Archives of Oral Biology 93 (2018) 47–55

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Archives of Oral Biology

journal homepage: www.elsevier.com/locate/archoralbio

Review

The overview of channels, transporters, and calcium signaling molecules T

during amelogenesis
⁎
Hee-Eun Kima, Jeong Hee Hongb,
a
Department of Dental Hygiene, College of Health Science, Gachon University, 191 Hambangmoe-ro, Yeonsu-gu, Incheon, 21936, South Korea
b
Department of Physiology, College of Medicine, Lee Gil Ya Cancer and Diabetes Institute, GAIHST, Gachon University, Incheon, 21999, South Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Enamel is a highly calcified tissue. Its formation requires a progressive and dynamic system for the regulation of
Ion transporters electrolyte concentration by enamel epithelia. A critical function of enamel epithelial cells, ameloblasts, is the
Calcium signaling secretion and movement of electrolytes via various channels and transporters to develop the enamel tissue.
Magnesium signaling Enamel formation generates protons, which need to be neutralised. Thus, ameloblasts possess a buffering system
Tooth development
to sustain mineral accretion. Normal tooth formation involves stage-dependent net fluctuations in pH during
Amelogenesis
amelogenesis. To date, all of our information about ion transporters in dental enamel tissue is based solely on
immunostaining-expression techniques. This review critically evaluates the current understanding and recent
discoveries and physiological role of ion channels and transporters, Mg2+ transporters, and Ca2+ regulatory
proteins during amelogenesis in enamel formation. The ways in which ameloblasts modulate ions are discussed
in the context of current research for developing a novel morphologic-functional model of enamel maturation.

1. Introduction differentiation of the tooth germ continues. Odontogenesis then pro-

Tooth formation is a dynamic system involving the regulation of
electrolyte concentrations by enamel epithelia. A cardinal function of
enamel epithelial cells is electrolyte secretion facilitated by various
channels and transporters. The enamel organ requires fidelity of ionic
regulation for its tissue to physically harden. For example, the abnormal
tooth formation and development in patients with cystic fibrosis (CF)
provides strong evidence that electrolyte regulation via ion channels
and transporters is involved in normal tooth formation (Duan, Mao,
Wen, Yang, & Xue, 2011). While the secretion of electrolytes such as
HCO3− and Cl− by non-mineral tissues including pancreatic and sali-
vary acinar and ductal cells has been studied and reviewed extensively
(Lee, Ohana, Park, Yang, & Muallem, 2012), the molecular mechanisms
of ion channels and transporters in ameloblasts is only partially un-
derstood, despite much progress in our overall understanding of enamel
formation.
2. Overview of tooth development
Odontogenesis takes place in a continuous process. Initiation of
odontogenesis leads to identifiable stages in tooth development, in-
cluding the bud stage, the cap stage, and the bell stage, while systematic
gresses to the stage of secretory with formation of the hard dental tis-
sues, such as enamel, dentin, and cementum, and then finally to the
stage of maturation for these structures. Especially, amelogenesis is the
process of enamel matrix formation and maturation that occurs in two
stages of tooth development, the stages of secretory and maturation.
During the secretory stage of amelogenesis, enamel matrix which is
produced by ameloblast initially is partially mineralized because it is
protein-rich matrix which is composed of only a small amount of hy-
droxyapatite crystals (Simmer et al., 2010). However, during the ma-
turation stage, enamel matrix completes its mineralization process after
the secretory of enamel matrix. Ameloblasts modulate their mor-
phology between ruffle-ended ameloblast (RA) and smooth-ended
ameloblast (SA) forms. Ruffle-ended ameloblasts add mineral to the
enamel; smooth-ended ameloblasts allow removal of water and de-
graded matrix proteins (Hand & Frank, 2014).
3. Major ions for pH regulation
A unique phenomenon during the maturation stage is that amelo-
blasts modulate. They are involved in several cellular activities in-
cluding pH regulation and ion transport (Smith, 1998). The enamel is
formed by ameloblasts containing many ion transporters and channels,

⁎
Corresponding author at: Department of Physiology, College of Medicine, Gachon University, Lee Gil Ya Cancer and Diabetes Institute, 155 Getbeolro, Yeonsu-gu, Incheon, 21999,
South Korea.
E-mail address: minicleo@gachon.ac.kr (J.H. Hong).

https://doi.org/10.1016/j.archoralbio.2018.05.014
Received 11 January 2018; Received in revised form 18 May 2018; Accepted 19 May 2018
0003-9969/ © 2018 Elsevier Ltd. All rights reserved.
H.-E. Kim, J.H. Hong
which are well known from studies of related diseases conducted in the
past two decades (Duan, 2014). HCO3−, the biological pH buffer re-
sponsible for maintaining intracellular and extracellular pH fluctua-
tions, is present in the enamel fluid. Depending on the cell morphology
found during maturation stage, the pH is mildly acidic in RAs and
nearly neutral in SAs. Since ion transporters have been addressed in
ameloblasts, we have begun to uncover the exact details of the mole-
cular mechanism of enamel-crystal growth. The ion transporters that
secrete HCO3− to the enamel matrix involve two kinds of HCO3−-
carrier proteins, the electrogenic Na+−HCO3− cotransporter 1
(NBCe1) and anion exchanger 2 (AE2) (Lacruz, Nanci, White et al.,
2010; Paine et al., 2008). Studies of NBCe1-knockout animals, in which
the enamel is extremely hypo-mineralised and architecturally weak,
showed that NBCe1 is strongly involved in the development of normal
enamel (Lacruz, Nanci, White et al., 2010). NBCe1 protein levels are
significantly increased in ameloblasts of Cftr-null and Ae2-null mice to
import HCO3− to compensate for dysregulated Cl−-dependent buf-
fering effect (Jalali et al., 2014). Such studies support the concept that
the regulation of HCO3− secretion into the enamel space is important
for understanding the molecular mechanisms of the enamel organ.
