Anoctamin-6 Controls Bone Mineralization by Activating the Calcium Transporter NCX1

Background: Deletion of Ano6 leads to reduced skeleton size and skeletal deformities.

Results: Ca²⁺-activated anion currents are missing in primary osteoblasts from Ano6 null mice. Ano6 is required for the Na⁺/Ca²⁺ exchanger NCX1 to operate in osteoblasts.

Conclusion: The Na⁺/Ca²⁺ exchanger NCX1 is essential for bone mineralization and requires Ano6 to fully operate.

Significance: Ano6 supports NCX1, which is essential for bone mineralization.

Abstract

Anoctamin-6 (Ano6, TMEM16F) belongs to a family of putative Ca²⁺-activated Cl⁻ channels and operates as membrane phospholipid scramblase. Deletion of Ano6 leads to reduced skeleton size, skeletal deformities, and mineralization defects in mice. However, it remains entirely unclear how a lack of Ano6 leads to a delay in bone mineralization by osteoblasts. The Na⁺/Ca²⁺ exchanger NCX1 was found to interact with Ano6 in a two-hybrid split-ubiquitin screen. Using human osteoblasts and osteoblasts from Ano6^−/− and WT mice, we demonstrate that NCX1 requires Ano6 to efficiently translocate Ca²⁺ out of osteoblasts into the calcifying bone matrix. Ca²⁺-activated anion currents are missing in primary osteoblasts isolated from Ano6 null mice. Our findings demonstrate the importance of NCX1 for bone mineralization and explain why deletion of an ion channel leads to the observed mineralization defect: Ano6 Cl⁻ currents are probably required to operate as a Cl⁻ bypass channel, thereby compensating net Na⁺ charge movement by NCX1.

Introduction

During vertebrate skeletal development, specialized cells, the osteoblasts, produce a mineralized bone matrix by deposition of hydroxyapatite crystals in the extracellular collagen matrix, where they become embedded and differentiate into osteocytes (1–3). This process requires the organized transport of high concentrations of Ca²⁺ and phosphate ions into the extracellular matrix without inducing premature crystallization. Although of fundamental importance, the process of calcification is not well understood. Different concepts currently exist. (i) A cell-independent mechanism has been suggested, whereby charged non-collagenous proteins associate with the collagen matrix to form mineralization foci (4). (ii) The amorphous calcium phosphate theory predicts that initial amorphous calcium phosphate is deposited, which gradually converts to pure crystalline hydroxyapatite (5, 6). (iii) A cell-controlled mechanism that requires budding of vesicles from the plasma membrane has also been proposed. These vesicles accumulate ions and organize the formation of calcium phosphate crystallization seeds containing hydroxyapatite particles. Subsequently, rupture of these vesicles spreads calcium phosphate onto the extracellular matrix (7, 8). (iv) Recent work identified intracellular vesicles that contain mineral in a mostly amorphous phase form (9, 10).

Whatever the precise mechanism is, mineralization requires Ca²⁺ transport. The mechanisms of Ca²⁺ transport by osteoblasts are not well understood at the molecular level. The Ca²⁺-permeable transient receptor potential channels TRPV4 and TRPV6 are expressed in bone cells, but whether they affect bone mineralization has not yet been elucidated (11). Another group of Ca²⁺ transporters, the plasma membrane Ca²⁺-ATPases, are strong candidates to transport Ca²⁺ into the extracellular compartment. PMCA1, PMCA2, and PMCA4 are expressed in osteoblasts, but their role in mineralization in mammals in vivo has not been demonstrated. In zebrafish, the PMCA2 homolog Atp2b1a seems to be required for mineralization by odontoblasts and possibly the adjacent bone structures (11–14). A third class of Ca²⁺ transporters are the Na⁺/Ca²⁺ exchangers (NCX). Of these, NCX1 and NCX3 are expressed in mouse osteoblasts, and NCX3 has been demonstrated to regulate Ca²⁺ transport in osteoblastic cell lines (14–17).

Recently, anoctamin-6 (Ano6, TMEM16F) has been reported as another protein required for proper bone mineralization (1). Ano6 belongs to a family of putative Ca²⁺-activated Cl⁻ channels (18–21). Several reports demonstrated that Ano6 generates an outwardly rectifying Cl⁻ current when activated by a large increase in local intracellular Ca²⁺ concentrations (22–24). Moreover, Ano6 operates as a Ca²⁺-activated phospholipid scramblase that leads to movement of phosphatidylserine from the inner to the outer leaflet of the plasma membrane bilayer (25). Impaired Ca²⁺-dependent phospholipid scrambling of phospholipids in platelets leads to Scott syndrome, a rare inherited defect in primary hemostasis due to mutations in Ano6 (25, 26). The defect in Ca²⁺-dependent phospholipid scrambling in cells from patients with Scott disease is paralleled by lack of Ca²⁺-dependent activation of Cl⁻ currents, which is necessary for full platelet phosphatidylserine exposure and procoagulant activity (26, 27). Recently, reconstitution of a purified Ano6 homolog from Aspergillus fumigatus confirmed that Ano6 has both ion channel and scramblase function and that transport of both ions and lipids is controlled by a single Ca²⁺-binding site (28).

