Search PubMed⌕ Search

Biomedical subjects

C Kung

Publications and source records attributed to C Kung.

At least 91 records · Page 5Linked to original sources
PubMed1990

Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes.

The patch-clamp technique was used to examine the plasma membranes of sensitive yeast spheroplasts exposed to partially purified killer toxin preparations. Asolectin liposomes in which the toxin was incorporated were also examined. Excised inside-out patches from these preparations often revealed at 118 pS conductance appearing in pairs. The current through this conductance flickered rapidly among three states: dwelling mostly at the unit-open state, less frequently at the two-unit-open state, and more rarely at the closed state. Membrane voltages from -80 to 80 mV had little influence on the opening probability. The current reversed near the equilibrium potential of K+ in asymmetric KCl solutions and also reversed near O mV at symmetric NaCl vs. KCl solutions. The two levels of the conductance were likely due to the toxin protein, as treatment of spheroplasts or liposomes with extracellular protein preparations from isogenic yeasts deleted for the toxin gene gave no such conductance levels. These results show that in vivo the killer-toxin fraction can form a cation channel that seldom closes regardless of membrane voltage. We suggest that this channel causes the death of sensitive yeast cells.

Cell Membrane↗
PubMed1989

Voltage-sensitive ion channel of Escherichia coli.

A voltage-sensitive, cation-selective ion channel of Escherichia coli has been reconstituted into liposomes and studied with the patch-clamp method. The single channel conductance was 91 pS in symmetric solutions of 150 mM KCl. Many channels were open most of the time, with frequent brief transitions to closed levels. Multiple conducting units could close and reopen simultaneously, and this apparent cooperativity in gating was increased with depolarizing voltages. Above a voltage threshold, the channels closed irreversibly, often in groups.

Bacterial Outer Membrane Proteins↗
PubMed1989

Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels.

We have modified the procedure of Criado and Keller (1987) to study ion channels of Escherichia coli reconstituted in liposomes. The modifications include (a) excluding the use of any detergent and (b) inducing blisters from liposomes with Mg2+. These blisters, which appear to be unilamellar, are stable for hours. They could be repeatedly sampled with different patch-clamp pipettes each achieving seal resistance greater than 10 GOhms. Activities of three types of ion channels are often observed by use of this method, including two voltage-sensitive cation channels of different conductances. Even the mechanosensitive channel, previously recorded from live E. coli cells (Martinac et al., 1987), was also detected in these blisters. Apparently the channel protein and any accessory structures, postulated to be needed for mechanotransduction, can be reconstituted together by this method.

Cell Membrane↗
PubMed1989

In vivo mutations of calmodulin: a mutant Paramecium with altered ion current regulation has an isoleucine-to-threonine change at residue 136 and an altered methylation state at lysine residue 115.

The Paramecium tetraurelia mutants termed pantophobiacs have altered behavior due to perturbed calcium activation of ion channel activity. The calmodulin from pantophobiac A1 (pntA1) was shown in previous studies to have a single amino acid change at residue 101 that is selective in its effects on activity. This change has no effect on posttranslational modifications. However, the calmodulin from the phenotypically related mutant pantophobiac A2 (pntA2) has a threonine residue at position 136, in the fourth calcium-binding domain, instead of an isoleucine or valine like all other calmodulins. This region of the calmodulin structure is within 4 A of a complementary hydrophobic structure in the third calcium-binding domain, raising the possibility of a perturbation of interdomain interactions in the pntA2 mutant. This possibility is supported by the heterogenous methylation state of lysine-115 in the pntA2 calmodulin. This lysine residue, located in the peptide connecting calcium-binding domains three and four, is fully trimethylated in the wild-type and pntA1 calmodulins. The functional selectivity of these structural changes is demonstrated by the conservation of calmodulin activator activity with a calmodulin-regulated protein kinase that has been used as a standard of comparison. Overall, these results indicate the degree to which the calmodulin can be mutated in vivo without being lethal to the organism, and they provide genetic evidence suggesting that the post-translational methylation state of residue 115 requires the appropriate conformation in addition to the local amino acid sequence.

Amino Acid Sequence↗
PubMed1988

A mechanosensitive ion channel in the yeast plasma membrane.