4. Ca2+ and Mg2+ for enamel growth
Ca2+ signalling is finely integrated for the performance of various
cell functions including muscle contraction, transcription, and exocy-
tosis. Excessive activation of the Ca2+-release pathway is extremely
toxic and is the nodal point of diseases associated with cell stress or
death. Although enamel is the most highly calcified tissue in mammals,
it is unclear how cells avoid the cytotoxic effects of excess Ca2+ while
supplying sufficient bulk Ca2+ for enamel growth. Recently, under-
standing of Ca2+ transcytosis, a new concept for Ca2+ transport in the
mineral-formation process, has been strengthened (Lacruz et al., 2011;
Lacruz, Smith, Kurtz, Hubbard, & Paine, 2013); however, further in-
vestigation of the precise mechanism is needed. In terms of Ca2+ sig-
nalling in tooth biology, the down regulation of Ca2+ transport could
contribute to multiple developmental defects in the enamel. The fun-
damental cellular mechanism of Ca2+ transport is a potential target for
drugs to improve enamel quality (Hubbard, 2000). In addition to Ca2+
transport, it is also noteworthy that Mg2+ also involved in amelogenesis
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Archives of Oral Biology 93 (2018) 47–55
Fig. 1. Schematic representation of ion transporters re-
lated to the modulation of the physiological micro-
environment of matured ameloblasts.
This model depicts our current knowledge of the ion
channels associated with the main activities of ameloblasts
during maturation. AE2: anion exchanger2; CFTR: cystic
fibrosis transmembrane conductance regulator; SLC26 A:
solute carrier 26 family A; ClC: Cl− channel; V-H+
ATPase: V (vacuole) type-H+ ATPase; NHE1: Na+-H+
exchanger 1; NBCe1: Electrogenic Na+−HCO3− co-
transporter 1; NKCC: Na+-K+-2Cl− cotransporter 1; CA:
carbonic anhydrase. PL: papillary layer.
(Nakano et al., 2016; Ogata et al., 2017; Simmer et al., 2014; Yamazaki
et al., 2013). Briefly we discuss below. Ameloblasts coordinate the
transport of ions and molecules to mature the enamel, allow crystal
growth, and resist excess acidity by biological pH buffer such as HCO3−
(Simmer et al., 2010). It is therefore critical to understand how ame-
loblasts neutralise the acidity during the maturation stage, which is a
typical by-product of hydroxyapatite formation. The goal of this article
is physiologically to evaluate the current understanding of how the ion
channels and transporters in ameloblasts handle fundamental electro-
lytes.
5. Amelogenesis and the polarity of ameloblasts
Various epithelial and enamel cells require a specialised structure
with directionality from the basal pole to the apical pole. The amelo-
blast is one of prototypical example of polarized cells. The polarised
cells compartmentalise the expression of proteins such as Ca2+-signal-
ling proteins and ion transporters. The polarised expression at the
apical and basolateral membranes causes the unidirectional secretion of
enzymes and fluid into the lumen (Kasai & Augustine, 1990). Ca2+-
signalling proteins and ion transporters are involved in the polarised
signalling pattern and directional electrolyte secretion during enamel
formation. In the basolateral membrane, proteins such as NBCe1,
NHE1, AE2, and Na+-K+-ATPase that transport HCO3− and Na+ are
important for Na+ influx, HCO3− secretion, and pH regulation. On the
other side, in the apical membrane, proteins such as V-H+ ATPase,
NHE1, NKCC1, CFTR, SLC26 A, NCX, and NCKX that transport H+,
HCO3−, Cl−, and Ca2+ adjust the pH and the ion concentration in a
maturation-dependent manner. The channels and transporters mainly
involved in enamel formation during maturation stage and these are
illustrated in Fig. 1.
The polarised expression of Ca2+-related proteins and receptors,
which evoke polarised Ca2+ signals, is well established in non-enamel
soft tissues such as those of the pancreas and salivary glands. Of the
many proteins associated with Ca2+ signalling in various tissues,
plasma membrane Ca2+ ATPase (PMCA) pumps (Borke, Zaki,
Eisenmann, & Mednieks, 1995), sarcoplasmic/endoplasmic reticulum
Ca2+ ATPase (SERCA) pumps (Franklin, Winz, & Hubbard, 2001), in-
ositol 1,4,5-trisphosphate receptors (IP3Rs) (Nurbaeva, Eckstein,
H.-E. Kim, J.H. Hong
Concepcion, et al., 2015), stromal interaction molecule 1 (STIM1) and
Orai protein (Hong et al., 2011; Wang et al., 2014), NCX (Okumura
et al., 2010), and NCKX (Hu et al., 2012; Wang et al., 2014) are directly
demonstrated in enamel cells. In the light of current knowledge, these
Ca2+ signalling proteins will be highlighted in this review.
