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.

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.

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.

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. I. Regulatory volume decrease.

Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types.

Cl channel blockers inhibit the volume-activated efflux of Cl and taurine in cultured neurons.

The effects of the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 1,9-dideoxyforskolin (DDF), dipyridamole, and niflumic acid and of the polyunsaturated fatty acids arachidonic, linolenic, and linoleic acids on regulatory volume decrease (RVD) and associated 125I and [3H]taurine fluxes in cultured rat cerebellar granule neurons were examined. Dose-response curves of NPPB, DDF, and dipyridamole showed 20-100% inhibition of RVD and osmolyte fluxes. Niflumic acid was less potent, requiring 150-600 microM to show effects of this magnitude. The polyunsaturated fatty acids (5-20 microM) inhibited 80-90% RVD and osmolyte fluxes, with arachidonic acid exhibiting the most potent effect. The volume-associated taurine efflux was somewhat higher in younger neurons, but the pharmacological sensitivity was essentially the same in immature and mature cells. The effects of all tested drugs on 125I and [3H]taurine fluxes were remarkably similar, indicating a close pharmacological sensitivity of the transport mechanism for the two osmolytes. This is in line with the suggestion of a common pathway for the volume-associated release of Cl and amino acids functioning as osmolytes.

Volume-associated osmolyte fluxes in cell lines with or without the anion exchanger.

To investigate the involvement of a red cell-type anion exchanger in the volume-sensitive amino acid release, the hyposmolarity-evoked release of D-[3H]aspartate and [3H]taurine was examined in three cell lines: 1) wild-type Chinese hamster ovary (CHO-K1) cells, expressing an anion exchanger activity (Cl-/SO4(2-)) functionally similar to the erythroid band 3; 2) a mutant CHO cell type (CHO 605) lacking this anion exchanger activity; and 3) 293 cells in which the Cl-/HCO3(-) anion exchanger is absent. All cell types accumulated D-[3H]aspartate and [3H]taurine under isosmotic conditions, and, similarly, in the three cell lines, cell swelling evoked by hyposmolarity induced a rapid and transient increase in the amino acid efflux. Blockers of the anion exchanger and/or Cl- channels [niflumic acid, dipyridamole, diphenylamine-2-carboxylate,5-nitro-2-(3-phenylpropylamino)-benzoi c acid, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid] were potent inhibitors of amino acid efflux in the three cell lines. 125I- efflux, used as a marker for Cl- fluxes, was also markedly increased in response to cell swelling in all cell lines, and this efflux was inhibited by the anion exchanger/Cl- channel blockers. These results do not support a role for an anion exchanger in the hyposmolarity-induced amino acid efflux and suggest that amino acids and Cl- may be transported by the same or a similar mechanism, presumably an anion channel-like structure.

Inhibition by Cl- channel blockers of the volume-activated, diffusional mechanism of inositol transport in primary astrocytes in culture.

[3H]Inositol accumulated by rat brain cultured astrocytes is released when cells swell by exposure to solutions of decreased osmolarity. Activation of inositol efflux was proportional to reductions in osmolarity from 30%-70%. This volume-activated inositol efflux pathway was increased (27%) in Na(+)-free medium and decreased (22%) in Cl(-)-free medium. It was independent of extracellular Ca2+ and was reduced (30%) in the presence of the intracellular chelator [1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester] (BAPTA-AM). The inositol efflux pathway was markedly inhibited by Cl- channel blockers, which at maximal inhibitory concentrations decreased inositol efflux by 70%-83%. The potency range of the drugs was: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > 1-9, dideoxyforskolin > 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) > niflumic acid. Inositol efflux was strongly inhibited by the SH blocker N-ethyl maleimide (NEM), which at 100 microM abolished inositol release. Inositol efflux can be reversed by increasing its extracellular concentration, suggesting that the efflux is mediated by a diffusional pathway whose direction is given by the concentration gradient. The inhibition of volume-associated fluxes of inositol by Cl- channel blockers supports the suggestion of an anion channel as the common pathway for inorganic and organic osmolytes in cultured astrocytes.

Inhibition by polyunsaturated fatty acids of cell volume regulation and osmolyte fluxes in astrocytes.

