Ion fluxes inAcetabularia: Vesicular shuttle

Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (²²Na⁺,⁴²K⁺,³⁶Cl^– and⁸⁶Rb⁺) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K⁺ with influx and efflux of about 100 nmol·m^–2·sec^–1×K⁺ passes three- to sevenfold more easily than Rb⁺ does. Under normal conditions, Cl^– (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m^–2·sec^–1, and a cytoplasmic as well as vacuolar [Cl^–] of about 420mm ([Cl^–] _o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl^–], are significantly reduced. Na⁺ ([Na⁺] _i : about 70mm, [Na⁺] _o : 461mm), which is of minor electrophysiological relevance compared to K⁺, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m^–2·sec^–1 for influx and efflux). Some results with Na⁺ and Cl^– are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.     

Hair morphogenesis inAcetabularia mediterranea: Temperature-dependent spacing and models of morphogen waves

Summary Whorls of sterile hairs inA. mediterranea show, at the moment of first appearance of hair initials, a spacing independent of number of hairs in the whorl but dependent on temperature. By changing the temperature at various times before appearance of hair initials, the pattern-forming event can be located at about 3–4 hours before initials become visible.The temperature dependence of spacing is like that of a chemical rate parameter: In (spacing)versus 1/T is linear. This suggests that the spacing is controlled by kinetic rather than structural factors, and correlates well with reaction-diffusion theory.Mathematical analysis and computer simulation have been used to show that the observed sequence of tip-flattening followed by whorl initiation can be interpreted in terms of published models for generation of dissipative structures by reaction and diffusion, and that at least two sequential processes must occur, the first of which shifts growth activity from extremity to circumference of the growing tip, permitting the second to operate around the circumference.Submitted to workshop on Morphogenesis inAcetabularia, Berlin (West), September 1980.     

Transient reduction of responsiveness of blue-light-mediated hair-whorl morphogenesis inAcetabularia mediterranea induced by blue light

After a prolonged period of red light the formation of a new whorl of lateral hairs can be induced inAcetabularia mediterranea Lamouroux (=A. acetabulum (L.) Silva) by a pulse of blue light. It has previously been shown that the response to blue light obeys the law of reciprocity. In this paper we demonstrate that the responses to blue light are additive only within 10 min after the onset of blue-light treatment, since the responsiveness of the cells is also affected by blue light. One hour after a short blue-light pulse the response to a second blue-light pulse has come to a minimum. After that, the responsiveness is restored in a refractory period of several hours. The fluenceresponse curves for hair-whorl formation at the time of minimum responsiveness are shifted parallel to the original fluence-response curves without preirradiation. Again, the law of reciprocity applies. This indicates an increased light requirement only for the same degree of hair-formation response. The sensitivity to blue light of the reduction of responsiveness response is higher by a factor of about 50 than the induction of hairformation response.     

Nuclear division in the cyst,and white spot nuclei preceding cyst formation,inAcetabularia wettsteinii

Summary Electron microscopy of nuclear division in young cysts ofAcetabularia wettsteinii shows that the dividing nucleus hat two additional cisternae of endoplasmic reticulum immediately outside the nuclear envelope. These additional cisternae are attached to, and apparently formed from a membrane body which develops outside the nucleus in early prophase. The interphase nucleus does not have the additional cisternae. The nucleoli are extruded from the nucleus at anaphase, the nucleolar bodies remaining in the peri-nuclear cytoplasm. The chromosomes have localized centromeres; the stratified ultrastructure characteristic of some chlorophycean and animal kinetochores has not been found inAcetabularia, although the kinetochore appears distinct, projecting from the chromatid, and has attached microtubules. The condensed bodies of the white spot nucleus are discussed.     