Ameloblasts are highly polarised columnar cells. The polarity per-
sists from the secretory stage to late in the maturation stage. Cyclical
morphological changes between the SA and RA phenotypes during
maturation have been addressed. The repetitive alternation between
the SA and RA phenotypes occurs within the ameloblast layer. The RA
region is presumed to be involved in ion movements across the cell
membrane, whereas the SA region is presumed to be an impermeable
cell layer (Lacruz, Nanci, White et al., 2010), suggesting that the dy-
namic modulation of permeability is associated with bidirectional ion
movement in the enamel organ.
6. Acid-base balance in enamel maturation
The modulation of ameloblast phenotypes seems to play a central
role in extracellular pH regulation and HCO3− transport for enamel
formation. Delayed modulation leads to enamel hypo-mineralisation
(Smith, Nanci, & Denbesten, 1993). Based on this evidence, tight con-
trol of the extracellular pH is required for normal enamel maturation.
Secreted ions such as H+ and HCO3− regulate the extracellular pH
around ameloblasts to generate the special acid-base balance in enamel
maturation. The net extracellular pH becomes more acidic in a stage-
dependent manner through a balance between H+ production and
HCO3− secretion during amelogenesis. HCO3− is generated by carbonic
anhydrase (CA), which is strongly expressed by ameloblasts and es-
sential for enamel mineralization (Bronckers, Lyaruu, Jalali, &
DenBesten, 2016; Toyosawa, Ogawa, Inagaki, & Ijuhin, 1996). The co-
localisation of V-H+ ATPase with CA II (a cytosolic form of the enzyme)
at the apical membrane of maturation-stage RAs suggests that the
ameloblasts secrete H+ and this mechanism is facilitated by CA II for
enamel formation (Lin, Nakamura, Noda, & Ozawa, 1994). Recently,
Sarkar et al. reported that V-H+ ATPase plays an important role in pH
regulation during enamel development (Sarkar, Wen, Simanian, &
Paine, 2016). It was shown that increased CA II activity produces an
acidic intracellular pH during ameloblast differentiation (Wang et al.,
2010). The decreased intracellular pH triggers the activation of a c-Jun
N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK)
signalling cascade. Other members of the CA protein family such as CA
III (cytosolic), CA VI (secreted extracellular), and CA XII (membrane-
associated) also exhibit enhanced expression during the maturation
stage, suggesting involvement in enamel maturation (Lacruz, Brookes
et al., 2013). CA VI is a candidate for the modulation of extracellular pH
fluctuation. DNA-fragment matching for rodent CA VI revealed CA VI
expression during the maturation stage of amelogenesis (Smith, Nanci,
& Moffatt, 2006). It is unclear, however, whether the CA VI is secreted
into the enamel layer to assist in the modulation of extracellular pH
fluctuations. Although the role of CA proteins is not discussed further in
this review, CAs commonly interact with and modulate the activity of
several ion transporters such as AE2, NHE, and NBCe1 (Becker, Klier, &
Deitmer, 2014). The differential expression of CAs and the associated
pH changes might provide a useful strategy for regulating amelogen-
esis.
In one of the best examples describing the acid-base balance in the
human body, the salivary glands secrete bulky electrolytes and pro-
teins. The delicate regulation of the electrolyte composition was
mediated by various ion transporters and channels (Lee et al., 2012).
The ionic balance and intracellular pH are tightly modulated by the
neutralisation of H+ by HCO3− through a mechanism similar to that of
HCO3−excretion from the salivary ducts into the saliva. The molecular
mechanisms of pH regulation by HCO3− and fluid secretion through
trans-epithelial transporters into the saliva are beyond the scope of this
review but are well reviewed elsewhere (Lee et al., 2012). During the
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ameloblast differentiation, alkaline conditions of extracellular space
predominate in the SA stage, whereas acidic conditions predominate in
the RA stage (Lacruz, Nanci, Kurtz, Wright, & Paine, 2010). It could be
hypothesised that ion transporters involved in H+ and HCO3− move-
ment modulate the extracellular pH fluctuations in each stage of dif-
ferentiation; however, the precise regulatory mechanisms of pH control
by pH-associated ion transporters and channels during ameloblast dif-
ferentiation are not fully understood. Specifically, Lin et al. addressed
that the RA secreted H+ to the enamel matrix through the V-H+ ATPase
(Lin et al., 1994). More recently, the cytoplasmic V-H+ATPase is sig-
nificantly upregulated in maturation stage of ameloblasts compared to
secretory-stage cells for lysosomal acidification by ameloblast-mediated
endocytosis of degraded enamel matrix proteins (Sarkar et al., 2016).
However, precise role of V-H+ ATPase in pH modulation needs to be
studied. The molecular mechanism of pH modulation is still not clear.
This review will briefly explore the current understanding of the ion
transporters expressed during or involved in amelogenesis. That un-
derstanding will then be used to identify new interventional strategies
for the treatment of tooth-development diseases.