A role of Ano6 in activating phosphatidylserine scrambling in osteoblasts has been demonstrated previously (1). However, its function as a Ca²⁺-activated Cl⁻ channel has not been investigated in osteoblasts. In this study, we therefore examined the role of Ano6 Cl⁻ currents in the mineralization process. Notably, in a yeast two-hybrid split-ubiquitin screen using Ano6 as bait, we found the Na⁺/Ca²⁺ exchanger NCX1 to be a potential binding partner, suggesting a molecular and functional link between Ano6 and NCX1.

EXPERIMENTAL PROCEDURES

Ano6-deficient Mice, RT-PCR, and Two-hybrid Analysis

Generation of Ano6 (TMEM16F)-deficient mice has been described (1). Total RNA was isolated from human and mouse osteoblasts using NucleoSpin RNA II columns (Macherey-Nagel GmbH & Co. KG, Düren, Germany). Total RNA (1 μg/50-μl reaction) was reverse-transcribed using random primers (Promega, Mannheim, Germany) and Moloney murine leukemia virus reverse transcriptase (RNase H Minus; Promega). Each RT-PCR contained sense and antisense primers for anoctamins (0.5 μm; see supplemental Tables 1 and 2) or for GAPDH (0.5 μm), 0.5 μl of cDNA, and GoTaq polymerase (Promega). After 2 min at 95 °C, cDNA was amplified in 30 cycles for 30 s at 95 °C, 30 s at 57 °C, and 1 min at 72 °C. PCR products were visualized by loading on ethidium bromide-containing agarose gels and analyzed using MetaMorph Version 6.2 (Molecular Devices). The two-hybrid split-ubiquitin screenings were performed using a normalized human universal NubG-x library with human Ano6 as a bait (Dualsystems Biotech AG, Zurich, Switzerland).

Primary Osteoblasts, Mineralization Assay, and HEK293 Cells

Frontal and parietal parts of mouse calvariae were dissected from day 18.5 embryos, and primary osteoblasts were isolated by two 30-min digestion steps in 0.1% collagenase IV (Sigma). Cells from individual animals were cultured separately, seeded at a density of 10⁴ cells/cm², and cultured in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin. Upon confluence, cells were reseeded at a density of 10⁴ cells/cm² and cultured in the presence of 10 mm glycerophosphate and 5 mm ascorbate phosphate to induce osteogenic differentiation and mineralization (day 0). After establishing a nontoxic effective concentration, cells were treated with 5 μm tannic acid or KB-R7943 (29). The potential toxicity of these inhibitors was excluded by negative trypan blue staining and by assessing the effect of these inhibitors on cell morphology (data not shown). Moreover, previous in vivo and in vitro studies demonstrated the nontoxicity of these inhibitors (30–32).

During differentiation, triplicate wells of each culture were fixed at days 3, 10, 17, and 20 in methanol and washed in deionized water. Staining intensity was quantified as described by Gregory et al. (33). Human primary osteoblasts were purchased from PromoCell (Heidelberg, Germany). Cells were grown in either osteoblast growth medium or osteoblast mineralization medium (PromoCell) according to the manufacturer's protocol. Mineralization was visualized by Alizarin Red staining, which was subsequently quantified (33). Briefly, Alizarin Red S was extracted from the cell layer in 10% acetic acid, and cell debris was removed by centrifugation and quantified at 405 nm in a microplate reader (NOVOstar, BMG LABTECH, Offenburg, Germany). HEK293 human embryonic kidney cells were grown in DMEM supplemented with 10% FBS. Cells were incubated in 5% CO₂ at 37 °C. pcDNA3.1 encoding human Ano6 and human NCX1 was transfected into HEK293 cells using standard methods (Lipofectamine, Invitrogen). Cells were examined 48 or 72 h after transfection.

