Biomedical subjects

Alexandre Hinzpeter

Publications and source records attributed to Alexandre Hinzpeter.

8 recordsLinked to original sources

PubMed2006

Membrane cholesterol content modulates ClC-2 gating and sensitivity to oxidative stress.

ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl-beta-cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.

Animals↗

PubMed2005

The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p.

The mammalian chloride channel ClC-2 is a member of the CLC voltage-gated chloride channels family. This broadly expressed protein shows diverse cellular locations and despite numerous studies, its precise function is poorly understood. Disruption of ClC-2-encoding gene in mouse leads to retinal and testicular degeneration and mutations in CLC2 (gene encoding the ClC-2 channel) are associated with idiopathic generalized epilepsies. ClC-2 may also be responsible for Cl- transport in mouse salivary glands. The only CLC homologue of the yeast Saccharomyces cerevisiae, Gef1p, exhibits CLC activity. We expressed the mammalian ClC-2 protein in S. cerevisiae devoid of Gef1p in an attempt to identify yeast proteins influencing the functioning of ClC-2. The presence of such proteins in yeast could indicate the existence of their homologues in mammalian cells and would greatly aid their identification. Expression of ClC-2 in yeast required optimization of the sequence context of the AUG translation initiation codon. After obtaining an efficient translation, we found that rat ClC-2 cannot directly substitute for yeast Gef1p. Functional substitution for Gef1p was, however, achieved in the presence of an increased level of intact or C-terminally truncated yeast Kha1 protein. Based on the deduced amino acid sequence, the Kha1 protein can be classified as a Na+/H+ transporter since it has a large N-terminal domain similar to the family of NHEs (Na+/H+ exchangers). This suggests that the Kha1p may take part in the regulation of intracellular cation homoeostasis and pH control. We have established that Kha1p is localized in the same cellular compartment as Gef1p and yeast-expressed ClC-2: the Golgi apparatus. We propose that Kha1p may aid ClC-2-dependent suppression of the Deltagef1-associated growth defects by keeping the Golgi apparatus pH in a range suitable for ClC-2 activity. The approach employed in the present study may be of general applicability to the characterization of poorly understood proteins by their functional expression in yeast.

Animals↗

PubMed2005

Blue native/SDS-PAGE analysis reveals reduced expression of the mClCA3 protein in cystic fibrosis knock-out mice.

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

Amino Acid Sequence↗

PubMed2005

Association between Hsp90 and the ClC-2 chloride channel upregulates channel function.

The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl-, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl- concentration [Cl-]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl-]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents.

Animals↗

PubMed2005

Cell-specific posttranscriptional regulation of CFTR gene expression via influence of MAPK cascades on 3'UTR part of transcripts.

Expression of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, which contains the mutations responsible for CF, is regulated by cytokines (TNF-alpha and IL-1beta) in a cell-specific manner. TNF-alpha decreases CFTR mRNA in human colon cell lines (HT-29), but not in pulmonary cell lines (Calu-3), and IL-1beta increases it only in Calu-3 cells. We looked for the cytokine-induced posttranscriptional regulation of CFTR gene expression and studied the modulation of CFTR mRNA stability linked to its 3' untranslated sequence (3'UTR) in HT-29 and Calu-3 cells. The stability of CFTR mRNA was analyzed by Northern blot after in vitro incubation of total RNAs from CFTR-expressing cells with cytosolic proteins extracted from control or cytokine-treated HT-29 and Calu-3 cells. CFTR mRNA was degraded only by extracts of TNF-alpha-treated HT-29 cells and not by cytosolic proteins from untreated or IL-1beta-treated HT-29 cells. In contrast, extracts of untreated Calu-3 cells enhanced CFTR mRNA degradation, and IL-1beta treatment inhibited this; TNF-alpha had no significant effect. The 3'UTR part of CFTR mRNA was found to be required for this posttranscriptional regulation. The 5' part of the 3'UTR (the 217 first bases), which contains two AUUUA sequences, was implicated in CFTR mRNA destabilization and the following 136 bases, containing several C-repeats in U-rich environment, in its protection. The proteins, which reacted with the U- and C-repeats of CFTR mRNA 3'UTR, were mainly controlled by stimulation of the p42/p44 and p38 MAP kinase cascades with interaction between these pathways. This posttranscriptional control of gene expression is a common feature of CFTR and many proteins of inflammation.

3' Untranslated Regions↗

PubMed2004

Immunohistochemistry of CFTR in native tissues and primary epithelial cell cultures.

Studies on CFTR protein expression and localization in native tissues or in primary cultures of human epithelial cells are scarce due to the intrinsic instability of this protein, its low expression in most tissues and also to technical difficulties. However, such data are of the highest importance to understand the pathophysiology of CF. The purpose of this article is to outline several assays for the characterization of primary epithelial cultures and to review different CFTR immunostaining protocols.

Cell Culture Techniques↗

PubMed2004

Biochemical methods to assess CFTR expression and membrane localization.

Detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein is usually a difficult task to accomplish due to the low levels of expression and high turnover that this membrane protein is submitted to in the cell. Common biochemical methods can be used for the detection of CFTR but several critical points must be taken into account. The scope of this article is to outline biochemical methods commonly used to assess CFTR expression, processing and membrane localization.

Biotinylation↗

PubMed2003

Effect of ouabain on CFTR gene expression in human Calu-3 cells.

We have previously shown that ouabain, which changes the electrochemical properties of cell membranes by inhibiting Na(+),K(+)-ATPase, induces the expression of multidrug resistance (MDR-1) gene in several human cell lines. Because the expressions of the MDR-1 and CFTR (which encodes the cAMP-activated Cl(-) channel associated with cystic fibrosis) genes are physiologically regulated in opposing directions, we wanted to determine whether ouabain also decreases CFTR transcripts and subsequently to analyze its mechanism of action. We found that the submicromolar concentrations of ouabain that increase MDR-1 mRNAs decrease the CFTR transcripts with analogous time-dependency in human pulmonary Calu-3 cells. By altering or reproducing the ouabain-induced changes in intracellular ionic activities (decreasing in external Na(+) or K(+) or using Na(+) ionophore), we show that the ouabain-induced regulations of both CFTR and MDR-1 transcripts depend on the Na(+)/K(+) pump inhibition but that the decrease in CFTR mRNAs also proceeds from cytoplasm reactions simultaneously activated by ouabain. These data, which emphasize the complex mechanism of action of ouabain, suggest that changes in intracellular ionic activities modulate CFTR/MDR-1 gene expressions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