7. Ion channels and transporters
7.1. Cystic fibrosis transmembrane conductance regulator (CFTR)
CFTR, a central regulator of fluid and electrolyte transport, is a
cyclic AMP-regulated Cl− channel with HCO3− permeability in epi-
thelial tissues. Mutations of the CFTR gene cause CF, a lethal hereditary
disease resulting in dysfunction of multiple tissues and organs such as
the pancreas, airways, gastrointestinal tract, and salivary tissues (Grubb
& Boucher, 1999). A CF mouse model demonstrated abnormal incisor
enamel caused by abnormal CFTR expression and the premature de-
generation of ameloblasts (Wright, Kiefer, Hall, & Grubb, 1996). CFTR-
null mice show defective pH regulation during the enamel-maturation
stage (Sui, Boyd, & Wright, 2003). The mice have enamel with normal
thickness but reduced mineral volume; the enamel proteins are re-
tained, and the enamel chips away from the incisal edges following
eruption. The protein retention might be caused by a decrease in en-
docytosis due to excessive fluoride stimulation resulting from the loss of
CFTR function (Duan et al., 2011). In addition, between 5% and 44% of
patients with CF have variable enamel hypoplasia (Jagels & Sweeney,
1976).
The precise physiological role of CFTR in enamel and ameloblast
maturation is not known. CFTR mediates HCO3− efflux and is regulated
by the intracellular and extracellular Cl− concentrations. In the pan-
creatic and salivary duct systems, CFTR activation is associated with
several regulatory factors such as the 1,4,5-trisphosphate (IP3) receptor-
binding protein released with IP3 (IRBIT) (Yang et al., 2009). The mode
of CFTR function has two stage-dependent phases and is addressed well
in the proposed ductal HCO3− efflux system (Lee et al., 2012). The roles
of CFTR and regulatory factors such as IRBIT are still not fully known in
enamel tissue. Hence, the mode of CFTR regulation and the identifi-
cation of CFTR regulatory factors in endocytosis and amelogenesis
await further study.
7.2. Anion exchanger 2 (AE2)
The molecular identity and function of AE2 are most likely encoded
by the SLC4A2 gene (Nguyen, Stuart-Tilley, Alper, & Melvin, 2004).
AE2 is a Cl−/HCO3− exchanger that plays a crucial role in anion se-
cretion and absorption and triggers a protective mechanism against
alkali load. Cl− uptake in the salivary glands depends on the co-
ordinated mechanisms of basolateral AE2 and Na+-H+ exchanger1
(NHE1) (Catalan, Nakamoto, & Melvin, 2009). The content of Cl− in
enamel formation is essential for normal tooth modulation. It has been
reported that AE2 is expressed in the basolateral membrane of ma-
turation-stage ameloblasts and is essential for normal enamel
H.-E. Kim, J.H. Hong
maturation to build proper tooth strength. AE2 regulates the micro-
environment by a redisposition of tight junctions (Bronckers et al.,
2009; Josephsen et al., 2010; Lyaruu et al., 2008). Although expression
of AE was discovered in apical epithelia and debatable, the molecular
identity and function of AE in dental tissue have remained a mystery.
Lyaruu et al. showed that the incisor enamel was absent in AE2-
knockout mice, suggesting that the physiological and developmental
roles of AE2 must be integrated in amelogenesis (Lyaruu et al., 2008).
Studies of the AE2 expression patterns in ameloblasts have shown
somewhat contradictory results, however (Bronckers et al., 2009;
Josephsen et al., 2010). Defective mineralisation caused by SLC4A2
deletion appears to be critical in enamel tissue but not in other mi-
neralising tissues such as bone. The regulatory role of AE2, in addition
to that of ClC buffers, against pH fluctuations during ameloblast mod-
ulation cycles will be determined in the coming years. Another pH
regulation system in ameloblasts is NHE1. This exchanger will discuss
below section.
7.3. ClC channels (ClCs)
ClCs were characterised from nine members of the mammalian CLC
gene family, which can be clustered as four plasma-membrane Cl−
channels (ClC-1, ClC-2, ClC-Ka, and ClC-Kb), three Cl− channels pre-
dominantly expressed in intracellular vesicles (ClC-3, ClC-6, and ClC-7),
and two voltage-dependent Cl−-H+ exchangers in the plasma mem-
brane (ClC-4 and ClC-5) (Jentsch, 2007). Clc gene family provides
neutralizing anion currents for V-H+ ATPases that acidify the en-
dosomal/lysosomal compartments (Scheel, Zdebik, Lourdel, & Jentsch,
2005). Few studies have elucidated the physiological role of ClCs in
bones and teeth. ClC-7 is highly expressed in osteoclasts, where it can
be inserted into the ‘ruffled border’ to secrete acid to the extracellular
compartment to aid in bone resorption (Weinert et al., 2010). The
acidic pH in the extracellular compartment is required both for the
dissolution of inorganic bone and the secretion of lysosomal enzymes.
The mRNAs of ClC-1∼7 express in the tooth germ (Hou, Situ, &
Duan, 2008). ClC-5 and ClC-7 have been found in the inner and outer
enamel epithelium and in ameloblasts (Duan et al., 2009; Duan, 2014).
ClC-5 deficiency induces the enhanced expression of TGF-ß1, which is
directly associated with abnormal dentin changes (Duan et al., 2009).
