Calcium (Ca2+) extrusion is an essential function of the enamel-forming ameloblasts, providing Ca2+ for extracellular mineralization. The plasma membrane Ca2+ ATPases (PMCAs) remove cytosolic Ca2+ (cCa2+) and were recently shown to be efficient when ameloblasts experienced low cCa2+ elevation. Sodium–calcium (Na+/Ca2+) exchange has higher capacity to extrude cCa2+, but there is limited evidence on the function of the two main families of Na+/Ca2+ exchangers in enamel formation. The purpose of this study was to analyze the function of the NCX (coded by SLC8) and the K+-dependent NCKX (coded by SLC24) exchangers in rat ameloblasts and to compare their efficacy in the two main stages of enamel formation: the enamel forming secretory stage and the mineralizing or maturation stage. mRNA expression profiling confirmed the expression of Slc8 and Slc24 genes in enamel cells, Slc24a4 being the most highly upregulated transcript during the maturation stage, when Ca2+ transport increases. Na+/Ca2+ exchange was analyzed in the Ca2+ influx mode in Fura-2 AM–loaded ameloblasts. We show that maturation-stage ameloblasts have a higher Na+/Ca2+ exchange capacity than secretory-stage cells. We also show that Na+/Ca2+ exchange in both stages is dominated by NCKX over NCX. The importance of NCKX function in ameloblasts may partly explain why mutations in the SLC24A4 gene, but not in SLC8 genes, result in enamel disease. Our results demonstrate that Na+/Ca2+ exchangers are fully operational in ameloblasts and that their contribution to Ca2+ homeostasis increases in the maturation stage, when Ca2+ transport need is higher.

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