Visualization of cytoskeletal changes through the life cycle inAcetabularia

Summary The cytoskeleton in the siphonous, marine green algaAcetabularia is visualized by immunocytochemistry using antibodies against plant alfa tubulin and animal smooth muscle actin. In the vegetative phase of the life cycle, when the cell grows a cylindrical stalk and until the reproductive cap is completed, actin forms continuous, parallel bundles that extend through the entire length of the stalk and cap rays respectively. Microtubules (MTs) cannot be detected until the primary nucleus, located in the rhizoid of the giant cell, divides to form thousands of secondary nuclei. MTs can then be seen radiating from each secondary nucleus that is encountered in the stalk on its migration upwards into the cap rays. They are oriented mostly parallel to the long axis of the cell. At arrival in the cap rays up to the white spot stage, when nuclei assume equidistant positions in the cap ray cytoplasm, a radiating system of MTs forms around each nucleus and dramatically increases until impressive radial arrays have developed. This phase coincides with a disappearance of actin bundles in the cap rays, but they are retained in the stalk cytoplasm. Shortly after that additional MTs appear around the disk like partitions of cap ray cytoplasm. Concomitantly, bundles of actin reappear colinearly with the circumferrential MTs eventually forming complete rings around each disk of cap ray cytoplasm. During this process the compartments of the future cysts are gradually bulging outwards and simultaneously the rings of actin sink inwards until domes are formed with the nuclei fixed in the top centers of the domes. At this stage the peripheral areas of the radiating MT systems around the nuclei start to break down, whereas the circumferrential MT systems remain intact. Subsequently, the rings of both actin and MTs decrease in diameter, and finally contract to a spot opposite the nucleus, while the cysts continue to develop their oval shape. After the cysts have become separated, they round up and enter several rounds of nuclear divisions. MTs form short radial arrays around each nucleus with minor changes due to a reduction of MTs during division followed by a reappearance after completion of each division. Actin is rearranged in the cysts to a cortical network of randomly oriented, short bundles, that is maintained until gamete formation sets in.These findings accentuate the involvement of Cytoskeletal elements in the key steps of morphogenesis inAcetabularia to an extent that is unknown in higher plants.     

Modeling and simulation of ion channels and action potentials in taste receptor cells

Based on patch clamp data on the ionic currents of rat taste receptor cells, a mathematical model of mammalian taste receptor cells was constructed to simulate the action potentials of taste receptor cells and their corresponding ionic components, including voltage-gated Na⁺ currents and outward delayed rectifier K⁺ currents. Our simulations reproduced the action potentials of taste receptor cells in response to electrical stimuli or sour tastants. The kinetics of ion channels and their roles in action potentials of taste receptor cells were also analyzed. Our prototype model of single taste receptor cell and simulation results presented in this paper provide the basis for the further study of taste information processing in the gustatory system.     

An interconnected plastidom inAcetabularia: Implications for the mechanism of chloroplast motility

Summary In the unicellular green algaAcetabularia, the vital fluorochrome 3,3′-dihexyloxacarbocyanine (DiOC₆) readily accumulates in chloroplasts and mitochondria at low concentrations, suboptimal for the visualization of the endoplasmic reticulum (ER). These organelles align along motility tracks and partially obscure each other, resulting in the loss of image information in conventional fluorescence microscopy. However, superior imaging of organelles was achieved by confocal laser scanning microscopy, which was particularly evident in areas where mitochondrial profiles overlap with chloroplasts. In addition to the tubular mitochondria, a new type of tubular membrane profiles was discovered inAcetabularia which connects the chloroplasts with each other. These tubules may either form short bridges or may stretch over hundreds of micrometers before connecting to the next chloroplast. Because staining intensity, size and overall shape of mitochondria and the connecting membrane tubules were very similar, pharmacological treatments have been applied to differentiate more clearly between the two compartments. Inhibitors of mitochondrial function are shown here to affect mitochondrial shape but not that of the chloroplast tubules. Finally, electron microscopic analysis of thin sectioned materials revealed long tubular emanations from the chloroplasts proving their plastidal origin. The function of these hitherto unknown plastidal membrane tubules is not known, but their behaviour suggests that they interact with the cytoskeleton and effectively modify chloroplast behaviour.     

