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C Kung

Publications and source records attributed to C Kung.

At least 19 recordsLinked to original sources
PubMed1992

Membrane-derived oligosaccharides (MDO's) promote closing of an E. coli porin channel.

The outer membrane of Escherichia coli is a diffusion barrier for macromolecules, but allows the passage of small hydrophilic solutes through non-specific channels, the porins. Some electrophysiological studies find reconstituted porins in a mostly open state, while those done with the patch-clamp technique performed on live cells suggest that the vast majority of the native channels are closed. We present here current measurements through porins from reconstituted outer membrane, which demonstrate that bacterial metabolites, the MDO's, which bathe the periplasmic side of the outer membrane, induce the channels to close. These findings illustrate that the degree of openness of porins can be regulated by compounds naturally found in bacteria.

Bacterial Outer Membrane Proteins↗
PubMed1992

Yeast casein kinase I homologues: an essential gene pair.

We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. Increased dosage of YCK1 suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The two predicted protein products share 77% overall amino acid identity and contain sequence elements conserved among protein kinases. Partial sequence obtained for rabbit casein kinase I shares 64% identity with the two yeast gene products. Moreover, an increase in casein kinase I activity is observed in extracts from cells overexpressing YCK2. Thus YCK1 and YCK2 appear to encode casein kinase I homologues.

Amino Acid Sequence↗
PubMed1992

A mechanosensitive ion channel in Schizosaccharomyces pombe.

Protoplast protuberances (blebs) of Schizosaccharomyces pombe were examined using the patch-clamp technique. In addition to several voltage-gated ion channels, we encountered the activities of a mechanosensitive ion channel with a conductance of 180 pS. Microscopic currents of one or two units were observed in some excised patches and ensemble currents of several tens of units were observed in all blebs examined in whole-bleb configuration. This channel opens at pressures of cm Hg applied to whole blebs and it passes cations, including Ca2+. It is inactivated by membrane depolarizations and blocked by Gd3+. We discuss the possible functions of such a channel, including its activation upon cell cycle dependent cytoskeletal reorganizations.

Cell Membrane↗
PubMed1992

Receptor binding of asialoerythropoietin.

The interaction of 125I-asialoerythropoietin (asialoepo) with receptors has been characterized both by binding assay and affinity cross-linking. Purified spleen cells from mice infected with the anemia strain of Friend virus (FVA cells) have receptors for 125I-asialoepo with two classes of affinity constant: one with Kd = 0.02-0.03 nM and 300-400 per cell, the other with lower affinity (Kd = 0.9-1.2 nM) and 1,000-1,200 per cell. The Kd value for the high affinity site is one-third of that for the binding of native 125I-erythropoietin (125I-epo) to the same FVA cells (Kd = 0.08-0.1 nM). Using 125I-asialoepo or 125I-epo affinity cross-linking methods, we find two components with apparent molecular weights of 88 kDa and 105 kDa in FVA cells, and in the transformed mouse cell lines, 201, IW32, and NN10, in agreement with earlier studies using 125I-epo. These results indicate that 125I-asialoepo binds to the same receptors as 125I-epo, but with greater affinity for the high affinity site. Since 201 cells contain only a single class of lower affinity receptors for erythropoietin (epo), finding the same two components as found for FVA cells by cross-linking experiment indicates that the two components do not represent the two classes of receptor.

Animals↗
PubMed1992

Primary mutations in calmodulin prevent activation of the Ca(++)-dependent Na+ channel in Paramecium.

Paramecium tetraurelia behavioral mutant cam12 displays a "fast-2" behavioral phenotype: it fails to respond to Na+ stimuli. Electrophysiologically, it lacks a Ca(++)-dependent Na+ current. Genetics and DNA sequencing showed the primary defect of cam12 to be in the calmodulin gene (Kink et al., 1990). To correlate calmodulin structure and function in Paramecium, we elucidated the primary structure of cam12 calmodulin. Peptide sequencing confirmed the two point mutations predicted by the DNA sequence: a glycine-to-glutamate substitution at position 40 and an aspartate-to-asparagine substitution at position 50. Our results further showed that lysine 13 and lysine 115 were methylated normally in cam12. It is likely that the electrophysiological abnormalities of cam12 are a direct reflection of the amino-acid substitutions, as opposed to improper posttranslational modification.

Amino Acid Sequence↗
PubMed1992

Calcium current activated upon hyperpolarization of Paramecium tetraurelia.

