Mini Review: Enamel: Molecular identity of its transepithelial ion transport system

Abstract

Enamel is the most calcified tissue in vertebrates. It differs from bone in a number of characteristics including its origin from ectodermal epithelium, lack of remodeling capacity by the enamel forming cells, and absence of collagen. The enamel-forming cells known as ameloblasts, choreograph first the synthesis of a unique protein-rich matrix, followed by the mineralization of this matrix into a tissue that is ~95% mineral. To do this, ameloblasts arrange the coordinated movement of ions across a cell barrier while removing matrix proteins and monitoring extracellular pH using a variety of buffering systems to enable the growth of carbonated apatite crystals. Although our knowledge of these processes and the molecular identity of the proteins involved in transepithelial ion transport has increased in the last decade, it remains limited compared to other cells. Here we present an overview of the evolution and development of enamel, its differences with bone, and describe the ion transport systems associated with ameloblasts.

Graphical abstract

Introduction

Tooth enamel is pretty close to being just a mineral in the mouth. The inorganic content in enamel is about 95% by weight [1], much higher than in bone. The basic structure of tooth enamel is the formation of millions of nanometer wide hydroxyapatite-like (Hap) crystals. Each of these crystals run for hundreds of micrometers bundled into groups called enamel prisms or rods [2]. The Hap-like crystals contain large amounts of Ca²⁺, being in fact the most highly calcified structure found in vertebrates containing about 60% Ca²⁺ by mass [2, 3]. However, despite its complex dependency on Ca²⁺, dental research seldom reaches the scientific attention that other topics in the Ca²⁺ signaling field receive.

This is in part associated with the fact that enamel research has largely focused on the chemical events and interactions of the ions involved in the formation of the enamel crystals, that is, the extracellular milieu, rather than the cellular events involved. As in any biological system, it is important to identify how cells orchestrate the functions that are relevant for developing a tissue. For example, recognizing that Ca²⁺ is essential for cell differentiation and survival, gene expression and other processes [4], should beckon us to consider how the physiology of the enamel generating cells is designed to cope with handling potentially highly toxic levels of Ca²⁺ before this cation is delivered outside the cell [5]. Ameloblasts are the master makers of enamel. Their capacity to transport Ca²⁺ to mineralize the extracellular matrix can be distinguished from the potential role of Ca²⁺ as an intracellular signaling messenger. Little is known about either role. Consequently, the potential effects of changes in the intracellular concentration of Ca²⁺ in regulating enamel crystal growth are poorly understood.

Ca²⁺ and PO4³⁻ are the dominant ions contained in mineralized enamel crystals. For these crystals to develop properly, their often variable stoichiometric composition also requires other ions including bicarbonate, Cl⁻, K⁺, Na⁺ and Mg²⁺ [6]. The delivery of these ions to the extracellular milieu is closely coordinated by the ameloblasts [7]. Yet knowledge of the ameloblast’s decision making process to switch functions from a tissue-forming to a mineralizing cell is extraordinarily poor and their physiology is ill-defined. Although recent studies have improved our overall knowledge of the ion transport system in ameloblasts, it remains limited. Here we succinctly describe the evolutionary origins of enamel and its differences with bone, and review what is known about the process of enamel formation, or amelogenesis, and then discuss the molecular identity of ion transport proteins that have thus far been identified in ameloblasts.

