Membrane potential responses controlling chemodispersal of Paramecium caudatum from quinine

Membrane potential responses of a ciliate protozoan Paramecium caudatum to the external application of quinine were investigated in relation to its motile activities. Wild-type specimens swimming in the reference solution did not enter into a quinine-containing (0.5 mM) test solution due to avoiding responses exhibited at the border between the two solutions, and therefore stayed in the reference solution (chemodispersal). Squirting of a quinine-containing test solution over a wild-type specimen evoked a train of action potentials superimposed on a depolarizing chemoreceptor potential. Squirting of a quinine-containing test solution over a CNR-mutant specimen defective in voltage-gated Ca²⁺ channel evoked only chemoreceptor potentials, which consisted of an initial transient depolarization, a following transient hyperpolarization and a sustained depolarization. A current-evoked action potential became larger in its amplitude and longer in its duration with the external application of quinine. Under the voltage-clamp condition, the fast inward current did not change whereas the delayed outward current decreased with the external application of quinine. It is concluded that quinine is a potent repellent for Paramecium because it produces a depolarizing chemoreceptor potential which evokes action potentials and prolongs the duration of the action potential.     

Ionic mechanisms of depolarizing and hyperpolarizing quinine receptor potentials in Paramecium caudatum

The ionic mechanisms of the depolarizing and the hyperpolarizing quinine receptor potentials in the ciliate Paramecium caudatum were examined by using a behavioral mutant strain. The depolarizing receptor potential was induced by stimulating the anterior end of the specimen, and the hyperpolarizing receptor potential by stimulating the posterior end. The amplitude of both the depolarizing and the hyperpolarizing receptor potentials increased linearly with logarithmic increase in quinine concentration applied. Threshold concentration for inducing the depolarizing receptor potential was lower than that for the hyperpolarizing one. The peak level of the depolarizing receptor potential shifted towards the depolarizing direction with increasing external Ca²⁺ concentration while that of the hyperpolarizing receptor potential shifted in the depolarizing direction with increasing external K⁺ concentration. Under voltage-clamp conditions, the specimen produced an inward current in response to anterior stimulation, and an outward current in response to posterior stimulation. Both the peak inward and the peak outward currents showed a linear relationship with membrane potential. Current-voltage relationships of the receptor currents indicated conductance increase during the application of quinine. The depolarizing quinine receptor potential appears to be produced by an activation of Ca²⁺ channels, and the hyperpolarizing quinine receptor potential by an activation of K⁺ channels. Accepted: 3 October 1997     

K<Superscript>+</Superscript>-induced Ca<Superscript>2+</Superscript> Conductance Responsible for the Prolonged Backward Swimming in K<Superscript>+</Superscript>-agitated Mutant of <Emphasis Type="Italic">Paramecium caudatum</Emphasis>

The K⁺-agitated (Kag) mutant of Paramecium caudatum shows prolonged backward swimming in K⁺-rich solution. To understand the regulation mechanisms of the ciliary motility in P. caudatum, we examined the membrane electrical properties of the Kag mutant. The duration of the backward swimming of the Kag in K⁺-rich solution was about 10 times longer than that of the wild type. In response to an injection of the outward current, the wild type produced an initial action potential and a subsequent membrane depolarization due to I-R potential drop, while the Kag exhibited repetitive action potentials during the depolarization. Under voltage-clamp conditions, the depolarization-activated transient inward current exhibited by the Kag was slightly smaller than that exhibited by the wild type. In response to an application of K⁺-rich solution, both the wild type and the Kag exhibited a depolarizing afterpotential representing the activation of the K⁺-induced Ca²⁺ conductance. The inactivation time course of the K⁺-induced Ca²⁺ conductance of Kag was about 10 times longer than that of the wild type. This difference corresponds well with the difference in behavioral responses between Kag and wild type to K⁺-rich solution. We conclude that the overreaction of the Kag mutant to the K⁺-rich solution is caused by slowing down of the inactivation of the K⁺-induced Ca²⁺ conductance.     