ClC-5-knockout mice display dentinogenesis imperfecta (Wang et al.,
2000). Clcn7, the gene encoding ClC-7, is expressed in maturation-stage
ameloblasts (Duan et al., 2009; Lacruz, Brookes et al., 2013). ClC-7-
deficient mice display an osteopetrosis phenotype and abnormal tooth
eruption, suggesting that ClC-7 is involved in the development of
dentition and calvaria (Hou et al., 2008). A similar phenotype was
observed in anion exchanger (AE2)-knockout mice (Josephsen et al.,
2009).
7.4. Solute carrier 26 A (SLC26 A) transporters
−
A new era in the study of Cl absorption and HCO3− secretion
began with the discovery of the down-regulated in adenoma (DRA)
protein (designated SLC26 A3) (Hoglund et al., 1996; Ko et al., 2004).
SLC26 A6 is a predominant pathway for Cl−/HCO3− exchange in the
pancreatic and salivary ducts (Lee et al., 2012). The mode of regulation
of Cl−/HCO3− exchange is still unknown in enamel biology. The role of
the SLC26 A family in dental-enamel formation was addressed recently.
SLC26 A4 (also called pendrin)-null mice conveyed normal dental
phenotype (Bronckers et al., 2011), and both SLC26 A1 and SLC26 A6
were found by genome-wide transcript profiling to be up regulated
during enamel maturation (Lacruz et al., 2012). Another breakthrough
for expression and functional studies of the SLC26 transporters in
ameloblasts was reported: SLC26 A3, SLC26 A4, SLC26 A6, and
SLC26 A7 are expressed in maturation-stage ameloblasts (Jalali et al.,
2015; Yin et al., 2015). The protein levels of SLC26 A3 and SLC26 A4
were increased in SLC26 A6-null mice. The data suggest that the
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SLC26 A isoforms compensate for one another to produce normal en-
amel phenotype in null mice (Jalali et al., 2015; Yin et al., 2015).
7.5. Electrogenic Na+-HCO3− cotransporter 1 (NBCe1)
NBC was first identified in the proximal tubule of the kidney (Boron
& Boulpaep, 1983). Electrogenic NBC1 has three splice variants,
NBCe1-A, NBCe1-B, and NBCe1-C, which differ at the N-terminus or the
C-terminus. NBCe1-A, known as kNBC1, is primarily expressed in the
kidney and plays an important role in HCO3− reabsorption (Abuladze
et al., 1998). The pNBC1 variant, renamed NBCe1-B, is expressed in the
pancreas, duodenum, and several other tissues (Abuladze et al., 1998;
Bok et al., 2001; Roussa, Nastainczyk, & Thevenod, 2004). NBCe1-C is
mainly expressed in the brain (Bevensee, Schmitt, Choi, Romero, &
Boron, 2000). NBC1 mutations, which cause proximal renal tubular
acidosis in humans and are also associated with ocular abnormalities
such as band keratopathy, glaucoma, and cataracts (Soleimani &
Burnham, 2000; Usui et al., 2001), have been proposed to mediate
HCO3− influx across the basolateral membrane to support trans-epi-
thelial secretion (Jacob et al., 2000).
The NBCe1 family are electrogenic transporters with 1Na+:2HCO3−
or 1Na+:3HCO3− stoichiometry expressed in a cell type-dependent
manner (Gross et al., 2001). The salivary duct expresses an electro-
neutral NBC, which, similar to cloned NBCn1-A, moves 1Na+ and
1HCO3− in the luminal membrane. Pancreatic and salivary ducts have a
mechanism for Na+ and HCO3− absorption at the luminal membrane,
which serves as an HCO3− salvage mechanism for the maintenance of
acidic conditions (Luo et al., 2001). Maturation-stage papillary cells
express NBCe1 and NBCn1, while the acid-loader AE2 and the acid-
extruder NHE1 were mainly found in ameloblasts (Josephsen et al.,
2010). Perhaps the specialised expression of HCO3−-transport proteins
in papillary cells is based on the cellular capacity for oscillatory pH
fluctuations in the extracellular environment. Lacruz et al. addressed
the expression of NBCe1 at the basal pole of ameloblasts (Lacruz, Nanci,
White et al., 2010); however, several studies of NBCe1 expression in-
dicate that the expression pattern is not restricted to the basal pole but
exists broadly in the entire basolateral membrane. With that expression
pattern, the basal-pole expression of NBCe1 in ameloblasts should be
influenced by the strong expression of NBCe1 in papillary cells. The
NBCs are not only important for the secretory process; they should have
a fundamental role in regulating pH homeostasis and HCO3− con-
centration for normal ameloblast development. Future studies could
look to investigate the expression levels of NBCs in ameloblasts and the
precise regulatory role of those proteins in each developmental stage.
7.6. Na+-H+ exchanger (NHE1)
NHE1 mediates the Na+ influx and H+ efflux and ubiquitously
expresses at the basolateral membrane of secretory epithelia. NHE1 as
an acid extruder was revealed in the lateral membrane during both the
secretory and the maturation stages of ameloblasts (Josephsen et al.,
2010). NHE1 might be involved in the maintenance of pH homeostasis
at all stages of enamel development. In vitro amelogenesis model, HAT-
7 rat ameloblast cell line was functionally studied in pH regulation
(Bori et al., 2016). The NH4+ pulse technique in the presence of
amiloride was used to identify the NHE1 activity in polarized transwell
system. Amiloride-sensitive basolateral rapid pH recovery mediated by
the H+ efflux of NHE1 (Racz et al., 2017; Varga, DenBesten, Racz, &
Zsembery, 2017). Although precise immunostaining of each develop-
mental stage of enamel tissue is yet to be performed, the pH fluctuation
in the extracellular microenvironment can be assumed to be caused by
the differential expression of ion transporters such as acid extruders or
alkali extruders.
