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C Kung

Publications and source records attributed to C Kung.

At least 127 records · Page 7Linked to original sources
PubMed1981

Ionic channels of Paramecium: from genetics and electrophysiology to biochemistry.

This paper reviews the combined genetical, electrophysiological and biochemical analysis of excitation that has been carried out in Paramecium. Paramecium cells display graded Ca++ action potentials in response to a variety of stimuli. These action potentials regulate the orientation of the ciliary beat hence the cell's swimming behaviour. A large array of mutants displaying altered behaviour have been isolated and mapped to over 20 loci. Detailed electrophysiological analyses have been carried out on several classes of mutants revealing defects in specific ion channels in some cases. Mutants have proven very useful to analyze channel properties, to unravel interactions between channels and to discover the function of these channels in a variety of cellular processes. Some important channels are located in the ciliary membranes and cilia as well as ciliary membranes can now be purified in high purity and reasonable yield. These fractions have been used recently in a variety of biochemical approaches to gain insight into the molecular components of the excitation machinery. Specific alterations in some minor membrane proteins have been found in two mutants as well as a specific defect in sphingolipids in a third mutant. Those alterations had to be distinguished from large scale variations in membrane proteins and lipids that occur in this organism in response to modifications in growth conditions. Several other recent biochemical developments are reviewed and the advantages as well as the difficulties of the genetic approach to the molecular study of biological processes are discussed.

Animals↗
PubMed1980

Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes.

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.

Animals↗
PubMed1980

Rotation and twist of the central-pair microtubules in the cilia of Paramecium.

The orientation and configuration of the central-pair microtubules in cilia were studied by serial thin-section analysis of "instantaneously fixed" paramecia. Cilia were frozen in various positions in metachronal waves by such a fixation. The spatial sequence of these positions across the wave represents the temporal sequence of the positions during the active beat cycle of a cilium. Systematic shifts of central-pair orientation across the wave indicate that the central pair rotates 360 degrees counterclockwise (viewed from outside) with each ciliary beat cycle (C. K. Omoto, 1979, Thesis, University of Wisconsin, Madison; C. K. Omoto and C. Kung, 1979, Nature [Lond.] 279:532-534). This is true even for paramecia with different directions of effective stroke as in forward- or backward-swimming cells. The systematic shifts of central-pair orientation cannot be seen in Ni++-paralyzed cells or sluggish mutants which do not have metachronal waves. Both serial thin-section and thick-section high-voltage electron microscopy show that whenever a twist in the central pair is seen, it is always left-handed. This twist is consistent with the hypothesis that the central pair continuously rotates counterclockwise with the rotation originating at the base of the cilium. That the rotation of the central pair is most likely with respect to the peripheral tubules as well as the cell surface is discussed. These results are incorporated into a model in which the central-pair complex is a component in the regulation of the mechanism needed for three-dimensional ciliary movement.

Animals↗
PubMed1980

Membrane currents of pawn mutants of the pwA group in Paramecium tetraurelia.

Membrane currents were recorded from the wild type and two pawn mutants of the pwA complementation group in Paramecium tetraurelia under a voltage clamp. Most currents are not changed by the mutations. Transient inward currents of a leaky mutant, pwA132, upon step depolarizations are less than those in the wild type. The inward transient is completely lacking in a non-leaky mutant, pwA500. The time course of the residual inward currents in the leaky mutant is not significantly different from that of wild type. The voltage sensitivity of the Ca channels in the leaky mutant is also similar to that of wild type. The inward currents upon membrane hyperpolarizations in the mutants show normal characteristics in the presence or absence of external K+. With sufficiently large, prolonged depolarization, outward currents progressively develop in the wild type but decay in the mutants. The simplest conclusion we can draw is that the pwA mutations reduce the number of functional Ca channels but do not change the channel characteristics. From the conductance measurements, 45% of the Ca channels remain in the leaky mutant pwA132, and none remain in the non-leaky mutant pwA500. By subtracting the outward currents of pwA500 from the slow and prolonged outward currents of the wild type, we have tentatively separated a Ca-induced K+ current from the voltage-dependent K+ current. The time courses of these two currents differ by two orders of magnitude.

Animals↗
PubMed1980

Ba2+ influx measures the duration of membrane excitation in Paramecium.

