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

Abstract

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 of alternative splicing that comprised four exons and encoded a stretch of 113 amino acids near the beginning of the large cytosolic loop. In comparison with other NCKX1 molecules and the rat NCKX2 protein, rat NCKX1 was highly conserved within the hydrophobic regions but was quite divergent in the two large hydrophilic loops. The only exception was the region of the cytosolic loop encoded by the second alternatively spliced exon, which was approximately 60% identical. Similar to bovine, but different from human, rat NCKX1 possessed an acidic motif that was repeated 14 times in the cytoplasmic loop. Analysis of NCKX1 expression in different rat tissues by Northern blot revealed 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 potassium-dependent sodium/calcium exchange activity were observed for each spliced variant.

Molecular Cloning of a Third Member of the Potassium-dependent Sodium-Calcium Exchanger Gene Family,NCKX3

“…Lower levels were found in a variety of other tissues, whereas kidney and liver were essentially negative. This pattern of size and distribution clearly distinguishes NCKX3 from the other two known members of the SLC24 gene family, NCKX1 (6 kb), and NCKX2 (ϳ11 kb), whose expression is almost exclusively restricted to eye and brain (20). Larger transcripts of somewhat variable size were evident in several tissues, as well as an ϳ1.8-kb pair species that was present in all rat tissues but not in mouse or human.…”

“…NCKX1 has been modeled to have a similar arrangement of transmembrane-spanning segments as NCX1, although actual amino acid sequence similarity is very limited and restricted only to the two ␣-repeats, as mentioned above. The cloning of NCKX1 from rat eye (20) also revealed the presence of alternatively spliced isoforms in this species, which differ by the arrangement of four exons at the N terminus of the large intracellular loop. Interestingly, the equivalent region of bovine NCKX1, but apparently not the alternatively spliced region in rat NCKX1, is responsible for producing a functionally silent protein when expressed in heterologous systems (20,21).…”

“…2 Two of these have recently been characterized functionally (30,31). In addition, analysis of NCKX mRNA expression using probes from regions of sequence conservation under conditions of reduced stringency has revealed evidence for further, as yet uncharacterized, mammalian members of the NCKX family (20). 3 These findings lend support to the idea that the NCKX gene family predates the evolution of vertebrate vision and that the encoded proteins are an essential component of intracellular Ca 2ϩ homeostasis in many different cells and tissues.…”

Calcium regulation in photoreceptors

“…Photoreceptors express the NCKX1 isoform (40)(41). The cloning of NCKX1 from rat eye (42) revealed the presence of alternatively spliced isoforms in this species, which differ in the arrangement of four exons at the N terminus of the large intracellular loop. Another isoform was recently isolated from cones (43).…”

A flagellar K + -dependent Na + /Ca 2+ exchanger keeps Ca 2+ low in sea urchin spermatozoa

“…Three NCKX subfamilies have been found in vertebrates, exchanging 1 K ϩ and 1 Ca 2ϩ (out) for 4 Na ϩ (in) (11). NCKX1 has been cloned from bovine (13), dolphin (14), chicken (15), rat (16), and human (17) retina rod cells. NCKX2 was originally identified in rat brain (18) and also retinal cone cells of chickens and humans (15).…”