Mechanosensitive ion channels use mechanical energy to gate the dissipation of electrochemical gradients across cell membranes. This function is fundamental to physiological processes such as hearing and touch. In electrophysiological studies of ion channels in the plasma membrane of the yeast Saccharomyces cerevisiae, channels were observed that were activated by, and adapted to, stretching of the membrane. Adaptation of channel activity to mechanical stimuli was voltage-dependent. Because these mechanosensitive channels pass both cations and anions, they may play a role in turgor regulation in this walled organism.

Adaptation, Physiological↗
PubMed1988

Osmotaxis in Escherichia coli.

The escape of motile organisms from high concentrations of chemicals was studied in Escherichia coli. We have found all chemicals tested to be osmorepellents. It was shown in both a spatial assay and a temporal assay that the known sensory receptors for chemotaxis are not used for osmotaxis, so a different sensory mechanism appears to be employed. According to the temporal assay, the mechanism between sensory receptors and flagella is also not used for tumbling response (at least in solutions above 0.4 osmolar).

Chemotaxis↗
PubMed1987

Biochemical characterization of a genetically altered calmodulin in Paramecium.

Recent evidence proposes that the calcium-binding protein, calmodulin, plays a crucial role in the regulation or modulation of the calcium-dependent potassium conductance in Paramecium tetraurelia (Hinrichsen, R.D., Burgess-Cassler, A., Soltvedt, B.C., Hennessey, T. and Kung, C. (1986) Science 323, 503-506). We purified the calmodulins from both the wild type and pantophobiac A (a mutant lacking the above-mentioned conductance and whose phenotypic defect is traceable to its calmodulin) by hydrophobic interaction and immunoaffinity chromatographies, and examined them biochemically. In this paper we address the preliminary characterization of the two calmodulins and discuss the consequences of the genetic alteration. The differences described here are in their electrophoretic mobilities in polyacrylamide gel electrophoresis and in their binding characteristics to monoclonal antibodies raised against calmodulin from wild-type paramecia. Also, we present data which indicate a difference in the stimulation of the calmodulin-dependent enzyme bovine brain phosphodiesterase under certain conditions.

Amino Acid Sequence↗
PubMed1987

A calcium-dependent potassium current is increased by a single-gene mutation in Paramecium.

The membrane currents of wild type Paramecium tetraurelia and the behavioral mutant teaA were analyzed under voltage clamp. The teaA mutant was shown to have a greatly increased outward current which was blocked completely by the combined use of internally delivered Cs+ and external TEA+. This, along with previous work (Satow, Y., Kung, C., 1976, J. Exp. Biol. 65:51-63) identified this as a K+ current. It was further found to be a calcium-activated K+ current since this increased outward K+ current cannot be elicited when the internal calcium is buffered with injected EGTA. The mutation pwB, which blocks the inward calcium current, also blocks this increased outward K+ current in teaA. This shows that this mutant current is activated by calcium through the normal depolarization-sensitive calcium channel. While tail current decay kinetic analysis showed that the apparent inactivation rates for this calcium-dependent K+ current are the same for mutant and wild type, the teaA current activates extremely rapidly. It is fully activated within 2 msec. This early activation of such a large outward current causes a characteristic reduction in the amplitude of the action potential of the teaA mutant. The teaA mutation had no effect on any of the other electrophysiological parameters examined. The phenotype of the teaA mutant is therefore a general decrease in responsiveness to depolarizing stimuli because of a rapidly activating calcium-dependent K+ current which prematurely repolarizes the action potential.

Animals↗
PubMed1987

A mutant Paramecium with a defective calcium-dependent potassium conductance has an altered calmodulin: a nonlethal selective alteration in calmodulin regulation.

The Paramecium mutant, pantophobiac A, has a defect that results in an in vivo loss of calcium-dependent potassium efflux channel activity. This defect is corrected fully by the microinjection of wild-type Paramecium calmodulin into pantophobiac A cells and is partially restored by calmodulins from other organisms, but it cannot be restored by microinjection of pantophobiac calmodulin. Overall, these results suggested that wild-type Paramecium calmodulin has unique features that allow it to restore fully a normal phenotype and that the defect in pantophobiac A might be an altered calmodulin molecule. Previous studies established the amino acid sequence of wild-type calmodulin and showed that Paramecium calmodulin has several differences from other calmodulins, including the presence of dimethyllysine at residue 13. To test directly the possibility that calmodulin from the pantophobiac mutant might be altered, we purified the mutant calmodulin and compared its properties to those of wild-type Paramecium calmodulin. We found one amino acid sequence difference between the two Paramecium calmodulins: a phenylalanine in the mutant protein, instead of a serine, at residue 101. This change is at a calcium-liganding residue in the third calcium-binding loop. These and previous studies demonstrate that comparatively subtle changes in the structure of calmodulin can result in quantitative alterations in in vivo activity, provide insight into the in vivo roles of calmodulin and the regulation of ion channels, and demonstrate that functional alterations of calmodulin are not necessarily lethal.