Western Blotting of Ano6 in Human Primary Osteoblasts and Co-immunoprecipitation of NCX1/Ano6 in HEK293 Cells

Cells were collected and lysed in Nonidet P-40 lysis buffer (25 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 5% glycerol, and 1× protease inhibitor mixture). Proteins (20 μg) were analyzed by 10% SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween 20 (NFM/TBST) for 1 h at room temperature and incubated overnight at 4 °C with rabbit anti-human Ano6 polyclonal antibody (diluted 1:500 in 1% NFM/TBST; P78, Davids Biotechnologie GmbH, Regensburg, Germany). Subsequently, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (diluted 1:10,000 in 1% NFM/TBST) for 2 h at room temperature. The immunoreactive signals were visualized using a SuperSignal chemiluminescence substrate detection kit (Pierce). To compare amounts of proteins, the membranes were probed with rabbit anti-β-actin antibody (diluted 1:1000 in 1% NFM/TBST; Sigma). The intensity of bands was quantified by densitometric scanning and normalized to β-actin.

Cell lysates in Nonidet P-40 lysis buffer were precleaned with protein G-agarose at 4 °C for 1 h. Cell lysates were incubated with 5 μg of primary antibody (goat anti-GFP or rabbit anti-NCX1 polyclonal antibody) overnight at 4 °C on a rotator. Cell lysates (70 μl containing 50 μg of protein) were incubated with protein G-agarose at 4 °C for 3 h, followed by centrifugation three times at 1000 × g for 1 min. Pellets were collected and resuspended in 1× loading buffer. The supernatant was collected, separated by 10% SDS-PAGE, and transferred to PVDF membrane. The membrane was blocked with 5% NFM/TBST for 1 h at room temperature and incubated overnight at 4 °C with goat anti-GFP polyclonal antibody (diluted 1:3000 in 1% NFM/TBST) or rabbit anti-NCX1 polyclonal antibody (diluted 1:1000 in 5% NFM/TBST). Subsequently, the membrane was incubated with HRP-conjugated donkey anti-goat IgG (diluted 1:2000 in 5% NFM/TBST) at room temperature for 1 h or with HRP-conjugated goat anti-rabbit IgG (diluted 1:10,000 in 1% NFM/TBST) at room temperature for 2 h. Immunoreactive signals were visualized using the SuperSignal chemiluminescence substrate detection kit.

Patch Clamping

Cells grown on coverslips were mounted in a perfused bath on the stage of a Zeiss IM35 inverted microscope and kept at 37 °C. The bath was perfused continuously with Ringer's solution (145 mm NaCl, 0.4 mm KH₂PO₄, 1.6 mm K₂HPO₄, 6 mm d-glucose, 1 mm MgCl₂, and 1.3 mm calcium gluconate (pH 7.4)) at ∼10 ml/min. Patch clamp experiments were performed in the fast whole cell configuration. Patch pipettes had an input resistance of 4–6 megohms when filled with an intracellular-like solution containing 30 mm KCl, mm 95 potassium gluconate, 1.2 mm NaH₂PO₄, 4.8 mm Na₂HPO₄, 1 mm EGTA, 0.758 mm calcium gluconate, 1.034 mm MgCl₂, 5 mm d-glucose, and 3 mm ATP. The pH was 7.2, and the Ca²⁺ activity was 0.1 μm or 1 mm. The access conductance was measured continuously and was 90–140 nanosiemens (EPC-9 amplifier, List Medical Electronics, Darmstadt, Germany). In regular intervals, membrane voltages were clamped in steps of 20 mV from −100 to +100 mV from a holding potential of −60 mV.

Intracellular Ca²⁺ Concentrations in HEK293 Cells Coexpressing Ano6 and NCX1

Cells were seeded on glass coverslips and loaded with 2 μm Fura-2/AM and 0.02% Pluronic F-127 (Invitrogen) in Ringer's solution (145 mmol/liter NaCl, 0.4 mmol/liter KH₂PO₄, 1.6 mmol/liter K₂HPO₄, 5 mmol/liter glucose, 1 mmol/liter MgCl₂, and 1.3 mmol/liter calcium gluconate) for 1 h at room temperature. Fluorescence was detected in cells perfused with Ringer's solution at 37 °C using an inverted microscope (Axiovert S100, Zeiss) and a high speed polychromator system (VisiChrome, Puchheim, Germany). Fura-2 was excited at 340/380 nm, and emission was recorded between 470 and 550 nm using a CoolSNAP HQ camera (Visitron Systems). [Ca²⁺]_i was calculated from the 340/380 nm fluorescence ratio after background subtraction. The formula used to calculate [Ca²⁺]_i was as follows; [Ca²⁺]_i = K_d × (R − R_min)/(R_max − R) × (S_f2/S_b2), where R is the observed fluorescence ratio. The values R_max and R_min (maximum and minimum ratios) and the constant S_f2/S_b2 (fluorescence of free and Ca²⁺-bound Fura-2 at 380 nm) were calculated using 1 μm ionomycin (Calbiochem), 5 μm nigericin, 10 μm monensin (Sigma), and 5 mm EGTA to equilibrate intracellular and extracellular Ca²⁺ in intact Fura-2-loaded cells. The dissociation constant for the Fura-2·Ca²⁺ complex was taken as 224 nmol/liter. Experiments, imaging acquisition, and data analysis were controlled with the software packages MetaFluor (Molecular Devices) and Origin (OriginLab Corp.).

