Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Alternative splicing of six exons of the sodium/calcium exchanger, NCX1 provide the diversity in the mature protein which can be observed in many mammalian cell types. We have previously described that one of two exons, exon A or B must be present in transcript to produce a functioning exchanger protein. In order to identify and quantitate the isoform transcripts present in various cell types, we developed a novel method called QERT-PCR. cDNA which is made from the RNA is amplified with end-labeled oligonucleotides. The labeled products are separated on a standard DNA sequencing gel and the size of the labeled products indicated the exon composition and the intensity the amount of that isoform. We show that astrocytes express three predominant isoforms all containing exon B, while neurons express two different isoforms expressing exon A. To demonstrate functional differences between these different isoforms present in astrocytes or neurons, cDNA clones containing these isoforms were expressed in Xenopus oocytes. We show that only A exon containing isoforms of NCX1 upregulate function when compared to B exon containing isoforms after activation by PKA. Full-length cDNA containing chimeric constructs of exons A and B have been used to identify a restricted part of exon A which is essential to provide the increase in NCX1 function observed after PKA activation. Additionally predominate isoforms of NCX1 found in cardiac and kidney cells have been differently affected by intracellular calcium and voltage dependence when these isoforms are expressed in Xenopus oocytes.

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Presentation Number SAschulze0881
Keywords: Na/Ca exchanger, ion transport, calcium, sodium, splicing