Structural and functional analysis of Na+/Ca2+ exchange in distal convoluted tubule cells.
Renal distal convoluted tubules (DCT) are a major site of hormone-regulated, active calcium absorption. Calcium exit across basolateral plasma membranes is thought to be mediated by Na+/Ca2+ exchange and a Ca(2+)-ATPase. In this report the presence and function of Na+/Ca2+ exchangers in DCT cells were assessed. cDNAs encoding a conserved region and the variable regions of three alternatively spliced isoforms of the Na+/Ca2+ exchanger, NACA2, NACA3, and NACA6, were isolated in a ratio of 7:12:1 using homology-based reverse transcription-polymerase chain reaction (RT-PCR) with RNA from an immortalized mouse DCT cell line. Northern blots probed with a 32P-labeled PCR product from a conserved region of the exchanger were positive for a single transcript of 7 kb in primary cultures of distal tubule cells (cortical ascending limb + DCT cells), consistent with the reported size of the exchanger in other tissues. Na+/Ca2+ exchange was assessed by measuring sodium-dependent changes of intracellular calcium ([Ca2+]i), in single cells. In the presence of an outward Na+ gradient, [Ca2+]i increased by 240%. Collapsing the Na+ gradient with monensin inhibited the rise of [Ca2+]i. Removal of extracellular Ca2+ or the addition of an Na+ ionophore inhibited the rise of [Ca2+]i. The intracellular Na+ concentration decreased upon removal of extracellular Na+ in parallel with the rise of [Ca2+]i. Western analysis performed on membranes prepared from DCT cells or primary cultures of distal tubule cells with a polyclonal antibody revealed bands at approximately 125 and 85 kDa, consistent with reported sizes for exchanger protein. These findings show that Na+/Ca2+ exchanger transcripts, protein, and activity are present in DCT cells and that Na(+)-dependent Ca2+ efflux may be mediated by NACA2, NACA3, and NACA6.
White
K E
KE
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835, USA.
Gesek
F A
FA
Friedman
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004285:Dogs; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D019831:Sodium-Calcium Exchanger; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger