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E. F. Talarico, Jr., B. G. Kennedy, N. J. Mangini; Alternative Splice Variants of Plasma Membrane Calcium-ATPases in Human Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2715.
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Plasma Membrane Calcium-ATPases (PMCAs) play a critical role in the regulation of intracellular calcium concentration ([Ca2+]i). Four genes encode PMCA proteins with alternative splicing of messenger RNA at three splice sites (A, B and C) serving to increase isoform diversity. Our previous work shows that all four PMCAs are expressed and have specific locations in human corneal epithelium (hCE). The present work examined which alternate splice variants of PMCAs are expressed in hCE.
Total RNA was extracted from hCE scraped from cadaver corneas of five different donors (two females and three males, age range 55 to 76 years). RT-PCR was performed using PMCA isoform-specific primers designed to amplify transcript that included either splice site A or splice sites B and C. PMCA cDNAs were directly sequenced or cloned, then sequenced.
Every donor expressed PMCA4 transcripts (4x at site A and 4b at site B/C). At splice sites B/C, every donor expressed PMCA1b and PMCA1kb (a variant previously only reported in rat intestine). One donor expressed PMCA2a and a novel PMCA2 variant we termed 2(i). A single, different donor expressed PMCA3a. For all the donors, PCR products were not detected at site A for PMCA1, PMCA2 and PMCA3.
This investigation shows that hCE expresses multiple splice variants of PMCA isoforms and that PMCA isoform-specific profiles vary among donors. Furthermore, this study describes a novel PMCA2(i) splice variant and documents the expression in hCE of the PMCA1k variant (PMCA1kb) previously only described in rat intestine. Finally, this study suggests that the molecular configuration of PMCA 1, 2 and 3 in the region of splice site A in hCE must be different than in other tissues since the same primers that produced site A transcripts in several other tissues were ineffective in priming PCR in hCE.
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