H.-E. Kim, J.H. Hong
7.7. Na+-K+-2Cl− cotransporters (NKCCs)
NKCCs are cation-coupled Cl− transporters which mainly involve in
volume regulation (Evans et al., 2000). Real time PCR and im-
munostaining analysis revealed the expression of NKCC1 in the mouse
dental papillary layer, not in ameloblast layer. Study of Nkcc1-null mice
revealed disorganized ameloblast maturation and reduced mineral
density (Jalali et al., 2017). HAT-7 ameloblast cell line has been studied
in functional NKCC1. NKCC1 inhibitor bumetanide attenuated the vo-
lume of HAT-7, suggesting that in line with volume regulation of
NKCC1. Above all, non-ameloblast epithelial cells express NKCC1 and
regulate cell volume and fuel ameloblasts to regulate cell volume of
dental epithelium.
8. Calcium (Ca2+) regulatory transport proteins in amelogenesis
Dental enamel is highly calcified in mammals. Enamel-forming
epithelial cells supply Ca2+ in bulk for mineralisation. Commonly,
Ca2+ mobilisation mediates various cell functions such as muscle
contraction, neuronal excitation, gene expression, secretion, and the
release of hormones and neurotransmitters. Ca2+ channels can be
classified according to their operative mode (e.g., receptor-operated
channels or voltage-operated channels). Diverse aspects of Ca2+ sig-
nalling in general have been reviewed in soft-tissue organs such as the
pancreas and salivary glands; however, relatively little work has spe-
cifically addressed Ca2+ signalling in enamel epithelial cells. For ex-
ample, it is unclear how enamel-forming cells exposed to high levels of
Ca2+ avoid the cytotoxic effects of Ca2+. It seems likely that an energy-
requiring Ca2+ regulatory mechanism is needed to avoid the Ca2+
toxicity. Many questions remain with regard to Ca2+ signalling in
ameloblasts. To date, several publications have shown that Ca2+ mo-
bilisation will implicate physiologically relevant information in ame-
loblast biology.
8.1. Plasma membrane Ca2+ ATPase (PMCA) and sarcoplasmic/
endoplasmic reticulum Ca2+ ATPase (SERCA)
The PMCA pump is a plasma-membrane transport protein that
mainly regulates the intracellular Ca2+ concentration, whereas SERCA
operates in the ER membrane. In addition to maintaining the in-
tracellular Ca2+ level, PMCA regulates the Ca2+ concentration in the
extracellular space (Strehler & Zacharias, 2001). It was reported that
PMCA1 and PMCA4 are expressed in parallel with the progression of
enamel mineralisation (Borke et al., 1995). The capacity for Ca2+
transport is greater in the maturation stage than in the secretory stage.
The threefold up-regulated expression of SERCA2b during enamel ma-
turation provides evidence that enamel cells possess a more effective
Ca2+ sequestering system than other cells (Franklin et al., 2001;
Nurbaeva, Eckstein, Concepcion, et al., 2015). To date, it is unclear how
the expression levels of Ca2+-sequestering proteins are regulated
during maturation. It is noteworthy; however, that energy-requiring
PMCA and SERCA are highly expressed in the maturation stage. Ame-
loblasts might require a mechanism to meet the excessive energy re-
quirements associated with the avoidance of Ca2+ toxicity. Further
studies are needed to address the regulatory roles of PMCA and SERCA
and energy metabolism in stage-dependent ameloblasts.
8.2. Na+-Ca2+ exchangers (NCX) and Na+-Ca2+-K+ exchangers
(NCKX)
In addition to the ubiquitous expression of L-type Ca2+ channels,
cellular Ca2+ influx and efflux mediated by NCX and NCKX have been
recently studied in teeth. NCX proteins are encoded by three members
of the SLC8A family (for NCX1–3, respectively). NCKX proteins are
encoded by six SLC24A genes (for NCKX1–6, respectively).
Bidirectional transport is accomplished by exchanging Na+ and Ca2+
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through gradient-driven ion transport (Uehara, Iwamoto, Nakamura, &
Imanaga, 2004). NCX proteins demonstrate tissue-specific expression.
NCX1 and NCX3 localise in the apical ameloblastic membrane
(Okumura et al., 2010). Genome-wide transcript profiling indicated
that NCKX4 is highly up regulated in maturation-stage enamel
(Josephsen et al., 2010). Increased NCKX4 mRNA expression occurs in
the late stages of amelogenesis, suggesting that it might be involved in
the modulation of Ca2+ transport during the enamel-maturation stage.
Recently, it was reported that NCKX4 is primarily expressed in the
apical and lateral membranes during the maturation stage, modulating
and transporting Na+, Ca2+, and K+ during amelogenesis (Hu et al.,
2012; Wen, Lacruz, Smith, & Paine, 2014). The expression of NCKX4 in
the enamel organ is restricted to maturation-stage ameloblasts (Hu
et al., 2012), suggesting an increased necessity for Ca2+ export during
maturation.
