Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

SLC20

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101880

Synonyms

Historical Background

All living beings need inorganic phosphate for many essential functions, including metabolism (high-energy bonds, signal transduction, pH control, etc.) and molecular components (membrane phospholipids, nucleotides, minerals, etc.). Because Pi is a polyprotic acid, it is ionized at life-compatible pHs with one or two negative charges, a characteristic that impairs the free entrance to the cell through lipid membranes. Therefore, all cells and living beings need Pi transporters in their plasma membranes, requiring energy to overcome the transmembrane potential (negative inside). The universal need of Pi explains the presence of the solute carrier family 20 (Slc20) of phosphate in all organisms, from bacteria and plants to fungi and animals, using either proton or sodium gradients as driving forces. A detailed evolutionary description of the Slc20 family can be found elsewhere (Forster et al. 2011).

In metazoans, two members of the Slc20 family are also present: Slc20A1 or PiT1 (Kavanaugh et al. 1994) and Slc20A2 or PiT2 (Olah et al. 1994). They are also retroviral receptors: the Slc20A1 functions as the gibbon ape leukemia virus receptor (Glvr-1), and Slc20A2 as the rat amphotropic leukemia virus receptor (Ram-1). In mammals, both transporters are encoded by different genes (SLC20A1 and SLC20A2 in man), and in contrast to the more specific epithelial Pi transporters belonging to the Slc34 family (see chapter by Forster in this Encyclopedia), they are ubiquitously expressed in tissues (Forster et al. 2013). In spite of this generalized expression, both the presence of the two Slc20 transporter paralogs in the same cells, the differential regulation by hormonal and nonhormonal factors, and the different involvement in pathophysiological conditions, as shall be shown in this chapter, question the role of these carriers as only housekeeping transporters.

Research on the Slc20 transporters has been modest in comparison to the Slc34 members, but the accumulated information is sufficient to reveal important roles in physiology and pathophysiology. Unfortunately, two commonly used but misleading experimental strategies in vitro have also helped to the slow progress in this knowledge. First, phosphonoformic acid (PFA, foscarnet) has been systematically used to inhibit Slc20-mediated Pi transport and to demonstrate their involvement of these transporters in cell signaling, Pi-mediated calcification, etc. However, both PiT1 and PiT2 are resistant to PFA (Ravera et al. 2007; Villa-Bellosta and Sorribas 2009), and PFA rather prevents calcification by acting as an analog of pyrophosphate. Therefore, the involvement of the Slc20 proteins in bone formation, ectopic calcification, and in some signaling routes remains unclear. Second, the use of high concentrations of Pi in bicarbonate-based culture medium to study physiological or ectopic calcifications causes alkalinity-mediated supersaturation and homogeneous precipitation of calcium phosphates in culture medium from the first minutes of incubation, rather than the heterogeneous/cell-mediated calcium deposition and calcification that occurs in vivo (Hortells et al. 2015). Therefore, the conclusions raised in the corresponding studies using these approaches have not been considered in this chapter and, for the sake of brevity, will not be further discussed.

Biochemical and Molecular Characteristics

There is no 3D model available for the Slc20 transporters, only 2D models have been developed using in silico hydropathic analysis and experimental support (Fig. 1). Also, the high identity of the two paralogs (62% amino acids in human) has helped to the establishment of a single 2D model. Nevertheless, it is still not definitive whether these transporters have 10, 11, or 12 transmembrane domains (TMD), as shown by the different proposals of the UniProt database (http://www.uniprot.org) about the human PiT1 (accession number Q8WUM9) and PiT2 (Q08357), or the transporter Classification Database (http://www.tcdb.org; 2.A.20.2.7 for PiT1 and 2.A.20.2.3 for PiT2). In the case of PiT1, the conclusion after substituted cysteine accessibility mutagenesis and treatment with sulphydryl reagents pointed to the presence of 12 TMD (Farrell et al. 2009). Similarly, both the hemagglutinin/c-myc epitope tagging of the N- and C-termini, and the demonstration of a glycosylation site at asparagine 81 strongly support the 12-TMD model of PiT2, with both termini located extracellularly (Salaün et al. 2001). In addition, both paralogs contain two repeated ProDrom (http://prodom.prabi.fr) domains, named PD001131, near the amino- and carboxy-termini (Fig. 1). This domain is common to all PiT members, with a smallest alignment length of 38 amino acids. PiT1 and PiT2 have also a large intracellular domain between TMD 7 and 8, encompassing amino acids 251–510 in the case of PiT1, and 235–482 in PiT2. This large loop is not necessary for Pi transport function (Bøttger and Pedersen 2011), but it contains several potential phosphorylation sites. Nevertheless, when the region encompassing the TMD domains 6 and 7 plus the large intracellular loop of the human PiT2 are deleted, a dramatic decrease of Pi transport activity is observed. Similarly, the external link between TMD 2 and 3 of Xenopus laevis PiT1 is also critical for Pi transport affinity, cation selectivity, and substrate selectivity (Ravera et al. 2013).
SLC20, Fig. 1

