The Control of Na Uptake into Nitella translucens

Movement of Na into cells of Nitella translucens is a ‘downhill’process; the ions move across the plasmalemma down an electrochemicalpotential gradient. Nevertheless, measurements of Na influxesunder a wide range of experimental conditions have shown thatthere must be links between Na uptake and processes controlledby metabolism. When Ca ions are present in the bathing solution,Na influxes are greatly increased by light under conditionswhere photosynthesis can proceed (i.e. when both photosystemsare active). In the presence of Ca, the influx of Na increasesonly slightly when the external Na concentration is raised above1 mM, and the light-promoted Na influx is considerably inhibitedwhen Cl is removed from the bathing solution. When the Cl concentrationis kept constant, the Na influx in light is determined by theconcentrations of other cations present in solution (K, Ca,or NH₄). In the absence of Ca from the cell wall and solution,the influx is stil enhanced by light, but does not saturatewhen the external Na concentration is raised above 1 mM. Itis suggested that the Na influx in light is partly linked tothe inward Cl pump, but there is also a separate (Cl-independent)effect of light on the permeability of the plasmalemma to Na.Links between Na and Cl uptake could be maintained by effectsof Cl on electrochemical driving forces controlling Na entry;alternatively, chemical coupling between the two processes maybe involved.     

Ion Fluxes to the Vacuole of Nitella translucens

The time course of the appearance in the vacuole of Nitellatranslucens and of Tolypella intricata of tracer from the outsidesolution has been studied over short periods of uptake. Thereare two components of chloride transfer to the vacuole, a fastcomponent linear with time and a second component at longertimes whose behaviour is reasonably well described in termsof a single rate constant for exchange; a constant fractionof the total entry is in the fast component and the apparentrate constant for the second component is proportional to theinflux. In Nitella the path of rapid transfer involves chlorideand sodium, and may also involve a small but variable amountof potassium, but in Tolypella potassium has a significant componentof rapid transfer; these correspond to the cations for whichchloride-linked components of cation influx have been shownby another worker. Over both parts of the time course the level of activity inthe cytoplasm specifies, not the rate of transfer to the vacuoleas would be expected, but the rate as a fraction of the influx;the processes of influx to the cell and transfer to the vacuoleare intimately linked. It is difficult to explain the results in terms of static membranesand fixed compartments. An explanation in terms of the sequence,entry of salt by pino-cytotic vesicles at the plasmalemma, fusionof these vesicles with the endoplasmic reticulum after someloss of tracer to the surrounding cytoplasm, and transfer tothe vacuole in minivacuoles formed from the endoplasmic reticulum,is consistent with the time course found. A model of this kind,involving transport by a dynamic membrane system, seems necessaryto explain the results.     

Quantized Fluxes of Chloride to the Vacuole of Nitella translucens

It has been shown previously that the transfer of tracer chloridefrom the outside solution to the vacuole of Nitella translucensis initially linear with time. In this paper the relations betweenthe initial rate of chloride transfer to the vacuole and thetotal influx to the cell are further examined. In the individualcells in each experiment the ratio (initial rate of transferto the vacuole/rate of entry to the cell), M_ov/M_T, is quantized;that is, in each experiment the ratio takes values close to, 2, 3,... etc. Formation of pinocytotic vesicles is a processwhich could be imagined to be quantized, but the fact that itis a flux ratio, rather than a flux, which is quantized suggeststhat entry to a small cytoplasmic phase, such as the endoplasmicreticulum, must precede a quantized discharge to the vacuole.It is suggested that the kinetics of tracer movement to thevacuole are consistent with transfer in small vacuoles buddedoff the endoplasmic reticulum which then fuse with the centralvacuole.     

Electrical Potentials and Na, K, and Cl Concentrations in the Vacuole and Cytoplasm of Nitella translucens

The potential differences across the tonoplast and plasmalemmamembranes have been measured in the single cells of Nitellatranslucens, the cells being immersed in an artificial pondwater (composition: NaCl _1.0 mM., KC1 _0.1 mM., CaCl₂, _0.1 mM.).The potential of the cytoplasm is –138 m V with respectto the bathing medium and –18 mV with respect to the vacuole.The concentrations of Na, K, and Cl have been measured in thetwo cell fractions. The concentrations in the flowing cytoplasmare: Na 14 mM., K 119 mM., and Cl 65 mM.; the vacuolar concentrationsare: Na 65 mM., K 75 mM.,and Cl 160 mM. The observed potential differences across the two membranesare compared with the Nernst potentials for all three ions.This analysis shows that all three ions are actively transportedat the plasmalemma: Na is pumped outwards while K and Cl arepumped inwards. At the tonoplast Na is pumped into the vacuolewhile K and Cl are close to electrochemical equilibrium. The inhibitor, ouabain, has no effect on the cell resting potential.     

