Expression of the Sodium/Calcium/Potassium Exchanger, NCKX4, in Ameloblasts

Abstract

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⁺/Ca²⁺) exchangers, which include the NCX and NCKX [sodium/calcium-potassium (Na⁺/Ca²⁺-K⁺) exchanger] proteins. During enamel maturation, when crystals expand in thickness, Ca²⁺ requirements vastly increase but exactly how Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ transport during amelogenesis.

Store-operated Ca2+ entry controls ameloblast cell function and enamel development

“…In our study, by limiting Ca 2+ influx as a consequence of disrupting SOCE, it is possible that Ca 2+ itself, which is known to be involved in protein trafficking (30), had an effect of NCKX4. Alternatively, given that NCKX4 localizes to the ruffled-distal border of WT ameloblasts (3,17), disruptions in the ameloblast cytoskeleton noted here dysregulated NCKX4 localization in Stim1/2 K14cre ameloblasts, likely disrupting its role in Ca 2+ efflux. We have previously noted that NCKX4 is found in the intracellular compartment of WT smooth-ended ameloblasts (3).…”

“…protein involved in Ca 2+ extrusion expressed by ameloblasts, the exchanger NCKX4 (3,(17)(18)(19), was highly upregulated ( Figure 4A). In WT ameloblasts, NCKX4 is localized at the apical pole of ruffled-ended ameloblasts, thus serving as a key extrusion path for Ca 2+ ( Figure 4B).…”

“…In our study, the observed changes in the elemental composition in the enamel of Stim1/2 K14cre mice reflect the relevance of maintaining a functional Ca 2+ transport system and the coupling of Ca 2+ availability with other ions in the overall physiology of enamel formation. Moreover, loss of appropriate levels of Ca 2+ influx via SOCE affected the localization of NCKX4, the main Ca 2+ exchanger involved in Ca 2+ efflux in ameloblasts (17). The critical role of NCKX4 in enamel has been determined recently by studies showing that mutations in the gene encoding NCKX4 in humans and mice result in abnormal enamel that is also hypomineralized (18).…”

A Functional Study of Mutations in K+-dependent Na+-Ca2+ Exchangers Associated with Amelogenesis Imperfecta and Non-syndromic Oculocutaneous Albinism

“…Na ϩ /Ca 2ϩ exchangers belong to either the SLC8A 2 gene family of K ϩ -independent Na ϩ /Ca 2ϩ exchangers, encoding three distinct NCX1-3 proteins (7), or the SLC24A gene family of K ϩ -dependent Na ϩ / Ca 2ϩ exchangers encoding the five distinct NCKX1-5 proteins (8). Transcripts of both NCXs and NCKXs were expressed at different stages of the developing enamel (9,10), specifically NCX1, NCX3, and NCKX4. Moreover, the highest SLC24A4 gene (accession number AF520705) transcript levels were observed in maturation-stage ameloblasts and the NCKX4 protein was localized at the apical pole suggesting that NCKX4 is a key Ca 2ϩ transporter during amelogenesis (10).…”

“…Transcripts of both NCXs and NCKXs were expressed at different stages of the developing enamel (9,10), specifically NCX1, NCX3, and NCKX4. Moreover, the highest SLC24A4 gene (accession number AF520705) transcript levels were observed in maturation-stage ameloblasts and the NCKX4 protein was localized at the apical pole suggesting that NCKX4 is a key Ca 2ϩ transporter during amelogenesis (10). The importance of the SLC24A4 gene is corroborated by recent genetic analyses linking three unique SLC24A4 missense mutations to amelogenesis imperfecta (AI) (4,(11)(12)(13), a genetic disease presented with abnormal teeth formation and development.…”