2D consensus model of the Slc20 family of Pi transporters. Twelve transmembrane domains (TMD) are shown, with both amino- and carboxy-termini located extracellularly, and an N-glycosylation site between TMD 2 and 3. The extracellular domain between TMD 10 and 11 is very short, and it can mask the presence of two TMDs. The two domains PD001131 are shown in transparent green areas. Blue linkers and blue TMD represent regions found necessary for Pi transport. Two sodium ions are cotransported with every monovalent Pi

The Slc20 family has some biochemical characteristics that are exclusive to these transporters. In contrast to the other Pi transport family (Slc34, which preferentially transports divalent Pi, HPO4) PiT1 and PiT2 transport monovalent phosphate, H2PO4 (Ravera et al. 2007), in spite of the fact that in the blood and most extracellular fluids, only 20% of Pi is monovalent, and the 80% is divalent. However, this is not an inconvenience because Pi concentration in plasma is 0.9–1.4 mM, and therefore 0.18–0.28 mM is H2PO4, which is a sufficient concentration for transmembrane transport, given the apparent Pi affinity , of about 0.1 mM Pi at pH 7.4 for both PiT1 and PiT2 (Villa-Bellosta et al. 2007). The preferential transport of monovalent Pi is a metabolic advantage for the cell because the energy expense of the Slc20 is lower than that of Slc34a1 (NaPiIIa) and Slc34a2 (NaPiIIb). The reason is that both PiT1 and PiT2 are electrogenic using a low 2:1 stoichiometry for Na: Pi (Ravera et al. 2007), whereas the electrogenic NaPiIIa and NaPiIIb function with a stoichiometry 3:1 for Na:Pi at the expense of higher Na+/K+-ATPase activity because they carry divalent Pi (Forster et al. 2013). The physiological reason for this elevated expense of energy of NaPiIIa and NaPiIIb is, most likely, that these transporters are not responsible for supplying Pi to the cells but of the control of Pi homeostasis in the organism by regulating the epithelial transport rate of, mainly, the intestine (Pi absorption) and kidney (reabsorption or urine waste of Pi).

The preference for monovalent Pi explains the fact that Pi transport function of PiT1 and PiT2 is maximal at pH 6.0, and it decreases uniformly till pH 8.5 (Ravera et al. 2007). This is not caused by a reduced affinity for substrate specificity, but by a decrease of H2PO4 concentration.

Another hallmark of the Slc20 members that differentiate from Slc34 is the resistance to PFA. This is a competitive inhibitor of Slc34 with a high inhibition constant (Ki; 0.3–0.6 mM) compared to the K0.5Pi of Pi (about 0.1 mM Pi; Forster et al. 2013). PiT1 and PiT2, however, are not inhibited with 1 mM PFA (Ravera et al. 2007), and partial inhibition of Slc20 transport was only observed at very high PFA concentrations, with indirectly determined Ki values in the 2.7–4.6 mM range (Villa-Bellosta et al. 2007). Nevertheless, this inhibition is, most likely, the consequence of a noncompetitive, allosteric mechanism.

Cell Biology

The ubiquitous expression of PiT1 and PiT2 led to the conclusion that the Slc20 transporters were expressed in the cell membrane with the only function of supplying Pi to the cells. However, not only there is strong evidence of additional important functions for these transporters (see below), but the subcellular distribution is also more complex. In the plasma membrane, PiT2 is physically associated with the ß-actin cytoskeleton/stress fibers in CHO cells, with a distribution that depends on the concentration of Pi in culture medium (Rodrigues and Heard 1999). The nature and relevance of this association is still unknown, as it is the finding that PiT1 and PiT2 are also expressed intracellularly, in the endoplasmic reticulum of vascular smooth muscle cells (VSMC) (Villa-Bellosta et al. 2009a). A summary of this expression characteristic is depicted in Fig. 2.
SLC20, Fig. 2

Cartoon of Slc20 family of Pi transporters in a nonepithelial cell. Both PiT1 and PiT2 (purple icons) are either located in the plasma membrane or the endoplasmic reticulum (green). Both carriers cotransport 2 sodium ions per monovalent Pi, which is 20% of total Pi at pH 7.4. During Pi depletion, PiT2, forms homo-oligomers (yellow star) to increase Pi transport, and it is related to ß-actin (blue straight lines). Signaling (red rays) to the nucleus for Pi transport regulation or proliferation could be initiated by the Slc20 transporters in the plasma membrane, in the endoplasmic reticulum or by the cytosolic concentration of Pi. Exit from the cell takes place through a sodium-independent Pi carrier