Teeth and bones

The skeletonized structures of vertebrates are recognized in the fossil record dating back to the Cambrian period well in excess of 500 million years ago. Early vertebrates developed bones and teeth separately [8]. It is considered that the bony-like skeletons of early vertebrates originally derived as a reservoir for calcium and phosphate. From these, and with the availability of collagen fibers already present in invertebrate organisms, the earliest mineralization of skeletonized structures appeared [8]. The earliest vertebrates do not show evidence of internal bony elements, which likely appeared millions of years after the outer skeleton [8]. Teeth may have developed ahead of the inner skeleton but the origin of teeth is not fully understood. Teeth either evolved from dermal denticles that migrated into the rudimentary oral space, or from specialized oropharyngeal denticles which in this case provided insulation for the primitive electrosensory organ [9, 10]. Besides differences in evolutionary origin, bones and teeth also differ in the combination of cell types that assemble to provide their embryonic origin. Enamel develops from ectodermal epithelium whereas bone cells derive from mesenchyme [11]. Moreover, although both tissues are formed via matrix-mediated mineralization, bone is much less mineralized than enamel and contains a large percentage of collagen which is nearly absent in enamel [12]. Bone becomes mineralized as it is formed, whereas enamel is mineralized in two stages, as described below. Enamel shows complex patters of hardness and resistance to fracture [13]. Importantly, bone is a living tissue and the cells that form and maintain this tissue (osteoblast, osteoclast, osteocytes) are active for the majority of the person’s life in non-pathological conditions. However, enamel does not contain any living cells and once formed, it does not regenerate.

Life and death of a very tall cell

Ameloblast cells are complex. They develop from ectodermal epithelium at the interface with mesenchymal cells which, upon condensation, instruct the epithelium to undergo cytodifferentiation [14, 15]. From here on, the post-mitotic epithelial cells will increase in height, develop a protein synthesis apparatus and a distal cell process (Tomes’ process) that is important for the organization of the crystals [16]. The secretory stage defines the first stage of amelogenesis [17]. Secretory ameloblasts are tall, reaching heights around 70 μm but maintaining a narrow 4–5 μm diameter and form a semipermeable barrier [18] (see also Fig 1). At this stage, enamel is only partially mineralized and the ameloblasts primary function is to synthesize and secrete a number of structural enamel matrix proteins to build the volume of the tissue [19]. In the next stage of amelogenesis coined the maturation stage, enamel undergoes two main processes: degradation and removal of organic matrix, and increased mineralization [7, 20]. Maturation stage ameloblasts retransform from the previous stage by changing their morphology in a number of ways. First, they decrease their height to about 40 μm or less and lose the Tomes’ process [21]. Instead of the Tomes’ process, the bulk of the cells in the maturation stage show a ruffled-border [1]. Ameloblasts at this stage will lose and reform this ruffled-border in alternating waves of ruffled-ended to smooth-ended morphology a number of times (Fig 1). The mechanisms and signals triggering these waves are poorly understood but recent evidence suggests that changes in [Ca²⁺]cyt or extracellular pH might be associated with the modulation from RA to SA [22, 23]. At the end of the maturation stage, ameloblasts reduce their height and regress forming a layer of epithelial cells around the enamel that is lost during eruption [1] . In rodents, the most widely used model in tooth development, the secretory stage lasts about 7 days and the maturation stage twice as long [1]. Shortly after, the teeth can be seen emerging through the gingiva into the oral cavity. In the erupted teeth of all mammals, there are no living ameloblasts, so enamel cannot remodel or self-repair.

Figure 1. Schematic of the histology of the enamel organ.

Ameloblasts undergo a number of important morphological changes. Ameloblasts originate from undifferentiated epithelium at the cervical loop (CL) before elongating and developing a secretory Tomes’ process (TP) distally. These tall polarized cells are the secretory ameloblasts. The adjacent stratum intermedium (SI) and stellate reticulum (SR) cells also form part of the enamel organ during the secretory stage. At this stage the matrix is poorly mineralized (light blue enamel). Secretory ameloblasts will transition to maturation stage ameloblasts increasing their capacity for ion transport to fully mineralize the enamel (dark blue enamel). The SR and SI cells also give rise to the papillary layer (PL) of cells. Organics are removed in maturation stage while Ca²⁺ and PO4³⁻ transport increases. The maturation stage contains two distinct cell morphologies. The dominant ruffle-ended ameloblasts (RA) cycle into smooth-ended cells (SA). The former has been associated with active transport. Ameloblasts regress and undergo apoptosis at the end of the life cycle prior to eruption of the tooth into the oral cavity.

