Plasma membrane Ca2+-ATPase expression in the human lens.
The focus of the study was to characterize plasma membrane calcium-ATPase pump (PMCA) isoform expression in the human lens and cultured lens epithelial cells as a basis for future studies of calcium homeostasis in the lens. Proteins and mRNA expression were analysed using Western Immunoblotting and reverse transcription polymerase chain reaction (RT-PCR), respectively. Clear human lenses from the Kentucky Lions Eye Bank and an immortalized human lens epithelial cell line (HLE B-3) were used. RT-PCR products of PMCA1, PMCA2, and PMCA4 primers were detected at 429, 557, and 849bp, respectively. All these products were identified as PMCA isoforms by sequence analysis. Protein bands at approximately 130, 115, and 135kDa were detected by Western blot analysis for PMCA1, PMCA2 and PMCA4, respectively. PMCA3 was not detected at protein or mRNA level in any human lens sample or cell culture, but was detected in the rat brain cortex used as a control. Several bands with lower molecular weights, especially for PMCA2, were detected in the epithelial samples and probably represent break down products of PMCA2. No PMCA proteins or breakdown products were detected in the nuclear or cortical fractions from human lenses. PMCA1, 2, and 4 proteins and mRNAs are expressed in human lens epithelium and cultured epithelial cells; PMCA3 is not. PMCA was not detected at all in the lens fibre cells. The calcium pump must be selectively processed, independent of other membrane proteins such as the Na-K-ATPase pumps, because the distribution of the Na-K-ATPase pump is asymmetrical in the epithelium and present throughout the lens whereas the calcium pumps are not. The findings of this study provide a basis for further studies to examine the role and modulation of PMCA isoforms in calcium homeostasis and in the development of cataract.
Department of Biochemistry and Molecular Biology, University of Louisville, 301 E. Muhammad Ali Blvd., Louisville, KY 40202, USA.
M J
MJ
Marian
H
H
Li
D
D
Borchman
C A
CA
Paterson
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000201:enzymology; D004847:Epithelial Cells / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007908:Lens, Crystalline / Q000201:enzymology*; D053499:Plasma Membrane Calcium-Transporting ATPases; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
C506952:ATP2B1 protein, human; D027682:Cation Transport Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D053499:Plasma Membrane Calcium-Transporting ATPases; C506958:ATP2B2 protein, human; C506962:ATP2B3 protein, human; C506955:Atp2b1 protein, rat; D000252:Calcium-Transporting ATPases