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Biomedical subjects

C Kung

Publications and source records attributed to C Kung.

At least 19 recordsLinked to original sources
PubMed2001

A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca(2+)-permeable channel in the yeast vacuolar membrane.

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca(2+) activates the YVC1 current carried by cations including Ca(2+) across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

Amino Acid Sequence↗
PubMed2001

Chemically charging the pore constriction opens the mechanosensitive channel MscL.

MscL is a bacterial mechanosensitive channel that protects the cell from osmotic downshock. We have previously shown that substitution of a residue that resides within the channel pore constriction, MscL's Gly-22, with all other 19 amino acids affects channel gating according to the hydrophobicity of the substitution (). Here, we first make a mild substitution, G22C, and then attach methanethiosulfonate (MTS) reagents to the cysteine under patch clamp. Binding MTS reagents that are positively charged ([2-(trimethylammonium)ethyl] methanethiosulfonate and 2-aminoethyl methanethiosulfonate) or negatively charged (sodium (2-sulfonatoethyl)methanethiosulfonate) causes MscL to gate spontaneously, even when no tension is applied. In contrast, the polar 2-hydroxyethyl methanethiosulfonate halves the threshold, and the hydrophobic methyl methanethiolsulfonate increases the threshold. These observations indicate that residue 22 is in a hydrophobic environment before gating and in a hydrophilic environment during opening to a substate, a finding consistent with our previous study. In addition, we have found that cysteine 22 is accessible to reagents from the cytoplasmic side only when the channel is opened whereas it is accessible from the periplasmic side even in the closed state. These results support the view that exposure of hydrophobic surfaces to a hydrophilic environment during channel opening serves as the barrier to gating.

Biophysical Phenomena↗
PubMed2001

Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Animals↗
PubMed2001

K(+)-channel transgenes reduce K(+) currents in Paramecium, probably by a post-translational mechanism.

PAK11 is 1 of more than 15 members in a gene family that encodes K(+)-channel pore-forming subunits in Paramecium tetraurelia. Microinjection of PAK11 DNA into macronuclei of wild-type cells results in clonal transformants that exhibit hyperexcitable swimming behaviors reminiscent of certain loss-of-K(+)-current mutants. PAK2, a distant homolog of PAK11, does not have the same effect. But PAK1, a close homolog of PAK11, induces the same hyperexcitability. Cutting the PAK11 open reading frame (ORF) with restriction enzymes before injection removes this effect entirely. Microinjection of PAK11 ORF flanked by the calmodulin 5' and 3' UTRs also induces the same hyperexcitable phenotype. Direct examination of transformed cells under voltage clamp reveals that two different Ca(2+)-activated K(+)-specific currents are reduced in amplitude. This reduction does not correlate with a deficit of PAK11 message, since RNA is clearly produced from the injected transgenes. Insertion of a single nucleotide at the start of the PAK11 ORF does not affect the RNA level but completely abolishes the phenotypic transformation. Thus, the reduction of K(+) currents by the expression of the K(+)-channel transgenes reported here is likely to be the consequence of a post-translational event. The complexity of behavioral changes, possible mechanisms, and implications in Paramecium biology are discussed.

3' Untranslated Regions↗
PubMed2000

The role of tyrosine 15 in erythropoietin action.

The results of iodination inactivation of erythropoietin suggest that tyrosine 15 is required for biological activity. This was confirmed by site-directed mutagenesis. Substitution of tyrosine by alanine or isoleucine resulted in mutants with no biological activity, whereas substitution by phenylalanine yielded an active mutein. Protein footprinting using trypsin showed that the N-terminal residues 1 to 46 and the C-terminal residues 155 to 165 linked by the 7 to 161 disulfide bond, includes one active site of the hormone.

Antibodies, Monoclonal↗
PubMed2000

Mutations in the tyrosine phosphatase CD45 gene in a child with severe combined immunodeficiency disease.

The hematopoietic-specific transmembrane protein tyrosine phosphatase CD45 functions to regulate Src kinases required for T- and B-cell antigen receptor signal transduction. So far, there have been no reports to our knowledge of a human deficiency in a tyrosine-specific phosphatase. Here, we identified a male patient with a deficiency in CD45 due to a large deletion at one allele and a point mutation at the other. The point mutation resulted in the alteration of intervening sequence 13 donor splice site. The patient presented at 2 months of age with severe combined immunodeficiency disease. The population of peripheral blood T lymphocytes was greatly diminished and unresponsive to mitogen stimulation. Despite normal B-lymphocyte numbers, serum immunoglobulin levels decreased with age. Thus, CD45 deficiency in humans results in T- and B-lymphocyte dysfunction.

Antigens, CD↗
PubMed2000

Expression of the green fluorescent protein in Paramecium tetraurelia.

In this paper we describe the expression of green fluorescent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV. Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins.

Animals↗
PubMed2000

The cloning and molecular analysis of pawn-B in Paramecium tetraurelia.

Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca(2+) current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca(2+) current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5' neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

Animals↗
PubMed1999

A new multigene family encoding calcium-dependent calmodulin-binding membrane proteins of Paramecium tetraurelia.

