Ion Channels and Bone Homeostasis Imbalance

To sustain the bone weight, there is a delicate balance between the osteoblast bone formation and osteoclast bone resorption known as bone homeostasis. The imbalance of bone homeostasis will induce bone disorder, such as osteoporosis and osteopetrosis. Numerous studies have shown that ion channels play critical roles in maintaining the bone homeostasis. Dysfunction of ion channels accompanied with disorder of the osteoblast bone formation and osteoclast bone resorption. In the present paper, the authors discussed the physiological roles of bone homeostasis on human body and summarized the ion channels expressing on the membrane of osteoblasts and osteoclasts. Finally, the limitations of the prior art and the prospects in this field were discussed.


Introduction
The human bone environment undergoes remodeling through bone formation and bone resorption throughout life to maintain bone homeostasis [1], which means bone resorption of osteoclasts is always accompanied with bone formation of osteoblasts [2]. The tight balance between bone formation and bone resorption regulates bone development and bone homeostasis. When the balance between bone formation and bone resorption is disrupted, it may lead to pathological conditions such as osteoporosis and osteopetrosis, whose characteristic may respectively be low bone mass and high bone mass, resulting in increased bone fragility and increased risk of fracture. As transmembrane proteins on cell membrane, ion channels are the basic excitatory units on the tissue cell membrane and participate in the process of transmitting electrical signals, which can be regarded as an excitable protein molecule reacts to specific stimuli. Depending on the permeability of the ions, the ion channels can be classified according to specific ions.

Sodium Ion Channels
Sodium ions widely exist in extracellular fluids and are the most abundant cations in human body, which are most closely related to liquid osmotic pressure. The voltage-gated sodium ion channel is a highly glycosylated complex consisting of an alpha subunit and two beta subunits. When the membrane potential is maintained at the resting membrane potential, the channel is closed, when the membrane depolarization potential is above the threshold, channel opened. The voltage-gated sodium ion channel is opened by depolarization, which allows sodium ions to flow in, causing membrane depolarization and inducing action potential [3]. It has been reported that there are two sodium channels in osteoblasts, namely voltage-sensitive sodium channels (Nav) and epithelial sodium channels (ENaC). In 1994, Killick R first discovered the ENaC channel expresses on cartilage and osteoblasts, plays a critical role in perceptual acidosis, maintenance of sodium homeostasis and transduction of mechanical stimulation. As a novel non-voltagedependent sodium channel, ENaC is a member of the ENaC/ degenerin family and transports sodium ions to critical pathways in epithelial cells, vascular endothelial cells and other tissues, which can be inhibited by amiloride. At the molecular level, ENaC consists of three homologous subunits, α, β, and γ [4]. Wherein each subunit consists of two transmembrane helices and one extracellular loop, and has a similar secondary structure, through a large extracellular domain, which linked to the two membrane spanning domains (TM1 and TM2), and short intracellular N-and C-terminal composition [5,6]. The crystal structure and site-directed mutagenesis of ENaC indicate that ENaC has a central ion channel located on the central axis of symmetry of the three subunits [7].
In many excitable cells, the Na+ channel is responsible for the depolarization phase of the action potential. It is now known to identify rapidly activated Na + conductance in chicken osteoclasts [8]. This inward current is rapidly activated at membrane potential, more positive than -30 mV (peak amplitude is 1-2ms) and then rapidly inactivated (within 5-7ms), the kinetics of ENaC is faster than other voltage-gated channels in osteoclasts. The Na + current can be inhibited by nanomolar concentrations of tetrodotoxin, a blocker of certain voltage-gated Na + channels. In chicken osteoclasts, voltage-activated Na + conductance at the whole cell level is relatively small compared to conductance of other channels.
This low density channel and its transient activation do not allow the generation of action potentials. In other cells, Na + conductance plays a vital role in secretion [9] and may play a role in the regulation of cell proliferation [10]. It is conceivable that larger Na + conductance plays a role in the proliferation of pre-osteoclasts and that only residues remaining in differentiated chicken osteoclasts.
The researchers also found that the ENaC-α were expressed in rat osteoblast-like cell line (UMR-106) and primary human osteoblasts [11] and after the ENaC gene was silenced on rat osteoblasts by siRNA technique, they found that osteogenic gene-related genes' expressions decreased (Alp, Col1); after the osteogenic estradiol added to the primary cultured rat osteoblasts and mouse osteoblasts, the expression of ENaC mRNA was increased, which proved that ENaC in osteoblasts participated in the bone formation [4]. Some scholars have also studied the effects of ferulic acid acting on rat skull proliferation and differentiation by CCK-8 and alkaline phosphatase (ALP) staining, Cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKGII) expression was silenced by small interfering RNA (siRNA) and then mRNA expression was detected by semi-quantitative PCR. The researchers found that ferulic acid promotes proliferation, differentiation and mineralization in rat calvarial osteoblasts in vitro, via cGMP-PKGII-ENaC signaling pathway, which also enhanced expressions of osteogenic genes [12]. JL G et al. [13] also confirmed in 2017 that epithelial sodium channels can promote osteogenic differentiation through the cGmp/PKGII/ENaC signaling pathway.
In 2016, Hu Songyan found that an inhibitor of ENaC, amiloride, which reduced the number of TRAP-positive osteoclasts and inhibited the formation of osteoclasts by staining with tartrateresistant acid phosphatase (TRAP) and bone resorption, founding the expression of the osteoclast-specific gene CK reduced, thereby demonstrating the expression on ENaC osteoclasts at the cellular level and regulating osteoclast differentiation and bone resorption, further indicating that ENaC may be involved in osteoclast function adjust [14]. Sandra J et al. [15] using immunocytochemistry and RNA sequencing found that voltage-gated sodium channel NaV channel was expressed in primary osteoblasts infant mouse skull.
The carbamazepine and phenytoin, which are the NaV channel blockers, sensitive sodium currents were recorded by whole cell patch clamp recording.

