In general high intracellular concentrations of calcium (Ca²⁺) catalyze cell death signaling cascades, so cells maintain a gradient of ~10⁻³ M Ca²⁺ concentration outside the cell, in the mitochondria, and in the endoplasmic reticulum (ER) where Ca²⁺ is stored; while in the cytoplasm, the concentration is ~10⁻⁷ M (Brini and Carafoli, 2011). The plasma membrane contains a variety of Ca²⁺ channels that transiently open to allow Ca²⁺ influx in response to plasma membrane voltage changes, ligand-receptor interaction, or emptying Ca²⁺ stores of the ER and mitochondria (Brini and Carafoli, 2011). Calcium is removed from the cytoplasm through a number of mechanisms including the SERCA pump that replenishes ER stores, the mitochondrial Ca²⁺ uniporter, that replenishes mitochondrial stores, the plasma membrane low-affinity high capacity Na⁺/Ca²⁺ exchanger proteins (NCX), the Na⁺/Ca²⁺ K⁺ exchanger proteins (NCKX), and the high-affinity low-capacity plasma membrane Ca²⁺-ATPase (PMCA) pump proteins (Berridge et al., 2003; Brini and Carafoli, 2011; Hu et al., 2012; Bronckers et al., 2015).

Calcium (Ca²⁺) transport is crucial to understand the process of amelogenesis because not only is unbound Ca²⁺ a major component of hydroxyapatite (Hap), Ca²⁺ can also act as a major signaling molecule capable of regulating cell processes in eukaryotic cells such as cell division, cell attachment, motility, survival, differentiation as well as gene expression (Hubbard, 1996; Blair et al., 2011).

The SLC8A (sodium/calcium exchangers or NCX), SLC24A (potassium-dependent sodium/calcium exchangers or NCKX), and ATP2B (ATPase plasma membrane Ca²⁺ transporting pumps or PMCA pumps) gene families of Ca²⁺ transporters mediate Ca²⁺ extrusion in most cell types (Brini and Carafoli, 2011), and proteins from all three of these families have been reported in enamel organ cells (Sasaki and Garant, 1986c; Borke et al., 1995; Zaki et al., 1996; Okumura et al., 2010; Hu et al., 2012; Wang et al., 2014). The SLC8A gene family has 3 members (NCX1-3) and all have a generally accepted stoichiometry of the extrusion of 1 Ca²⁺ in exchange for the intrusion of 3 Na⁺ (Brini and Carafoli, 2011), while the SLC24A gene family has 5 members (NCKX1-5) and extrudes 1 Ca²⁺ and 1 K⁺ in exchange for 4 Na⁺, typically against the Ca²⁺ gradient; however, the directionality both NCX and NCKX exchangers can be reversed depending on the Na⁺ and Ca²⁺ gradients (Jalloul et al., 2016b; Zhekova et al., 2016). SLC8A and SLC24A gene families are electrogenic because there is a translocation of net charge across the plasma membrane, have a low Ca²⁺ affinity, and are capable of transporting Ca²⁺ in bulk rapidly across the plasma membrane (Brini, 2009). They are reversible but in ameloblasts they likely operate in extruding Ca²⁺ from the cytoplasm facilitated by transport of Na⁺ and K⁺ down their gradients (Brini and Carafoli, 2011; Hu et al., 2012). PMCA pumps/proteins have 4 members (PMCA1-4, coded by genes ATP2B1-4) and are part of a larger family of genes, called P-type primary ion transport ATPases, that catalyze the auto-phosphorylation of a conserved aspartyl residue within the pump from ATP (Palmgren and Nissen, 2011).

The SLC8A and SLC24A families are primarily expressed in excitable tissues such as muscle and heart, as their rapid bulk transport of Ca²⁺ is important in, for example, muscle and heart contraction (Brini and Carafoli, 2011). The SLC8A/NCX and SLC24A/NCKX transporters are Na⁺/Ca⁺ exchangers and can be either K⁺-dependent (NCKX) or K⁺-independent (NCX) (Shumilina et al., 2010).

