PMCA Ca 2+ clearance in dental enamel cells depends on the magnitude of cytosolic Ca 2 .

Enamel formation (amelogenesis) is a two-step process whereby crystals partially grow during the secretory stage followed by a significant growth expansion during the maturation stage concurrent with an increase in vectorial Ca 2+ transport. This requires tight regulation of cytosolic Ca 2+ ( c Ca 2+ ) concentration in the enamel forming ameloblasts by controlling Ca 2+ influx (entry) and Ca 2+ extrusion (clearance). Gene and protein expression studies suggest that the plasma membrane Ca 2+ -ATPases (PMCA1-4) are likely involved in c Ca 2+ extrusion in ameloblasts, yet no functional analysis of these pumps has been reported nor whether their activity changes across amelogenesis. PMCAs have high Ca 2+ affinity and low Ca 2+ clearance which may be a limiting factor in their contribution to enamel formation as maturation stage ameloblasts handle high Ca 2+ loads. We analyzed PMCA function in rat secretory and maturation ameloblasts by blocking or potentiating these pumps. Low/moderate elevations in c Ca 2+ measured using the Ca 2+ probe Fura-2-AM show that secretory ameloblasts clear Ca 2+ faster than maturation stage cells through PMCAs. This process was completely inhibited by an external alkaline (pH 9.0) solution or was significantly delayed by the PMCA blockers vanadate and caloxin 1b1. Eliciting higher c Ca 2+ transients via the activation of the ORAI1 Ca 2+ channel showed that the PMCAs of maturation ameloblasts were more efficient. Inhibiting PMCAs decreased the rate of Ca 2+ influx via ORAI1 but potentiation with forskolin had no effect. Our findings suggest that PMCAs are functional Ca 2+ pumps during amelogenesis regulating c Ca 2+ upon low and/or moderate Ca 2+ stimulus in secretory stage, thus participating in amelogenesis.