8.3. Na+-K+-ATPase
Although Na+-K+-ATPase cannot be categorised as a part of Ca2+
signalling, real-time PCR revealed that the expression of Na+-K+-
ATPase in ameloblasts increases from the secretory stage to the ma-
turation stage (Wen et al., 2014). Against electrochemical gradient of
Na+, three Na+ are exported and two K+ are imported by using ATP.
The Na+-K+-ATPase is expressed at the basolateral membrane as a
potent counterpart channel to the apical and lateral NCKX4 channel
(Hu et al., 2012), suggesting that the transcellular movements of both
Na+ and K+ involve Ca2+. The α subunit of Na+-K+-ATPase interacts
with the inositol IP3 receptor (IP3R), which mediates Ca2+ release from
the endoplasmic reticulum (ER). It could therefore be postulated that
interaction with Na+-K+-ATPase alters the gating property of IP3R,
releasing Ca2+ (Wen et al., 2014).
8.4. Inositol 1,4,5-trisphosphate receptors (IP3Rs)
Increases in intracellular Ca2+ are mediated by Ca2+ release from
ER stores via IP3Rs and ryanodine receptors (RyRs) or by Ca2+ influx
from the extracellular space. Rodent ameloblasts in the secretory and
maturation stages express both IP3Rs and RyRs (Nurbaeva, Eckstein,
Concepcion, et al., 2015). Although IP3Rs and RyRs are mainly asso-
ciated with the ER Ca2+-release mechanism, immunostaining patterns
showed a dominant signal for IP3Rs and a very weak signal for RyRs.
IP3R2 was expressed only in the nuclei, whereas IP3R1 and IP3R3 lo-
calised in the cytoplasm during the secretory and maturation stages.
The strong staining patterns of IP3R2 in the nuclei and IP3R3 in the
cytoplasm suggest that nuclear Ca2+ signalling related to protein se-
cretion is necessary in the secretory stage and Ca2+ transport is ne-
cessary in the maturation stage. Although the differential cytoplasmic
expression of IP3R1 and IP3R3 might be associated with the localized
Ca2+ signals, that issue should be clarified.
8.5. Stromal interaction molecule (STIM) and Orai
STIM functions as an ER Ca2+ sensor. Although STIM itself is not a
channel, we briefly summarized the role of STIM as an activating pro-
tein of Ca2+ entry channels, such as Orai. STIM1 has a single trans-
membrane span with a Ca2+-binding EF hand in the ER that is oligo-
merised and activated by the depletion of ER Ca2+ stores (Liou et al.,
2005; Spassova et al., 2006; Zhang et al., 2005). After the depletion of
Ca2+ stores following Ca2+-evoked stimuli, the dissociation of Ca2+
from the EF hand results in STIM1 clustering at the ER-plasma mem-
brane junctions, where the clusters activate store-operated channels
composed of Orai subunits (Putney, 2009). Protein-protein interactions
involving both STIM1 and Orai mediate store-operated Ca2+ entry
(SOC entry), which controls numerous physiological functions in many
cell types including ameloblasts (Wang et al., 2014). Although STIM1 is
not considered an ion channel, the failure of STIM1 function impairs
H.-E. Kim, J.H. Hong
Ca2+ influx and thus sequentially impairs enamel maturation (S. Wang
et al., 2014). Elevated intracellular Ca2+ levels caused by dominantly
constitutive activation of STIM1 do not appear to influence enamel
maturation (Bohm et al., 2013).
Ameloblasts in the maturation stage might not be sensitive to basal
Ca2+ levels, or else excessive Ca2+ might be moved into the enamel
area. Because STIM1 is critical for the maturation stage, there must be
an important Ca2+ entry pathway in amelogenesis. More recently, SOC
entry via the Ca2+ release-activated Ca2+ (CRAC) channel was shown
to lead to increased expression of enamel matrix-protein genes such as
Amelx, Ambn, Enam, and Mmp20 in ameloblast-like LS8 cells and
murine primary enamel cells (Nurbaeva, Eckstein, Snead, Feske, &
Lacruz, 2015). Orai1 expression was dramatically increased in ma-
turation stage (Nurbaeva, Eckstein, Concepcion, et al., 2015; Nurbaeva,
Eckstein, Snead et al., 2015). Orai1 is an essential transmembrane
subunit of the CRAC channel, forming the pore in the membrane. Pa-
tients with deficient Orai1 display severe immune deficiency, including
ectodermal dysplasia with defective dental-enamel calcification
(McCarl et al., 2009). Enamel deposition in the Orai1 knockout mice
was reduced and irregular as observed in patients with Orai1 defects
(Robinson et al., 2012). Orai1 knockdown attenuates the expression of
ameloblast-differentiation markers such as dentin sialoprotein, enamel-
matrix proteins such as amelogenine and ameloblastin, and PMCA1
(Zheng et al., 2015).