Enamel crystal formation and enamel matrix proteins

Enamel is a remarkable example of cell-directed mineralization. Enamel crystals form in the extracellular compartment by supersaturation of minerals guided or mediated by matrix proteins [24]. Crystal growth requires nucleation events as a first step, or the clustering of minerals into recognizable structures, of which the smallest unit is referred to as a unit cell [6]. For dental enamel, the chemical formula of the unit cell of these carbonated hydroxyapatite (Hap) crystals that are formed is: Ca10 (PO4)6 (OH)2. Hexagonally-shaped enamel crystallites are formed as ameloblasts develop their Tomes’ process. These crystallites of just a few nanometers in diameter grow in length to some hundreds of micrometers and are surrounded by organic matrix [25]. These events occur in the secretory stage. The maturation of the crystals involves removal of matrix proteins and the increase in transport of Ca²⁺ and PO3⁻ which enables an increase in width and thickness of the crystals to the point that there is almost no separation between them [2, 26]. The crystals are packed into units called enamel prisms or rods of ~5um in diameter [2, 25]. The arrangement of these enamel rods can vary from species to species. When fully mineralized, enamel hardness has been likened to that intermediate of iron and carbon steel, while maintaining an unusual level of elasticity [6].

At least three main proteins are recognized as the main structural mediators of enamel crystal growth [19, 27]. These proteins are: amelogenin (AMELX), ameloblastin (AMBN) and enamelin (ENAM). They all have in common that mutations in their coding genes affect enamel development to varying degrees. The resulting phenotypes are directly associated with amelogenesis imperfecta (AI) [28]. AI is a clinical term that broadly defines abnormal enamel formation. As will be discussed later, a number of mutations in many other genes that are widely expressed in the body organs besides ameloblasts which also impact enamel [29].

AMELX is by far the most abundant product synthesized by ameloblasts with as much as 90% of the total output of proteins secreted by ameloblasts being AMELX [30]. Secreted AMELX (25 kDa) is quickly degraded into smaller units of lower molecular weight which may be required for its binding to crystals and direct their growth direction [19]. The details of this interaction between AMELX and Hap-like crystal still fuels scientific debate (i.e. [31]). AMBN function remains unclear but includes acting as a nucleator during crystal formation, as a cell adhesion molecule that maintains ameloblasts in a differentiated state, or required for the formation and maintenance of a mineralization front [32, 33]. ENAM is associated with the elongation of enamel crystals [34] or shaping minerals into enamel ribbons (earliest formed crystal precursors) [35].

Calcium and Phosphate

Analysis of the elemental composition of human enamel shows that Ca²⁺ account for ~37% of the mineral content by weight with PO4³⁻ being the second most abundant at ~ 17% [36]. Other elements found in enamel include Na⁺, Mg²⁺ and K⁺ all of which are present is less than 1% [36]. Cl⁻ and F⁻ are found as trace elements in enamel. These elements reach the forming enamel crystals by crossing the epithelial barrier formed by the ameloblasts from the blood circulation. Radiolabel studies have shown that both ⁴⁵Ca²⁺ and ³²P can move quickly (within minutes) from blood circulation into enamel [37, 38]. In both cases, the inclusion of these elements is heightened during the maturation stage. While this fast movement has been associated with passive transepithelial transport following the injection of radiolabelled compounds, it is now recognized that ameloblasts themselves actively control ionic transport in a carefully orchestrated manner [1, 39]. It was originally proposed that Ca²⁺ uptake into ameloblasts was as a passive process facilitated by the extracellular to intracellular [Ca²⁺] gradient [40]. Ca²⁺ could then be removed from the cytoplasm via the plasma membrane Ca²⁺-ATPases (PMCA) [40]. It is now evident that store-operated Ca²⁺ entry (SOCE) is a major entry path for Ca²⁺ particularly in maturation stage ameloblasts and also to some extent in cells of the secretory stage [41]. SOCE is a common mechanism for the uptake of extracellular Ca²⁺ in response to receptor stimulation, generation of the IP3 and release of endoplasmic reticulum (ER) Ca²⁺ stores via the IP₃ receptors [42]. SOCE is mediated by the Ca²⁺ release activated Ca²⁺ (CRAC) channels [43]. The molecular components of CRAC channels are the ER Ca²⁺ sensor stromal interaction molecule 1 (STIM1) and STIM2, and the plasma membrane pore subunit of the CRAC channel known as ORAI1 [44, 45]. CRAC channels are the main mediators of Ca²⁺ influx in enamel cells as determined by a reduction in [Ca²⁺]cyt in thapsigargin stimulated cells pre-treated with CRAC channel inhibitors [41, 46]. Thapsigargin passively depletes ER Ca²⁺ stores by blocking the sarco-endoplasmic reticulum Ca²⁺-ATPases (SERCA) therefore stimulating SOCE. These data are in keeping with reports that mutations in the STIM1 and ORAI1 genes cause hypomineralized enamel and AI-like phenotypes, which strongly suggests that CRAC channels are essential for the normal mineralization of enamel [47–49]. Differences in Ca²⁺ handling between the secretory and maturation stage enamel cells are important to better understand their roles in enamel mineralization. Overall, secretory stage enamel cells showed decreased capacity to handle Ca²⁺ than maturation stage cells (see also below) [41]. This is also consistent with increased expression of STIM1 and ORAI1 expression in rodent maturation stage ameloblasts [41]. Importantly also, we have reported that a SOCE mediated increase in [Ca²⁺]cyt can up-regulate the expression of the enamel genes [46], although the identity of transcription factors mediating this change remains unknown. Thus we surmise that CRAC channels not only mediate Ca²⁺ influx but that changes in [Ca²⁺]cyt modulate gene expression in enamel cells. This is in line with previous reports of an ameloblast cell line in which extracellular stimulation with Ca²⁺ showed a positive response in the expression of the amelogenin gene [50].

