cDNA cloning and functional expression of the dolphin retinal rod Na-Ca plus K exchanger NCKX1: Comparison with the functionally silent bovine NCKX1

cDNAs of human and bovine retinal rod Na+-Ca2++K+ exchanger (NCKX1) have pr eviously been cloned, but potassium-dependent Na-Ca exchange activity upon heterologous expression has not been demonstrated. We have cloned NCKX1 cDN A from dolphin, examined function upon transfection in HEK293 cells, and co mpared the dolphin sequence encoded by the cDNA with those of human and bov ine. The dolphin NCKX1 cDNA encodes 1013 amino acid residues. Comparison to bovine and human NCKX1 revealed strong conservation in the transmembrane d omains (>95%), but relatively low conservation in the large extracellular ( similar to 50%) and cytosolic (similar to 50%) domains. The dolphin cytosol ic domain differs from the bovine sequence by the absence of a stretch of 1 14 amino acids. HEK293 cells transfected with dolphin NCKX1 cDNA showed K+- dependent Na-Ca exchange in >95% of the experiments, whereas transfection w ith bovine NCKX1 yielded no function. The bovine NCKX1 phenotype was impart ed on dolphin NCKX1 when the dolphin cytosolic loop was replaced by that fr om bovine. Conversely, deletion of 114 amino acids from the bovine sequence to match the dolphin sequence resulted in a mutant bovine NCKX1 which perf ormed K+-dependent Na-Ca exchange. These results suggest that domains withi n the large cytosolic loop of NCKX1 control functional activity when expres sed in heterologous systems.