Materials and Statistics

All compounds used were the highest grade from Sigma (ionomycin, cyclopiazonic acid, tannic acid, KB-R7943, and CaCCinh-A01) or Merck. Student's t test (for paired or unpaired samples as appropriate) was used for statistical analysis. A p value <0.05 was accepted as significant.

RESULTS

Ca²⁺-dependent Ion Currents and Mineralization by Mouse Primary Osteoblasts Require Ano6 and NCX

In two-hybrid split-ubiquitin screenings of a normalized human universal NubG-x library with human Ano6 as bait, we identified NCX1 as a potential Ano6-interacting protein. Although two-hybrid screening identified a number of potential Ano6 targets, NCX1 appeared most interesting (Table 1). We further analyzed the potential interaction partner NCX1 because Ano6-deficient mice have been shown to suffer from a bone mineralization defect, whereas NCX1 has been reported previously to control bone mineralization (1, 34). Moreover, Ano6 has been shown to form a Ca²⁺ channel (35) or may alternatively control Ca²⁺ transport through NCX1. Further evidence for a possible direct coupling of NCX1 and Ano6 was provided by co-immunoprecipitation of both proteins (see Fig. 2c). We hypothesized that both Ano6 and NCX1 may be required for Ca²⁺ transport by osteoblasts. To test if Ano6 and NCX1 are coexpressed in primary osteoblasts, cells were isolated from WT and Ano6^−/− mice and grown in the presence of β-glycerophosphate and ascorbate phosphate to induce osteogenic differentiation. By RT-PCR, we observed expression of Ano6, Ano8, and Ano10 in WT osteoblasts. Expression of Ano6 and Ano10 was up-regulated during differentiation (see Fig. 2a). No Ano6 mRNA was detected in osteoblasts from Ano6^−/− animals (see Fig. 2a) (1). We found that an increase in [Ca²⁺]_i by the Ca²⁺ ionophore ionomycin activated Cl⁻ currents only in WT (but not Ano6^−/−) osteoblasts (Fig. 1). Apart from Ano6, also Na⁺/Ca²⁺ exchangers (NCX1 and NCX3) were expressed in primary osteoblasts (Fig. 2b). Using Alizarin Red staining, we detected prominent mineralization at days 17 and 20 of mouse primary calvarial cultures (Fig. 2d). Mineralization was delayed after treatment of these osteoblasts with the NCX inhibitor KB-R7943, supporting a role of Na⁺/Ca²⁺ exchange in the mineralization process (36). Mineralization was also significantly inhibited by the Ano6 inhibitor tannic acid (30), demonstrating the role of Ano6 Cl⁻ currents in mineralization.

TABLE 1.

Potential partners for interaction with the Na⁺/Ca²⁺ exchanger NCX1 as identified in a two-hybrid split-ubiquitin analysis

OS, organism name; GN, gene name; PE, protein existence; SV, sequence version.

UniProt ID	Protein
O60613	15-kDa selenoprotein
Q13686	Alkylated DNA repair protein
Q96GG9	DCN1-like protein 1
P06746	DNA polymerase β
Q9Y253	DNA polymerase η
P61978	Heterogeneous nuclear ribonucleoprotein KO
P01903	HLA class II histocompatibility antigen, DR α chain
P01033	Metalloproteinase inhibitor 1
P00912	N- (5′-Phosphoribosyl)anthranilate isomerase
P61758	Prefoldin subunit 3
O75934	Pre-mRNA-splicing factor SPF27
Q8IVG9	Putative humanin peptide
P32418	Sodium/calcium exchanger 1
P48643	T-complex protein 1 subunit ϵ
Q8N6G2	Testis-expressed sequence 26 protein
Q96B02	Ubiquitin-conjugating enzyme E2
Q9Y3C8	Ubiquitin-fold modifier-conjugating enzyme 1
Q8N6I4	UPF0694 transmembrane protein C14orf109
Q6UX73	UPF0764 protein C16orf89
Q9H0V9	VIP36-like protein
Q8WU90	Zinc finger CCCH domain-containing protein 15
P17028	Zinc finger protein 24
P08708	40 S ribosomal protein S17
Q9Y3U8	60 S ribosomal protein L36
P08548	LINE-1 reverse transcriptase homolog
P10451	Osteopontin
Q86U02	Putative uncharacterized protein C14orf165
Q8WTZ3	Zinc finger protein ENSP00000375192
P00846	ATP synthase subunit a
P00156	Cytochrome b (OS = Homo sapiens, GN = MT-CYB, PE = 1, SV = 1)
P00395	Cytochrome c oxidase subunit 1
P02792	Ferritin light chain
P03886	NADH-ubiquinone oxidoreductase chain 1
P03901	NADH-ubiquinone oxidoreductase chain 4L
P0CG47	Polyubiquitin-B (OS = H. sapiens, GN = UBB, PE = 1, SV = 1)
Q9Y6A9	Signal peptidase complex subunit 1

