difluorodinitrobenzene on Induced Electrical Modifications by External pH Changes in Nitella

The action of mono- (FNB) or difluorodinitrobenzene (DFNB) onion permeability is mainly attributed to its interactionwithamino groups of the membrane by dinitrophenylation. Nitella cells were dimtrophenylated at pH 7.3 and the membranepotential and electrical resistance were then measured in acidicor basic solutions. No matter what the pH value was, FNB andDFNB induced a depolarization of the membrane potential andcaused a diminution of resistance. However these effects ofFNB and DFNB were more drastic at alkaline pH and in the presenceof a weak concentration of potassium. Neither the addition of0.1 mM calcium nor the substitution of chlorides by nitratesmodified the DFNB effect. These results are compatible withthe assumption that the DFNB binding to the membrane leads toan augmentation of the negative charges of the membrane bringingabout an increased cation conductance and a modification ofthe affinity of a K⁺/H⁺exchange pump. The transient responseof the membrane potential at the time of dinitrophenylationwas used to roughly estimate the total density of amino groupsof the membrane of Nitella.     

Oscillations in Extracellular Current, External pH and Membrane Potential and Conductance in the Alkaline Bands of Nitella and Chara

Oscillations with a period of approximately 2 min were observedin the membrane potential of Chara and Nitella upon illuminationof dark-treated cells, as well as in the extracellular currentpattern and pH values. A 2-min oscillation in the membrane potentialwas observed when the voltage electrode was placed close tothe border of an alkaline and acid region. Comparison of resultsfrom Chara and Nitella revealed an identical control mechanismfor external pattern stabilization in the effect of light onmembrane potential and conductance. Vibrating probe experimentsindicated that oscillations in the extracellular current occurredonly at the border of the alkaline band. Ion-specific pH micro-electrodesplaced within the alkaline band detected oscillations associatedwith light reactivation of the banding phenomenon. These resultsindicate that the oscillations represent a localized phenomenoninvolving spatially-dependent time-constants. More evidencefor the spatial dependence of time constants is gained fromsingle active acid regions in Nitella. Using this combinationof techniques, we have established that a light-dependent H⁺transporter is involved in this oscillation. Current-voltagecurves taken during these oscillations and relaxation, afterchanging the light intensity, confirmed this identification. Key words: Oscillation, vibrating probe, pH micro-electrode, time-constant. I/V curve, Chara, Nitella     

苦参碱对棉铃虫幼虫神经细胞钠通道的影响

用全细胞膜片钳技术研究了生物碱类植物杀虫剂苦参碱对棉铃虫Helicoverpa armigera幼虫离体培养中枢神经细胞钠离子通道门控过程的影响。结果表明： 苦参碱对棉铃虫幼虫神经细胞所表达的TTX （tetradotoxin, 河豚毒素)敏感钠通道具有浓度依赖性阻滞作用,1,10和100 μmol/L的苦参碱作用5 min后,分别使钠电流峰值较给药前下降（12.49±1.67）%、（18.79±2.16）%和（43.15±8.17）% (n=8, P<0.05)。苦参碱使钠电流的电流 电压关系曲线上移,但并不改变其激活电压、峰电压和电流电压关系曲线的形状。苦参碱对钠通道的阻滞作用可能是其具有某些毒理效应的离子基础。     

The effect of external pH on the membrane potential of Nitella and its linkage to metabolism

The membrane potential of Nitella is highly sensitive to externalpH in the neutral region (pH 5.5 to 8) in the light. We foundthat the sensitivity usually was reduced by darkness, metabolicinhibitors (DCMU, DNP) or cooling. Under some circumstancesthe high pH-sensitivity of the membrane potential was observedeven in the dark. However, even in this case, it was reducedby DNP or cooling. These results indicate that the externalpH-dependence of the membrane potential of Nitella is closelylinked to metabolism. (Received May 8, 1973; )     

