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Biomedical subjects

X B Chang

Publications and source records attributed to X B Chang.

33 records · Page 2Linked to original sources
PubMed1994

Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion.

Technical difficulties in obtaining three-dimensional structures of intrinsic membrane proteins continues to limit understanding of their function. However, considerable insight can be gained from their two-dimensional topological arrangement in the lipid bilayer. Efficient molecular genetic approaches are available to discern the topology of prokaryotic but not of eukaryotic membrane proteins. The absolute asymmetry of the sidedness of their N-glycosylation was employed here to develop such a method using the cystic fibrosis transmembrane conductance regulator (CFTR). Insertion by in vitro mutagenesis of N-glycosylation consensus sequences (NXS/T) in predicted cytoplasmic and extracytoplasmic loops between hydrophobic sequences capable of traversing the membrane established the membrane topology of CFTR. This provides the first experimental evaluation of the original topological model of CFTR based solely on hydropathy algorithms and a method which may be generally applicable for the in vivo evaluation of the topology of other mammalian membrane proteins.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Multi-ion pore behaviour in the CFTR chloride channel.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a non-rectifying, low-conductance channel regulated by ATP and phosphorylation, which mediates apical chloride conductance in secretory epithelia and malfunctions in cystic fibrosis (CF). Mutations at Lys 335 and Arg 347 in the sixth predicted transmembrane helix of CFTR alter its halide selectivity in whole-cell studies and its single channel conductance, but the physical basis of these alterations is unknown and permeation in CFTR is poorly understood. Here we present evidence that wild-type CFTR can contain more than one anion simultaneously. The conductance of CFTR passes through a minimum when channels are bathed in mixtures of two permeant anions. This anomalous mole fraction effect can be abolished by replacing Arg 347 with an aspartate and can be toggled on or off by varying the pH after the same residue is replaced with a histidine. Thus the CFTR channel should provide a convenient model in which to study multi-ion pore behaviour and conduction. The loss of multiple occupancy may explain how naturally occurring CF mutations at this site cause disease.

Amino Acid Sequence↗
PubMed1993

The delta F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. Determination of functional half-lives on transfected cells.

Deletion of the phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most prevalent mutation in cystic fibrosis (CF). This mutation (delta F508CFTR) leads to a reduced cAMP-sensitive Cl- conductance in epithelial cells. While the mutant protein can function as a Cl- channel, it seems to be misprocessed and unable to accumulate at normal levels in the plasma membrane. Under conditions where the biosynthetic block of delta F508CFTR is not complete, the residence time of delta F508CFTR in the plasma membrane is a critical determinant of the cAMP-sensitive Cl- conductance. To assess the stability of the mutant and wild-type CFTR, we compared their functional half-lives at the plasma membrane of transfected Chinese hamster ovary cells. The plasma membrane Cl- conductance was assessed by patch-clamp recordings and/or by fluorimetric determinations of the membrane potential. Accumulation of delta F508CFTR in the plasma membrane was promoted by growing the transfected cells at reduced temperature (24-28 degrees C), and was verified by immunoblotting and by detecting the appearance of a plasmalemmal cAMP-activated Cl- conductance. Subsequently increasing the temperature to 37 degrees C inhibited further delivery of newly synthesized delta F508CFTR to the surface membrane. By studying the time dependence of the disappearance of the Cl- conductance, the functional half-life of the mutant protein at the plasma membrane was determined to be < 4 h, which is considerably shorter than the half-life of wild-type CFTR (> 24 h). The latter was estimated by terminating protein synthesis or secretion with cycloheximide or brefeldin A, respectively. Inhibition of protein synthesis did not alter the rate of disappearance of delta F508CFTR at 37 degrees C, validating the difference in turnover between mutant and wild-type CFTR. These results indicate that the structural abnormality of delta F508CFTR affects not only the delivery of the protein to the plasma membrane, but also its stability therein. Moreover, they suggest that overcoming the processing block at the endoplasmic reticulum may not suffice to restore normal Cl- conductance in CF.

Animals↗
PubMed1993

Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.