Chloride Ion Channels
As the most important anions in human body, chloride ion plays an important role in the electrochemical balance between the intracellular and extracellular side. It also participates in regulating intracellular and extracellular functions through the active transport of chloride channel, which can regulate the physiological process through the change of its channel current, such as liquid secretion, cell volume regulation, transmembrane transport, excitatory conduction and intracellular acidification. It is reported that a variety of chloride channels exist in the cell membrane or organelles. Abnormal chloride channels can cause a variety of physiological diseases, such as cystic fibrosis, congenital myotonia, epilepsy, Barter syndrome, etc [16]. Chloride channels associated with bone metabolism have been reported to include chloride Lysosomal function changes in mice lacking ClC-7, which leads to severe lysosomal storage [21]. Mice lacking Clcn7 gene presents severe osteopetrosis due to the inability of osteoclasts to secrete acid, retinal degeneration and death within 7 weeks. Clcn mutation was identified in human, and homozygous mutations resulted in osteosclerosis in malignant infants [22]. Clcn7 -/mice show lysosomal storage diseases, however, their osteosclerosis is mild and they lack the coat color phenotype, only certain effects of ClC-7 mediated Cl -/H + exchange and it can be taken over by Cl-conductance. The data show that ClC-7 may mediate Cl -/H + exchange during lysosomal acidification [24].  [25]. And Le Heron L et al. [26] found that common bone deficiency disease in patients with cystic fibrosis, Cftr mRNA and protein were expressed in primary human osteoblasts.

ATP-Gated Channel
ATP is a key energy currency and a ubiquitous extracellular messenger, and it also plays an important role in bone tissue.
Based on its dose and the participating purinergic receptor (P2R) subtypes, ATP can trigger many different cellular responses, including cell proliferation, differentiation, and apoptosis [27]. In the biological activities of bone tissue, ATP can be involved in bone growth, development and repair [28]. Depending on the mode of receptor transduction, P2 receptors can be divided into two families, P2X and P2Y. Among them, P2X is a family of ion receptors, which is a ligand-gated ion channel, while P2Y is a family of metabotropic receptors, belonging to G protein-coupled receptors.
P2X receptor is also expressed in human and mediates a variety of functions, including muscle contraction, neuronal excitation and bone formation, with subunits of intracellular N-and C-termini, two transmembrane domains and an extracellular ligand bind to the loop, but has no crystal structure. The effect of P2X on osteoblast is mainly to regulate the release of cytokines. P2X also regulates the start of osteoblast apoptosis on osteoclast [29]. It is reported that seven different P2 receptor subtypes were expressed in osteoblasts, including P2X2, P2X5, P2X7, P2Y1, P2Y2, P2Y4 and P2Y6. In the same time, Gallagher and Buckley reported that P2Y2 was strongly expressed in human osteoblast, while P2Y1 receptors were more predominant in rat osteoblast [30]. acid] transiently attenuates bone cancer-induced pain in mice but has no effect in the later stages of the malignant process [32]. It has also been found that the P2X3 receptor is upregulated in the dorsal root ganglion function in a rat model of bone cancer [33]. It has also been reported that cancer-related bone pain is associated with osteoclast activation and Ca 2+ imbalance [34,35].

Transient Receptor Potential Channel, TRP
It is well known that both the external Ca 2+ and intracellular Ca 2+ signaling are critical to bone homeostasis.
First, the normal function of bone depends on normal serum calcium levels, while bone also plays an important role in maintaining systemic calcium homeostasis. In fact, 99% of calcium in body is stored in bone, which contributes to its mechanical structural properties. Therefore, bone needs enough calcium to maintain bone integrity. What's more, intracellular Ca 2+ is also an important second messenger in bone. Intracellular Ca 2+ signaling in osteoblasts, osteoclasts, chondrocytes, and nerve endings has been shown to regulate many functions, including differentiation, signal transduction and mechanical transport, permeation and perception of painful stimuli. Therefore, fine-tuning of intracellular Ca 2+ levels is critical for normal bone homeostasis, at the same time, transporters abnormalities, which are also involved in Ca 2+ signaling, will clearly lead to diseases that also affect bone structure or function [36]. Maintenance of extracellular and intracellular Ca 2+ homeostasis is important for bone biology and is largely dependent on Ca 2+ channels. There are several types of Ca 2+ channels, including a) The ryanodine receptor (RyR) and the inositol-1,4,5triphosphate receptor (IP3R), mediates the release of Ca 2+ from the endoplasmic reticulum (ER).  [42]. It is also found that the cortical bone mass of TRPV5-/-mice reduced, which was associated with an increase in the number of osteoclasts, although bone resorption parameters are reduced [43]. These results convincingly demonstrate that TRPV5 is important for systemic calcium homeostasis by fine-tuning renal calcium reabsorption. Skeletal defects in  thinking, these problems will eventually be solved.