Data reported by Nurbaeva et al. showed that secretory stage cells show a two-fold lower basal [Ca²⁺]cyt than maturation stage enamel cells [41]. Moreover, the Ca²⁺ content leaked out of the ER by blocking SERCA with thapsigargin shows that maturation stage cells have higher ER Ca²⁺ storage than secretory stage. Maturation stage cells are also more responsive to the re-addition of extracellular Ca²⁺ showing significantly higher uptake than secretory cells. Although Ca²⁺ dynamics are clearly of higher magnitude in maturation stage cells, secretory cells are also responsive to SOCE stimulation [41]. This might be important for a number of reasons. First, enamel crystal growth is initiated in the secretory stage, so at this stage a supply of Ca²⁺ is also required. Given that STIM1 is nearly absent at this early stage, it might be the case that STIM2 could interact with ORAI1 or other ORAI homologues and enable sufficient Ca²⁺ influx. Stim2 mRNA transcripts and protein were expressed in secretory stage cells, as were the mRNA levels of Orai1-3 [41]. Currently, only mutations in STIM1 and ORAI1 are associated with abnormal enamel in patients [47–49]. This however, does not exclude the possibility that other members of these protein families may have a role in enamel development. In fact, it would not only be important to identify the effects of new potential mutations but also to recognize how interactions between these two proteins or between other homologues can lead to differences in Ca²⁺ dynamics. The use of enamel cells from animal models with altered expressions of STIM1 and ORAI proteins will help elucidate their roles in the secretory stage and evaluate the effects of increased Ca²⁺ dynamics in maturation stage.

Cells exert control over [Ca²⁺]cyt by chelating it, sequestering it into intracellular stores (ER or mitochondria), or by extruding it from the cell [51]. Ameloblasts express common cytosolic buffers including calmodulin, parvalbumin, calretinin, calcineurin as well as two calbindins (9Dka, 28Dka) (reviewed in [39]). Ca²⁺ sequestration into ER is modulated by the ATP-dependent SERCA2 pump in enamel cells [39, 52], whereas both the mitochondrial Ca²⁺ uniporter (MCU) and the mitochondrial Ca²⁺ exchanger NCLX are up-regulated in maturation stage enamel cells (our unpublished data). The Ca²⁺ extrusion system seems to be dominated by the exchanger NCKX4 (encoded by SLC24A4 gene) which we identified from a genome-wide screening of mRNA expression in enamel cells [7, 53, 54]. NCKX4 co-transports 1K⁺ and 1Ca²⁺ in exchange for 4 Na⁺ and it is the only exchanger to date associated with abnormal enamel phenotypes as reported in patients with mutations in SLC24A4 and in Slc24a4-deficient mice [49, 55]. NCKX4 expression is high during the maturation stage and nearly absent in secretory stage ameloblasts [49, 53, 54, 56]. It should be noted that other NCKX isoforms are also expressed in enamel cells [53] but no mutations in their coding sequences are known to disrupt enamel formation. Other contributors to clearing Ca²⁺ from ameloblasts include the Na⁺/Ca²⁺ exchanger NCXs and PMCA as mentioned above [40, 57]. The expression and potential function of PMCA’s at each stage of enamel formation should be revisited given that the proposed cellular localization discussed in various studies is contradictory [58]. No dysfunctions in either NCX or PMCA are known to affect enamel.