Amino Acid Sequence↗
PubMed1992

The cystic fibrosis transmembrane regulator is present and functional in endosomes. Role as a determinant of endosomal pH.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to defective Cl- conductance in epithelial cells. While the CFTR gene product has been detected in the plasma membrane, its presence and functional role in the membranes of intracellular compartments remain to be established. The purpose of the present experiments was to functionally localize CFTR in the endosomal membrane and to test the role of the associated Cl- conductance in the regulation of endosomal pH (pH(en)). When using conductive protonophores, the net H+ flux across the endosomal membrane of Chinese hamster ovary (CHO) cells is limited by the movement of counterions. Thus, ionic permeability could be estimated indirectly, from the changes in pH(en) determined fluorimetrically. Measurements in situ and in a cell-free microsomal preparation indicate the presence of a protein kinase A (PKA)-activated anion conductance in endosomes from CHO cells transfected with CFTR, but not in endosomes from wild-type or mock-transfected cells. In endosomes isolated from CFTR-expressing cells, the stimulatory effect of PKA was diminished by a specific peptide inhibitor of PKA, by alkaline phosphatase treatment or by a monoclonal antibody against the second nucleotide binding fold of CFTR. Increasing counterion permeability by phosphorylation of CFTR or by addition of valinomycin failed to alter the rate or extent of endosomal acidification in situ. Our observations indicate that functional CFTR, susceptible to activation by PKA, is present in endosomes of transfected CHO cells. More importantly, the data suggest that factors other than counterion permeability are the major determinants of pH(en).

Alkaline Phosphatase↗
PubMed1992

CFTR, a channel with the structure of a transporter.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a superfamily of active transport molecules. However, when expressed in a wide variety of heterologous cell systems and when purified to homogeneity and reconstituted in planar lipid bilayers, it exhibits low conductance chloride channel activity. We postulate that the active transport capability of the molecule has been adapted to provide very stringent metabolic control of this channel which is responsible for chloride secretion and hydration of wet epithelial surfaces.

Biological Transport, Active↗
PubMed1992

Right-angle light scattering to assay basal and regulated plasma membrane Cl- conductances.

We describe a simple and rapid technique for assaying both constitutive and regulated plasma membrane Cl- conductances. The method uses right-angle light scattering to measure the rate of swelling of cells in suspension, in which the anion conductance is rate limiting for swelling, due to introduction of high plasma membrane cation conductance using gramicidin. The technique was verified using Chinese hamster ovary cells and mouse L cells, both stably transfected with the cystic fibrosis transmembrane conductance regulator (CFTR), to confer a specific cAMP-activated Cl- conductance not normally present in these cell types. In agreement with results obtained using other methods for assaying Cl- permeability in these cells, forskolin stimulated a significant increase in plasma membrane Cl- conductance in CFTR-expressing cells, as indicated by an increase in light scattering. That the enhanced light scattering by the cells was the result of cell swelling due to NaCl influx was shown by ion substitution experiments, in which no forskolin-induced increase in light scatter occurred in N-methyl-D-glucamine Cl- or Na+ gluconate medium. Enhanced light scattering was also observed in both CFTR-expressing and control cells stimulated with the Ca2+ ionophore, ionomycin. Extracellular anion substitution, to exploit the inwardly directed halide gradient utilized in this protocol, enabled determination of the anion selectivities of both the cAMP- and Ca(2+)-activated Cl- channels. Thus this technique provides a simple optical method for rapidly assaying not only constitutive and regulated Cl- conductance pathways but also their anion selectivities.

Animals↗
PubMed1991

Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene.

A cyclic AMP-stimulated chloride conductance appears when the cystic fibrosis gene is expressed in non-epithelial cells by infection with recombinant viruses. Cyclic AMP-stimulated conductance in this system is mediated by the same ohmic, low-conductance Cl- channel as in human secretory epithelia, but control of this channel by phosphorylation has not been directly demonstrated. Here we report the appearance of the low-conductance Cl- channel in Chinese hamster ovary cells after stable transfection with the cystic fibrosis gene. The channel is regulated on-cell by membrane-permeant analogues of cAMP and off-cell by protein kinases A and C and by alkaline phosphatase. These results are further evidence that the cystic fibrosis transmembrane regulator is a Cl- channel which can be activated by specific phosphorylation events and inactivated by dephosphorylation; they reveal an unsuspected synergism between converging kinase regulatory pathways.

Animals↗
PubMed1989

Primary cells and established cell lines join DNA ends with the same efficiency relative to homologous recombination.

