Biomedical subjects

L P Cid

Publications and source records attributed to L P Cid.

4 recordsLinked to original sources

PubMed2000

Splice variants of a ClC-2 chloride channel with differing functional characteristics.

We identified two ClC-2 clones in a guinea pig intestinal epithelial cDNA library, one of which carries a 30-bp deletion in the NH(2) terminus. PCR using primers encompassing the deletion gave two products that furthermore were amplified with specific primers confirming their authenticity. The corresponding genomic DNA sequence gave a structure of three exons and two introns. An internal donor site occurring within one of the exons accounts for the deletion, consistent with alternative splicing. Expression of the variants gpClC-2 and gpClC-2Delta77-86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2Delta77-86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2Delta77-86 is not responsible for the observed change. The deletion-carrying variant is found in most tissues examined, and it appears more abundant in proximal colon, kidney, and testis. The presence of a splice variant of ClC-2 modified in its NH(2)-terminal domain could have functional consequences in tissues where their relative expression levels are different.

Alternative Splicing↗

PubMed1999

Separate taurine and chloride efflux pathways activated during regulatory volume decrease.

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl- by anions that are more permeant through the volume-activated Cl- channel, as indicated by electrophysiological measurements, significantly decreased taurine efflux. In the presence of less-permeant anions, an increase in taurine efflux was observed. Simultaneous measurement of the 125I, used as a tracer for Cl-, and [3H]taurine efflux showed that the time courses for the two effluxes differed. In Cl--rich medium the increase in I- efflux was transient, whereas that for taurine was sustained. Osmosensitive Cl- conductance, assessed by measuring changes in cell volume, increased rapidly after hypotonic shock. The influx of taurine was able to counteract Cl- conductance-dependent cell shrinkage but only approximately 4 min after triggering cell swelling. This taurine-induced effect was blocked by DIDS. Differences in anion sensitivity, the time course of activation, and sensitivity to DIDS suggest that the main cell swelling-activated permeability pathways for taurine and Cl- are separate.

Anions↗

PubMed1997

Modulation by extracellular Cl- of volume-activated organic osmolyte and halide permeabilities in HeLa cells.

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by osmotically induced cell swelling were studied using electrophysiological and radiotracer efflux techniques. On hypotonic challenge, HeLa cells responded by activating an efflux pathway for [3H]taurine and a swelling-induced outwardly rectifying Cl- channel. Removal of extracellular Cl-, or its replacement by a less permeable anion, enhanced taurine efflux and decreased the inward current (Cl- efflux). The effect of Cl- removal on taurine efflux was not a consequence of changes in membrane potential. The degree of deactivation of the Cl- current at depolarized potentials was also Cl- dependent, suggesting that external Cl- is necessary for channel activity. The Cl- channel inhibitors 1,9-dideoxyforskolin, tamoxifen, and 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited swelling-activated taurine efflux, with DIDS being the most potent, at variance with the sensitivity of the Cl- channel. DIDS effect was dependent on external Cl-; concentrations of DIDS that inhibited 50% of taurine efflux were 0.2 and 4 microM at low and high Cl-, respectively. The results could be interpreted on the basis of separate pathways for swelling-activated taurine efflux and Cl- current differentially affected by Cl-. Alternatively, taurine and Cl- flux might occur through a common channel, with the two solutes interacting within the pore and being affected differentially by Cl- replacement.

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

PubMed1995

Cloning of a putative human voltage-gated chloride channel (CIC-2) cDNA widely expressed in human tissues.

We have cloned a cDNA from the human epithelial cell line T84 whose predicted amino acid sequence shows 93.9% identity with rat CIC-2. Mapping by somatic cell hybrids and polymerase chain reaction localizes the gene corresponding to this cDNA to chromosome 3q26-qter. The major transcription start site assessed by RNA primer extension is 100 nt upstream of the putative translation initiation codon. Analysis of the 5' flanking sequence revealed a high GC content and lack of common transcriptional elements such as TATA and CCAAT boxes. Northern blot analysis indicated wide organ distribution including tissues affected in cystic fibrosis (CF) and expression in an airway epithelial cell line derived from a CF patient. The high degree of sequence similarity and similar tissue distribution to rat CIC-2 suggests that this cDNA encodes the human CIC-2 voltage-gated chloride channel. Since this chloride channel is present in epithelial tissues it may be amenable to manipulation to circumvent the chloride secretion defect observed in CF.

Amino Acid Sequence↗