Minimal domain requirement for cation transport by the potassium-dependentNa/Ca-K exchanger - Comparison with an NCKX paralog from Caenorhabditis elegans

The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion prot ein utilizing both inward sodium gradient and outward potassium gradient. T hree mammalian rod NCKX cDNAs have been cloned to date, but quantitative an alysis of NCKX function in heterologous systems has proven difficult, Here, we describe a simple system for quantitative analysis of NCKX function; st able transformation of cultured insect cells with the novel pEA1/153A vecto r containing NCKX cDNAs was combined with measurements of potassium-depende nt Ca-45 uptake in sodium-loaded cells, We carried out structure-function s tudies on NCKX with the following results: 1) two-thirds of the full-length sequence of bovine NCKX could be deleted without affecting potassium-depen dent calcium transport and without affecting key properties of the potassiu m binding site; 2) the affinity of NCKX for potassium was about 10-fold gre ater in choline medium when compared with lithium medium; this shift was ob served in rod outer segments or in cells expressing fall-length rod NCKX, t he above deletion mutant, or a distantly related NCKX paralog cloned from C aenorhabditis elegans. We conclude that the potassium binding site is highl y conserved among members of the NCKX family and is formed by residues loca ted within the two sets of transmembrane spanning segments in the NCKX sequ ence.