NCX1 is expressed in heart, brain, bladder, kidney, and cells of the enamel organ; NCX2 is expressed in brain and skeletal muscle; and NCX3 is expressed in brain, skeletal muscle and cells of the enamel organ (Lytton, 2007; Okumura et al., 2010; Lacruz et al., 2012b; Sharma and O'halloran, 2014). Okumura et al. demonstrated NCX1 and NCX3 expression at the apical pole of both secretory and maturation ameloblasts, and expression of NCX1 was also observed in cells of the stratum intermedium and papillary layer (Okumura et al., 2010). In addition, protein levels of NCX1 and NCX3 throughout amelogenesis remained relatively unchanged (Okumura et al., 2010). Using real-time PCR, Lacruz et al. confirmed that the mRNA levels of both NCX1 and NCX3 did not significantly change from secretory- to maturation-stage enamel organ cells (Lacruz et al., 2012b).

NCKX1 is expressed primarily in retinal rod photoreceptors and platelets (Schnetkamp, 2004; Lytton, 2007). NCKX2 is expressed in cone photoreceptors and is involved in mouse motor learning and memory (Schnetkamp, 2004; Lee et al., 2009, 2013), and NCKX3 is expressed in the brain and the kidneys (Schnetkamp, 2004; Lee et al., 2009) though it is expressed in the kidneys at higher levels in female mice than in male mice (Lee et al., 2009). NCKX3 is also highly expressed in the human endometrium during the menstrual cycle, where its expression is partially regulated by the steroid hormone 17β-estradiol (Yang et al., 2011). NCKX4 is expressed in olfactory neurons (Stephan et al., 2011), and also in the maturation-stage ameloblasts (Hu et al., 2012). NCKX5 is expressed in skin melanocytes, retinal epithelium, and brain (Schnetkamp, 2004; Lytton, 2007; Sharma and O'halloran, 2014; Jalloul et al., 2016a,b). NCKX6/NCLX was originally considered a member of the NCKX family but is now considered part of the Ca²⁺ cation (CCX) exchanger branch (Cai and Lytton, 2004; Sharma and O'halloran, 2014) as a mitochondrial membrane Ca²⁺, Li⁺/Na⁺ exchanger with a wide tissue distribution (Schnetkamp, 2004; Lytton, 2007; Sharma and O'halloran, 2014).

The ATPase plasma membrane Ca²⁺ transporting (or PMCA) gene family is postulated to be involved in Ca²⁺ homeostasis, as it has a high affinity for Ca²⁺ but cannot transport Ca²⁺ as rapidly as either the NCX or NCKX transporters (Brini and Carafoli, 2011). The PMCA family is part of the superfamily of P-type ATPase pumps that form a stable phosphorylated intermediate as it hydrolyzes one molecule of ATP for each Ca²⁺ transported (Strehler and Zacharias, 2001; Cai and Lytton, 2004). The phosphorylated enzyme intermediate of the P-type ATPases, which include the SERCA family of transporters in the ER membrane (Giacomello et al., 2013), occurs between γ-phosphate of a hydrolyzed ATP with a D-residue in a highly conserved region of the ATPase pump (Brini and Carafoli, 2011).

PMCA1 is expressed in most tissues throughout development. Its expression is highest in the nervous system, heart, skeletal muscle, and intestine (Zacharias and Kappen, 1999), and regulated by growth factors such as glucocorticoids and Vitamin D (Zacharias and Kappen, 1999; Giacomello et al., 2013). PMCA2 is expressed mainly in the brain, heart, mammary glands and ear, and decreased expression of PMCA2 causes increased apoptosis in breast cancer cells (Curry et al., 2012; Giacomello et al., 2013). PMCA3 has the highest calmodulin affinity and is detected primarily in the brain and skeletal muscles (Krebs, 2009; Giacomello et al., 2013). PMCA4 is involved in the fertilization process and cardiac function, and has been found to associate with lipid rafts, which often function to aggregate protein complexes important in signaling pathways (Giacomello et al., 2013). It has been suggested that PMCA4 is more involved in cell-specific Ca²⁺ signaling than as a pump for bulk Ca²⁺ export (Strehler, 2013; Brini et al., 2017). PMCA4 interacts with nitric oxide synthase and with calcineurin, which regulates NFAT signaling (Brini, 2009; Kim et al., 2012; Strehler, 2013).