Theoretical and simulation studies on voltage-gated sodium channels

Voltage-gated sodium (Na_v) channels are indispensable membrane elements for the generation and propagation of electric signals in excitable cells. The successes in the crystallographic studies on prokaryotic Na_v channels in recent years greatly promote the mechanistic investigation of these proteins and their eukaryotic counterparts. In this paper, we mainly review the progress in computational studies, especially the simulation studies, on these proteins in the past years.     

Effects of membrane potential on calcium fluxes of Pelvetia eggs

⁴⁵Ca²⁺ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K⁺]) increases, and depolarization (with high [K⁺]) decreases the influx of Ca²⁺ over the range of [K⁺] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of ⁴⁵Ca²⁺ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of ⁴⁵Ca²⁺ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca²⁺].     

Action spectrum for the blue-light-dependent morphogenesis of hair whorls in Acetabularia mediterranea

In young Acetabularia mediterranea Lamouroux (=A. acetabulum (L.) Silva) the formation of the lateral hair whorls can be induced by a short pulse of blue light after continuous red preillumination. In this paper we describe the experimental conditions for optimum response and the properties of the action spectrum. The probit of the cells which eventually form hair whorls is linearly correlated to the logarithm of the incident quanta of blue light. Parallel fluence-response curves for all wavelengths indicate the involvement of only one photoreceptor pigment. The action spectrum shows no effectiveness of wavelengths above 520 nm, a high action peak at 470 nm and two lower ones at 425 and 370 nm, and is in accordance with those of cryptochrome-like photoreceptors.     

Action potential-like responses due to the inward rectifying potassium channel

Summary This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a pathch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5mm), and were suppressed by barium (0.5mm). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed. The voltage responses described in this paper look similar to the slow action potentials on heart, which are sensitive to modifications of the calcium channels, but also of the potassium channels. Some implications in cardiac pharmacology are discussed.     

Short-term influence of recolonisation by the polycheate worm [2pt] Nereis succinea on oxygen and nitrogen fluxes and [2pt] denitrification: a microcosm simulation

The short-term effects of sediment recolonisation by Nereis succinea on sediment-water column fluxes of oxygen and dissolved inorganic nitrogen, and rates of denitrification, were studied in microcosms of homogenised, sieved sediments. The added worms enhanced oxygen uptake by the sediments, due to the increased surface area provided by the burrow walls and the degree of stimulation was stable with time. Similarly, ammonium fluxes to the water column were stimulated by N. succinea, but declined over the 3 day incubation in all microcosms including the controls. Nitrate fluxes were generally greater in the faunated microcosms, but highly variable with time. Denitrification rates were positively stimulated by N. succinea populations, denitrification of water column nitrate was stimulated 10-fold in comparison to denitrification coupled to nitrification in the sediments. Rates of denitrification of water column nitrate were not significantly different from rates in undisturbed sediment cores with similar densities of N. succinea, whereas rates of coupled nitrification–denitrification were 3-fold lower in the experimental set-up. These results may reflect the relative growth rates of nitrifying and denitrifying bacteria, which allow more rapid colonisation of new burrow surfaces by denitrifier compared to nitrifier populations. The data indicate that recolonisation by burrowing macrofauna of the highly reduced sediments of the Sacca di Goro, Lagoon, Italy, following the annual dystrophic crisis, may play a significant role in the reoxidation and detoxification of the sediments. The increased rates of denitrification associated with the worm burrows, may promote nitrogen losses, but due to the low capacity of nitrifying bacteria to colonise the new burrow structures, these losses would be highly dependent upon water column nitrate concentrations.     

Action potential shape change in an electrically coupled network during propagation: a computer simulation

We applied compartmental computer modeling to test a model of spike shape change in the jellyfish, Polyorchis penicillatus, to determine whether adaptive spike shortening can be attributed to the inactivation properties of a potassium channel. We modeled the jellyfish outer nerve-ring as a continuous linear segment, using ion channel and membrane properties derived in earlier studies. The model supported action potentials that shortened as they propagated away from the site of initiation and this was found to be largely independent of potassium channel inactivation. Spike broadening near the site of initiation was found to be due to a depolarization plateau that collapsed as two spikes spread from the point of initiation. The lifetime of this plateau was found to depend critically on the inward current flux and the space constant of the membrane. These data suggest that the spike shape changes may be due not only to potassium channel inactivation, but also to the passive properties of the membrane.     