Effect of Na3VO4 and membrane potential on the structure of sarcoplasmic reticulum membrane

Summary Two-dimensional crystalline arrays of Ca²⁺-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na₃VO₄ in a Ca²⁺-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3-diethyl-2,2-thiadicarbocyanine (Di–S–C₂(5)) to monitor inside positive or inside negative membrane, potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca²⁺-ATPase, while negative potential disrupts preformed Ca²⁺-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca²⁺-ATPase.     

The Electrical Resistance and Capacitance of the Membranes of Nitella translucens

The resistance and capacitance of the membranes of Nitella translucenshave been measured by direct current and alternating currentmethods. Current of the order of 10^-7 amp. was injected intothe cell by means of a conventional Ag, AgCl_-3N KCl glass microelectrodeinserted into the vacuole of the cell. The change of potentialacross the membrane was recorded by two other internal microelectrodeswhich had been inserted into the cell at known distances fromthe current-injecting electrode. In the direct-current experimentsthe input current was in the form of a square pulse, while sinusoidalcurrents of frequency 25 cycles per second were used in thealternating current experiments. The cell was treated as a shortlength of coaxial cable and from the measurements the followingparameters could be obtained: the space constant (), the membraneresistance (R_m) and the membrane capacitance (C_m). The valuesof R_m ranged from 6.7 to 36 K ohm cm.² (mean of 21.4 K ohm cm.²)and those of ranged from 1.5 to 5.7 cm. (mean of 2.6 cm.).The capacitance value was about I µF cm.^-2 These results are discussed within the framework of our knowledgeof these parameters for other cells, particularly plant cells.The measured electrical resistance is shown to be at least tentimes less than the value estimated from the passive fluxesof the principal ions K, Na, and Cl. It is suggested that thisdiscrepancy, which is usually attributed to non-independentmovement of these ions, could be partially explained on electro-osmoticgrounds. The value of the capacitance is very close to thatwhich is usually obtained for other cell membranes. One exceptionallylow value for Nitella has been quoted in the literature. Thereason for the gross error in this particular measurement isgiven.     

Axial and Transnodal Movement of 14C, 22Na, and 36Cl in Nitella translucens

The time courses of axial movement in Nitella translucens ofa number of ¹⁴C-labelled compounds and of ³⁶C1 and ²²Na havebeen determined together with simultaneous microscopical observationsof the rates of visible protoplasmic streaming. In general,the translocation profiles suggest the irreversible absorptionof tracer though there are examples of reversible absorption.The coupling between visible streaming and the front of theaxial transport is weak. Light, DCMU (dichlorophenyldimethylurea),and temperature affect the translocation but visible streamingis affected only by temperature and the passage of an actionpotential. There is evidence, from a number of compounds, ofa faster-thari-streaming translocation of small amounts of tracerin the form of ‘pulses’ which are completely detachedfrom the main body of tracer. Either vesicles or microperistalticpackets are thought to account for this rapid moiety. Observations of transnodal transfer suggest that an active mechanismis involved in the transference of carbonate, urea, acetate,and chloride. The influxes of the various compounds have been calculated andshown to be in good agreement with existing published values.Action potentials reduce the influx of carbonate, chloride,and possibly acetate, as do lack of light and DCMU. It is suggestedthat the influx of these anions is coupled with the active anionpump and with loading sites on the plasma membrane which becomeinoperative during and immediately following an action potential.     

The Reappearance of An Extremely Rare and Critically Endangered Nitella translucens (Charophyceae) in Poland

We report the reappearance of the rare charophyte Nitella translucens in Poland. It was identified in the soft‐water lobelian Lake Jeleń (North Poland) during 2013 and 2018 phytolittoral surveys. This species is considered critically endangered in various European countries and was previously classified as extinct in Poland. Its occurrence was confirmed using morphological and molecular data (ITS1‐18S, ITS2‐28S, rDNA, and rbcL). The N. translucens occupied ~20% of the lake bottom, at depths of 1.5–6.5 m, water pH 7.5–8.6, conductivity of 59–66 μS · cm^?1, and total nitrogen and phosphorus during growing season in the range of 1.1–1.4 mg · L^?1 and 0.07–0.1 mg · L^?1, respectively. It co‐occurred mainly with plant species typical for lobelia lakes: Isoetes lacustris, Littorella uniflora, and Myriophyllum alterniflorum, as well as Ceratophyllum demersum and Elodea canadensis, which are characteristic for eutrophicated waters. It appears that N. translucens may thrive in lobelia lakes during their transformation to more eutrophic states.     