We have developed an assay for the duration of membrane excitation in Paramecium tetraurelia by tracing the influx of 133Ba2+. This assay is performed at physiological temperatures and in physiological solutions. Ba2+ enters the paramecia through the Ca channels, since mutants defective in Ca channels show no significant Ba2+ influx. Ba2+ enters only when the Ca channels are opened during excitation which can be triggered by Na+ or Ba2+ itself. The ionic species and relative concentrations determine the duration of the action potentials and hence the duration of spinning or backward swimming. The longer the average period of excitation, the larger the Ba2+ influx. In a Ba-Ca solution the cells spend 30% of their time in the excited state. The rate of Ba2+ entry into the paramecia in that state is 1.3 mM/min. Ba2+ influx occurs over a 50-fold range of Ba2+ concentration. There is very little Ba2+ efflux. The fate of the entered Ba2+, the consequences of the large and rapid influx, the advantages and drawbacks of the Ba2+ influx assay, and the possible use of the assay for Ca channel function in cell-free preparations are discussed.

Animals↗
PubMed1980

Ca-induced K+-outward current in Paramecium tetraurelia.

Late K-outward currents upon membrane depolarization were recorded in Paramecium tetraurelia under a voltage clamp. A Ca-induced K-outward component is demonstrated by subtracting the value of the outward current in a pawn A mutant lacking functional Ca-channels (pwA500). The Ca-induced K-outward current activates slowly, reaching a peak after 100 to 1000 ms. The current then remains steady or reaches the steady state after a decline of several seconds. EGTA2-injection experiments show that the Ca-induced K-outward current is dependent on the internal Ca2+ concentration. The current is shown to depend on the voltage-dependent Ca conductance, by study of the leaky pawn A mutant (pwA132), which has a lowered Ca conductance as well as a lowered Ca-induced K-current. The Ca-induced GK is thus indirectly dependent on the voltage. The maximal GK is about 40 nmho/cell at +7 mV in 4 mM-K+. The Ca-induced K current is sustained throughout the prolonged depolarization and the prolonged ciliary reversal.

Animals↗
PubMed1980

A Ca-induced Na-current in Paramecium.

Under a voltage clamp, step depolarization and repolarization can induce a sustained inward current and a tail inward current in Paramecium tetraurelia bathed in a solution containing 8 mM-Na+. These currents are best seen in the 'paranoiac' mutant. The I-V plot of the sustained inward current can have a region of negative resistance around -20 mV. This current is absent when Na+ is excluded from the bath solution, and it increases as the Na+ concentration increases from 2 to 8 mM. Injection of Na+ into the cell suppresses this inward current. This current develops very slowly, reaching its maximum seconds after the step depolarization and decays with a time constant of hundreds of milliseconds after the repolarization. This slow current is dependent on Ca2+. It can be suppressed by reduction or deletion of external Ca2+ or by iontophoretic injection of EGTA. 'Pawn' mutants with defective Ca-conductance also lack this current. We conclude that Paramecium has a Ca-induced conductance through which the Na-current flows. Although more prominent in the 'paranoiac' mutant, this Ca-induced Na-current is also seen in the wild type. This conductance may function in generating plateau depolarizations lasting seconds or even minutes and the corresponding prolonged backward swimming away from sources of irritation and stress.

Animals↗
PubMed1978

A potassium conductance activated by hyperpolarization in paramecium.

Voltage clamp studies show that the wild-type membrane of Paramecium tetraurelia contains a conductance component which is sensitive to hyperpolarization. This component manifests itself as "anomalous", or "inward going", rectification of membrane voltage in response to applied constant current pulses and as a "hyperpolarizing spike" when no K is added to the external solution (Y. Satow, C. Kung, 1977. J. Comp. Physiol. 119:99). Like the conductances which underlie anomalous rectification in other cells, the hyperpolarization-sensitive conductance in Paramecium is specific for K, and the magnitude of the voltage-dependent conductance change depends not only on voltage but also on external potassium concentration. The internal potassium ion concentration of Paramecium is calculated to be between 17 and 18mM.

Animals↗
PubMed1978

K-resistant mutants and "adaptation" in Paramecium.

A class of mutants of Paramecium tetraurelia has been isolated by adding a critical amount of K(+) to mutagenized, autogamized cultures. Adding 35 mM K(+) to the medium kills the wild type. The mutants can grow at 35 mM K(+) and some survive an addition of up to 80 mM K(+). Thirty-three lines of such mutants have been studied. These mutants have few or no behavioral abnormalities in culture medium.Paramecia can "adapt." Hildebrand and Dryl showed that P. caudatum incubated in 15 mM K(+) for over 15 min fail to give avoiding reactions when challenged with Ba(2+) or a thermal gradient [Hildebrand, E. & Dryl, S. (1976) Bioelectrochem. Bioenerg. 3, 543-544]. We have observed a similar phenomenon in wild-type P. tetraurelia. However, the K(+)-resistant mutants show little or no such "adaptation," i.e., they perform avoiding reactions when challenged with Ba(2+) after prolonged incubation in 15 mM K(+). The K(+) resistance and the lack of "adaptation" are strictly correlated. Because all 33 lines of mutants show this correlation, "adaptation" and killing by K(+) must share a mechanism. This mechanism may be studied by finding the basis of the K(+) resistance in the mutants. The possible ionic bases of "adaptation" are discussed.

