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. CONCLUSION: These findings show that Na+-dependent cellular Ca2+ efflux in DCT cells is primarily mediated by NACA2 and NACA3.
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. CONCLUSION: These findings show that Na+-dependent cellular Ca2+ efflux in DCT cells is primarily mediated by NACA2 and NACA3.