Supplementary Material for: Expression of the Sodium/Calcium/Potassium Exchanger, NCKX4, in Ameloblasts

Transcellular calcium transport is an essential activity in mineralized tissue formation, including dental hard tissues. In many organ systems, this activity is regulated by membrane-bound sodium/calcium (Na<sup>+</sup>/Ca<sup>2+</sup>) exchangers, which include the NCX and NCKX [sodium/calcium-potassium (Na<sup>+</sup>/Ca<sup>2+</sup>-K<sup>+</sup>) exchanger] proteins. During enamel maturation, when crystals expand in thickness, Ca<sup>2+</sup> requirements vastly increase but exactly how Ca<sup>2+</sup> traffics through ameloblasts remains uncertain. Previous studies have shown that several NCX proteins are expressed in ameloblasts, although no significant shifts in expression were observed during maturation which pointed to the possible identification of other Ca<sup>2+</sup> membrane transporters. NCKX proteins are encoded by members of the solute carrier gene family, Slc24a, which include 6 different proteins (NCKX1–6). NCKX are bidirectional electrogenic transporters regulating Ca<sup>2+</sup> transport in and out of cells dependent on the transmembrane ion gradient. In this study we show that all NCKX mRNAs are expressed in dental tissues. Real-time PCR indicates that of all the members of the NCKX group, NCKX4 is the most highly expressed gene transcript during the late stages of amelogenesis. In situ hybridization and immunolocalization analyses clearly establish that in the enamel organ, NCKX4 is expressed primarily by ameloblasts during the maturation stage. Further, during the mid-late maturation stages of amelogenesis, the expression of NCKX4 in ameloblasts is most prominent at the apical poles and at the lateral membranes proximal to the apical ends. These data suggest that NCKX4 might be an important regulator of Ca<sup>2+</sup> transport during amelogenesis.