Translocation of pI_Cln in a pig kidney cell line, LLC-PK1, by low osmotic stress

Abstract

Background. There has been no conclusive explanation regarding the function of pI_Cln (a 26- to 27-kDa acidic protein) on an osmo-sensitive chloride channel responsible for an outwardly rectifying anion current. We observed the effects of the hypotonic treatment of LLC-PK1 cells on the intra-cellular dynamic state of pI_Cln.

Methods. LLC-PK1 cells were cultured, and pI_Cln in cells was observed immunohistochemically. The cells were fractionated into nuclei, mitochondrial, microsomal, and soluble fractions biochemically, and pI_Cln was detected by an immunoblotting method after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.

Results. pI_Cln in cells was observed on nuclei and their surroundings, but not on cell membranes. pI_Cln was present in soluble and insoluble forms. The molecular masses of the oligomeric forms in the soluble fractions were different from those previously reported with Madin-Darby canine kidney (MDCK) cells, indicating the differences in the pI_Cln-oligomer depending on cell type. On analysis with SDS-polyacrylamide gel electrophoresis, the exposure of cells to hypotonic media elevated the ratio of soluble to insoluble forms within 5 min. This result also conflicted with those previously reported with MDCK cells. This finding suggests that the function of pI_Cln and the signaling mechanism differ depending on the cell species. Both extracellular ATP and NaN₃ inhibited this elevation of the soluble/insoluble ratio, coinciding with previous reports that extracellular nucleotides and depletion of intracellular ATP inhibited the volume-sensitive chloride channel. Dihydrocytochalasin B, an F-actin-disrupting drug, inhibited the elevation of the soluble/insoluble ratio.

Conclusions. The soluble form of pI_Cln was increased within 5 min by exposure of LLC-PK1 cells to hypotonic media. This translocation was inhibited by extracellular ATP, NaN₃, and dihydrocytochalasin.