L-Glutamate-induced membrane hyperpolarization and behavioural responses inParamecium tetraurelia

Summary Paramecium tetraurelia is attracted to L-glutamic acid concentrations of 10^–9 M to 10^–4 M in a behavioural assay. Electrophysiological studies show thatP. tetraurelia responds to L-glutamate application with hyperpolarization. This response is transient, even in the continued presence of the stimulus. The concentration dependence of the membrane potential response is similar to that of the behavioural responses, although the threshold concentration of L-glutamate required for hyperpolarization is three orders of magnitude lower than for attraction. The membrane potential response to L-glutamate persists following artificial deciliation ofP. tetraurelia.While application of L-glutamate toP. tetraurelia invariably elicits a hyperpolarization, withdrawal of the stimulus frequently results in a second transient membrane response, in the form of either a hyperpolarization or a depolarization. It is suggested that these off-responses may have a significant role in maintaining a behavioural response to L-glutamate.Abbreviation I_che index of chemokinesis     

Membrane potential responses of paramecium caudatum to external Na+

The membrane potential responses of Paramecium caudatum to Na+ ions were examined to understand the mechanisms underlying the sensation of external inorganic ions in the ciliate by comparing the responses of the wild type and the behavioral mutant. Wild-type cells exhibited initial continuous backward swimming followed by repeated transient backward swimming in the Na+-containing test solution. A wild-type cell impaled by a microelectrode produced initial action potentials and a sustained depolarization to an application of the test solution. The prolonged depolarization, the depolarizing afterpotential, took place subsequently after stimulation. The ciliary reversal of the cell was closely associated with the depolarizing responses. When the application of the test solution was prolonged, the wild-type cell produced sustained depolarization overlapped by repeated transient depolarization. A behavioral mutant defective in the Ca2+ channel, CNR (caudatum non reversal), produced a sustained depolarization but no action potential or depolarizing afterpotential. The mutant cell responded to prolonged stimulation with sustained depolarization overlapped by transient depolarization, although it did not show backward swimming. The results suggest that Paramecium shows at least two kinds of membrane potential responses to Na+ ions: a depolarizing afterpotential mediating initial backward swimming and repeated transient depolarization responsible for the repeated transient backward swimming.     

Dynamic behaviour of stationary pronuclei during their positioning in Paramecium caudatum

During conjugation of Paramecium caudatum, there are two well-known stages when nuclear migration occurs. What happens to the nuclei is closely related to their localisations in cells. The first of these stages is the entrance of one meiotic product into the paroral region. This nucleus survives, while the remaining three outside this area degenerate. The second stage is the antero-posterior localisation of eight synkaryon division products. Four posterior nuclei are differentiated into macronuclear anlagen, whereas four anterior nuclei remain as the presumptive micronuclei. In this experiment, the process of the third prezygotic division of P. caudatum was studied with the help of protargol staining. Here, a third nuclear migration was discovered. By two spindle turnings and two spindle elongations, stationary pronuclei were positioned near migratory pronuclei. This positioning of stationary pronuclei could shorten the distance for transferred migratory pronuclei to recognise and reach the stationary pronuclei. This fosters the synkaryon formation of P. caudatum.     

Electrophysiological properties ofDictyostelium derived from membrane potential measurements with microelectrodes

Summary Electrical membrane properties of the cellular slime moldDictyostelium discoideum were investigated with the use of intracellular microelectrodes. The rapid potential transients (1 msec) upon microelectrode penetration of normal cells had a negative-going peak-shaped time course. This indicates that penetration of a cell with a microelectrode causes a rapid depolarization, which can just be recorded by the microelectrode itself. Therefore, the initial (negative) peak potential transient valueE _p (–19 mV) should be used as an indicator of the resting membrane potentialE _m ofD. discoideum before impalement, rather than the subsequent semistationary depolarized valueE _n (–5 mV). Using enlarged cells such as giant mutant cells (E _p=–39 mV) and electrofused normal cells (E _p=–30 mV) improved the reliability ofE _p as an indicator ofE _m. From the data we concluded thatE _m ofD. discoideum cells bathed in (mm) 40 NaCl, 5 KCl and 1 CaCl₂ is at least –50 mV. This potential was shown to be dependent on extracellular potassium. The average input resistanceR _i of the impaled cells was 56 M for normalD. discoideum. However, our analysis indicates that the membrane resistance of these cells before impalement is >1 G. Specific membrane capacitance was 1–3 pF/cm². Long-term recording of the membrane potential showed the existence of a transient hyperpolarization following the rapid impalement transient. This hyperpolarization was associated with an increase inR _i of the impaled cell. It was followed by a depolarization, which was associated with a decrease inR _i. The depolarization time was dependent on the filling of the microelectrode. The present characterization of the electrical membrane properties ofDictyostelium cells is a first step in a membrane electrophysiological analysis of signal transduction in cellular slime molds.     

