Biomedical subjects

R Sanchez-Olea

Publications and source records attributed to R Sanchez-Olea.

5 recordsLinked to original sources

PubMed1998

Characterization of pI(Cln) binding proteins: identification of p17 and assessment of the role of acidic domains in mediating protein-protein interactions.

pICln is a ubiquitous and abundant 27 kDa soluble protein that is localized primarily to the cytoplasm. The protein has been proposed to be a swelling-activated anion channel or a channel regulator. Recent studies, however, have cast significant doubt on these hypotheses, and the function of pI(Cln) therefore remains unknown. To further characterize the physiological role of pI(Cln), we have begun to identify the proteins that bind to it and the amino acid domains that mediate pICln protein-protein interactions. Using affinity assays and immunoprecipitation we have identified three proteins in C6 glioma cells with molecular masses of 17 kDa, 29 kDa and 72 kDa that bind selectively to pI(Cln). Microsequencing revealed that p17 is the non-muscle isoform of the alkali myosin light chain. pI(Cln) contains three acidic amino acid domains termed AD1, AD2 and AD3. Mutation of AD1 and/or AD2 had no effect on p17, p29 and p72 binding. However, binding of p72 was lost when four acidic amino acid residues were mutated in AD3, which is located at the carboxy terminus. A truncation peptide containing the last 29 amino acids of pI(Cln) was able to bind p72 normally. These results indicate that the carboxy terminus is necessary for p72-pI(Cln) interaction. Based on these and other findings, we propose that pI(Cln) is a protein responsible for regulating the structure and function of the cytoskeleton, and/or a protein involved in mediating interactions between components of intracellular signal transduction pathways.

Amino Acid Sequence↗

PubMed1998

Characterization of pICln phosphorylation state and a pICln-associated protein kinase.

pICln is a ubiquitous cellular protein that has been proposed to be a volume-sensitive Cl- channel or a channel regulator. Detailed biochemical, cellular and molecular characterization of pICln is required to understand its function. Our goal in the present investigation was to define further the biochemical properties of pICln and the proteins that associate with it. Immunoprecipitation of pICln from 32P-orthophosphoric acid-labeled C6 glioma cells revealed that the protein is phosphorylated constitutively, primarily on serine residues. Protein kinase activity was detected in pICln immunoprecipitates, revealing that a constitutively active protein kinase co-precipitates with pICln. A specific association between pICln and a protein kinase was also observed in affinity assays using a recombinant GST-pICln fusion protein. The pICln-associated kinase displayed broad substrate specificity and was inhibited in a concentration-dependent manner by heparin, zinc and 5,6-dichloro-1-beta-D-ribofuranosylbenose (DRB). These characteristics resembled those of casein kinase I and II. The pICln-associated kinase was not recognized, however, by antibodies against these two enzymes. Association of the kinase with pICln was disrupted by increasing concentrations of NaCl in the washing buffer, suggesting that electrostatic interactions are involved in kinase binding. Mutagenesis experiments corroborated this observation. Truncation of pICln demonstrated that two highly charged clusters of acidic amino acid residues are both necessary and sufficient for kinase binding. Phosphopeptide mapping demonstrated that pICln contains at least two phosphorylated serine residues that are located on trypsin cleavage fragments rich in acidic amino acid residues. We propose that the kinase or a kinase binding protein binds to acidic amino acids located between D101 and Y156 and phosphorylates nearby serine residues.

Amino Acid Sequence↗

PubMed1998

Recombinant pICln forms highly cation-selective channels when reconstituted into artificial and biological membranes.

pICln has been proposed to be the swelling-activated anion channel responsible for ICl, swell, or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pICln into artificial and biological membranes. Single channels were observed when pICln was reconstituted into planar lipid bilayers. In the presence of symmetrical 300 mM KCl, the channels had a high open probability and a slope conductance of 48 pS, and were outwardly rectifying. Reduction of trans KCl to 50 mM shifted the reversal potential by -31.2 +/- 0.06 mV, demonstrating that the channel is at least seven times more selective for cations than for anions. Consistent with this finding, channel conductance was unaffected by substitution of Cl- with glutamate, but was undetectable when K+ was replaced by N-methyl-D-glucamine. Reconstitution of pICln into liposomes increased 86Rb+ uptake by three- to fourfold, but had no effect on 36Cl- uptake. Phosphorylation of pICln with casein kinase II or mutation of G54, G56, and G58 to alanine decreased channel open probability and 86Rb+ uptake. When added to the external medium bathing Sf9 cells, pICln inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pICln and not minor contaminant proteins. However, these findings do not support the hypothesis that pICln is the anion-selective ICl, swell channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pICln, or may be a consequence of in vitro reconstitution of purified, recombinant protein.

Animals↗

PubMed1993

Volume-activated Rb+ transport in astrocytes in culture.

The involvement of K+ on the volume regulatory process in astrocytes was investigated by characterizing the hyposmolarity-induced efflux of K+ using 86Rb as a tracer. About 70 and 30% of the intracellular content of 86Rb was released after reductions in osmolarity from 320 to 160 or 220 mosM, respectively, during the time in which cells exhibit a volume regulatory response subsequent to swelling. No significant increase in 86Rb efflux was observed with lower reductions in osmolarity. The 86Rb efflux was Ca2+ independent and insensitive to temperature. It was inhibited by furosemide but not by bumetanide and was unaffected when nitrate, but not gluconate, replaced intracellular Cl-. The efflux was markedly inhibited by quinidine and by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Quinidine also prevented the volume regulatory decrease of cells, and this effect was overcome when a large cation permeability was imposed by gramicidin. In isosmotic conditions 86Rb efflux was not activated by N-ethylmaleimide, but this drug strongly inhibited the hyposmolarity-activated release. These findings suggest that 86Rb efflux from astrocytes associated to cell swelling is not mediated by an electroneutral cotransporter and rather favor the implication of a conductive exit pathway that may be a Ca(2+)-independent K+ channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1991

Hyposmolarity-activated fluxes of taurine in astrocytes are mediated by diffusion.

In order to obtain information about the mechanism responsible for swelling associated taurine release in astrocytes, the kinetics of taurine uptake in cultured astrocytes from mouse cerebral cortex was studied under isosmotic and hyposmotic (50% osmolarity) conditions. It was found that the Vmax for the high affinity component of taurine uptake was unaffected by exposure of the astrocytes to hyposmotic conditions and that the Km value was somewhat increased. Contrary to Vmax, the non-saturable component of the uptake was greatly increased (2.5-fold) after exposure of the cells to hyposmotic media leading to cell swelling. In addition to the kinetic characterization of taurine uptake the actual intracellular taurine content after incubation (15 min) in isosmotic or hyposmotic media with different taurine concentrations (0-100 mM) under Na(+)-free conditions was determined. At taurine concentrations less than 30 mM corresponding to the intracellular content in cells not exposed to taurine, exposure to hyposmotic media led to a decrease in the intracellular taurine content. At higher external taurine concentrations (greater than 30 mM) the intracellular taurine contents were dramatically increased after exposure to hyposmotic conditions. The increase in intracellular taurine seen under hyposmotic conditions at 100 mM external taurine could be significantly reduced by 100 microM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate). Altogether these results suggest that a diffusional process rather than the high affinity taurine carrier is involved in the swelling induced increase in astrocytic taurine influx and efflux.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