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    Molecular characterization of the plasma membrane sodium/calcium exchanger

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    Author
    Kofuji, Paulo
    Advisor
    Lederer, W. Jonathan
    Date
    1993
    Type
    dissertation
    
    Metadata
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    Abstract
    In many cells including cardiac myocytes, cytoplasmic calcium is controlled by the plasma membrane Na/Ca exchanger. The tissue diversity and differences in cellular environment raises the question whether the same exchanger is found in all tissues. I have examined the diversity of the Na/Ca exchanger message in various rat, rabbit and human tissues by library screening, Northern blot analysis, RNase protection assays and Polymerase Chain Reaction (PCR). Initially, the full length Na/Ca exchanger cDNA from a human cardiac library was cloned by screening with a homologous probe generated using PCR. The full length clone encoded a deduced polypeptide sequence of 973 amino acids with eleven potential membrane spanning domains and a large intracellular loop. Upon transfection of the cDNA in a human kidney fibroblast cell line (HEK 293 cells) the expressed polypeptide displayed normal functional characteristics of Na/Ca exchanger. Using this full length clone as a probe in Northern blots, a major common transcript of 7.2 kb was detected at different levels in brain, kidney, liver, skeletal muscle, placenta, pancreas and lung. A difference between the Na/Ca exchanger message in the coding region of the brain and heart cDNAs was observed in the carboxyl end of the intracellular loop, a region recently linked to ionic and metabolic regulation of the Na/Ca exchanger. This variable region of the exchanger was examined by PCR analysis revealing the presence of additional isoforms in other tissues. Examination of the gene structure of the Na/Ca exchanger indicates that a single gene encodes for the Na/Ca exchanger message and is alternatively spliced to give rise to the multiple isoforms. Specifically, sequence analysis of the intron-exon boundaries revealed the presence of two "mutually exclusive" exons in conjunction with four "cassette" type exons in the region of the Na/Ca exchanger gene that codes for the carboxyl end of the predicted intracellular loop region. This unusual arrangement of exons in the Na/Ca exchanger gene could allow for the generation of up to 32 different Na/Ca exchanger mRNAs and accounts for the isoforms identified to date.
    Description
    University of Maryland, Baltimore. Ph.D. 1993
    Keyword
    Biology, Molecular
    Biology, Cell
    Biology, Animal Physiology
    plasma membrane sodium/calcium exchanger
    Exons--genetics
    Sodium-Calcium Exchanger--genetics
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1695
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