More recently, novel functions have been suggested for PiT1, in relation to cell proliferation and apoptosis. Depletion of PiT1 in HeLa and HepG2 cells and in tumor cells in vivo decreases cell proliferation (Beck et al. 2009). This effect is independent of the Pi transport function and exclusive of PiT1 because it is not observed during PiT2 depletion. HeLa cells depleted of PiT1 are also more sensitive to the proapoptotic activity of tumor necrosis factor α, and again, this effect is independent of Pi transport function (Salaün et al. 2010). The relevance of these effects on physiology and pathology still remains unknown.

Little is known about the signaling mediated by PiT1 in the transport-independent function (cell proliferation, apoptosis, attachment, etc.), but the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), increased expression of the survival-related kinase Akt-1, and phosphorylation of p38 mitogen-activated protein (MAP) kinase upon PiT1 depletion have been described in several culture models.

Physiological Roles and the Regulation

Whereas the ubiquitous expression of the Slc20 Pi transporters in the plasma membrane strongly suggests that they have the unique function of providing Pi to the cells, the fact that the two Slc20 members are simultaneously present in many cells suggests that additional functions are entrusted to these transporters, apart from the Pi uptake. Several studies have demonstrated the regulation of the Slc20 transporters by the concentration of Pi, insulin, insulin-like growth factor-I (IGF-1), platelet-derived growth factor, 1,25-dihydroxyvitamin D3, etc. A summary of different regulatory mechanisms described to date is shown in Table 1. Upregulation of PiT1 and PiT2 expressions and Pi transport has been described by several groups in several cell lines and tissues, and this is at least mediated by increased mRNA and protein concentrations, but also by conformational changes with increased formation of homooligomers (Fig. 2) whose molecular characteristics and relevance are still unknown (Salaün et al. 2002).
SLC20, Table 1

Summary of regulatory mechanisms of Slc20A1 and Slc20A2

Slc20 target

Mechanism

Tissues and organs tested

Increased P i transport

PiT1, PiT2

Low Pi concentration, dietary Pi deprivation

Small intestine, kidney, fibroblasts, several cell cultures, parathyroid gland

PiT1

Platelet-derived growth factor B homodimer (PDGF-BB)

Aortic smooth muscle cells

PiT1, PiT2

Insulin

Aortic smooth muscle cells

PiT1

Bone morphogenetic protein 2

MC3T3-E1 osteoblast-like cells

PiT1, PiT2

1,25-dihydroxy vitamin D3

Parathyroid hormone, intestine

PiT1

Insulin-like growth factor I (IGF-I)

SaOS-2 osteoblast-like cells

PyMS rat osteoblastic cells

Decreased Pi transport

Slc20 target

Mechanism

Tissues and organs tested

PiT1, PiT2

High Pi concentration and dietary Pi

Small intestine, kidney, several cell cultures

PiT2

Parathyroid hormone

Kidney

Many publications have studied the expression of the Slc20 transporters in a variety of tissues, and a selection discussed below.

Intestine and Kidney

Although it was initially thought that the Pi transport through the apical membrane of enterocytes and proximal tubular epithelial cells was exclusively mediated by the Slc34 transporters, and that the Slc20 was only expressed basolaterally to supply Pi from the blood, it is now known that these transporters are also expressed apically and similarly regulated by Pi deprivation (Giral et al. 2009; Villa-Bellosta et al. 2009b). A precise knowledge of the effect of parathyroid hormone and phosphatonins (FGF23, MEPE, and soluble FRP4) on Slc20 transporters in the kidney and other organs is still unknown (Picard et al. 2010). However, regardless of the regulatory mechanisms, the contribution of the Slc20 transporters to the renal reabsorption should be small.

Bone

PiT1 transcript is expressed in early hypertrophic chondrocytes from developing embryonic murine metatarsals, but no expression was observed in osteoblasts (Palmer et al. 1999). This finding suggested the involvement of PiT1 in matrix calcification, but this important role has not been confirmed. Several studies in vitro using bone-related cell lines have shown the modulating role of endocrine agents on PiT-1 expression and Pi transport. Bone morphogenetic protein 2 increases Pi transport, PiT1 expression, and matrix calcification in MC3T3-E1 osteoblast-like cells, and this is prevented by PiT1 knockdown and inhibition of the c-Jun-N-terminal kinase (JNK) pathway (Suzuki et al. 2006). The relationship between PiT1 and bone formation has not been demonstrated in transgenic animals, but on the contrary, hypomorphic mice with 85% downregulation of PiT1 show no difference in skeleton structure and microstructure, just a 6% reduction of femur length compared to wild-type mice (Bourgine et al. 2013). These hypomorphic mice also exhibited, however, an increased expression of PiT2, which could compensate for the reduced expression of PiT1.