Cesium,Na+-K+ pump and pacemaker potential in cardiac purkinje fibers

The mechanisms of the hyperpolarizing and depolarizing actions of cesium were studied in cardiac Purkinje fibers perfused in vitro by means of a microelectrode technique under conditions that modify either the Na⁺-K⁺ pump activity or I_f. Cs⁺ (2 mM) inconsistently increased and then decreased the maximum diastolic potential (MDP); and markedly decreased diastolic depolarization (DD). Increase and decrease in MDP persisted in fibers driven at fast rate (no diastolic interval and no activation of I_f). In quiescent fibers, Cs⁺ caused a transient hyperpolarization during which elicited action potentials were followed by a markedly decreased undershoot and a much reduced DD. In fibers depolarized at the plateau in zero [K⁺]_o (no I_f), Cs⁺ induced a persistent hyperpolarization. In 2 mM [K⁺]_o, Cs⁺ reduced the undershoot and suppressed spontaneous activity by hyperpolarizing and thus preventing the attainment of the threshold. In 7 mM [K⁺]_o, DD and undershoot were smaller and Cs⁺ reduced them. In 7 and 10 mM [K⁺]_o, Cs⁺ caused a small inconsistent hyperpolarization and a net depolarization in quiescent fibers; and decreased MDP in driven fibers. In the presence of strophanthidin, Cs⁺ hyperpolarized less. Increasing [Cs⁺]_o to 4, 8 and 16 mM gradually hyperpolarized less, depolarized more and abolished the undershoot. We conclude that in Purkinje fibers Cs⁺ hyperpolarizes the membrane by stimulating the activity of the electrogenic Na⁺-K⁺ pump (and not by suppressing I_f); and blocks the pacemaker potential by blocking the undershoot, consistent with a Cs⁺ block of a potassium pacemaker current.     

Inositol trisphosphate-induced hyperpolarization in rat dorsal root ganglion neurons.

Inositol 1,4,5-trisphosphate (1,4,5-InsP3) was perfused into rat dorsal root ganglion (DRG) neurons by whole-cell patch-clamp electrodes, while measuring the membrane potential. This operation evoked a transient (2-3 min) membrane hyperpolarization of about -15 mV (from -42 mV) followed by a depolarization. The membrane hyperpolarization was abolished when 30 mM EGTA was perfused together with 1,4,5-InsP3 or when 0.2 mM quinine was added to the bath solution. The hyperpolarizing response was enhanced when a low-Ca2+ EGTA-free intracellular solution was used. Two InsP2 isomers induced a different response. Our results suggest that the hyperpolarization is due to 1,4,5-InsP3-induced Ca2+ release which may trigger Ca-sensitive K+ channels to open. Present results show that cultured DRG neurons are able to respond to 1,4,5-InsP3 perfusion in the whole-cell configuration.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

Effects of anoxia and high CO2 concentration on the electrogenic activity of leaf cell membrane in the dark

Resting membrane potential of a single cell in Antirrhinum leafdepolarized rapidly by about 40% under anoxia in the dark andrecovered to the normal level with reaeration. V_m in the dark comprised two fractions; one was passive diffusionpotential and the other was intimately dependent on aerobicmetabolism. The rapidity of the depolarization due to anoxiasuggested the possible functioning of an electrogenic mechanism,although the level of V_m was not much higher than the equilibriumpotential of K⁺ across membrane. 50% CO₂ in air brought about slightly more complex responses:a slight transient hyperpolarization and a subsequent depolarization.Sometimes one more transient depolarization took place as a"CO₂-off" effect. The total depolarization span was equal tothat caused by anoxia. 50% CO₂ under anoxia did not evoke furtherdepolarization but only hyperpolarization. (Received July 7, 1976; )     

A mutant of Paramecium with increased relative resting potassium permeability

Fast-2, a membrane mutant of Paramecium aurelia, is due to a single-gene mutation and has behavioral abnormalities. Intracellular recordings through changes of external solutions were made. The mutant membrane hyperpolarized when it encountered solutions with low K⁺ concentration. This hyperpolarization and other associated activities were best observed in Ca- or Na-solutions devoid of K⁺. Membrane potential was plotted against the concentration of K⁺ (0.5 to 16 mM) in solutions of fixed Na⁺ or Ca⁺⁺ concentration. The slopes of the curves for the mutant membrane were steeper than those for the wild type at the lower concentrations of K⁺. Inclusion of 2 mM tetraethylammonium chloride (TEA-Cl) counteracted the mutational effects. Spontaneous action potentials in Ba-solution and the electrically evoked action potentials in various solutions are normal in this mutant. We conclude that the resting permeability to K⁺ relative to the permeabilities to Na⁺ and Ca⁺⁺ has been increased by the mutation.     

