Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

We have investigated the structure of the rat eye sodium-calcium+potassium exchanger, NCKX1, by cDNA cloning and expression. Oligonucleotide primers based on sequences conserved between human and bovine NCKX1 at the amino and carboxyl termini were used to amplify oligo(dT)-reverse transcribed mRNA from rat eye using a high-fidelity polymerase. Nine independent clones were obtained and sequenced. All clones encoded an identical protein, with the exception of a stretch of 113 amino acids near the center of the large cytosolic loop, that were either partially or entirely deleted in certain clones. Further analysis of rat eye mRNA by RT-PCR confirmed the existence of four different alternatively spliced products of differing length. In comparison with the published bovine and human NCKX1 molecules, the rat protein is almost completely identical within the hydrophobic transmembrane regions, but quite divergent in the two large hydrophilic loops. Similar to bovine, but different from human, rat NCKX1 possesses an acidic motif, which is repeated fourteen times in the cytoplasmic loop. The rat NCKX1 protein was tagged in the extracellular loop with the FLAG epitope, and expressed in HEK293 cells. Surface delivery and potassiuim-dependent sodium-calcium exchange activity were observed for each spliced variant.

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Presentation Number SApoon0412
Keywords: sodium, calcium, potassium, exchanger, cloning