Hyperpolarization of Paramecium tetraurelia under conditions where K+ currents are suppressed elicits an inward current that activates rapidly toward a peak at 25-80 ms and decays thereafter. This peak current (Ihyp) is not affected by removing Cl ions from the microelectrodes used to clamp membrane potential, or by changing extracellular Cl- concentration, but is lost upon removing extracellular Ca2+. Ihyp is also lost upon replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Co2+, Mg2+, Mn2+, or Sr2+, suggesting that the permeability mechanism that mediates Ihyp is highly selective for Ca2+. Divalent cations also inhibit Ihyp when introduced extracellularly, in a concentration- and voltage-dependent manner. Ba2+ inhibits Ihyp with an apparent dissociation constant of 81 microM at -110 mV, and with an effective valence of 0.42. Ihyp is also inhibited reversibly by amiloride, with a dissociation constant of 0.4 mM. Ihyp is not affected significantly by changes in extracellular Na+, K+, or H+ concentration, or by EGTA injection. Also, it is unaffected by manipulations or mutations that suppress the depolarization-activated Ca2+ current or the various Ca(2+)-dependent currents of Paramecium. We suggest that Ihyp is mediated by a novel, hyperpolarization-activated calcium conductance that is distinct from the one activated by depolarization.

Animals↗
PubMed1992

Calcium-dependent inactivation of the calcium current activated upon hyperpolarization of Paramecium tetraurelia.

The Ca2+ current activated upon hyperpolarization of Paramecium tetraurelia decays over a period of 150-200 ms during sustained steps under voltage clamp. At membrane potentials between -70 and approximately -100 mV, the time course of this inactivation is described by a single exponential function. Steps negative to approximately -100 mV elicit currents that decay biexponentially, however. Three lines of evidence suggest that this current's inactivation is a function of intracellular Ca2+ concentration rather than membrane potential: (a) Comparing currents with similar amplitudes but elicited at widely differing membrane potentials suggests that their time course of decay is a sole function of inward current magnitude. (b) The extent of current inactivation is correlated with the amount of Ca2+ entering the cell during hyperpolarization. (c) The onset and time course of recovery from inactivation can be hastened significantly by injecting cells with EGTA. We suggest that the decay of this current during hyperpolarization involves a Ca(2+)-dependent pathway.

Animals↗
PubMed1992

COT1, a gene involved in cobalt accumulation in Saccharomyces cerevisiae.

The COT1 gene of Saccharomyces cerevisiae has been isolated as a dosage-dependent suppressor of cobalt toxicity. Overexpression of the COT1 gene confers increased tolerance to cobalt and rhodium ions but not other divalent cations. Strains containing null alleles of COT1 are viable yet more sensitive to cobalt than are wild-type strains. Transcription of COT1 responds minimally to the extracellular cobalt concentration. Addition of cobalt ions to growth media results in a twofold increase in COT1 mRNA abundance. The gene encodes a 48-kDa protein which is found in mitochondrial membrane fractions of cells. The protein contains six possible membrane-spanning domains and several potential metal-binding amino acid residues. The COT1 protein shares 60% identity with the ZRC1 gene product, which confers resistance to zinc and cadmium ions. Cobalt transport studies indicate that the COT1 product is involved in the uptake of cobalt ions yet is not solely responsible for it. The increased tolerance of strains containing multiple copies of the COT1 gene is probably due to increased compartmentalization or sequestration of the ion within mitochondria.

Amino Acid Sequence↗
PubMed1991

Efficient transformation of cam2, a behavioral mutant of Paramecium tetraurelia, with the calmodulin gene.

An Ile-136----Thr substitution in calmodulin reduces the Ca(2+)-dependent K+ currents of cam2, a behavioral mutant of Paramecium tetraurelia, and renders it overly susceptible to BaCl2. DNA fragments carrying the wild-type CAM gene injected into cam2 macronuclei reverted these phenotypes in the clonal descendants of the recipients. Tetrahymena telomeric sequences, added in vitro to the fragment termini before injection, enhanced the efficiency and quality of transformation. Five times 10(4) copies of such fragments consistently restored the phenotypes to near normal; even 10(3) or fewer copies could still effect weak transformation. The restored phenotypes were stable for greater than 20 fissions in many clones and were lost after autogamy. We examined the fate of the injected fragments in the transformed clones and discuss the possible application of this efficient transformation in the cloning of other genes of P. tetraurelia.

Amino Acid Sequence↗
PubMed1991

A mechanosensitive channel in whole cells and in membrane patches of the fungus Uromyces.

Bean leaf stomata provide a topographical signal that induces germlings of the phytopathogen Uromyces appendiculatus to develop specialized infection structures. Protoplasts from germ tubes of this fungus, when examined with patch-clamp electrodes, displayed the activities of a 600-picosiemen mechanosensitive ion channel. This channel passes a variety of cations, including Ca2+, and is blocked by Gd3+ at 50 micromolar. This channel could transduce the membrane stress induced by the leaf topography into an influx of ions, including Ca2+, that may trigger differentiation.

