Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

The Saccharomyces cerevisiae phosphotyrosyl phosphatase activator proteins are required for a subset of the functions disrupted by protein phosphatase 2A mutations

In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaDelta mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21Delta genetic background. Moreover, ypaDelta mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypaDelta mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.     

TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells

Using patch clamp and Ca(2+) imaging techniques, we have studied Ca(2+) entry pathways in human hepatoblastoma (HepG2) cells. These cells express the mRNA of TRPV1, TRPV2, TRPV3 and TRPV4 channels, but not those of TRPV5 and TRPV6. Functional assessment showed that capsaicin (10 microM), 4alpha-phorbol-12,13-didecanoate (4alphaPDD, 1 microM), arachidonic acid (10 microM), hypotonic stress, and heat all stimulated increases in [Ca(2+)](i) within minutes. The increase in [Ca(2+)](i) depended on extracellular Ca(2+) and on the transmembrane potential, which indicated that both driving forces affected Ca(2+) entry. Capsaicin also stimulated an increase in [Ca(2+)](i) in nominally Ca(2+)-free solutions, which was compatible with the receptor functioning as a Ca(2+) release channel. Hepatocyte growth factor/scatter factor (HGF/SF) modulated Ca(2+) entry. Ca(2+) influx was greater in HepG2 cells incubated with HGF/SF (20 ng/ml for 20 h) compared with non-stimulated cells, but this occurred only in those cells with a migrating phenotype as determined by presence of a lamellipodium and trailing footplate. The effect of capsaicin on [Ca(2+)](i) was greater in migrating HGF/SF-treated cells, and this was inhibited by capsazepine. The difference between control and HGF/SF-treated cells was not found in Ca(2+)-free solutions. 4alphaPDD also had no greater effect on HGF/SF-treated cells. We conclude that TRPV1 and TRPV4 channels provide Ca(2+) entry pathways in HepG2 cells. HGF/SF increases Ca(2+) entry via TRPV1, but not via TRPV4. This rise in [Ca(2+)](i) may constitute an early response of a signalling cascade that gives rise to cell locomotion and the migratory phenotype.     

Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6

TRPV6 (CaT1/ECaC2), a highly Ca(2+)-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg(2+). Mg(2+) blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg(2+) is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg(2+), outward conductance is still approximately 10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg(2+)-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg(2+) sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg(2+). The effects of intracellular Mg(2+) on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K(+) channels and may represent a novel regulatory mechanism for TRPV6 function in vivo.     

Identification and characterization of B"-subunits of protein phosphatase 2 A in Xenopus laevis oocytes and adult tissues.

Protein phosphatase 2A is a phosphoserine/threonine phosphatase implicated in many cellular processes. The core enzyme comprises a catalytic and a PR65/A-subunit. The substrate specificity and subcellular localization are determined by a third regulatory B-subunit (PR55/B, PR61/B' and PR72/130/B"). To identify the proteins of the B" family in Xenopus laevis oocytes, a prophase Xenopus oocyte cDNA library was screened using human PR130 cDNA as a probe. Three different classes of cDNAs were isolated. One class is very similar to human PR130 and is probably the Xenopus orthologue of PR130 (XPR130). A second class of clones (XN73) is identical to the N-terminal part of XPR130 but ends a few amino acids downstream of the putative splicing site of PR130. To investigate how this occurs, the genomic structure of the human PR130 gene was determined. This novel protein does not act as a PP2A subunit but might compete with the function of PR130. The third set of clones (XPR70) is very similar to human PR48 but has an N-terminal extension. Further analysis of the human EST-database and the human PR48 gene structure, revealed that the human PR48 clone published is incomplete. The Xenopus orthologue of PR48 encodes a protein of 70 kDa which like the XPR130, interacts with the A-subunit in GST pull-down assays. XPR70 is ubiquitously expressed in adult tissues and oocytes whereas expression of XPR130 is very low in brain and oocytes. Expression of XN73 mainly parallels XPR130 with the exception of the brain.     

Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms

Exposure of rat pulmonary artery smooth muscle cells (rPASMC) to cytokines leads to nitric oxide (NO) production by NO synthase 2 (NOS2). NO stimulates cGMP synthesis by soluble guanylate cyclase (sGC), a heterodimer composed of alpha(1)- and beta(1)-subunits. Prolonged exposure of rPASMC to NO decreases sGC subunit mRNA and protein levels. The objective of this study was to determine whether levels of NO produced endogenously by NOS2 are sufficient to decrease sGC expression in rPASMC. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) increased NOS2 mRNA levels and decreased sGC subunit mRNA levels. Exposure of rPASMC to IL-1beta and TNF-alpha for 24 h decreased sGC subunit protein levels and NO-stimulated sGC enzyme activity. L-N(6)-(1-iminoethyl)lysine (NOS2 inhibitor) or 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (sGC inhibitor) partially prevented the cytokine-mediated decrease in sGC subunit mRNA levels. However, cytokines also decreased sGC subunit mRNA levels in PASMC derived from NOS2-deficient mice. These results demonstrate that levels of NO and cGMP produced in cytokine-exposed PASMC are sufficient to decrease sGC subunit mRNA levels. In addition, cytokines can decrease sGC subunit mRNA levels via NO-independent mechanisms.     