PMCA1 and PMCA4 are important in osteoclast differentiation, maturity, and survival, and Atp2b1^+/− and Atp2b4^−/− mice have decreased bone density due to an increased number of mature osteoclasts and increased osteoclast apoptosis (Kim et al., 2012). The PMCA family members can also influence IP₃-mediated calcium signaling by binding to phosphatidylinositol-4,5-bisphosphate (PIP₂) on the plasma membrane as well as removing Ca²⁺ necessary for phospholipase C (PLC) activity, which prevents cleaving by PLC and thereby prevents Ca²⁺ release from the ER (Penniston et al., 2014). Altered PMCA expression is a characteristic of many cancers (Curry et al., 2011) and many other human diseases (Brini et al., 2013), but the diversity in isoforms and splicing and lack of specificity of small molecules to target PMCAs present challenges in therapeutic agent development (Strehler, 2013).

There now are a number of reports that show expression and localization data for NCX1 and NCX3 (Okumura et al., 2010; Lacruz et al., 2012b), and NCKX4 (Hu et al., 2012; Wang et al., 2014) in the enamel organ. Reports on PMCA expression and activities throughout amelogenesis are scant (Sasaki and Garant, 1986c; Borke et al., 1995; Zaki et al., 1996), and to the authors' knowledge, the only investigations into the role of PMCA proteins in amelogenesis date back decades. Data presented here better defines the mRNA profiles of all SLC8A, SLC24A, and ATP2B gene family members, and adds additional insight into the protein and spatiotemporal expression profiles of NCKX3, PMCA1, and PMCA4.

A number of the ATP2B, SLC8A, and SLC24A gene family members are linked to mammalian disease, but notably mutations to SLC24A4 are associated with non-syndromic amelogenesis imperfecta (AI) (Parry et al., 2013; Seymen et al., 2014; Wang et al., 2014; Herzog et al., 2015). A comprehensive list of the PMCA, NCX, and NCKX pumps and exchangers, their links to human pathologies, and mouse models of each gene is found in Table 1.

All vertebrate animal manipulation was carried out in accordance with Institutional and Federal guidelines. The animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Southern California (Protocol #20461).

Secretory-stage and maturation-stage enamel organ cells from mandibular incisors of 4-week old Wistar Hanover rats were collected as previously described (Lacruz et al., 2012b; Wen et al., 2014), and RNA extraction was performed using a QIAshredder, an RNeasy Protect Mini Kit, and DNase I solution from Qiagen (Valencia, CA, USA). Reverse transcription and real-time PCR were performed using the iScript cDNA Synthesis kit and SYBR Green Supermix from BioRad, respectively. Real-time PCR was performed on the CFX96 system (BioRad Laboratories, Hercules, CA, USA) in 10 μl volumes with a final primer concentration of 100 nm, for 40 cycles at 95°C for 10 s and 58°C for 45 s. Six independent real-time PCR analyses were conducted using samples from a total of 6 rats, 3 males, and 3 females, for each gene of interest (primers are listed in Table 2), and for both stages of amelogenesis. The male and female data were analyzed separately and no significant differences were noted between the sexes, so the data presented in the graph were generated from all 6 animals (n = 6). Rat enamel organ is preferred to mouse enamel organ for real-time PCR and western blot studies because separating secretory and maturation stage from adult mouse incisors is technically difficult and yields less RNA and protein per animal.