PO4³⁻ transport in enamel cells is less understood than Ca²⁺ transport. In fact, much of the molecular data available are limited to the potential role of members of the solute carrier gene family SLC34A [54] and SLC20A [59]. The former gene encodes a pH dependent Na⁺/ PO4³⁻ transporter called NaPi that appears to increase phosphate transport at lower pH. Our report that Slc34a2 transcripts were high in rat maturation stage enamel organs is in keeping with an increased need for phosphate at this stage as enamel crystals expand their growth and require increased PO4³⁻ availability [7, 54]. A more recent genome wide analysis in rat enamel organ cells also identified the phosphate transporters PiT-1 and PiT-2 (encoded by Slc20a1 and Slc20a2 genes) although their expression profile did not change significantly from secretory to maturation stage, which might indicate that they have more of a housekeeping role in enamel [59]. Beyond knowledge of the expression patterns of these two phosphate transporters (NaPi and PiT), little else is known about their putative roles in enamel. It should be noted that hypophosphatasia related to mutations in the tissue-non-specific alkaline phosphatase (TNALP) gene (ALPL) also causes enamel abnormalities [60].

Transport of other elements

Despite the relevant role of F⁻ in enamel as its incorporation in the crystal lattice increases the stability of the crystal assembly [6], how fluoride is transported by ameloblasts remains unknown. One possible option is that F⁻ is co-transported with other elements, possibly chloride (Cl⁻), a hypothesis based on data originated from bacterial studies [61].

The cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed to transport Cl⁻ in ameloblasts [62]. CFTR is expressed in ameloblasts most commonly on the apical side in proximity to the growing enamel crystals and likely moves Cl⁻ out of the ameloblasts [63] although it may also contribute to HCO₃⁻ transport [64]. Other reports indicate the expression of several Ca²⁺-dependent and independent Cl⁻ channels some of which (i.e. CLCN7) were expressed in the lysosomal/endosomal apparatus of ameloblasts [54, 65, 66]

Knowledge on the transport of other elements present in enamel such as Mg²⁺, Na⁺ and K⁺ is also limited. As discussed above, the expression of NCX and NKCX in ameloblasts points to a potential role not only in Ca²⁺ transport but in the exchange of Na⁺ for both, and also K⁺ for the latter. NCX removes 1Ca²⁺ in exchange for 3Na⁺ whereas NCKX exchanges 1K⁺ and 1Ca²⁺ for 4Na⁺. The better known Na⁺-K⁺-ATPases are also expressed largely in the cytosolic compartment of late maturation-stage ameloblasts [67]. Other proteins associated with Na⁺ transport are discussed below. The direct role of Na⁺ and K⁺ in enamel crystal growth is not well understood but it can be said that Ca²⁺ content in enamel negatively correlates with K⁺ [68]. By contrast, Mg²⁺ may compete with Ca²⁺ at some growth sites although Mg²⁺ has a smaller atomic radius and higher affinity for water which limits its incorporation in the crystal structure [6]. Recent clinical data provide some clues concerning Mg²⁺ transport in enamel. Hypercalciuria and hypomagnesaemia with nephrocalcinosis (FHHNC) related to CLDN16 mutations causes an AI-like phenotype [69]. Moreoever, Jalili syndrome, associated with ocular deficiencies, is linked to a mutation in the CNNM4 (cyclin and CBS domain divalent metal cation transport mediator 4) gene. Patients with Jalili syndrome present with hypomineralized and thinner enamel with lower Ca²⁺ but increased Mg²⁺ content relative to healthy individuals [70]. In addition, the expression of transient receptor potential cation channel subfamily M member 7 (TRPM7), an ion channel and protein kinase associated with the regulation of Mg²⁺ homeostasis, has been identified in maturation stage ameloblasts [71]. Trpm7-deficient mice show hypomineralized enamel [71]. These data highlight the impact of Mg²⁺ in enamel mineralization, which is negatively affected by an increase in Mg²⁺ [6].

