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 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 150kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100kDa band in HLE-B3 cells. In the human lens tissues, a 115kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100kDa 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.
Misri
Sandeep
S
Cell Biophysics Group, Boonshoft School of Medicine, Wright State University, 054 Biological Sciences Building, Dayton, OH 45435, USA.
Chimote
Ameet A
AA
Adragna
Norma C
NC
Warwar
Ronald
R
Brown
Thomas L
TL
Lauf
Peter K
PK
D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D005136:Eye Proteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007120:Immunochemistry / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D007536:Isomerism; D007908:Lens, Crystalline / Q000737:chemistry*; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods; D011188:Potassium / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D027981:Symporters / Q000032:analysis / Q000378:metabolism*; D014020:Tissue Extracts / Q000737:chemistry; D000096922:K Cl- Cotransporters
D002712:Chlorides; D005136:Eye Proteins; D008565:Membrane Proteins; D011188:Potassium; D012333:RNA, Messenger; D027981:Symporters; D014020:Tissue Extracts; D000096922:K Cl- Cotransporters