Breakdown phenomena in the Chara membrane

The breakdown phenomenon in the Chara internodal cell was studiedusing the voltage clamp technique. When a slowly hyperpolarizingramp potential pulse was applied to the Chara membrane, thebreakdown occurred with hyperpolarization of about 220 mV. Thebreakdown was observed by less hyperpolarization, if the externalK⁺ concentration was increased. Such a breakdown phenomenonin the Chara membrane was caused principally by a large shiftof the membrane electromotive force toward depolarization. Thisshift frequently exceeded the peak level of the action potential. (Received July 26, 1976; )     

GTP和GDP类似物对棉铃虫神经细胞高电压敏感钙通道的调节作用

以Ba²⁺为载流子,采用全细胞膜片钳法,研究了在电极液中分别加入G蛋白稳定 激活剂GTPγS（GTP类似物）和抑制剂GDPβS（GDP类似物）对棉铃虫Helicoverpaarmigera 3龄幼虫神经细胞高电压敏感钙通道的调节作用。Ba²⁺电流记录时间为20 min。对照组 和实验组的Ba²+电流在记录的初期均出现电流的增加现象,随后电流衰减,即“rundown ”。对照组峰电流在第20 min时降为初始值的(72.09±12.80)%。电极内液中加入2 mmol/L GTPγS可缓解电流的衰减现象,在第20 min时,峰电流为初始值的(95.99±7.93)%,明显大 于对照组的峰电流(P<0.01),而且电流 电压（I-V）关系曲线向正电压方向移动。相反 ,电极内液加入2 mmol/L GDPβS则导致峰电流衰减更加严重,第20 min时,峰电流仅为初始水 平的(41.95±9.32)%,显著小于对照组（P<0.01）,但未见电流 电压（I-V）关系曲 线的明显漂移。结果表明,棉铃虫神经细胞钙通道活动受G蛋白激活剂GTPγS和G蛋白抑制剂GDPβS的影响,提示G蛋白活动水平的改变调节钙通道的电流幅值和电压依赖性。     

REPETITIVE ACTION POTENTIALS IN NITELLA INTERNODES

Typical spontaneous action potentials can be elicited in 10–100mM NaCl or LiCl solution. The period of repetition is 0.5–2seconds and the action potential generally consists of a rapidspike alone. Similar spontaneous action potentials are alsodemonstrated by adding either 1 mM EDTA (pH 6.6) or 2 mM ATP(pH 6.6) to the artificial pond water. In these cases, however,the period of repetition is much longer and the action potentialis of a normal shape, a rapid spike being followed by a slowtransient depolarization. The period of repetition and the sizeof the action potential decrease with the elevation of the vacuolarpotential level. The cause of the spontaneous firing is supposedto be the removal of Ca⁺⁺ from the outer surface of the Nitellamembrane. (Received May 18, 1966; )     

The Influence of Temperature on a K+-Channel and on a Carrier-Type Transporter in Nitella

Fisahn, J. and Hansen, U-P. 1986. The influence of temperatureon a K⁺ -channel and on a carrier type transporter in Nilella—J.exp. Bot. 37. 440–460. In Nitella, the effects of temperature on membrane potentialand on resistance consist of several components. The evaluationof their associated time-constants measured in linear(ized)temperature responses at a resting potential of–120 mVprovides an approach to their identification. For changes slowerthan c. 1 s, the temperature effect on membrane potential andresistance does not originate from temperature action on theinvolved transporter, but is mediated by signals from temperaturesensitive metabolic processes. In the case of potential, theseprocesses seem to be identical to those which also mediate thelight effect: pH-regulation, and two direct signals from photosynthesis,as indicated by the similarities of the related time-constants( respectively). The temperature effect on resistance displays only one time-constant of 40 sinmost experiments. The related process is unknown. The non-coincidenceof the time-constants of the effect on potential and on resistanceimplies the involvement of a carrier-type transporter (H+-pumpor cotransporter) in the effect on potential, and of a K+channelin the effect on resistance. The K+-channel is identified bythe reversal potential of the effect on membrane potential measuredin cells depolarized or hyperpolarized by an injected electricalcurrent Under these conditions the temperature effect on resistancedominates the effect on potential. Key words: H⁺-pump, K⁺-channel, kinetic analysis, Nitella, oscillation, pH-regulation, reversal, potential, temperature, time-constants     

Effect of pH on the Membrane Potential and Electrical Resistance of Nitella flexilis in the Presence of Calcium