The polyunsaturated fatty acids, arachidonic, linoleic, and linolenic acids, were potent blockers of regulatory volume decrease (RVD) and of the swelling-activated efflux of [3H]taurine, D-[3H]aspartate, [3H]inositol, and 125I (used as marker of Cl) from rat cerebellar astrocytes in culture. The monounsaturated oleic and ricinoleic acids and saturated fatty acids were ineffective. The amino acid and 125I fluxes were similarly inhibited by fatty acids, whereas inositol release was less sensitive. Polyunsaturated fatty acids appear to directly affect RVD in trypsinized astrocytes as the inhibition was immediate and fully reversible. Blockers of the arachidonic acid metabolic pathways, indomethacin (cyclooxygenase), esculetin (lipoxygenases), and metyrapone (P-450 monooxygenases), did not prevent the effect of arachidonic acid, suggesting that further metabolism is not required for displaying the effects of arachidonic acid on RVD and osmolyte fluxes. Some blockers of arachidonic acid metabolic pathways, such as nordihydroguaiaretic acid (lipoxygenases) and naphthoflavone (P-450 monooxygenases), also exhibited marked inhibitory effects on RVD and on osmolyte fluxes. The predominant arachidonic acid metabolite in astrocytes, 12-hydroxyeicosatetraenoic acid, did not affect RVD or osmolyte fluxes. These results suggest that arachidonic acid and other polyunsaturated fatty acids directly inhibit the permeability pathways correcting cell volume after swelling in cultured astrocytes.

Inhibition by dihydropyridines of regulatory volume decrease and osmolyte fluxes in cultured astrocytes is unrelated to extracellular calcium.

The 1,4-dihydropyridines (DHP), nimodipine (NMD) and nitrendipine (NTD) were potent blockers of regulatory volume decrease (RVD) and the volume-associated release of [3H]taurine and chloride (measured as 125I) in 2-weeks cultured rat cerebellar astrocytes. The IC50 were 30 microM and 29 microM for taurine efflux and 26 and 27 microM for C1 efflux for NMD and NTD, respectively. Inhibition by DHP was independent of extracellular Ca, as the effect was the same in media with 1 mM Ca or without Ca and 0.5 mM EGTA. DHP did not affect the basal (isosmotic) release of [3H]taurine or 125I inhibition by DHP (measured only on [3H]taurine efflux) was the same in 3-4 weeks cultured cerebellar astrocytes, 2-4 weeks cultured cortical astrocytes and 2-weeks cultured cerebellar astrocytes treated with dibutyril cAMP. Diltiazem (50 microM) and verapamil (100 microM) failed to inhibit RVD or osmolyte efflux.

Regulatory volume decrease in cultured astrocytes. II. Permeability pathway to amino acids and polyols.

The permeability of the hyposmolarity-activated pathway to amino acids and polyols in cultured astrocytes was examined following the change in rate and direction of regulatory volume decrease (RVD) when the extracellular concentration of the osmolytes was increased to reverse their intracellular-extracellular concentration gradient. Activation of the pathway by swelling would allow those permeable osmolytes to enter the cell and inhibit RVD. The pathway was found to be permeable to neutral amino acids, with beta-amino acids (beta-alanine = taurine > gamma-aminobutyric acid) more permeable than alpha-amino acids. Glycine, alanine, threonine, phenylalanine, and asparagine, but not glutamine, were permeable through this pathway. Aspartate was more permeable than glutamate, and K+ and not Na+ must be the accompanying cation. Basic amino acids were excluded. The dimension of the amino acid pore activated by hyposmolarity seems to be at the limit of glutamate-glutamine size. Influx rather than efflux of amino acids was observed when extracellular concentration was greater than intracellular concentration, with differences in the amount accumulated by cells correlating with their efficiency as RVD blockers. Influx of taurine (as representative of permeable amino acids) was inhibited by the Cl- channel blockers/exchangers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (40%) and dipyridamole (85%) , and it is suggested that amino acids permeate through an anion channel. Sorbitol and mannitol, but not inositol, exhibited a small inhibitory effect on the later phase of RVD, whereas inositol slightly accelerated RVD.

