Abstract
The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion protein utilizing both inward sodium gradient and outward potassium gradient. Three mammalian rod NCKX cDNAs have been cloned to date, but quantitative analysis of NCKX function in heterologous systems has proven difficult. Here, we describe a simple system for quantitative analysis of NCKX function; stable transformation of cultured insect cells with the novel pEA1/153A vector containing NCKX cDNAs was combined with measurements of potassium-dependent (45)Ca uptake in sodium-loaded cells. We carried out structure-function studies on NCKX with the following results: 1) two-thirds of the full-length sequence of bovine NCKX could be deleted without affecting potassium-dependent calcium transport and without affecting key properties of the potassium binding site; 2) the affinity of NCKX for potassium was about 10-fold greater in choline medium when compared with lithium medium; this shift was observed in rod outer segments or in cells expressing full-length rod NCKX, the above deletion mutant, or a distantly related NCKX paralog cloned from Caenorhabditis elegans. We conclude that the potassium binding site is highly conserved among members of the NCKX family and is formed by residues located within the two sets of transmembrane spanning segments in the NCKX sequence.
Highlights
The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion protein utilizing both inward sodium gradient and outward potassium gradient
Three NCKX cDNAs have been cloned from different mammalian retinal rod photoreceptors (4 – 6), one from rat brain [7], and several putative members have been identified in lower eukaryotes as well as in several prokaryotes on the basis of sequence similarity within the two sets of transmembrane-spanning segments [2, 8]
In situ NCKX protein in retinal rod outer segments was shown to act as a Na/Ca exchanger that both requires and transports potassium; i.e. calcium extrusion is driven by both the inward sodium gradient and the outward potassium gradient at a stoichiometry of 4
Summary
(Received for publication, August 30, 1999, and in revised form, October 18, 1999). Robert T. Studies on functional properties of the “in situ” Na/Ca-K exchanger have been limited far to NCKX1 found in the plasma membrane of the outer segments of retinal rod photoreceptors We tested the hypothesis that the cation binding sites and residues involved in potassium-dependent Na/Ca exchange transport reside within the above two sets of conserved transmembrane spanning segments. We cloned the cDNA of an NCKX paralog identified in the Caenorhabditis elegans genomic sequencing project (ceNCKX; GenBankTM accession number AJ005701) and tested for potassium-dependent Na/Ca exchange. Potassium-dependent Na/Ca exchange activity was measured in High Five insect cells after stable transformation with the various NCKX cDNAs using a novel expression vector [12].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