Effects of pH on the membrane potential and electrical resistanceof Nitella were investigated in a bathing medium with or withoutcalcium. The membrane potential became more negative as theexternal pH was raised, at a faster rate in the presence ofcalcium than in its absence. The value then achieved by thepotential could be reversed by restoring the original pH whilstin a Ca-free medium the cell remained ‘hyperpolarized’.Tenfold changes of the external concentration of potassium broughtabout larger modifications of the membrane potential when thepH of the solution was high and calcium concentration low. Theelectrical resistance was lowest in alkaline and calcium-freesolutions. We conclude that calcium prevents the mediation ofsome changes in the membrane structure by lowering the concentrationof external H⁺ ions, and that the permeability of Nitella topotassium increases with rising pH.     

Electromotive force of Nitella membrane during excitation

Excitation of the Nitella membrane is analysed by assuming themembrane to be an electromotive force in series with a resistance,both being variables of time and of membrane potential. Duringstep depolarization beyond a threshold, conductance and electromotiveforce increase transiently, finally reaching their respectivesteady state levels. The conductance increase peak is attainedearlier than the peak for electromotive force increase. Wheneverelectromotive force increases beyond the level of clamped membranepotential, the ionic current flows inward. This is consideredto be the origin of the apparent negative resistance characteristicof the excitable membrane. Anodal break response and spontaneousfiring of Nitella membrane are also caused by transient increasesin electromotive force and conductance irrespective of whetherthe membrane potential is being held at its resting level. Thetransient increase in electromotive force reflects changes,like a phase transition, occurring during excitation. (Received May 6, 1968; )     

The N-Shaped Current-Potential Characteristic in Frog Skin: II. Kinetic Behavior during Ramp Voltage Clamp

Previous step voltage-clamp measurements on frog skin showed the presence of an N-shaped current-potential (I-V) relation in excitable skin. However, the collection and reconstruction of I-V data using discrete step changes of skin potential was tedious because of the long refractory period (up to 1 min) in frog skin. A direct and rapid (5 msec) method for recording the N-shaped I-V characteristic in real time is presented. Ramp functions are used as the command to the clamp system instead of a step function. Consequently the skin potential is forced to change in a linear manner (as commanded) and the skin current can be recorded as a continuous function of the controlled change of skin potential. With the ramp clamp, a low-resistance membrane state ( 10 Omega . cm(2)) resembling a breakdown phenomenon was observed at high skin potential ( 300 mv). Entry into the low resistance state resulted in a collapse of the N-shaped I-V relation to a nearly linear function. The utility of the ramp measurement is demonstrated by predicting (1) that the maximum rate of rise of the spike occurs at a voltage corresponding to the valley (local minimum) in the N-shaped I-V curve, (2) that the rate of rise of the spike increases with increasing clamp currents, (3) the voltage peak of the spike, and (4) the time course of the rising phase of the spike.     

The electromotive force of the Chara membrane during the hyperpolarizing response

The hyperpolarizing response of the Chara internodal cell wasstudied by applying the voltage clamp and constant current techniques.By assuming the membrane as an electromotive force (emf) inseries with a resistance r (which is the sum of a series resistancer_s and the membrane resistance r_m), it was shown that the hyperpolarizingresponse was brought about not only by the increase in membraneresistance but by the increase in membrane emf. The time dependentcurrent-voltage (I–V_m) curve obtained under the voltageclamp during the hyperpolarizing response showed a negativeresistance. The hyperpolarizing response is also an excitation,since it is a transition process of the membrane across a negativeresistance region. (Received July 22, 1974; )     

Two Light-Induced Processes in the Photoreceptor Cells of Limulus Ventral Eye

The dark-adapted current-voltage (I-V) curve of a ventral photoreceptor cell of Limulus, measured by a voltage-clamp technique, has a high slope-resistance region more negative than resting voltage, a lower slope-resistance region between resting voltage and zero, and a negative slope-resistance region more positive than 0 v. With illumination, we find no unique voltage at which there is no light-induced current. At the termination of illumination, the I-V curve changes quickly, then recovers very slowly to a dark-adapted configuration. The voltage-clamp currents during and after illumination can be interpreted to arise from two separate processes. One process (fast) changes quickly with change in illumination, has a reversal potential at +20 mv, and has an I-V curve with positive slope resistance at all voltages. These properties are consistent with a light-induced change in membrane conductance to sodium ions. The other process (slow) changes slowly with changes in illumination, generates light-activated current at +20 mv, and has an I-V curve with a large region of negative slope resistance. The mechanism of this process cannot as yet be identified.     

