Effects of hypotonic swelling on the cellular distribution and expression of pI(Cln) in human nonpigmented ciliary epithelial cells.
OBJECTIVE
It has been proposed that pI(Cln), a highly acidic protein, is a candidate gene product related to the swelling-activated chloride (Cl-) channel Icl.swell in mammalian cells. However, no consensus has been reached as to whether this relationship is direct or indirect. Recently the cDNA for pI(Cln) was isolated from human ciliary epithelial cells. To learn more about the structure-function of pI(Cln) we attempted to: i) overexpress pI(Cln) as a fusion protein in bacteria; ii) carry out its purification; iii) generate polyclonal antibodies to study its expression and cellular localization in the ciliary epithelial cells; and iv) determine whether cell-swelling affects pI(Cln) expression in ciliary epithelial cells.


METHODS
The open reading frame (ORF) of human pI(Cln) was subcloned in the pET-20b(+) plasmid and established as a recombinant vector in E. coli BL21(DE3)pLysS cells. Upon induction with iso-propyl-beta-thio-galactopyranoside (IPTG), pI(Cln) was isolated as a His-Tag fusion protein and purified to homogeneity. Polyclonal antibodies were raised in rabbits after immunization with pI(Cln) purified protein, and its expression and cellular distribution in ciliary epithelial cells determined by Western blot, immunoprecipitation and indirect immunofluorescence respectively. Cell-swelling effect on ciliary epithelial cells was carried out upon treatment of cultured cells with hypotonic solution up to 60 min and pI(Cln) expression measured by Northern and Western blot analysis.


RESULTS
By Western blot analysis or immunoprecipitation, pI(Cln) antisera recognized a main band of 37-kDa in total cell extracts from ciliary body or metabolically labeled ciliary epithelial cells. By indirect immunofluorescence, pI(Cln) antibodies stained the cytoplasm of NPE in the intact tissue, and the perinuclear region of cultured ciliary epithelial cells. When subjected to hypotonic treatment, NPE cells did not induce translocation of pI(Cln) protein from the cytoplasm into the plasma membrane, nor changes in pI(Cln) expression at the protein level, but did down regulate up to 30% the level of pI(Cln) mRNA in continued hypotonic treatment.


CONCLUSIONS
These observations indicate that, contrary to previous suggestions, the pI(Cln) protein is not likely to be in contact with the plasma membrane of ciliary epithelial cells, and its influence on Cl- -channel activity is more likely to be expressed indirectly, (i.e. through cytoskeletal restructuring).
Sánchez-Torres
J
J
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Huang
W
W
Civan
M M
MM
Coca-Prados
M
M
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology*; D007106:Immune Sera / Q000276:immunology; D007473:Ion Channels; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014018:Tissue Distribution / Q000502:physiology
C410035:CLNS1A protein, human; D018118:Chloride Channels; D007038:Hypotonic Solutions; D007106:Immune Sera; D007473:Ion Channels; D011993:Recombinant Fusion Proteins