Minimal domain requirement for cation transport by the potassium-dependent Na/Ca-K exchanger. Comparison with an NCKX paralog from Caenorhabditis elegans.
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.
Szerencsei
R T
RT
Department of Physiology, University of Calgary, Calgary, Alberta, T2N 4N1 Canada. pschnetk@ucalgary.ca
Tucker
J E
JE
Cooper
C B
CB
Winkfein
R J
RJ
Farrell
P J
PJ
Iatrou
K
K
Schnetkamp
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D017173:Caenorhabditis elegans / Q000235:genetics*; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D002417:Cattle; D004289:Dolphins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger; D013696:Temperature
D002352:Carrier Proteins; D002412:Cations; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D012964:Sodium; D011188:Potassium; D002118:Calcium