FIGURE 2.

Na⁺/Ca²⁺ exchange and Cl⁻ transport through Ano6 are required for mineralization by mouse primary osteoblasts. a, summary of RT-PCR analysis indicates up-regulation of Ano6 in differentiated WT osteoblasts and lack of expression of Ano6 in osteoblasts from Ano6^−/− animals. KO, knock-out. b, RT-PCR analysis indicating expression of NCX1 and NCX3 in WT osteoblasts. RT, reverse transcriptase. c, co-immunoprecipitation of Ano6 with NCX1 (upper panel) and NCX1 with Ano6 (lower panel). 20 μg of lysate was loaded as an input control. No protein was pulled down when immunoprecipitation (IP) was done without antibody (beads only; n = 6 experiments). d, in vitro mineralization assays using Alizarin Red staining. Differentiated (diff.) versus non-differentiated (non-diff.) refers to the presence and absence, respectively, of β-glycerophosphate and ascorbate phosphate. Only differentiated osteoblasts demonstrated matrix mineralization. Mineralization was inhibited by KB-R7943 (KBR; 5 μm), indicating the contribution of Na⁺/Ca²⁺ exchanger, and by the Ano6 blocker tannic acid (TA; 5 μm). All assays were performed in triplicates. e, optical density in shown arbitrary units (au). Values represent mean ± S.E. #, significantly different from non-differentiated cells (a) and differentiated cells (e) (analysis of variance).

FIGURE 1.

Missing Ca²⁺-activated whole cell currents in primary osteoblasts from Ano6^−/− mice. a, whole cell ion currents in WT osteoblasts (Ano6^+/+) measured under control conditions and after stimulation with the Ca²⁺ ionophore ionomycin (Iono; 1 μm). The holding voltage was −100 mV. The cells were voltage-clamped by ±100 mV in steps of 20 mV. Experiments were performed in the presence of 145 mm extracellular Cl⁻ (145Cl⁻; left) or after replacement of 140 mm Cl⁻ with impermeable gluconate (5Cl⁻; right). Removal of Cl⁻ ions inhibited whole cell currents, indicating activation of Cl⁻ currents by ionomycin. b, current/voltage relationships before (control (con)) and after stimulation with ionomycin in the presence of high (145 mm; left) or low (5 mm; right) extracellular Cl⁻. V_c, voltage clamp. c, whole cell currents were not enhanced by ionomycin in knock-out osteoblasts (Ano6^−/−). d, corresponding current/voltage relationships before and after stimulation with ionomycin. Values represent mean ± S.E. *, significant increase by ionomycin (paired t test). The number of cells is indicated in parentheses.

Ano6 Drives the Forward Mode of the Na⁺/Ca²⁺ Exchanger

We further examined the functional relationship between Ano6 and NCX1 in HEK293 cells (14). An increase in [Ca²⁺]_i by ionomycin activated whole cell Cl⁻ currents in HEK293 cells overexpressing Ano6 (Fig. 3a) (22). We observed a small negative (Na⁺ inward) current that was inhibited when extracellular Na⁺ was replaced with impermeable N-methyl-d-glucamine (NMDG⁺).² We suggested that the NMDG⁺-sensitive inward current is due to electrogenic transport by the Na⁺/Ca²⁺ exchanger NCX1 (Fig. 3, a and b). Endogenous expression of NCX1 in HEK293 cells was effectively knocked down by siRNA (Fig. 3, c and d). Inhibition of NCX1 expression attenuated NMDG⁺-sensitive Na⁺ inward currents and augmented Cl⁻ outward currents. Thus, ionomycin-induced increase in [Ca²⁺]_i appears to be larger in the absence of Na⁺ or after reduced expression of NCX1. Measurements of intracellular Ca²⁺ demonstrated that inhibition of NCX1 by NMDG⁺ augmented [Ca²⁺]_i (Fig. 3h, upper panel). In the presence of extracellular NMDG⁺, NCX1 operated in the reverse mode, thus transporting Ca²⁺ into the cell. Blocking NCX1 with KB-R7943 inhibited Ca²⁺ outward transport; therefore, the Ca²⁺ increase was less pronounced. Notably, NMDG⁺ had no effects on [Ca²⁺]_i in the presence of the NCX1 blocker KB-R7943 (Fig. 3h, lower panel).