The joining of DNA ends was compared in the established CV1 cell line and in African green monkey kidney primary cells, from which the CV1 cell line was established. Using a linear SV40 genome that carries a terminal repeat of 526 nucleotide pairs, we measured the efficiency of circularization by end joining relative to circularization by homologous recombination between the terminal repeats. The ratios of end joining to homologous recombination were identical in the two types of cells regardless of whether the DNA ends were sticky, blunt, or mismatched. These studies demonstrate that the efficient end joining observed in established cell lines is not a peculiar adaptation to life in culture, but rather reflects a normal aspect of DNA metabolism in mammalian cells. Furthermore, the similar results in primary and established cells support the general validity of using data from studies of cultured cells to formulate models of recombinational processes in primary cells and potentially in multicellular organisms as well.

Animals↗
PubMed1989

Comparison of filler DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation.

Extra nucleotides (termed filler DNA) are commonly found at the junctions of genetic rearrangements in mammalian cells. The filler DNA at immune system rearrangements, which are called N regions, are generated at VDJ joints primarily by terminal deoxynucleotidyl transferase. However, the origin of filler DNA at genetic rearrangements in nonlymphoid cells is uncertain. In an analysis of more than 200 junctions that arose by circularization of transfected linear DNA (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986), we found 18 junctions with extra nucleotides exactly at the point of circularization. Analysis of these 18 junctions indicated that nonlymphoid cells could add extra nucleotides to the ends of duplex DNA. The characteristics of the extra nucleotides at these junctions and at 31 other rearrangement junctions from nonlymphoid cells were quite similar, suggesting that many genetic rearrangements may pass through a stage with free DNA ends. A comparison of the filler DNA at these 49 nonimmune system rearrangements with 97 N regions derived from immune system rearrangements suggested that lymphoid and nonlymphoid cells use different mechanisms for insertion of filler DNA, as expected from the absence of detectable terminal deoxynucleotidyl transferase in cells from nonlymphoid tissues. The filler DNAs at a smaller group of 22 translocations associated with cancer had features in common with both immune and nonimmune system rearrangements and therefore may represent a mixture of these two processes. Mechanisms that might account for the presence of filler DNA in nonlymphoid cells are discussed.

Animals↗
PubMed1987

Modification of DNA ends can decrease end joining relative to homologous recombination in mammalian cells.

In animal cells, exogenous DNA recombines into random chromosomal sites much more frequently than it recombines into homologous sites. Free DNA ends are "recombinogenic" in both processes. To test the effects of specific ends on analogous extrachromosomal processes, we constructed a linear genome of simian virus 40 with terminal repeated sequences. After transfection into monkey cells, the model substrate can circularize by end joining (analogous to random integration) or by homologous recombination between its terminal repeats (analogous to targeted recombination). Since the two types of recombination are in competition with one another, the ratio of homologous-recombination to end-join products is a sensitive indicator of the differential effects of specific ends. Substrates with blunt ends, complementary sticky ends, or mismatched ends generated the same ratio of homologous-recombination to end-join products. However, addition of dideoxynucleotides to the 3' hydroxyls of the substrate decreased the frequency of end joining by a factor of 5-6 relative to homologous recombination. Thus, the frequency of end joining can be decreased relative to that of homologous recombination by modification of the ends of the input DNA. These results suggest an approach to altering the ratio of random to targeted integration in mammalian cells.

Animals↗
PubMed1986

Formation of deletions after initiation of simian virus 40 replication: influence of packaging limit of the capsid.

Transfected DNA is frequently broken and rejoined in mammalian cells by recombination processes that depend on minimal nucleotide sequence homology. Although measurements of breakage and joining account reasonably well for the frequent formation of deletions during transfection, they are inadequate to explain the high frequency of deletion formation by simian virus 40 (SV40) genomes that are slightly larger than the packaging limit of the capsid. To investigate this anomaly, we constructed and transfected into CV-1 cells a series of modified SV40 genomes containing 136, 284, 460, and 656 extra base pairs in the intron of the gene encoding T antigen. These experiments indicate that the effective packaging limit of an SV40 capsid lies between 284 and 460 extra base pairs. Further analysis of these transfections suggests that molecules just above the effective packaging limit may be encapsidated and transmitted between cells at low efficiency, thereby allowing multiple rounds of replication and multiple opportunities to generate and package genomes that contain deletions. The junctional sequences in several such deletions were determined; they were similar to the junctions in deletions that were formed before replication began, suggesting that the enzymatic machinery responsible for both types of deletion may be similar.

Animals↗