Therefore, in spite of the efforts to understand the involvement of the Slc20 in bone formation and physiology, our present knowledge is limited to the correlation of PiT1 expression and calcification of chondrocytes, in part as a consequence of the incorrect interpretation of PFA effects.

Parathyroid Gland

Only one study has addressed the role of the Slc20 in the parathyroid gland, in spite of being a major regulatory gland of Pi homeostasis through the secretion of PTH during hyperphoshatemia. PiT1 was identified in the parathyroid gland and the Slc20A1 RNA expression increased with Pi deprivation and 1,25-dihydroxyvitamin D3 administration (Tatsumi et al. 1998). It was proposed that PiT1 could serve as a sensor of plasma Pi concentration, possibly after changes in the quaternary structures described previously (Salaün et al. 2002) but this possibility has not been clarified to date.

Pathological Involvement

In spite of the apparent relevant functions of the Slc20 family of Pi transporters, a few diseases have been clearly related to their Pi transport malfunctioning, in contrast to the Pi transporters from the Slc34 family. An exception is the primary familial brain calcification, also known as familial idiopathic basal ganglia calcification (IBGC). This disease is manifested in adulthood, with progressive incoordination and psychiatric impairment. IBGC seems to be caused by mutations in several genes, 40% of them being on PiT2 which causes impaired Pi transport by haploinsufficiency, as shown after heterologous expression in Xenopus laevis oocytes (Wang et al. 2012). Some of these mutations also cause dominant negative downregulation of wild-type PiT2 (Larsen et al. 2016). The resulting impaired Pi transport in cells and choroid plexus would result in phosphate accumulation in cerebrospinal fluid and the environment of the ganglia, causing calcium phosphate deposition. Nevertheless, in the absence of detailed thermodynamic conditions, the reason for the limitation of calcium deposition to the basal ganglia is still unknown.

The involvement of PiT1 and PiT2 in the ectopic calcification of soft tissues, mainly in the medial vascular (Mönckeberg’s arteriosclerosis) and aortic valve calcifications, has also been proposed. Unfortunately, most of the conclusions have been raised from in vitro studies using PFA to inhibit the (PFA-insensitive) (see above) Slc20-mediated Pi transport, in combination with elevated concentrations of calcium and Pi that precipitate in high bicarbonate-based culture media. PiT1 is overexpressed in the highly calcified aortas of animals with hyperphosphatemia (Mizobuchi et al. 2006), therefore increasing the capacity (Vmax) of the transport system and enabling higher transport rate. This increased capacity is mandatory for higher transport rate because Pi transport is already saturated in vascular smooth muscle cells during normophosphatemia, and therefore Pi transport cannot increase during hyperphosphatemia unless its capacity also increases (Villa-Bellosta et al. 2007). However, the relationship between increased Pi transport and calcification is difficult to understand if the involvement of a malfunctioning PiT2 with reduced transport rate has been involved in basal ganglia calcification, as described above (Wang et al. 2012). It is also unknown if PiT1 is involved in the advanced ectopic calcification through Pi transport-independent functions, such as mitosis or apoptosis (Beck et al. 2009; Salaün et al. 2010), or if the increased expression is only involving the intracellular expression rather than an increased abundance in the plasma membrane.

Summary

Both members of the Slc20 family of Pi transporters, PiT1 and PiT2 are ubiquitously expressed and, therefore, most likely constitute the main pathway for Pi influx to the cells. We have started to understand the main structure and functional domains of the transporters, but most of the physiological questions are still open. We still need to answer the key question of the apparent redundancy, i.e. why two members of the Slc20 family are expressed in most cells, and how they complement with the Slc34 family members in epithelial tissues. The physiological regulation of these transporters with hormonal and nonhormonal agents also need to be clarified, as well as their role, if any, in physiological and pathological calcifications, cell proliferation, and predisposition to apoptosis. For these studies, high quality antibodies are necessary, which are not easy to obtain most likely because there is a high evolutionary conservation of protein sequences and topologies. However, research on regulation, signaling, and calcification also need to be performed using correct strategies, the finding and use of specific inhibitors of Slc20-mediated Pi transport, a clear understanding of phosphonoformate effects on the different Pi transporters, and the precise control of pH in culture media using low bicarbonate media and realistic concentrations of calcium and Pi.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Laboratory of Molecular ToxicologyUniversity of ZaragozaZaragozaSpain