FIGURE 3.

Ano6 activates the Na⁺/Ca²⁺ exchanger in HEK293 cells. a, current/voltage relationships from whole cell patch clamp experiments in Ano6-expressing cells. Ionomycin (Iono; 1 μm)-activated currents were enhanced after knockdown of NCX1. I/V curves are shown after replacement of extracellular Na⁺ with NMDG⁺ (blue triangles). con, control. b, NMDG⁺-sensitive inward currents (ΔI_NMDG at voltage clamp (V_c) of −100 mV) were reduced after knockdown of NCX1. siNCX1, NCX1 siRNA. c, RT-PCR analysis shows dominant expression of NCX1 in HEK293 cells. RT, reverse transcriptase. d, Western blot of NCX1 in cells transfected with empty plasmid (mock) or NCX1 or treated with scrambled RNA (scrbld) or siRNAs for NCX1. e, recordings with high (1 mm) Ca²⁺ in the patch pipette. Replacement of extracellular Na⁺ with NMDG⁺ blocked inward currents. f, NMDG⁺-sensitive inward currents were detected only after coexpression of Ano6 and activation by high Ca²⁺. Values represent mean ± S.E. #, significant effect of NMDG⁺ (unpaired t test). g, coexpression of NCX1 augments inward currents and reduces outward currents. h, upper panel, summary trace of cytosolic Ca²⁺ concentration that was enhanced by removal of extracellular Na⁺ (NMDG⁺) or inhibition of NCX1 by 10 μm KB-R7943 (KBR). Lower panel, summary of the NMDG⁺-induced increase in intracellular Ca²⁺ in the absence or presence of KB-R7943. Values represent mean ± S.E. The number of cells is indicated in parentheses. *, significant effects of NMDG⁺ or KB-R7943 (paired t test); #, significant difference compared with scrambled RNA or low Ca²⁺ (0.1 μm; unpaired t test). Blots were performed in triplicates.

Ion transport was also activated in NCX1/Ano6-expressing cells with high Ca²⁺ concentrations (1 mmol/liter) in the patch pipette. Notably, NMDG⁺-inhibitable Na⁺ currents (NCX1) were detected only in the presence of Ano6 (Fig. 3, e and f). Finally, when Ano6 and NCX1 were coexpressed, ionomycin-activated inward currents were larger, whereas outward currents were smaller compared with cells expressing only Ano6 (Fig. 3g). In contrast, inward currents decreased and outward currents increased after inhibition of NCX1 by KB-R7943 (Fig. 4). Both inward and outward currents were abolished by further removal of extracellular Cl⁻ (5Cl⁻). These results indicate that Cl⁻ transport through Ano6 is essential for NCX1 to operate in the forward mode, i.e. to transport Ca²⁺ out of the cell.

FIGURE 4.

Parallel transport by Ano6 and NCX1. a, schematic illustrating intracellular Ca²⁺ levels under various conditions. b, whole cell overlay currents activated by ionomycin (Iono) in HEK293 cells coexpressing Ano6 and NCX1 (holding voltage of −100 mV, steps of 20 mV, ±100 mV). Currents are shown under control conditions, in the presence of 10 μm KB-R7943 (KBR), and after replacement of extracellular Cl⁻ with gluconate (5Cl⁻). c, summary of current/voltage relationships for the conditions as shown above. Values represent mean ± S.E. *, significant increase by ionomycin (paired t test). The number of cells is indicated in parentheses. V_c, voltage clamp.

Ano6 Supports Outward Transport of Ca²⁺ through the Na⁺/Ca²⁺ Exchanger

We further demonstrated regulation of NCX1 by Ano6 in [Ca²⁺]_i measurements using Fura-2. Endoplasmic reticulum Ca²⁺ stores were emptied by cyclopiazonic acid in Ca²⁺-free bath solution (Fig. 5a, upper right scheme). Re-addition of extracellular Ca²⁺ increased [Ca²⁺]_i only transiently through store-operated Ca²⁺ influx because Ca²⁺ was transported out of the cell by NCX1 [Ca²⁺]_i. NCX1 was inhibited by KB-R7943, Na⁺ replacement with NMDG⁺, or inhibition of Ano6 currents by removal of extracellular Cl⁻ (5Cl⁻) (Fig. 5, a (lower right scheme), b, and c). Moreover, siRNA knockdown of Ano6 also inhibited outward transport of Ca²⁺ (Fig. 5, d and e). The results demonstrate the essential role of NCX1/Ano6 in controlling [Ca²⁺]_i in mouse osteoblasts and HEK293 cells.

