KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts

We recently reported potassium–chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium–chloride cotransport (KCC) isoforms by reverse transcriptase–polymerase chain re...

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Bibliographic Details
Published in:Experimental eye research 2006-11, Vol.83 (5), p.1287-1294
Main Authors: Misri, Sandeep, Chimote, Ameet A., Adragna, Norma C., Warwar, Ronald, Brown, Thomas L., Lauf, Peter K.
Format: Article
Language:English
Subjects:
Blotting, Western - methods
cataract
cation–chloride cotransporter
Cell Line
Chlorides - metabolism
Eye Proteins - analysis
Eye Proteins - metabolism
human lens
human lens epithelial B3 cell line
Humans
Immunochemistry - methods
immunofluorescence
Immunohistochemistry - methods
Isomerism
K Cl- Cotransporters
KCC isoform
Lens, Crystalline - chemistry
Membrane Proteins - metabolism
Microscopy, Confocal
Microscopy, Fluorescence - methods
NKCC1 isoform
Potassium - metabolism
Reverse Transcriptase Polymerase Chain Reaction - methods
reverse transcriptase–polymerase chain reaction
RNA, Messenger - analysis
Symporters - analysis
Symporters - metabolism
Tissue Extracts - chemistry
Western blot
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Summary:We recently reported potassium–chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium–chloride cotransport (KCC) isoforms by reverse transcriptase–polymerase chain reaction (RT–PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT–PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150 kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105 kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112 kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100 kDa band in HLE-B3 cells. In the human lens tissues, a 115 kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100 kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.
ISSN:0014-4835
1096-0007
DOI:10.1016/j.exer.2006.07.006