The Influence of the Cell Membrane on some Toxicity Effects of Growth Substances in Nitella

When cells of Nitella are placed in a solution of some plantgrowth substances there is a profound increase in the membraneresistance as measured by means of an internal silver/silverchloride micro-electrode. This impedance effect is accompaniedby some marked visible changes within the cell cytoplasm, thesechanges being dependent upon the concentration of undissociatedgrowth substance in the vicinity of the membrane, and upon thepH of the external environment. A detailed study is made of these visible changes and, takinginto account the negatively charged membrane, one possible interpretationof the results is that only undissociated molecules of the growthsubstance may enter the cytoplasm of Nitella from the environment,the diffusion obeying a simple Fick Law relationship. The substances are used in sufficient quantity to kill the Nitellacell, death following the visible effects, and they appear tobe unique in that their toxic effect produces a system of highmembrane impedance.     

Effects of Density and Gating of Delayed-Rectifier Potassium Channels on Resting Membrane Potential and its Fluctuations

The aim of this study is to evaluate directly, using a reduced experimental system, the nature of interactions between voltage-gated potassium channels and the resting membrane potential. Xenopus oocytes were injected with various concentrations of cRNA coding for a delayed-rectifier potassium channel Shaker-IR. The effects of the density and kinetics of the expressed channels on resting membrane potential is explored in isolated (``inside-out') patches. The channel density is given in terms of maximal conductance (G _max), measured from the maximal slope of the I-V curve under voltage clamp conditions. The capacitance of the experimental setup is approximately 1 pF. At high channel densities (G _max > 10 pA/mV) the mean membrane potential is stabilized at approximately −60 mV. This resting membrane potential is more than 35 mV positive to the reversal potential for potassium ions under the same experimental conditions. Analyses of voltage clamp experiments indicate that at high channel densities the mean membrane potential is determined by the rates of channel activation and deactivation, but is not affected by the rates involved in the process of slow (C-type) inactivation. In contrast, at lower channel densities membrane potential is very unstable, and its mean value and amplitude of fluctuations are strongly affected by the process of slow (C-type) inactivation. Received: 21 March 1996/Revised: 6 August 1996     

The electrogenic ion pump revealed by the external pH effect on the membrane potential of Nitella. Influences of external ions and electric current on the pH effect

The influences of external ions and electric current on thepH effect of the membrane potential of Nitella are described.Results are explained in terms of a pH-dependent electrogenicion pump. The reduction of membrane resistance with a pH rise is attributedto the pump current being potential-dependent. (Received July 9, 1974; )     

Strip-shaped Projections at the Cytoplasmic Face of the Outer Membrane of the Generative Cell in Amaryllis belladonna

Strip-shaped projections are present at the cytoplasmic faceof the outer membrane of the generative cell in Amaryllis belladonna.This outer membrane is actually the inner plasma membrane ofthe vegetative cell which surrounds the generative cell. Theprojections are situated in groups and arranged parallel toeach other. Their predominant orientation is perpendicular tothe long axis of the generative cell. The projections are approximately35 nm high, and on average equally spaced 40 nm apart. Theirmaximum observed length, estimated from grazing sections ofgenerative cells, is 250 nm. Generative cell, outer membrane, Amaryllis belladonna, ultrastructure     

Rhythmic change of membrane potential and cyclosis of Nitella internode

The membrane potential of Nitella internode shows a spontaneousrhythmicity. The period depends largely upon temperature andthe length of the cell. Moreover, the period is reversibly prolongedat low temperature and also by treatment with cytochalasin B.We concluded that this bioelectrical rhythm is closely relatedto cytoplasmic streaming. (Received August 11, 1975; )