Facial nerve sensory responses recorded from the geniculate ganglion ofGallus gallus var.domesticus

Summary The extracellular responses of single sensory afferent cell bodies were recorded from the geniculate ganglion of the chicken following chemical, mechanical and thermal stimulation of the oral cavity using glass coated tungsten microelectrodes. Forty eight chemoreceptive units were identified from the anterior and posterior palate, and from the anterior mandibular area of the lower jaw. Their response characteristics to tyrode Ringer solution, distilled water, 0.05M hydrochloric acid, 0.5M sodium chloride, 1M fructose and 0.05M quinine hydrochloride were investigated. Only 5 units responded to a single stimulus and all of the other units responded to 2 or more stimuli. Thirty seven of the units which did not show single stimulus specificity did however respond best to one of the stimuli tested. The firing rates of these chemoresponsive units was slow, they showed little or no spontaneous activity and showed variable response patterns.Rapidly adapting and slowly adapting mechanoreceptors were also identified together with thermoreceptors (cold and warm units) and ear units.The results show that the facial nerve plays the major role in gustatory physiology of the chicken and these results are discussed in relation to the mammalian gustatory system.Abbreviations AMA anterior mandibular area - AP anterior palate - PP posterior palate - QHCl quinine hydrochloride     

Transient and sustained effects of hormones and calcium on membrane potential in a bone cell clone

Measurements were made of the electrophysiological and cAMP response to changes in extracellular [Ca2+] and to hormone application in a bone cell clone. Both transient and long-term electrophysiological responses were studied. An increase in extracellular [Ca2+] usually resulted in a transient hyperpolarization of about 60-sec duration. In addition, increases in extracellular [Ca2+] from 0.9 to 1.8 mM and from 1.8 to 3.6 mM resulted in long-term hyperpolarization and increased potential fluctuations. Increasing bathing [Ca2+] until the membrane potential reached the K+ equilibrium level resulted in a significant decrease in fluctuations. Addition to the bathing medium of quinine, a putative blocker of the Ca2+-dependent K+ channel, resulted in long-term depolarization of the mean membrane potential, and a long-term decrease in potential fluctuations. Addition of Mg2+, a mild antagonist of Ca2+ entry into the cell, produced transient depolarization and reduction of potential fluctuations. These effects suggest that the potential fluctuations reflect cytoplasmic [Ca2+] fluctuations via Ca2+-dependent K+ membrane channels. Under an extracellular [Ca2+] of 1.8 mM, the application of prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone produced no significant effect on mean membrane potential or on the sustained potential fluctuations, but PGE2 did significantly raise intracellular cAMP. Under an increased bathing [Ca2+], significant changes in mean potential and fluctuations did occur in response to PGE2, but not in response to the other hormones, while the PGE2 effect on cAMP was not greatly changed. Hyperpolarizing transients of about 30-sec duration occurred in response to all of the hormones, particularly at an extracellular [Ca2+] of 3.6 mM. Thus, there are both transient and long-term electrophysiological responses to hormone application, with only the long-term response correlated with the production of cAMP. These electrophysiological responses may represent separate transient and long-term calcium transport responses to hormone application.     

Genetic control of lactate dehydrogenase isozymes in Paramecium caudatum

The LDH isozymes were surveyed in bacterized cultures of syngens 1, 3, 12, and 13 of Paramecium caudatum by polyacrylamide gel electrophoresis. All the examined stocks of different syngens except one stock in syngen 3 had a single common LDH isozyme, and intra- and intersyngenic variation was not observed except for the one stock in syngen 3. Breeding data using the exceptional stock indicated that the LDH isozymes of P. caudatum are controlled by two codominant alleles at a single locus whose products aggregate randomly, forming a dimer.     

Activation by Angiotensin II of Ca2+-dependent K+ and Cl− Currents in Zona Fasciculata Cells of Bovine Adrenal Gland

The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10⁻⁵ m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl⁻-free solution, the outward current reversed at −78.5 mV, a value close to E _K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K⁺-free solution, the transient outward current, sensitive to Cl⁻ channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E _Cl. Its magnitude changed in the same direction as the driving force for Cl⁻. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca²⁺-free solution (47% of cells) and was reversibly blocked by Cd²⁺ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca²⁺ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca²⁺-dependent K⁺ and Cl⁻ currents. These two membrane currents are co-activated in response to an internal increase of [Ca²⁺] _i originating from intra- and extracellular stores. Received: 29 May 1997/Revised: 4 November 1997     

Depolarization-induced changes in neurite elongation and intracellular Ca2+ in isolated Helisoma neurons

