Plasma membrane Ca2+-ATPase and NCX1 Na+/Ca2+ exchanger expression in distal convoluted tubule cells

Abstract

The plasma membrane Ca2+-ATPase (PMCA) and the NCX1 Na+/Ca2+ exchanger regulate intracellular Ca2+ concentrations and mediate Ca2+ efflux in absorptive epithelial cells. We characterized the PMCA isoforms and subtypes expressed in mouse distal convoluted tubule (mDCT) cells and Na+/Ca2+ exchanger protein expression in mDCT cells. In lysates of mDCT cells, immunoprecipitation and Western blot analysis, performed with a monoclonal antibody to PMCA, revealed a 140-kDa protein consistent with PMCA. Laser-scanning confocal fluorescence microscopy indicated that PMCA and NCX1 expression is restricted to basolateral membranes only in confluent mDCT cells, because subconfluent cultures predominately express intracellular localizations. PMCA isoform-specific PCR primers generated appropriately sized products only for PMCA1 and PMCA4 from DCT cells but PMCA1–4 from whole mouse kidney. Assessment of splice site C within the calmodulin-binding domain demonstrated the presence of PMCA1b and PMCA4b mRNAs in mDCT cells. Northern blot analysis of mDCT cell RNA revealed transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4. We conclude that DCT cells express PMCA transcripts encoding PMCA1b and PMCA4b. Basolateral localization of the Na+/Ca2+ exchanger and PMCAs support the idea that multiple PMCA isoforms, in concert with the Na+/Ca2+ exchanger, mediate basal or hormone-stimulated Ca2+ efflux by distal tubules.