Secretory (S) and maturation (M) enamel organ cells from mandibular incisors of 4-week old Wistar Hanover rats were collected. Brain (B) and heart (H) tissues were also collected as control tissues. Total protein extraction was performed with RIPA buffer (1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, 150 mm NaCl, 50 mm Tris, pH 8.0) and protease inhibitor cocktail, complete mini (Roche Applied Sciences, Indianapolis, IN, USA). Samples were homogenized manually with a pestle six times, then sonicated with a BRANSON digital sonifier Model 450 (All-Spec Industries, Wilmington, NC, USA; 10% intensity, 10 s on and 10 s off). Samples were then cleared by centrifugation (15,000 g, 15 min, 4°C). Proteins were quantified using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA) and equal quantities were loaded (15 μg per lane) onto 4–12% SDS–PAGE resolving gels. Protein was transferred to a PVDF membrane, then blocked with 5% milk in TBST. Antibodies against PMCA1 (AbCam, Cambridge, MA, USA; catalog #ab190355), PMCA2 (ab3529), PMCA3 (ab3530), PMCA4 (ab2783), NCKX3 (St. John's Laboratory, London, UK, catalog #STJ94358), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA, catalog #sc-32233), amelogenin (ThermoFisher Scientific, catalog #PA5-31286), and cardiac muscle actin (ACTC1) (GeneTex Inc., Irvine, CA, catalog #GTX101876) were used at dilutions of 1:500, 1:2,000, 1:300, 1:5,000, 1:500, 1:500, 1:3,000, and 1:500, respectively in 5% milk in TBST. Secondary antibody for PMCA1-4 from Cell Signaling (Danvers, MA, USA; catalog #7074 and #7076) was applied at a dilution of 1:10,000. Secondary antibodies for NCKX3, amelogenin, and ACTC1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA; catalog #sc-2004 and sc-2418) were applied at a dilution of 1:7500. Pierce ECL Plus Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA; catalog #32132) was used as the detection system for all antibodies. TBST (tris-buffered saline with.1% Tween-20) was used as a wash buffer.

Mandibular incisors were dissected from 9-day-old wild type mice and placed in 4% paraformaldehyde in PBS overnight. Mouse incisors were preferred to rat incisors in the immunofluorescence studies because mouse incisors decalcify more rapidly and all stages of amelogenesis are visible in one sagittal section. The incisors were then washed in PBS and decalcified in 10% EDTA in PBS pH 7.4 for 4 weeks at 4°C. The sample was embedded in paraffin and 4 μm sections were cut with a microtome. The sections were deparaffinized and rehydrated. The primary antibodies for PMCA1 and PMCA4 (AbCam, Cambridge, MA, USA; catalog #ab3528 and #ab2783, respectively) were used at dilutions of 1:40 and 1:200 in 1% BSA in PBS, respectively. The primary antibody for NCKX3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; catalog #sc-50129) was used at a dilution of 1:50. The secondary antibodies (Vector Laboratories, Burlingame, CA, catalog #DI-1088, DI-2488, DI-2594, DI-3094) were used at a dilution of 1:300 in 1% BSA in PBST. Sections were mounted with mounting medium with DAPI (Vector Laboratories, Burlingame, CA, catalog #H-1200) and imaged on a Leica TCS SP8 confocal microscope (Leica Biosystems). PBST (0.1% Tween-20) was used as a wash buffer for the experiments. Negative control sections, using secondary antibody only, under identical conditions, were included and showed negligible auto fluorescence—see Supplemental Figure 1.

