title: KCC2a expression in a human fetal lens epithelial cell line.
abstract: The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin.
authors.affiliation: Cell Biophysics Group, Department of Pathology, Wright State University, Dayton, OH 45435, USA. peter.lauf@wright.edu
authors.forename: Peter K
authors.initials: PK
authors.lastname: Lauf
authors.forename: Mauricio
authors.initials: M
authors.lastname: Di Fulvio
authors.forename: Vinita
authors.initials: V
authors.lastname: Srivastava
authors.forename: Neelima
authors.initials: N
authors.lastname: Sharma
authors.forename: Norma C
authors.initials: NC
authors.lastname: Adragna
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007908:Lens, Crystalline / Q000166:cytology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D020033:Protein Isoforms; D012333:RNA, Messenger; D000254:Sodium-Potassium-Exchanging ATPase; D027981:Symporters; D000096922:K Cl- Cotransporters; C499076:ATP1A1 protein, human