Structural and functional analysis of Na+/Ca2+ exchange in distal convoluted tubule cells

Kenneth E. White, Frank A. Gesek, Peter A. Friedman

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

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 Ca2+-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 4- 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 -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.

Original languageEnglish (US)
Pages (from-to)F560-F570
JournalAmerican Journal of Physiology
Volume271
Issue number3 PART 2
DOIs
StatePublished - 1996

Keywords

  • Calcium transport
  • Intracellular calcium
  • Kidney
  • Reverse transcriptionpolymerase chain reaction
  • Sodium/calcium exchanger

ASJC Scopus subject areas

  • Physiology (medical)

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