Effects of hypotonic swelling on the cellular distribution and expression of pI(Cln) in human nonpigmented ciliary epithelial cells

Purpose. It has been proposed that pI(Clin), a highly acidic protein, is a candidate gene product related to the swelling-activated chloride (Cl-) cha nnel I-cl.swell in mammalian cells. However, no consensus has been reached as to whether this relationship is direct or indirect. Recently the cDNA fo r pI(Clin) was isolated from human ciliary epithelial cells. To learn more about the structure-function ofpI(Clin) we attempted to: i) overexpress pI( Clin) as a fusion protein in bacteria; ii) carry out its purification; iii) generate polyclonal antibodies to study its expression and cellular locali zation in the ciliary epithelial cells; and iv) determine whether cell-swel ling affects pI(Clin) expression in ciliary epithelial cells. Methods. The open reading frame (ORF) of human pI(Clin) was subcloned in th e pET-20b(+) plasmid and established as a recombinant vector in E. coli BL2 1(DE3)pLysS cells. Upon induction with iso-propyl-beta-thio-galactopyranosi de (IPTG), pI(Clin) was isolated as a His-Tag fusion protein and purified t o homogeneity. Polyclonal antibodies were raised in rabbits after immunizat ion with pI(Clin) purified protein, and its expression and cellular distrib ution in ciliary epithelial cells determined by Western blot, immunoprecipi tation and indirect immunofluorescence respectively. Cell-swelling effect o n ciliary epithelial cells was carried out upon treatment of cultured cells with hypotonic solution up to 60 min and pI(Clin) expression measured by N orthern and Western blot analysis. Results. By Western blot analysis or immunoprecipitation, Plo, antisera rec ognized a main band of 37-kDa in total cell extracts from ciliary body or m etabolically labeled ciliary epithelial cells. By indirect immunofluorescen ce, pI(Clin) anti- bodies stained the cytoplasm of NPE in the intact tissue , and the perinuclear region of cultured ciliary epithelial cells. When sub jected to hypotonic treatment, NPE cells did not induce translocation of pI (Clin) protein from the cytoplasm into the plasma membrane, nor changes in pI(Clin) expression at the protein level, but did down regulate up to 30% t he level of pI(Clin) mRNA in continued hypotonic treatment. Conclusions. These observations indicate that, contrary to previous suggest ions, the pI(Clin) protein is not likely to be in contact with the plasma m embrane of ciliary epithelial cells, and its influence on Cl(-)channel acti vity is more likely to be expressed indirectly, (i.e. through cytoskeletal restructuring).