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 (Δ[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 Δ[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 Δ[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 potyclonal 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.