IMMUNOLOCALIZATION OF THE NA+ CA2+ EXCHANGER IN RABBIT KIDNEY/

We recently isolated a cDNA encoding a Na+/Ca2+ exchanger from rabbit kidney that was highly similar to the canine cardiac sarcolemmal Na+/C a2+ exchanger. In the present study, we used two different antibodies to the exchanger to identify the protein and establish its cellular an d subcellular localization in the kidney. The first antibody was prepa red against a fusion protein consisting of 190 amino acids of the larg e, presumably intracellular loop of the rabbit renal exchanger fused t o the maltose-binding protein. The second was a monoclonal antibody ge nerated against the isolated purified canine cardiac sarcolemmal excha nger. To identify the Na+/Ca2+ exchanger protein, we performed immunob lot analysis against a membrane vesicle preparation from rabbit kidney cortex. Both antibodies immunoblotted proteins of 120 and 70 kDa that are known to be associated with the exchanger. Indirect immunofluores cence revealed that both antisera labeled the basolateral surface of t he majority of cells in the connecting tubule (CNT). Since the phase-d ense (intercalated) cells in the CNT were not stained, this suggested that the labeled cells were CNT cells. No labeling was detected in oth er nephron segments with the exception of occasional faint staining of the majority cell population of the cortical collecting duct. The fac t that we did not detect labeling in other nephron segments is consist ent with either 1) the absence of expression of the Na+/Ca2+ exchanger in these segments, 2) the expression of the exchanger in levels below the threshold of detection of the two antibodies used in this study, or 3) the exchanger in these segments is represented by a different is oform. We conclude that the CNT is the primary site of expression of t he Na+/Ca2+ exchanger in rabbit kidney.