An increase in interest in the SOC entry system has led to the recent
discovery of key components of CRAC channels, such as Orai1–3 and
STIM1 and STIM2, in murine enamel cells (Nurbaeva, Eckstein,
Concepcion, et al., 2015). Experiments applying the CRAC-channel
blocker Synta-66 to enamel cells suggest that CRAC channels mediate
SOC entry. The results so far indicate that ameloblasts express excessive
Ca2+-modulatory proteins such as Orai1 and STIM1, enabling Ca2+
entry in bulk, and that SERCA2b and NCKX4 sequester Ca2+ into the ER
or the extracellular space during maturation, which suggests that the
ER can be a critical organelle for Ca2+ transcytosis (Hubbard, 2000;
Nurbaeva, Eckstein, Concepcion, et al., 2015). Recent growing evi-
dences address that SOCE via CRAC channels is critical for the main-
tenance of enamel crystals and appropriate enamel mineralization. The
STIM1/2K14cre mice are generated to investigate the effects of CRAC
channel deficiency in enamel, revealing associations among Ca2+-in-
duced ER stress, abnormal mitochondria, and disruption to the glu-
tathione system (Eckstein et al., 2017). STIM1 and STIM1/2 conditional
52
Archives of Oral Biology 93 (2018) 47–55
Fig. 2. Schematic representation of Ca2+ and Mg2+
transporting molecules in matured ameloblasts.
This model depicts our current knowledge of the Ca2+
signalling proteins and Mg2+ transporting molecules in
ameloblasts during maturation. NCX: Na+-Ca2+ ex-
changer; NCKX: Na+/Ca2+-K+ exchanger; STIM1:
Stromal interaction molecule 1; SERCA2b: sarcoplasmic/
endoplasmic reticulum Ca2+ ATPase 2b, IP3R: 1,4,5-tri-
phosphates (IP3) receptor; PMCA: plasma membrane Ca2+
ATPase; CNNM4: Mg2+/2Na+ exchanger; TRPM7: tran-
sient receptor potential subfamily M, member 7; N: nu-
cleus. ER: endoplasmic reticulum
knockout mice were observed inferior enamel mineralization with im-
paired structural integrity (Furukawa et al., 2017). Accordingly, any
improvement in the understanding of how Ca2+ signalling can be
regulated in ameloblasts could contribute to strategies for treating ab-
normal tooth formation.
9. Mg2+ transporting molecules: CNNM4 and TRPM7
Mg2+ homeostasis is critical ion in bone development and enamel
mineralization. Two types of Mg2+ transporting molecules, cyclin M4
(CNNM4) and transient receptor potential subfamily M, member 7
(TRPM7), are identified in ameloblasts. Amelogenesis imperfecta
spectrum has been the identification of mutations to the Mg2+/2Na+
exchanger CNNM4 (Parry et al., 2009). CNNM4 possess in basolateral
membrane of RA (Simmer et al., 2014; Yamazaki et al., 2013). Severe
hypomagnesemia is observed in Cnnm4-null mice in enamel (Yamazaki
et al., 2013). TRPM7, also Ca2+-permeable channel, expresses in ma-
tured ameloblasts and odontoblasts during tooth development and
mediates Mg2+ influx into the cells (Nakano et al., 2016; Ogata et al.,
2017). TRPM7 contains a serine/threonine protein kinase domain
merged to an ion channel. TRPM7 kinase domain, independent of
channel activity, involves in ameloblast differentiation through the
phosphorylation of Smad1/5/9, p38, and cAMP response element
binding protein (Ogata et al., 2017). Above all, the Ca2+ and Mg2+
transporting molecules addressed are crucial for maturation stage and
illustrated in Fig. 2.
10. Summary
This review summarised the current understanding of ion channels
and transporters in amelogenesis. Unfortunately, many questions re-
main unanswered with respect to pH regulation and ionic balance for
normal tooth development. Tooth formation is a highly dedicated
process in which the cellular mechanism is aligned in a single layer of
cells. The polarised distribution of the single layer of ameloblasts and
odontoblasts has a cardinal function in enamel maturation. The mod-
ulation of electrolytes balance by ion transporters apparently provides
both morphological and functional stability to achieve appropriate
tissue hardness. The ion transporters addressed in this review shows
that coordinated function is crucial for enamel maturation and its dif-
ferential expression based on immunostaining results is illustrated in
H.-E. Kim, J.H. Hong
Fig. 3. Schematic representation of differential expression of proteins in ameloblasts.
Indicated bars represent the expression profile of protein in ameloblasts with stage-dependent manner. Expression level of SERCA, SLC26 A3/4/6/7, STIM1, Orai1,
NCKX4, AE2, CA II/III/VI/XII, ClC-7, CFTR, cytoplasmic V-H+ ATPase, CNNM4, and TRPM7 was dependent on enamel maturation. NHE1, NBCe1, Na+-K+-ATPase,
and IP3R1/R3 are expressed in whole stage of amelogenesis. Am: ameloblasts; PC: papillary cell. SA: smooth-ended ameloblasts, RA:ruffle-ended ameloblasts.
Fig. 3. However, we really know very little about the detailed mod-
ulation of these proteins, depending on the stage and there is still rare
experimental system to mimic the dental microenvironment in vivo. The
further discovery of additional transporters or of the molecular char-
acteristics of ion transporters might become instrumental in under-
standing the details of tooth formation to overcome enamel dysplasia.
Conflict of interest
No conflicts of interest are declared by the authors.
Acknowledgement
This research was supported by National Research Foundation of
Korea (NRF) grant funded by the Korean Government (NRF-
2017R1D1A1B03029570).
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