Ca2+/calmodulin (CaM) regulates various physiological processes in a wide variety of organisms, metazoa and protists alike. To better understand Ca2+/CaM-dependent processes, particularly those with membrane-associated components, we studied Ca2+/CaM-binding membrane proteins in Paramecium tetraurelia, a unicellular model system. A CaM-binding protein, PCM1 (Paramecium CaM-binding membrane-bound protein), from a detergent-solubilized ciliary membrane fraction was identified and purified through Ca2+-dependent CaM-affinity chromatography. PCM1 has an apparent molecular mass of approx. 65kDa. It binds radiolabeled CaM in blot overlay assays and binds to CaM-affinity columns, both only in the presence of 10 microM or higher Ca2+. Three peptide sequences from PCM1 were obtained, and polymerase chain reaction (PCR) and Southern hybridization experiments were designed accordingly, leading to a partial cDNA clone for PCM1 and the discovery of three homologs: PCM2, PCM3 and PCM4. Amino acid sequences predicted by the full-length coding sequence for PCM3 and partial genes for PCM1, PCM2 and PCM4 are very similar (approx. 85% amino-acid identities). Their sequences indicate that they are hitherto novel proteins with beta/gamma-crystallin domains, cysteine-rich regions and potential CaM-binding domains. These protein motifs are suggested to mediate protein-protein interaction important for Ca2+/CaM signal transduction event(s) through the PCM family of proteins.

Amino Acid Sequence↗
PubMed1999

Potassium uptake through the TOK1 K+ channel in the budding yeast.

The current through TOK1 (YKC1), the outward-rectifying K+ channel in Saccharomyces cerevisiae, was amplified by expressing TOK1 from a plasmid driven by a strong constitutive promoter. TOK1 so hyper-expressed could overcome the K+ auxotrophy of a mutant missing the two K+ transporters, TRK1 and TRK2. This trk1Delta trk2Delta double mutant hyperexpressing the TOK1 transgene had a higher internal K+ content than one expressing the empty plasmid. We examined protoplasts of these TOK1-hyperexpressing cells under a patch clamp. Besides the expected K+ outward current activating at membrane potential (Vm) above the K+ equilibrium potential (EK+), a small inward current was consistently observed when the Vm was slightly below EK+. The inward and the outward currents are similar in their activation rates, deactivation rates, ion specificities and Ba2+ inhibition, indicating that they flow through the same channel. Thus, the yeast outwardly rectifying K+ channel can take up K+ into yeast cells, at least under certain conditions.

Fungal Proteins↗
PubMed1999

CD45-associated protein is not essential for the regulation of antigen receptor-mediated signal transduction.

CD45 is a transmembrane protein tyrosine phosphatase required for signaling through the T-and B-cell antigen receptors. In lymphocytes, CD45 interacts with CD45-associated protein (CD45AP), a 32 000 Mr phosphoprotein, through their respective transmembrane domains. To determine whether CD45AP affects the ability of CD45 to regulate antigen receptor signaling, CD45AP-deficient mice were generated. Thymocyte development was grossly normal. Moreover, the cellularity of the thymus and spleens were normal. CD45 expression on thymocytes and splenocytes, ascertained by flow cytometry, was comparable between CD45AP-deficient mice and littermate controls. In contrast to a previous report (Matsuda et al., J. Exp. Med. 1998 187: 1863 - 1870). CD45AP-deficient and normal thymocytes and splenocytes proliferated similarly in response to various mitogens or antigen receptor cross-linking. Furthermore, thymocyte CD45-associated p56(lck) kinase activity was similar between CD45AP-deficient and normal cells. We conclude that CD45AP is not essential for the regulation of Src-family kinase activity by CD45.

Animals↗
PubMed1999

Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity.

Mechanosensitive channel large (MscL) encodes the large conductance mechanosensitive channel of the Escherichia coli inner membrane that protects bacteria from lysis upon osmotic shock. To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly(22) with all other common amino acids. Gly(22) was highlighted in random mutagenesis screens of E. coli MscL (, Proc. Nat. Acad. Sci. USA. 95:11471-11475). By analogy to the recently published MscL structure from Mycobacterium tuberculosis (, Science. 282:2220-2226), Gly(22) is buried within the constriction that closes the pore. Substituting Gly(22) with hydrophilic residues decreased the threshold pressure at which channels opened and uncovered an intermediate subconducting state. In contrast, hydrophobic substitutions increased the threshold pressure. Although hydrophobic substitutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions correlated with decreased proliferation. These results suggest a model for gating in which Gly(22) moves from a hydrophobic, and through a hydrophilic, environment upon transition from the closed to open conformation.

Amino Acid Substitution↗
PubMed1999

Channel gate! Tension, leak and disclosure.

The crystal structure of a bacterial MscL shows how this homopentameric channel protein is held tightly shut to prevent leakage whilst at rest. By inference, the structure also shows how a stretch force in the lipid bilayer causes the channel to open. We now have a concrete picture as to how a stimulus 'gates' an ion channel.

Amino Acid Sequence↗
PubMed1999

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL.

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

Algorithms↗
PubMed1998

One face of a transmembrane helix is crucial in mechanosensitive channel gating.

MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore. To determine functionally important residues within this molecule, we have randomly mutagenized mscL, expressed the genes in living bacteria, and screened for gain-of-function mutants with hampered growth. Expression of these genes caused leakage of cytoplasmic solutes on little or no hypo-osmotic stress. In excised patches, the mutant channels gated at membrane tensions that are less than that required for the gating of the wild-type MscL. Hence, the data suggest that the slowed or no-growth phenotype is caused by solute loss because of inappropriate gating of the channel. Most of the mutations mapped to the first transmembrane domain. When this domain is modeled as an alpha-helix, the most severe mutations are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating.

Amino Acid Sequence↗