Abstract
We have isolated a novel cDNA clone from rat cerebral cortex encoding a protein of 670 amino acids (NCKX2) that has significant similarity to the 1199-amino acid-long Na/Ca-K exchanger of bovine rod outer segment (NCKX1). NCKX2 transcripts are 10.5 kilobase pairs in length and are expressed abundantly in neurons throughout the brain and with much lower abundance in selected other tissues. The predicted topology of the rat NCKX2 protein is very similar to that of bovine NCKX1, beginning with a solitary transmembrane segment (M0), which is removed as a "signal peptide" in bovine NCKX1, an extracellular loop, a cluster of five transmembrane spanning segments (M1 to M5), a long cytoplasmic loop, and a final hydrophobic cluster (M6 to M11). Within the hydrophobic clusters, rat NCKX2 shares 80% identity and 91% similarity with bovine NCKX1. The two larger hydrophilic loops are much shorter in NCKX2 than in NCKX1, accounting largely for the difference in length between the two proteins, and are dissimilar in sequence except for a 32-amino acid stretch with 69% identity in the cytosolic loop. NCKX2 was epitope-tagged in the extracellular domain and was shown to be expressed at the surface of transfected HEK cells. Analysis of NCKX2 function by fluorescent imaging of fura-2-loaded transfected cells demonstrated that NCKX2 is a potassium-dependent sodium/calcium exchanger.
Highlights
A plasma membrane sodium-calcium exchange process plays an important role in controlling cytosolic calcium concentrations in a broad number of tissues [1]
We identified and isolated a full-length cDNA, called NCKX2, from rat brain that encodes a novel protein with extensive similarity to bovine NCKX1 in the hydrophobic regions and displays potassium-dependent sodium-calcium exchange activity when expressed in HEK-293 cells
We have identified a novel Na/Ca exchanger molecule expressed in rat brain that shares a high degree of sequence similarity with the Na/Ca-K exchanger of bovine retinal rod outer segments (NCKX1) and requires potassium for function
Summary
A plasma membrane sodium-calcium exchange process plays an important role in controlling cytosolic calcium concentrations in a broad number of tissues [1]. Structural studies have revealed the existence of two classes of protein that underlie the sodium-calcium exchange process. One class, exemplified by the sodium-calcium exchanger from dog heart, NCX1,1 catalyzes the exchange of three sodium ions for one calcium ion [2]. The other class, exemplified by the sodium-calcium ϩ potassium exchanger of bovine retinal rod outer segments, NCKX1, transports four sodium ions in exchange for one calcium and one potassium ion [3]. NCX1 is the most abundantly and widely expressed sodiumcalcium exchanger gene, with products present in almost every tissue and present at a high level in heart, brain, and kidney. Two short stretches, one in each hydrophobic helix cluster, do show significant similarity These regions are the sites of highest conservation among sodium-calcium exchangers cloned from various organisms. It was recently noted that the two regions are similar to one another and may have arisen from an ancient gene duplication event, a finding that has led to the speculation that these sites may constitute the ion binding pocket required for transport across the membrane [11, 12]
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