This study focuses on the effects of K⁺ depolarization on neurite elongation of identified Helisoma neurons isolated into culture. Application of K⁺ to the external medium caused a dose-dependent suppression of neurite elongation. Lower concentrations of K⁺ were associated with a slowing in the rate of neurite elongation, whereas higher concentrations produced neurite retraction. Surprisingly, the effects of K⁺ depolarization were transient, and neurite elongation rates recovered towards control levels within 90 min even though the neurons remained in high-K⁺ solution. Identified neurons differed in the magnitude of their response to K⁺ depolarization; neurite elongation of buccal neuron B4 was inhibited at 5 mM K⁺, but elongation in B5 and B19 was not affected until concentrations of 25 mM. Electrophysiologically, K⁺ application evoked a brief period (5–10 s) of action potential activity that was followed by a steady-state membrane depolarization lasting 2 h or more. The changes in neurite elongation induced by K⁺ depolarization occurred in isolated growth cones severed from their neurites and were blocked by application of calcium antagonists. Intracellular free Ca²⁺ levels in growth cones of B4 and B19 increased and then decreased during the 90-min depolarization, corresponding to the changes in elongation. B4 and B19 showed differences in the magnitude, time course, and spatial distribution of the Ca²⁺ change during depolarization, reflecting their different sensitivities to depolarization.     

Light-induced membrane potential changes of epidermal and mesophyll cells in growing leaves of Pisum sativum

Light transiently depolarizes the membrane of growing leaf cells. The ionic basis for changes in cell membrane electrical potentials in response to light has been determined separately for growing epidermal and mesophyll cells of the argenteum mutant of pea (Pisum sativum L.). In mesophyll cells light induces a large, transient depolarization that depends on the external Cl^– concentration, is unaffected by changes in the external Ca²⁺ or K⁺ concentration, is stimulated by K⁺-channel blockers tetraethylammonium (TEA⁺) and Ba²⁺, and is inhibited by 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU). In isolated epidermal tissue, light induces a small, transient depolarization followed by a hyperpolarization of the membrane potential. The depolarization is enhanced by increasing the external Ca²⁺ concentration and by addition of Ba²⁺, and is not sensitive to DCMU. Epidermal cells in contact with mesophyll display a depolarization resembling the response of the underlying mesophyll cells. The light-induced depolarization in mesophyll cells seems to be mediated by an increased efflux of Cl^– while the membrane-potential changes in epidermal strips reflect changes in the fluxes of Ca²⁺ and in the activity of the proton-pumping ATPase.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - CCCP carbonylcyanide m-chlorophenylhydrazone - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - LID _e light-induced depolarization in epidermal cells - LID _m light-induced depolarization in mesophyll cells - LIH light-induced hyperpolarization - TEA⁺ tetraethylammonium Ecotrans paper #43. This research was supported by National Science Foundation grants DCB-8903744 and MCB-9220110 to E.V.     

Voltage-gated ion conductances corresponding to regenerative positive and negative spikes in the dinoflagellateNoctiluca miliaris

Depolarization-activated and hyperpolarization-activated ion conductances in the membrane of a marine dinoflagellateNoctiluca miliaris were examined under voltage-clamp conditions.Noctiluca exhibited a transient inward current in response to a step depolarization from a holding potential level of –80 mV to a potential level more positive than –50 mV. The I–V relationship for the current exhibited typical N-shaped characteristics similar to those of most excitable membranes. The current was inactivated by a membrane depolarization. The reversal potential of the current shifted in hyperpolarizing direction when the external Na⁺ concentration was lowered. The transient inward current is assumed to be responsible for the Na⁺-dependent positive spike in non-clamped specimens ofNoctiluca.Noctiluca exhibited a transient outward current in response to a step hyperpolarization from a holding potential level of –20 mV to a potential level more negative than –30 mV. The I–V relationship for the current was a typical N-shape as if it was turned 180° around its origin. The outward current showed a two-step exponential time-decay. The outward current was inactivated by a membrane hyperpolarization. The reversal potential shifted in the depolarizing direction when the external Cl^– concentration was lowered. The transient outward current is responsible for the Cl^–-dependent negative spike in non-clamped specimens ofNoctiluca.Abbreviations ASW artificial seawater - TRP tentacle regulating potentials - TTX tetrodotoxin     

Mating agglutination of cilia detached from complementary mating types of Paramecium

Cell-free preparations of mating-reactive cilia were obtained from complementary mating types of Paramecium caudatum with the treatment of the cells in a Triton X-100 solution and then in a Ca solution. When the cilia from both mating types thus obtained were mixed, strong agglutination was observed.