Quantitative PCR (qPCR) comparing mRNA expression levels in secretory and maturation enamel organ cells for all PMCA (Atp2b), NCX (Slc8) and NCKX (Slc24) gene family members indicate that: (1) PMCA1 (Atp2b1), 3 (Atp2b3), and 4 (Atp2b4), and NCKX3 (Slc24a3) expression is highest during secretory-stage amelogenesis; (2) NCX1 (Slc8a1) and 3 (Slc8a3), and NCKX6 (Slc24a6) were expressed during secretory and maturation stages; and (3) NCKX4 (Slc24a4) is most highly expressed during maturation-stage amelogenesis (Figure 1). The expression levels of PMCA2 (Atp2b2), NCX2 (Slc8a2), NCKX1 (Slc24a1), NCKX2 (Slc24a2), and NCKX5 (Slc24a5) are negligible throughout amelogenesis (Figure 1). These data for NCX (Slc8) and NCKX (Slc24) gene family members are consistant with previously published gene expression data (Okumura et al., 2010; Hu et al., 2012), and add novel information suggesting that PMCA1, PMCA4, and to a lesser extent PMCA3 (which is expressed in secretory enamel organ cells at a level an order of magnitude lower than seen for PMCA1 and PMCA4), play an important role in secretory-stage amelogenesis.

Western blot data indicate that PMCA1 and PMCA4 are more highly expressed in secretory stage than in maturation stage, and PMCA2 is not expressed at any appreciable level in amelogenesis, consistent with the qPCR data (Figures 2Ai,Aiv,Aii respectively). Contrary to the qPCR data, PMCA3 and NCKX3 appear to be expressed at similar levels during both secretory stage and maturation stage (Figures 2Aiii,B respectively). Rat brain and heart protein samples were used as control tissues and analyzed with the secretory- and maturation-stage protein samples. The expected molecular weights for PMCA1-4 are ~130, 133, 123, and 129 kDa respectively, and relate to the single bands seen at approximately the 150 kDa molecular weight mark (as indicated by an arrow, Figure 2A). The expected molecular weight of NCKX3 is ~60 kDa (Figure 2B). Gapdh has been included as a loading control for all samples, and additional controls include Western analysis for both amelogenin (Amelx) and cardiac muscle alpha actin (Actc) (Figure 2B).

In the enamel organ, PMCA1 expression is seen primarily on the basolateral membrane of both secretory- and maturation-stage ameloblasts, with stronger signals seen in secretory ameloblasts (green; Figures 3A–C). These data complement both the qPCR (Figure 1) and Western blot data (Figure 2) on the spatiotemporal expression of PMCA1 in enamel organ cells. When compared to ameloblasts, a weaker signal of PMCA1 is seen in the stratum intermedium (Figure 3A) and papillary layer cells of the enamel organ (Figure 3B); as reflected by the orange color observed in the merged images (Figures 3G–I). In the enamel organ, PMCA4 expression is also seen on the basolateral membrane of secretory- and maturation-stage ameloblasts, and also cells of the stratum intermedium and papillary layer cells (red; Figures 3D–F). Similar to the PMCA1 data, these PMCA4 immunolocalization data complement the qPCR and Western blot data (Figures 1, 2). The co-localization of both PMCA1 and PMCA4 in polarized ameloblasts can be appreciated in the merged image (yellow; Figures 3G–I), while PMCA4 (but not PMCA1) is also expressed in the stratum intermedium and papillary layer cells of the enamel organ (red; Figures 3G–I).

NCKX3 expression is highest in the Tomes' processes (Figure 3M) and the apical membrane of transition- and maturation-stage ameloblasts (Figures 3N,O), while some minor ameloblast-specific intracellular granular immune-reaction is also apparent (Figures 3M–O). We compared the expression profile for NCKX3 (red; Figures 3M–O) to the expression profile of the control PMCA1 (green; Figures 3J–L). As can be appreciated from the images (Figures 3M–R), the expression profile for NCKX3 in the enamel organ is highest at the distal/apical pole, and this is distinct from the expression profiles seen for PMCA1 and PMCA4 where expression is seen on the lateral membranes of polarized ameloblasts (for both PMCA1 and PMCA4) and stratum intermedium and papillary layer cells (only PMCA4) (Figures 3A–L).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.