FIGURE 5.

Ano6 supports outward transport of Ca²⁺ through the Na⁺/Ca²⁺ exchanger. a, time course (mean ± S.E.) of [Ca²⁺]_i in HEK293 cells. Shown are the emptying of Ca²⁺ stores with 10 μm cyclopiazonic acid (CPA) in Ca²⁺-free bath solution, the re-increase in [Ca²⁺]_i by Ca²⁺ influx in Ca²⁺-containing buffer, and the subsequent decrease by the Na⁺/Ca²⁺ exchanger NCX1 (dashed line). Schemes show transport activities and action of drugs. ER, endoplasmic reticulum. b and c, decrease in [Ca²⁺]_i in the absence or presence of NCX inhibitors (KB-R7943 (KBR), 10 μm), replacement of extracellular Na⁺ with NMDG⁺, and replacement of extracellular Cl⁻ with gluconate (5Cl⁻). d and e, decrease in [Ca²⁺]_i in cells treated with scrambled RNA (scrbld) or Ano6 siRNA (siAno6). Values represent mean ± S.E. #, significant difference compared with the control (con) or scrambled RNA (unpaired t test). The number of cells is indicated in parentheses.

Ano6 Supports Ca²⁺ Transport and Mineralization in Primary Human Osteoblasts

Mouse primary osteoblasts up-regulated Ano6 upon differentiation by β-glycerophosphate and ascorbate phosphate (Fig. 2a). Similar results were found in human primary osteoblasts. Human primary osteoblasts expressed predominately Ano6 and Ano10, with Ano6 being strongly up-regulated during differentiation (Fig. 6, a–c). An increase in intracellular Ca²⁺ by ionomycin activated whole cell currents, which were larger in differentiated cells. Similar, CaCCinh-A01-sensitive Cl⁻ currents were enhanced in differentiated cells (Fig. 6, d–f). These results suggest that similar to recombinant NCX1, also in mouse and human osteoblasts, NCX1 might be regulated by Ano6. We performed a mineralization assay in differentiated primary human osteoblasts, which demonstrated extracellular calcium phosphate deposition (Fig. 7a). Extracellular precipitation made patch clamp experiments often difficult because it limited the access of the patch pipette to the cell membrane. Similar to mouse osteoblasts, also in human osteoblasts, mineralization was strongly inhibited by inhibition of NCX1 with KB-R7943 or blocking of Ano6 with tannic acid (Fig. 7). The fact that blocking NCX1 reduced mineralization less than inhibition of Ano6 suggests additional Ca²⁺ export mechanisms unrelated to NCX1. Nevertheless, the data indicate that functional coupling of Ano6 and NCX1 is essential for bone mineralization, which explains the skeletal defect observed in Ano6 null mice (Fig. 8).

FIGURE 6.

Ca²⁺-activated Ano6 Cl⁻ currents in primary human osteoblasts. a, RT-PCR analysis indicates predominant expression of Ano6 and Ano10, as well as the Na⁺/Ca²⁺ exchanger NCX1, in human primary osteoblasts. b and c, Western blots indicating up-regulation of Ano6 in differentiated osteoblasts (day 20 after treatment with glycerophosphate and ascorbate phosphate). d, whole cell overlay currents from non-differentiated (non-diff.) and differentiated (diff.) osteoblasts (holding voltage of −100 mV, steps of 20 mV, ±100 mV). e, current/voltage relationships obtained in non-differentiated (upper panels) and differentiated (lower panels) osteoblasts. Cells were stimulated with ionomycin (Iono; 1 μm) in the absence (left panels) or presence (right panels) of the anoctamin inhibitor CaCCinh-A01 (AO1; 10 μm). Inhibition of ionomycin-activated currents by CaCCinh-A01 was more pronounced in differentiated cells. con, control; V_c, voltage clamp. f, density of CaCCinh-A01- and tannic acid (TA)-sensitive currents in non-differentiated and differentiated osteoblasts. Values represent mean ± S.E. #, significant difference compared with non-differentiated cells (unpaired t test); *, significant increase by ionomycin (paired t test). The number of cells is indicated in parentheses. pF, picofarads.

FIGURE 7.