The pH challenge

The initial nucleation events that lead to crystal formation have a complex chemistry. One of the main challenges that develop during the enamel crystal growth process is the handling of free protons (H⁺) that are released during the chemical events involved [6, 20]. If the concentration of these H⁺ increases, crystal growth will be disrupted as the local pH is lowered. As many as 8H⁺ may be generated during the formation of the Hap crystals [6]. Therefore, the ameloblasts must act in a number of ways to neutralize the acidic load and monitor extracellular pH. Buffering this acidic environment can be regulated by ameloblasts directly by secreting AMELX, which can bind H⁺, or by pumping bicarbonate (HCO₃⁻) into the extracellular compartment at the right time [1, 72–74]. This modulation is particularly important in the maturation stage when nucleation events increase as a result of the increase in thickness of the Hap crystals thus releasing a greater load of H⁺. During maturation, we have identified an increase in the expression of the HCO₃⁻ cotransporter NBCe1 [73]. Its localization at the basolateral pole of maturation stage ameloblasts points to a role as an importer of HCO₃⁻ into the ameloblasts given the proximity of blood vessels to the basal pole [7, 73]. The cellular localization of the anion exchanger AE2 at the lateral portion of the cell suggest that it may release HCO₃⁻ while moving Cl⁻ into the cytosol [75]. Similarly, at the apical pole of maturation stage ameloblasts, close to the enamel crystals, gene variants of the solute carrier member SLC26A, including those with the proteins names pendrin, PAT1 and SUT2 encoded by the SLC26A4, SLC26A6 and SLC26A7 genes respectively, could plausibly move HCO₃⁻ out of the cell in exchange for Cl⁻ [76, 77]. Recent data indicate that the levels of Cl⁻ in enamel could determine some aspects of HCO₃⁻ buffering and hence on the activity of SLC genes [78].

HCO₃⁻ might be be generated in the cytosol of ameloblasts by carbonic anhydrase 2 (CA2) with CA6, a secreted isozyme, being potentially involved in generating extracellular HCO₃⁻ [79–82]. CA enzymes are some of the more efficient and fast acting in biology and catalyze the reversible hydration of carbon dioxide to bicarbonate $\mathrm{C}{\mathrm{O}}_2+{\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{CA}}{\iff }\mathrm{H}\mathrm{C}{{\mathrm{O}}_3}^{-}+{\mathrm{H}}^{+}$. However, while acting as a provider of HCO₃⁻, the downside of these potent enzymes is that they generate H⁺ as a byproduct, both in the cytosol and in the enamel compartment. Ameloblasts remove intracellular H⁺ in at least two ways. The Na⁺/H⁺ exchanger NHE found at the basolateral pole of ameloblasts removes intracellular H⁺ in exchange for Na⁺ thus playing a role in intracellular pH homeostasis [83]. The V-type ATPase pump have been identified in the cytoplasm of ameloblasts likely removing H⁺ into lysosomal structures, and possibly also into the enamel compartment [83, 84]. H⁺-ATPases are also localized to the ruffled border of maturation stage ameloblasts pumping H⁺ into the enamel [83]. Both CA2 and CA6 isozymes are more highly expressed in maturation than in secretory stage cells [79, 83, 85]. Many of the remaining isozymes (there are 16 isozymes in mammals) of the CA family are also expressed in enamel cells, but their expression patterns and putative role at each stage is only just being recognized [79, 86].

