Enamel cells handle large amounts of calcium, particularly during the developmental phase (termed maturation) when dental enamel is hypermineralized. The extent of intracellular calcium burden, and the nature of calcium homeostasis machinery used to accommodate it, are largely unknown. Here, the calcium-binding capacity of enamel cell cytosol was found to increase during development, in parallel with the putative transcellular flux of calcium. At maturation, the abundance of calcium-binding proteins in enamel cells exceeded that in brain and other established calcium-oriented tissues, which implies a large calcium burden. A search for likely cytosolic calcium transporters revealed only one high-affinity calcium-binding protein (12 kDa, distinguished from alpha-parvalbumin) that was up-regulated during maturation, but its low abundance (0.02 % of soluble protein) precluded a major calcium transport or cytoprotective role. Two low-affinity calcium-binding proteins up-regulated during maturation (by 1.8-fold and 2.1-fold, respectively) were identified as calreticulin and endoplasmin, both residents of the endoplasmic reticulum. Together, calreticulin and endoplasmin constituted an exceptionally high proportion (5%) of soluble protein during maturation, which gives an inferred calcium capacity 67-fold higher than that of the principal cytosolic calcium-binding protein, 28-kDa calbindin. Evidence that endoplasmin expression varied inversely with serum calcium concentration, and that the inositol trisphosphate receptor also was maturation, supported the novel hypothesis that non-mitochondrial calcium stores play a major role in transcellular calcium transport. In conclusion: (a) enamel cells contain a general high abundance of calcium homeostasis proteins, consistent with a heavy intracellular calcium burden; (b) the expression pattern (phenotype) of calcium-binding proteins varies with enamel cell function; (c) enamel cells appear to contain unusually large non-mitochondrial calcium stores; (d) contrary to the prevailing view that calcium passes mainly through the cytosol of calcium-transporting cells, the findings imply a route through the endoplasmic reticulum. This study gives novel information about how a highly calcium-oriented tissue avoids calcium toxicity, and provides a new focus for investigations into the mechanisms of transcellular calcium transport.