Na⁺/Ca²⁺ exchange and Cl⁻ transport through Ano6 are required for mineralization by primary human osteoblasts. a, visualization of mineralization by primary human osteoblasts in vitro at day 20 of differentiated (diff.) cell culture using Alizarin Red staining. No mineralization was observed in non-differentiated (non-diff.) cells. Both tannic acid (TA; 10 μm; inhibition of Ano6) and KB-R7943 (KB; 10 μm; inhibition of Na⁺/Ca²⁺ exchanger) inhibited mineralization. Scale bar = 20 μm. b, summary of Alizarin Red assays indicating significant increase in mineralization in differentiated cells inhibited by TA and KB-R7943. The optical density in shown in arbitrary units (au). Values represent mean ± S.E. #, significant difference (unpaired t test). The number of assays are indicated in parentheses.

FIGURE 8.

Role of Ano6 and NCX1 in bone mineralization. Shown is a schematic model of the roles of Ano6 and NCX1 in bone mineralization. Ca²⁺ increases in osteoblasts activate outwardly rectifying Ano6 Cl⁻ currents and the outward transport of Ca²⁺ by the Na⁺/Ca²⁺ exchanger NCX1. The inward transport of Cl⁻ compensates the cytosolic positive transport potential by the electrogenic Na⁺/Ca²⁺ exchanger, thereby maintaining Ca²⁺ outward transport at a high rate. NCX1 may directly interact with Ano6 (yellow arrow). Scrambling of phosphatidylserine (PS; green) by Ano6 supports the mineralization process.

DISCUSSION

Our results indicate that decreased mineral deposition in the skeletons of Ano6 null mice (1) is at least in part due to defective regulation of plasma membrane Na⁺/Ca²⁺ exchange. Cl⁻ transport by Ano6 drives Ca²⁺ transport by NCX1, as indicated by inhibitors of Ano6 (CaCCinh-A01 and tannic acid), or removal of extracellular Cl⁻. All inhibited deposition of hydroxyapatite by osteoblasts. We propose a model in which the Cl⁻ channel function of Ano6 serves to activate NCX1 and possibly NCX3, thereby increasing extracellular Ca²⁺ deposition, which is required for formation of hydroxyapatite in the extracellular matrix. As NCX1- or NCX2-deficient mice have not yet been reported, it is difficult to estimate if other Ca²⁺ transporters are also regulated by Ano6. Likewise, other anion channels may be linked to Ca²⁺ secretion. It is noteworthy that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel is expressed in osteoblasts and in ameloblasts/odontoblasts (37). Cystic fibrosis patients carrying the common F508del mutation in the CFTR gene and mice carrying an inactivated CFTR allele suffer from bone mineralization defects leading to osteoporosis and osteopenia (37–40). The mechanisms of how CFTR operates in osteoblasts are only poorly understood. Nevertheless, the reduced bone mineral density underlines the importance of Cl⁻ transport in osteoblasts for proper bone formation.

Ano6-dependent Na⁺/Ca²⁺ transport may also be related to the nonselective cation conductance that has been reported for Ano6 (35). We propose that instead of directly transporting Ca²⁺ ions, Ano6 may support the activity of the Na⁺/Ca²⁺ exchanger.

The function of Ano6 in osteoblasts is reminiscent of the function of Ano1 in proximal tubular epithelial cells of the kidney (41). In the proximal tubule, protons are secreted by the vacuolar proton pump (V-ATPase). Translocation of H⁺ to the tubular lumen by the Ca²⁺-activated V-ATPase is an electrogenic process, and accumulation of charge may limit the transport activity of the pump. Parallel activation of Ano1 leads to Cl⁻ efflux that counterbalances charge movement, thereby facilitating H⁺ secretion (41).

The mineralization defect in Ano6-deficient mice has previously been discussed as being linked to the scramblase function of Ano6, which is significantly impaired in osteoblasts (1). Here, we have shown that Ano6 facilitates matrix mineralization by supporting the function of the Na⁺/Ca²⁺ exchanger (Fig. 8). However, the present data do not elucidate whether Na⁺/Ca²⁺ exchangers operate to directly transport Ca²⁺ into the extracellular collagen matrix, where it gradually converts to crystalline hydroxyapatite (5), or whether Na⁺/Ca²⁺ exchange takes place in budding vesicles or in intracellular calcium phosphate-containing vesicles (8–10). In this respect, it is of interest that exposure of phosphatidylserine has been proposed to be required for the formation of matrix vesicles and/or the localization of these vesicles to the collagen network (2). Ano6 might thus couple two essential functions: Ca²⁺ secretion and formation of matrix vesicles (Fig. 8).

Note Added in Proof

Figs. 2c and 6a were not formatted correctly in the version of this article that was published on-line on January 14, 2015, as a Paper in Press. These figures have been revised to conform with JBC policies.