The ion transport system of enamel cells

With a few exceptions, previous depictions of the transpeithelial ion transport system in ameloblasts are based on the molecular identification of proteins and their cellular localization, or on gene expression patterns (i.e.[7, 40, 58, 68]). Only a few studies have used pharmacological inhibitors or electrophysiology to test the functional roles of these channels in enamel cells [41, 57]. Hence most interpretations provide transport models that are based on knowledge of the biology of ameloblasts at each stage (see below) together with protein localization as determined by immunohistochemistry of tissue sections, or by the use of radiolabelled or immunogold labelling of molecules. These models thus await further functional testing to assess their validity. In addition, animal models have been used to assess the impact of alterations in the transport system by analyzing changes in the chemical composition of the enamel formed by these mutant mice (i.e. [68]). This latter approach however cannot ascertain whether the chemical changes observed are related to modifications in one (direct) or more (indirect) transport mechanisms.

Ion transport models have made use to the morphological changes that occur in the ameloblasts between the ruffled-ended (RA) and the smooth-ended (SA) cells. The localization of junctional complexes either at the proximal (basal) pole of some SA cells or at the distal (apical) pole of the RA cells [87, 88], has been used to formulate possible routes for the passive diffusion of molecules and minerals. When cells are in the RA phase and tight junctions are present distally, the enamel compartment is isolated from the intercellular space. Material that may have diffused from the blood vessels that invaginate the papillary layer at the basal pole, could pass between cells but cannot penetrate the distal junctions of RA cells and hence cannot reach the enamel space [1]. This situation changes during the transition to SA cells and the distal junctions disappear or become leaky and some junctional complexes may be found at the basal pole of some SA cells [87]. This gating mechanism is somewhat reminiscent of the passing of vessels through the Panama Canal as ships move through a complex system of gates when water fills the space between gates. In enamel cells, a limiting factor in this passive transcellular movement associated with RA and SA cells changes is that the majority of ameloblasts (~70%) are RA [1]. Thus the active system of ameloblasts is commonly defined in RA cells. Here we depict a summary schematic of the localization of transport proteins identified in ameloblasts and have been linked to such functions in ameloblasts (Figure 2).

Figure 2. Model of ion channels and transport mechanisms in ameloblasts.

The schematic represents a maturation-stage ruffled ended ameloblast. This model is based on reports of mRNA and protein expression as well as cellular localization. Thus in some cases the functional roles of these proteins have not been established. At the basal pole, HCO₃⁻ is transported into the cytosol by the Na⁺/ HCO₃⁻ exchanger NBCe1. Intracellular HCO₃⁻ can also be produced by carbonic anhydrases (CA2). The removal of H⁺ that are generated during CA2 activity and other sources of cytosolic H⁺ might be mediated by the Na⁺/H⁺ exchanger NHe1. Extrusion of HCO₃⁻ is mediated by the anion exchanger AE2 at the lateral membrane pole and by members of the SLC26a family (at least 3 members) at the apical pole. Chloride (Cl⁻) is transported in and out of the cytoplasm by a number of proteins. AE2 and SLC26a exchange Cl⁻ for HCO₃⁻ whereas the cystic fibrosis transmembrane conductance regulator (CFTR) removes cytosolic Cl⁻ at the distal pole. Ca²⁺ enters the cell largely via the activity of the Ca²⁺ release activated Ca²⁺ channel mediated by ORAI1 and STIM1 proteins. Removal of cytosolic Ca²⁺ into ER is modulated by the sarco-endoplasmic reticulum SERCA2a pump, and it is released from this organelle by inositol triphosphate receptors (IP3R). The expression of L-type voltage-gated Ca²⁺ channels has not been reported in enamel cells but patients with dysfunctional channel show abnormal enamel. It might be the case that it plays a role in the functioning of the cell’s membrane potential. The bulk of Ca²⁺ extrusion is likely mediated by the Na⁺/Ca²⁺/K⁺ exchanger NCKX4, but also to a lesser extent by the plasma membrane Ca²⁺-ATPases (PMCA) and the Na⁺/Ca²⁺ exchanger NCX. Phosphate transport is poorly known but the Na⁺/ PO₄³⁻ proteins NaPi have been localized at the distal cell pole. The cation Mg²⁺ can be moved into the cytosol by TRMP7 (transient receptor potential cation channel subfamily M member 7) whereas CNN4 (cyclin and CBS domain divalent metal cation transport mediator 4) can remove Mg²⁺ out of the cell in exchange for Na⁺. N= nucleus. ER= endoplasmic reticulum. GJ=gap junctions.