Barium↗
PubMed1991

A single amino acid substitution alters conductance and gating of OmpC porin of Escherichia coli.

We have reconstituted into liposomes outer-membrane fractions from Escherichia coli strains which express OmpC porins with altered pore properties. Single-channel experiments were performed with the patch-clamp technique on blisters generated from the reconstituted liposomes. Our goal was to identify positively the activity pattern of OmpC in our reconstituted system. The properties of the parent strain were compared to those of a strain whose OmpC porin has a single amino acid substitution in a postulated transmembrane segment. The parent and the mutant strain each exhibit a cation-selective channel of high open probability and gating to closed levels of various amplitudes. However, the mutant channel appeared to be 9 to 30% larger in unit conductance. It tended to close and reopen most often in groups of three units, as opposed to two units in the parent channel. The results are discussed in terms of the observed phenotype and of their implication as to the structure-function relationship of the porin channels.

Bacterial Outer Membrane Proteins↗
PubMed1990

Mechanosensitive ion channels of E. coli activated by amphipaths.

Mechanosensitive channels have been found in more than 30 cell types, including bacterial, yeast, plant and animal cells. Whether tension is transferred to the channel through the lipid bilayer and/or underlying cytoskeleton is not clear. Using the patch-clamp method, we found that amphipathic compounds, which are molecules having hydrophobic and hydrophilic character with positive, negative or no net electric charge at pH 7, could slowly activate the mechanosensitive channels of giant Escherichia coli spheroplasts, with effectiveness proportional to their lipid solubility. The cationic or anionic amphipaths were able to compensate for each other's effect. After a channel was activated by an amphipath of one charge, if that amphipath was gradually replaced by one with the opposite charge, the channel first inactivated before reactivating. These findings support the view that the mechanical gating force can come from the surrounding lipids.

Cell Membrane↗
PubMed1990

Mutations in paramecium calmodulin indicate functional differences between the C-terminal and N-terminal lobes in vivo.

We examined calmodulin and its gene from the wild-type and viable mutants of P. tetraurelia. The mutants, selected for their behavioral aberrations, have little or no defects in growth rates, secretion, excretion, or motility. They can be grouped according to whether they underreact or overreact behaviorally to certain stimuli, reflecting their respective loss of either a Ca2(+)-dependent Na+ current or a Ca2(+)-dependent K+ current. Sequence analyses showed that all three underreactors have amino acid substitutions in the N-terminal lobe of the calmodulin dumbbell, whereas all three overreactors have substitutions in the C-terminal lobe. No mutations fell in the central helix connecting the two lobes. These results may indicate that the sites defined by these mutations are important in membrane excitation but not in other biological functions. They also suggest that the two lobes of calmodulin may be used differentially for the activation of different Ca2(+)-dependent channels.

Amino Acid Sequence↗
PubMed1990

Ion channel activities in the Escherichia coli outer membrane.

The electrical properties of Escherichia coli cells were examined by the patch-clamp technique. Giant cells or giant spheroplasts were generated by five different methods. By electron micrographic and other criteria we determined that the patches are most likely from the outer membrane. We regularly observed currents through at least two types of channels in this membrane. The first current is mechanosensitive and voltage-dependent, and can be observed in single gene mutants of the known major porins (ompF, ompC, phoE, lamB); this channel may represent a minor porin or a new class of outer membrane protein. The possible identity of the second, voltage-sensitive channel with one of the known outer membrane proteins is being explored. The high-resistance seals consistently formed on these patches and the presence of gated ion channels suggest that most of the pores of the outer membrane are not statically open, as commonly held, but are closed at rest and may be openable by physiological stimuli.

Bacterial Outer Membrane Proteins↗
PubMed1990

Evidence for two K+ currents activated upon hyperpolarization of Paramecium tetraurelia.

Hyperpolarization of voltage-clamped Paramecium tetraurelia in K+ solutions elicits a complex of Ca2+ and K+ currents. The tail current that accompanies a return to holding potential (-40 mV) contains two K+ components. The tail current elicited by a step to -110 mV of greater than or equal to 50-msec duration contains fast-decaying (tau approximately 3.5 msec) and slow-decaying (tau approximately 20 msec) components. The reversal potential of both components shifts by 55-57 mV/10-fold change in external [K+], suggesting that they represent pure K+ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K+ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca2+ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K+ currents are inhibited by extracellular TEA+ in a concentration-dependent, noncooperative manner, whereas the fast K+ current alone is inhibited by 0.7 mM quinidine.

Animals↗