Quantitative analysis of Na -K'-2Cl cotransporter rna splice variant transcripts from human tissue

C. R T Vibat, J. J. Kang, M. J. Holland, Martha E O'Donnell

Research output: Contribution to journalArticle

Abstract

The Na-K-2Cl cotransporter (NKCC1) is crucial in the regulation of intracellular volume in a variety of cell types and in vectorial transport of ions across epithelia. We and others have evidence to suggest that cotransporter activity and cell volume changes in the trabecular meshwork of the eye modulate aqueous humor outflow facility. It is hypothesised that defectve TM cell volume regulation may underlie the increased resistance to aqueous humor outflow facility that is observed in primary open angle glaucoma. We have demonstrated that activity and regulation of NKCC1 is aberrant in glucomatous vs. normal TM cells. Recently, we have observed two RNA splice variants of NKCC1 in human TM cells. NKCCla contains 48 bases encoding a putative regulatory site which is not present in the shorter variant, NKCClb. While others have identified these two splice variants in mutine brain, relative abundance of these transcripts has not been previously evaluated. Our work aims to use kinetic RT-PCR techniques to explore the relative levels of NKCCla and NKCClb transcripts in human TM and other tissues. RNA splice variant discrimination and transcript levels are evaluated by using an automated system which measures accumulation of PCR product after each amplification cycle. Preliminary results suggest that NKCCla transcript predominates in normal human TM cells. The ratio of NKCClb/NKCCla appears to be much greater in human brain. Ongoing studies include evaluating NKCCClb/NKCCla RNA in glaucomatous TM cells and other human tissues and exploring possible functional roles of the NKCCla and NKCClb proteins. Supported by the Glaucoma Research Foundation and NIH HL07682.

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number8
StatePublished - 1998

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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