Systemic diseases that impact enamel

Having briefly described the transport proteins identified in ameloblasts, it should be recognized that dysfunction in a number of these proteins cause systemic diseases and can also impact enamel. A recent study published by Wright and colleagues showed that 91 hereditary conditions found in the Online Mendelian Inheritance in Man (OMIM), of which a subset of 71 had a known molecular basis, showed an association with dental defects [29]. The proteins in that subset that can cause systemic-AI can be grouped by function with enzymes being 30% of the identified proteins affecting enamel, regulatory proteins or transcription factors 20% and 14% were transmembrane proteins [29]. These data were limited as for example the OMIM database did not report enamel deficiencies in patients with cystic fibrosis [29], despite that about 50% of patients with mutations in the CFTR gene show enamel defects [89]. Other proteins, such as ORAI1, were not included at the time despite the reported mutations in the ORAI1 gene in 2009 affecting enamel [48]. A list of proteins linked to systemic diseases also affecting enamel is shown in Table 1.

Table 1.

List of genes linked to systemic diseases also affecting enamel

Gene	Protein	Disease
ORAI1	ORAI1- plasma membrane pore subunit of CRAC channel (Refs:	severe-combined immunodeficiency
STIM1	STIM1 (Stromal interacting molecule 1) ER Ca2+ sensor (Refs:	immune disorders
SLC24A4	NCKX4 (Na+/Ca2+/K+ exchanger) (Refs:	amelogenesis imperfecta
SLC4A4	NBCe1 (Bicarbonate co-transporter) uptake of HCO3− and Na+	proximal renal tubular acidosis
SLC4A2	AE2 (Anion exchanger) extrudes HCO3− in exchange for Cl-	primary biliary cirrhosis
CFTR	CFTR (Cystic fibrosis transmembrane conductance regulator) Extrudes Cl−	cystic fibrosis
TRPM7	TRPM7 (Transient receptor potential cation channel 7) Mg2+ channel CNNM4 (Cyclin and CBS domain divalent metal cation transport mediator 4)	?
CNNM4	Mg2+ channel	Jalili syndrome

The Future

Enamel, as a tissue, is unique. However, the molecular identity of proteins recognized in ameloblasts associated with transepithelial ion transport is similar to that described in other organs (i.e. kidney) [7]. Thus ion transport in enamel biology must be integrated within systems biology and hence it is equally affected by physiological anomalies [7] (after all, teeth form a functional part of the gastrointestinal tract). The study of enamel biology is complex for a number of reasons. The cells are difficult to access, there are technical challenges in isolating ameloblasts from surrounding cell types, primary cells do not proliferate in culture, and the limited options of available cell lines. Thus, historically, enamel cell biology has remained somewhat descriptive. Therefore, an important challenge for those involved in decoding the transport system of enamel cells is likely turning this descriptive biology of the cell and it’s physiology into an experiment-based science. This will require an effort to increase the availability of molecular tools. We think that this approach can only improve the relevance of studying enamel biology within the communities of researchers involved in cell biology/physiology at large. These interactions can be transformative for enamel biologists and will certainly broaden the scope of cell physiology. As these studies develop, and there are already some good recent examples [22, 74], ameloblasts will become a wider target in the study of general cell physiology.

Highlights.

• Enamel is the most calcified tissue in the vertebrate body

• Transepithelial ion transport is cell regulated

• Enamel cells are known as ameloblasts, form and mineralize hydroxyapatite-like crystals

• De novo crystal formation is mediated by ameloblasts as is extracellular pH

• Models for ion transport generally lack functional testing

Abbreviations

Hap

(hydroxyapatite)

NCKX

(sodium/calcium/potassium exchanger)

SLC

(solute carrier)

STIM

(stromal interaction molecule)

SOCE

(store-operated Ca²⁺ entry)

CRAC

(Ca²⁺ release activated Ca²⁺)