Journal Article↗
PubMed1978

Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia.

The morphology of the transition zone between the terminal plate of the basal body and the 9 + 2 region of the somatic (non-oral) cilium has been examined in Paramecium tetraurelia. Freeze-fracture and thin-section techniques disclosed both membrane specializations and various internal structural linkages. Freeze-fracture material revealed sets of particles interrupting the unit membrane. The more distal of these form plaquelike arrays while the proximal set of particles forms the ciliary "necklace." The plaque regions correspond to anionic sites on the outer membrane surface as revealed by binding of polycationic ferritin. Both the plaque particles and the necklace particles appear to be in contact with outer doublet microtubules via a complex of connecting structures. In the interior of the transition zone an axosomal plate supports an axosome surrounded by a ring of lightly packed material. Only one of the two central tubules of the axoneme reaches and penetrates the axosome. Below the axosomal plate four rings, each approx. 20 nm wide, connect adjacent outer doublets. An intermediate plate lies proximal to these rings, and a terminal plate marks the proximal boundary of this zone. Nine transitional fibers extend from the region of the terminal plate to the plasmalemma. The observations described above have been used to construct a three-dimensional model of the transition region of "wild-type" Paramecium somatic cilia. It is anticipated that this model will be useful in future studies concerning possible function of transition-zone specializations, since Paramecium may be examined in both normal and reversed ciliary beating modes, and since mutants incapable of reverse beating are available.

Animals↗
PubMed1977

Separation of membrane currents using a Paramecium mutant.

The net membrane current of the electrically excitable membrane of Paramecium during step depolarisations was measured using voltage-clamp techniques. The mutant, pawn B, lacks a functional Ca channel. Thus the difference between the total current measured in the wild type and the leakage and rectification currents measured in the pawn mutant is the Ca current. These findings were confirmed by ion substitution experiments. The possibility that inactivation may be apparent only, and caused by a hypothetical, superposed, Ca-induced, rectifying K+ current was eliminated.

Animals↗
PubMed1977

Genetic analyses of "paranoiac" mutants of Paramecium tetraurelia.

Six mutants of Paramecium tetraurelia with curious "Paranoiac" phenotypes have been isolated and examined. Instead of the normal transient avoiding reactions in Na+ solution, these mutants show "violent avoidances"--backing continuously for 10 to over 60 sec. This behavior corresponds to prolonged membrane excitation excitation.--Genetic analyses establish five genic loci at which mutations give the "Paranoiac" phenotype. Close linkage between two of these genes occurs. Allelic variants are found for two of the genes. In one case, the two alleles determine very different behavioral phenotypes ("Paranoiac" and "fast-2"). These results show that the mechanism(s) which shuts off excitation in the wild-type membrane is (are) complex, but in the future may be fruitfully pursued in mutants which are defective.

Animals↗
PubMed1976

Mutants with reduced Ca activation in Paramecium aurelia.

Two heat-sensitive "pawn" mutants of Paramecium aurelia are capable of avoiding reactions when grown at 23 degrees C but not at 35 degrees C. Electrophysiological analyses show that Ca activation is reduces in the mutants even when they are grown at 23 degrees C. The maximal rate of rise and the peak of the evoked action potential (Ca-spike) in the mutants are smaller than those of wild type in a K-solution. After suppression of K conductance by either TEA+ or Ba++, the action potentials of the mutants peak at the same level as that of wild type. However, the maximal rate of rise of the mutants remains only about half that of wild type. Thus, the mutations affect Ca activation but not K activation. Incubation at a high temperature (35 degrees C) further reduces Ca activation to almost zero in the mutants but has little or no effect on wild type. This almost complete loss of Ca activation explains the lack of avoiding reactions when the mutants are grown at high temperatures. A double mutant containing two heat-sensitive mutations shows extremely reduced Ca activation even when grown at 23 degrees C.

Action Potentials↗
PubMed1976

Defective ion regulation in a class of membrane-excitation mutants in Paramecium.

The "paranoiac" mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na+, unlike wild-type paramecia, which jerk back and forth in Na+ solutions. The paranoiac mutants in Na+ solutions also show large losses of cellular K+ and large influxes of Na+. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na+ -dependent loss of cellular K+ and a much smaller Na+ influx. In K+ -containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K+ content. The Na+ influx, the K+ loss, and the duration of backward swimming are all proportional to the extracellular Na+ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na+ influxes and the large K+ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.

Animals↗
PubMed1976

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.

Animals↗