Inhibition of volume regulation and efflux of osmoregulatory amino acids by blockers of Cl- transport in cultured astrocytes.

Regulatory volume decrease (RVD) in astrocytes was inhibited by the Cl- exchanger blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), dipyridamole and niflumic acid but not by the Cl- channel inhibitors diphenylamine-2-carboxylate (DPC) and anthracene-9-carboxylate (9-AC). The volume activated efflux of [3H]taurine and [3H]D-aspartate (as marker for glutamate) was similarly affected by these compounds. However, neither RVD nor osmolyte fluxes were significantly reduced by removal of external Cl-, suggesting that an anion exchanger activity is not required for the volume regulatory process. Alternatively, these results suggest that the anion exchanger molecule may function as an unidirectional Cl- channel possibly permeable also to amino acids.

Contribution of organic and inorganic osmolytes to volume regulation in rat brain cells in culture.

In this work we examined the time course and the amount released, by hyposmolarity, for the most abundant free amino acids (FAA) in rat brain cortex astrocytes and neurons in culture. The aim was to evaluate their contribution to the process of cell volume regulation. Taurine, glutamate, and D-aspartate in the two types of cells, beta-alanine in astrocytes and GABA in neurons were promptly released by hyposmolarity, reaching a maximum within 1-2 min. after an osmolarity change. A substantial amount of the intracellular pool of these amino acids was mobilized in response to hyposmolarity. The amount released in media with osmolarity reduced from 300 mOsm to 150 mOsm or 210 mOsm, represented 50%-65% and 13%-31%, respectively, of the total amino acid content in cells. In both astrocytes and neurons, the efflux of glutamine and alanine was higher under isosmotic conditions and increased only marginally during hyposmotic conditions. 86Rb+, used as tracer for K+, was released from astrocytes, 30% and 11%, respectively, in hyposmotic media of 150 mOsm or 210 mOsm but was not transported in neurons. From these results it was calculated that FAA contribute 54% and inorganic ions 46% to the process of volume regulation in astrocytes exposed to a 150 mOsm hyposmotic medium. This contribution was 55% for FAA and 45% for K+ and Cl- in cells exposed to 210 mOsm hyposmotic solutions. These results indicate that the contribution of FAA to the process of cell volume regulation is higher in astrocytes than in other cell types including renal and blood cells.

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.

Neurons respond to hyposmotic conditions by an increase in intracellular free calcium.

The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca2+]i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca2+ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca2+]i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca2+]i after exposure to 50 mM K+ which was of the same magnitude as the increase in [Ca2+]i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca2+ channels verapamil had no effect on the hyposmolarity induced increase in [Ca2+]i. On the contrary, this increase in [Ca2+]i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca2+ channels Mg2+ and La3+. Dantrolene which prevents release of Ca2+ from internal stores had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in taurine transport evoked by hyperosmolarity in cultured astrocytes.

Cultured astrocytes grown chronically (1-3 days) in medium made hyperosmotic (450 mOsm) with NaCl or sucrose showed an increase in taurine concentration from 294 to 501 nmol/mg protein in NaCl and to 382 nmol/mg protein in sucrose. The effect of hyperosmolarity on taurine uptake and release was examined to investigate whether or not changes in these processes may account for the increase observed in cell taurine content. Hyperosmolarity significantly affected the two components of taurine uptake (i.e., the Na(+)-dependent and the diffusional component). The Vmax of the Na(+)-dependent, active transport increased 50%, whereas no change was observed in the Km. The diffusion coefficient was markedly decreased by hyperosmolarity, being 2.2 x 10(-4) and 6.6 x 10(-6) ml/min/mg protein in isosmotic and hyperosmotic conditions, respectively, indicating a blockade of the leak pathway. These changes in the active and passive components of taurine transport were opposite to those induced by hyperosmolarity. The effect of hyperosmolarity increasing cell taurine content was insensitive to cycloheximide and colchicine. The basal efflux of taurine from astrocytes also decreased in cells exposed to hyperosmotic medium, indicating that alterations in both influx and efflux of taurine are involved in the mechanism responsible for the increase in taurine levels induced by hyperosmolarity in astrocytes.