NCX1 Na/Ca exchanger inhibition by antisense oligonucleotides in mouse distal convoluted tubule cells.
BACKGROUND
Plasma membrane NCX1 Na+/Ca2+ exchangers mediate cellular Ca2+ efflux. Renal distal convoluted tubule (DCT) cells express transcripts encoding three alternatively spliced NCX1 isoforms: NACA2 (exons B, C, D), NACA3 (exons B and D), and NACA6 (exons A, C, D). We used antisense oligodeoxynucleotides (ODNs) to determine the function of these NACA isoforms on Na+/Ca2+ exchanger activity and expression in DCT cells.


METHODS
Sense and antisense ODNs targeting exchanger transcripts were introduced into DCT cells permeabilized with streptolysin O. Na+/Ca2+ exchange activity was assessed by measuring Na+-dependent changes of free intracellular Ca2+ concentration (delta[Ca2+]i), in single cells, when the electrochemical gradient for Na+ was reversed.


RESULTS
The change of [Ca2+]i in cells treated with antisense ODNs to a downstream or upstream region common to all NCX1 isoforms was 173 nM (-66%) to the downstream region located in the putative ninth transmembrane domain, and 226 nM (-39%) with ODNs to an upstream region located 5' to the variable portion of the intracellular loop. Antisense ODNs to exon B, present in both NACA2 and NACA3, decreased delta[Ca2+]i by 209 nM (-44%), while antisense ODNs specific for NACA6 (exon A) were without effect. Antisense ODNs specific for exon C, present in NACA2 and NACA6, decreased delta[Ca2+]i by 226 nM (-39%). Northern analysis of mRNA prepared from primary cultures of distal tubule cells revealed exon B- but not exon A-containing transcripts. Immunofluorescence analysis using a polyclonal antibody that recognizes NCX1 confirmed that protein expression was inhibited after treatment with the exon B antisense ODNs.


CONCLUSIONS
These findings show that Na+-dependent cellular Ca2+ efflux in DCT cells is primarily mediated by NACA2 and NACA3.
White
K E
KE
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire, USA.
Gesek
F A
FA
Reilly
R F
RF
Friedman
P A
PA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005091:Exons; D007686:Kidney Tubules, Distal / Q000378:metabolism*; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D012964:Sodium; D002118:Calcium