Alternatively spliced isoforms of the rat eye sodium/calcium plus potassium exchanger NCKX1

We have investigated the structure, function, and expression of the rat eye sodium/calcium+potassium exchanger NCKX1. The sequence of independent rat NCKX1 clones and the analysis of rat eye mRNA by RT-PCR revealed a region o f alternative splicing that comprised four exons and encoded a stretch of 1 13 amino acids near the beginning of the large cytosolic loop. In compariso n with other NCKX1 molecules and the rat NCKX2 protein, rat NCKX1 was highl y conserved within the hydrophobic regions but was quite divergent in the t wo large hydrophilic loops. The only exception was the region of the cytoso lic loop encoded by the second alternatively spliced exon, which was simila r to 60% identical. Similar to bovine, but different from human, rat NCKX1 possessed an acidic motif that was repeated 14 times in the cytoplasmic loo p. Analysis of NCKX1 expression in different rat tissues by Northern blot r evealed a very high level of expression of a 7-kb transcript in the eye but also lower levels of transcripts of various lengths in other tissues. The recombinant rat NCKX1 protein was tagged in the extracellular loop with the FLAG epitope and expressed in HEK-293 cells. Surface delivery and potassiu m-dependent sodium/calcium exchange activity were observed for each spliced variant.