Computer simulation of the KvAP voltage-gated potassium channel: steered molecular dynamics of the voltage sensor

The recent crystal structures of the voltage-gated potassium channel KvAP and its isolated voltage-sensing 'paddle' (composed of segments S1-S4) challenge existing models of voltage gating and raise a number of questions about the structure of the physiologically relevant state. We investigate a possible gating mechanism based on the crystal structures in a 10 ns steered molecular dynamics simulation of KvAP in a membrane-mimetic octane layer. The structure of the full KvAP protein has been modified by restraining the S2-S4 domain to the conformation of the isolated high-resolution paddle structure. After an initial relaxation, the paddle tips are pulled through the membrane from the intracellular to the extracellular side, corresponding to a putative change from closed to open. We describe the effect of this large-scale motion on the central pore domain, which remains largely unchanged, on the protein hydrogen-bonding network and on solvent. We analyze the motion of the S3b-S4 portion of the protein and propose a possible coupling mechanism between the paddle motion and the opening of the channel. Interactions between the arginine residues in S4, solvent and chloride ions are likely to play a role in the gating charge.     

Mechanism of luminal Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> action on the vacuolar slowly activating channels

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca²⁺-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channels closed conformations. Vacuolar Ca²⁺ and Mg²⁺ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a permeable blocker. Vacuolar Ca²⁺—with a much higher affinity than Mg²⁺—slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca²⁺-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channels closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca²⁺ at the vacuolar side. The specificity for Ca²⁺ compared to Mg²⁺ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca²⁺ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.Abbreviation SV Slow vacuolar     

Reconstituted voltage-sensitive sodium channels from eel electroplax: Activation of permeability by quaternary lidocaine,N-bromoacetamide,and N-bromosuccinimide

Summary We have investigated the ion permeability properties of sodium channels purified from eel electroplax and reconstituted into liposomes. Under the influence of a depolarizing diffusion potential, these channels appear capable of occasional spontaneous openings. Fluxes which result from these openings are sodium selective and blocked (from opposite sides of the membrane) by tetrodotoxin (TTX) and moderate concentrations of the lidocaine analogue QX-314. Low concentrations of QX-314 paradoxically enhance this channel-mediated flux. N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), reagents which remove inactivation gating in physiological preparations, transiently stimulate the sodium permeability of inside-out facing channels to high levels. The rise and subsequent fall of permeability appear to result from consecutive covalent modifications of the protein. Titration of the protein with the more reactive NBS can be used to produce stable, chronically active forms of the protein. Low concentrations of QX-314 produce a net facilitation of channel activation by NBA, while higher concentrations produce block of conductance. This suggests that rates of modifications by NBA which lead to the activation of permeability are influenced by conformational changes induced by QX-314 binding.     

Aerolysin,a hemolysin fromAeromonas hydrophila,forms voltage-gated channels in planar lipid bilayers

Summary The cytolytic toxin aerolysin was found to form ion channels which displayed slight anion selectivity in planar lipid bilayers. In voltage-clamp experiments the ion current flowing through the channels was homogeneous indicating a defined conformation and a uniform size. The channels remained open between –70 to +70 mV, but outside this range they underwent voltage-dependent inactivation which was observed as open-closed fluctuations at the single-channel level. Zinc ions not only prevented the formation of channels by inhibiting oligomerization of monomeric aerolysin but they also induced a closure of preformed channels in a voltage-dependent fashion. The results of a Hill plot indicated that 2–3 zinc ions bound to a site within the channel lumen. Proaerolysin, and a mutant of aerolysin in which histidine 132 was replaced by an asparagine, were both unable to oligomerize and neither could form channels. This is evidence that oligomerization is a necessary step in channel formation.     

Studies on the tonoplast action potential ofNitella flexilis

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