Amino Acid Sequence↗
PubMed1987

Pressure-sensitive ion channel in Escherichia coli.

We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherichia coli and have discovered pressure-activated ion channels. The channels have the following properties: activation by slight positive or negative pressure; voltage dependence; large conductance; selectivity for anions over cations; dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.

Electric Conductivity↗
PubMed1986

Ion channels in yeast.

Voltage-dependent ion channels have been found in the plasma membrane of the yeast Saccharomyces cerevisiae. Ion channel activities were recorded from spheroplasts or patches of plasma membrane with the patch-clamp technique. The most prominent activities came from a set of potassium channels with the properties of activation by positive but not negative voltages, high selectivity for potassium over sodium ion, unit conductance of 20 picosiemens, inhibition by tetraethylammonium or barium ions, and bursting kinetics.

Cell Membrane↗
PubMed1986

Restoration by calmodulin of a Ca2+-dependent K+ current missing in a mutant of Paramecium.

A combination of genetics, biochemistry, and biophysics was used to show that calmodulin is involved in the regulation of an ion channel. Calmodulin restored the Ca2+-dependent K+ current in pantophobiac, a mutant in Paramecium that lacks this current. The restoration of the current occurred within 2 hours after the injection of 1 picogram of wild-type calmodulin into the mutant. The current remained for approximately 30 hours before the mutant phenotype returned. The injection of calmodulin isolated from pantophobiac had no effect. These results imply that calmodulin is required for the function or regulation of the Ca2+-dependent K+ current in Paramecium.

Calcium↗
PubMed1986

A mutation that increases a novel calcium-activated potassium conductance of Paramecium tetraurelia.

Under two-electrode voltage clamp, a mutant of P. tetraurelia, restless (rst/rst), showed a large increase in induced current and an outward tail current when compared to the wild-type cell for hyperpolarizing voltage steps. An increase in the induced and tail currents is also observed for depolarizing voltage steps. The larger current during voltage steps and tail in the mutant were eliminated by the use of CsCl-filled electrodes and tetraethylammonium ion (TEA+) in the bath solution, characterizing the lesion as affecting a K+ conductance. Ionophoretic injection of ethylene glycol bis-(beta-aminoethyl ether) n,n,n',n-tetraacetic acid (EGTA) to buffer internal Ca2+ concentration reduced the increased K+ current and tail of the restless cell, indicating Ca2+ activation of the K+ current. Time course and amplitude of remaining currents after blockage of K+ conductances with Cs+ and TEA+ were similar in wild-type and restless cells suggesting no restless defect in entry of calcium. The Ca2+-activated sodium current was similar in the mutant to that in wild type arguing against a defect in calcium regulation activating the K+ channel in the restless cell. We conclude that the restless mutation alters a Ca2+-activated potassium conductance other than the one previously described. The multiplicity of Ca2+-activated potassium conductances in Paramecium is discussed.

Animals↗
PubMed1986

Mutations resulting in resistance to polyene antibiotics decrease voltage-sensitive calcium channel activity in Paramecium.

In this report, the isolation of Paramecium tetraurelia mutants resistant to the polyene antibiotics amphotericin B and filipin are described. These antibiotics are known to specifically interact with membrane sterols to produce a cytotoxic effect. Four mutants resistant to amphotericin B and two mutants resistant to filipin have been isolated. In each case, an individual mutant shows resistance to both amphotericin B and filipin. Genetic analysis indicates that all 6 mutations map to the same complementation group and are not allelic to any of the 3 pawn mutations or to either of the two "barium shy" mutations. The behavioral analysis suggests that these mutations cause an alteration of normal ion channel function. Direct electrophysiological analysis of one mutant indicates mutations to polyene antibiotic resistance cause a specific decrease in voltage-sensitive Ca2+ channel activity.

Amphotericin B↗