Molecular cloning and characterization of fox testis kinectin

Kinectin was isolated and characterized from a fox testis cDNA library using a monoclonal antibody (FTA-1) raised against testis surface proteins. The cDNA sequence of 4,479 nucleotides encodes an ORF of 1,330 amino acids (aa) with high homology to mouse, human, and chicken kinectins (GenBank Accession Number AF095786). Southern analysis was used to show that genes homologous to kinectin are present in several mammal species and in at least one marsupial, but not in bacteria. Alternatively spliced forms of fox kinectin were identified, and one of these is uniquely expressed in brain and spleen tissues. Kinectin expression was highest in testis relative to other tissues examined. Sequence analysis and comparisons between species revealed that kinectin encodes multiple alpha-helical coiled coils predicted to form dimers, and is, therefore, likely to exist as a dimer. The results presented in this article suggest that kinectin is required for spermatogenesis, but is not a likely candidate for use in immunocontraceptive vaccines.     

Assessment of electroporation by flow cytometry

BACKGROUND: Electroporation accomplishes transient permeabilization of cells and thus aids in the uptake of drugs. The method has been employed clinically in the treatment of dermatological tumors with bleomycin. The conditions of electroporation are still largely empirical and information is lacking as to the interrelationships among voltage pulse height, pulse number and toxicity, cell permeation, drug uptake, and effects on drug toxicity. We used propidium iodide (PI) and flow cytometry to define cell permeation into cytoplasmic and nuclear compartments to determine the improvements of drug toxicity that can be accomplished by electroporation. METHODS: Human squamous carcinoma cells of defined TP53 status and normal human epithelial cells were subjected to electroporation using a square wave pulse generator in the range of 0-5,000 V/cm. Flow cytometry served to establish entry of the drug reporter, PI, into the cytoplasm and nucleus. A dye staining method served to establish cell survival and to determine the toxicity of bleomycin alone, electroporation alone, and electroporation with bleomycin. RESULTS: The electric field intensity (EFI) required to produce 50% permeabilization (EP(50)) is cell type dependent. The EP(50) varied from 1,465 to 2,027 V/cm. An EFI below 900 V/cm is growth stimulatory whereas an EFI in excess of 1,000 V/cm is growth inhibitory. An EFI of 1,000 V/cm is sufficient to increase bleomycin toxicity by a factor of 2-3. A differential electroporation efficiency is observed between normal and tumor cells. CONCLUSIONS: Tumor cells can be targeted preferentially at electroporation voltages where normal cells are less permeable.     

Folding properties of the hepatitis B core as a carrier protein for vaccination research

The hepatitis B core (HBc) protein has been used successfully in numerous experiments as a carrier for heterologous peptides. Folding and capsid formation of the chimeric proteins is not always achieved easily. In silico analyses were performed to provide further comprehension of the feasibility for predicting successful capsid formation. In contrast to previous work, we show that common in silico predictions do not ensure assembly into particles. We included new considerations regarding capsid formation of HBc fusion proteins. Not only the primary sequence and the length of the inserts seem important, also the rigidity, the distance between the N and the C-terminus and the presence of cysteines, which could form disulphide bonds, could influence proper capsid formation. Furthermore, new conformational insights were formulated when linkers were added to create extra flexibility of the chimeric particles. Different hypotheses were suggested to clarify the obtained results. To this extent, the addition of glycine-rich linkers could lower high rigidity of the insert, removal of the strain of the core protein or ease interaction between the HBc and the insert. Finally, we observed specific changes in capsid formation properties when longer linkers were used. These findings have not been reported before in this and other virus-like particle carriers. In this study, we also propose a new high-yield purification protocol for fusion proteins to be used in vaccination experiments with the carrier protein or in comparative studies of particulate or non-particulate HBc fusion proteins.     

The likely impact of elevated [CO2], nitrogen deposition, increased temperature and management on carbon sequestration in temperate and boreal forest ecosystems: a literature review

Temperate and boreal forest ecosystems contain a large part of the carbon stored on land, in the form of both biomass and soil organic matter. Increasing atmospheric [CO2], increasing temperature, elevated nitrogen deposition and intensified management will change this C store. Well documented single-factor responses of net primary production are: higher photosynthetic rate (the main [CO2] response); increasing length of growing season (the main temperature response); and higher leaf-area index (the main N deposition and partly [CO2] response). Soil organic matter will increase with increasing litter input, although priming may decrease the soil C stock initially, but litter quality effects should be minimal (response to [CO2], N deposition, and temperature); will decrease because of increasing temperature; and will increase because of retardation of decomposition with N deposition, although the rate of decomposition of high-quality litter can be increased and that of low-quality litter decreased. Single-factor responses can be misleading because of interactions between factors, in particular those between N and other factors, and indirect effects such as increased N availability from temperature-induced decomposition. In the long term the strength of feedbacks, for example the increasing demand for N from increased growth, will dominate over short-term responses to single factors. However, management has considerable potential for controlling the C store.