Electrophysiological Techniques and the Magnitudes of the Membrane Potentials and Resistances of Nitella translucens

The membrane resistance of internodal cells of Nitella translucensincreased by 50 per cent during the first 5 h after insertionof two microelectrodes into the vacuole even when precautionswere taken to eliminate external disturbances. The insertionof a third microelectrode into the cytoplasm did not affectthe resistance. In artificial pond water the final value forthe plasmalemma resistance was 112 k cm² and that for the tonoplastwas 6 k cm². The increase in the membrane potential after thefirst hour was less than 10 per cent. A recent suggestion that accurate measurements of the plasmalemmaresistance can be made with a microelectrode outside the plasmalemma,but in close contact with it, is criticized. Tests were made of the claim that leakage of current at thepoint where microelectrodes enter the cytoplasm gives rise toa local increase in current density at the tonoplast and henceleads to an overestimate of the tonoplast resistance. Valuesfor the tonoplast resistance obtained when the cytoplasmic microelectrodewas inserted through the plasmalemma were similar to those observedwhen it was pushed across the cell and inserted through thetonoplast at a point remote from the postulated current leakage.Furthermore, the tonoplast resistance stayed remarkably constantwhen the plasmalemma resistance varied in a way which wouldcause different proportions of the applied current to pass throughthe leak resistance and produce variations in the apparent tonoplastresistance. It is concluded that published values of the tonoplastresistance are not grossly inaccurate.     

The effect of cell excision and microelectrode perforation on membrane resistance measurements of Nitella translucens.

The effect of cell excision and microelectrode perforation on the membrane resistance measurements of Nitella translucens was determined by direct experiment. From the results it is concluded that perforation has no effect on cells as short as 1 cm. Current leakage though the node of an excised cell has however to be given some consideration. The method used for determining the resistance recovery to insertion has a wide application and its simplicity will encourage its use in other biological systems.     

Investigations into biophysical regulation mechanisms of physiological water exchange of the Nitella translucens cells with the surroundings

In the present paper, we have presented a theoretical discussion of specific biophysical mechanisms pertaining to the regulation (by means of internal factors) of physiological water exchange with the water medium by the cells of the Nitella translucens. The investigation has demonstrated that these cells have a capacity for water exchange regulation through changes in the values of transport parameters of the cell membrane (i.e. the filtration coefficient L_p and the reflection coefficient σ), as well as changes in the concentration C_si (inside the cells) of the osmotically active solute.     

Molecular determinants of PI(4,5)P2 and PI(3,4,5)P3 regulation of the epithelial Na+ channel

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na(+) channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P(3) and PI(4,5)P(2) to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in beta- and gamma- but not alpha-ENaC as necessary for PI(3,4,5)P(2) but not PI(4,5)P(2) modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in beta- and gamma-ENaC are critical to PI(3,4,5)P(3) augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of beta- and gamma-ENaC were identified as being critical to down-regulation of ENaC activity and P(o) in response to depletion of membrane PI(4,5)P(2). These regions of the channel played no identifiable role in a PI(3,4,5)P(3) response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P(2) to increase open probability. We conclude that beta and gamma subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P(3) and PI(4,5)P(2). This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.     

K Permeability of Nitella clavata in the Depolarized State

Membrane current responses to sudden potential changes were recorded in solutions of various [K]_o on 52 internodal cells of Nitella clavata. The membrane current after sudden depolarization had a component sensitive to [K]_o which increased with time from 0.3 to 2.0 s and remained steady thereafter. This late current became zero at values of E and [K]_o which suggests that the current was nearly all carried by K⁺. The potassium conductivity represented by this current increased with depolarization, with a half-maximum value at about -70 mV, and saturation at about -30 to -20 mV. The potassium conductance also increased with increasing [K]_o, but less rapidly than predicted for constant potassium permeability. This failure of the conductance to increase with [K]_o was relatively the same at all membrane potentials and may be explained by a model with a finite number of channels. No attempt was made to model the dependence of g_K on time after depolarization or on membrane potential. However, the finding that the membrane potential did not affect the way in which the permeability depended on [K]_o suggests that the membrane potential change does not affect the affinity of the sites, and that the increase in g_K with time after depolarization is brought about by an increase in the number of channels with such sites.