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Immunopurification and structural analysis of a putative epithelial Cl- channel protein isolated from bovine trachea
Insights
Researchers purified a 38-kDa protein (p38) from bovine tracheal epithelium that mediates iodide uptake. Antibodies against p38 identified a multimeric chloride channel complex, suggesting a heteromeric structure.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Bovine tracheal epithelium contains proteins involved in ion transport.
- Understanding ion channel structure is crucial for cellular function and disease research.
Purpose of the Study:
- To purify and characterize a protein mediating stilbene disulfonate-sensitive iodide uptake.
- To elucidate the structure of the native chloride channel complex.
Main Methods:
- Protein purification to homogeneity.
- Liposome reconstitution assays.
- Antibody production and immunopurification.
- Western blot analysis under reducing and non-reducing conditions.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Main Results:
- A 38-kDa protein (p38) was purified and shown to mediate iodide uptake in reconstituted liposomes.
- Antibodies against p38 recognized a 62-64 kDa doublet and higher molecular weight forms (140, 240 kDa) in membrane preparations.
- Reduction of disulfide bonds led to the disappearance of higher molecular weight forms and the appearance of 32- and 38-kDa proteins.
Conclusions:
- The native chloride channel is likely a heteromeric complex composed of at least four subunits.
- Subunit association is mediated by disulfide bridges, explaining the observed molecular weight shifts under reducing conditions.
Abstract:
We have purified to homogeneity a 38-kDa protein (called p38) from bovine tracheal epithelium. This protein, when reconstituted into liposomes, mediates stilbene disulfonate-sensitive 125I- conductive uptake. On nonreduced or partially reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this protein associates into a doublet of 62-64 kDa. In some experiments a multimer of 141 kDa was also observed. Rabbit polyclonal anti-P38 antibodies have been produced and used to immunopurify the native transporter. Upon reconstitution of the immunoaffinity-purified protein into liposomes, a 260-fold enhancement of 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate and valinomycin-sensitive 125I- uptake was observed as compared to proteoliposomes containing unseparated material. On Western blots of total solubilized tracheal membrane proteins or semipurified fractions, the antibody recognized the 62-64-kDa doublet much better than the original 38-kDa antigen. Similar protein bands were detected in T84 and CFPAC cells as well. However, if apical membrane proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, the antibody recognized major bands at 140 and approximately 240 kDa. Upon partial reduction, immunolabeling of these proteins diminished with the concomitant appearance of the 62-64-kDa doublet. Upon complete reduction, the appearance of 32- and 38-kDa proteins was evident with the disappearance of the 62-64-kDa doublet. We hypothesize that the native Cl-channel is a heteromer containing at least four subunits connected by S-S bridges.
