Molecular basis of epithelial Ca²⁺ and Mg²⁺ transport: insights from the TRP channel family

Abstract

Maintenance of plasma Ca²⁺ and Mg²⁺ levels is of vital importance for many physiological functions. This is achieved via a coordinated interplay between the intestine, bone and kidney by amending the rate of absorption, storage and excretion, respectively. Discovery of the transient receptor potential (TRP) family identified several new ion channels acting as gatekeepers of Ca²⁺ and Mg²⁺ transport in these epithelia, greatly increasing our understanding of the molecular processes that facilitate the movement of these minerals. In the intestine, TRP channels contribute to the saturable active transcellular movement of divalent cations from the lumen into the enterocyte. Furthermore, in bone, TRPV channels play important roles by influencing the osteoclastic resorption process, thereby contributing importantly to overall bone mineral content. The divalent cation-permeable TRPV5 and TRPM6 channels are located in the renal distal convolution, the main site of active transcellular Ca²⁺ and Mg²⁺ transport. The channels are regulated by a multitude of factors and hormones that contribute importantly to keeping the systemic concentrations of Ca²⁺ and Mg²⁺ within normal limits. Dysregulation of either channel impacts the renal reabsorptive capacity for these cations. This review summarizes the current knowledge related to TRP channels in epithelial Ca²⁺ and Mg²⁺ transport.

Henrik Dimke (right) is a PhD student in the Ion Transport group, Department of Physiology, Radboud University Nijmegen Medical Centre, which is headed by Prof. Dr Joost Hoenderop (middle) and Prof. Dr René Bindels (left). The group has a long-standing interest in elucidating the molecular mechanisms underlying regulation of ion transport processes in epithelia including the kidney and intestine. The major emphasis is on novel epithelial channels, which participate in Ca²⁺ and Mg²⁺ homeostasis.

Introduction

It is essential to maintain plasma calcium (Ca²⁺) and magnesium (Mg²⁺) concentrations within a tight physiological range. Failure to do so impairs neural excitability, causes arrythmia, alters bone formation and has many other pathological consequences. Ca²⁺ and Mg²⁺ balance is maintained by changing rates of transport across renal and intestinal epithelia and via storage and release from bone. These combined processes ultimately determine the final plasma concentration of these minerals. The cloning, characterization and study of genetic defects of TRP channels localized to specific cell subtypes within these organs has highlighted their important role in the active transcellular transport of divalent cations.

TRP channels

The transient receptor potential channel family is characterized by a similar channel structure. Typically, they contain six transmembrane domains, an intracellular N- and C- terminus, and a predicted pore region composed of a hydrophobic stretch between transmembrane domains 5 and 6. TRP channels assemble into tetramers with a postulated single central pore. There are six subfamilies in mammals denoted as TRPC, TRPM, TRPV, TRPA, TRPML and TRPP. Within subfamilies, TRP channels have been known to assemble into homomeric and heteromeric tetramers. For instance, in the TRPV subfamily, the individual channel subunits accumulate preferentially into homomeric complexes, with the exception of TRPV5 and TRPV6, which will also form heterotetramers (Hoenderop et al. 2003b). In contrast to other TRPV family members, TRPV5 and TRPV6 are highly selective for the permeation of Ca²⁺ ions, (Voets et al. 2002; Hoenderop & Bindels, 2008). Importantly, TRPV5 and TRPV6 freely permeate Ca²⁺ at physiological extracellular concentrations. Furthermore, both channels are expressed exclusively in organs involved in the transepithelial transport of Ca²⁺ (Hoenderop et al. 1999; Peng et al. 1999), indicating a potential role in these processes.

TRPM6 and TRPM7 are channels selective for divalent cations (Hoenderop & Bindels, 2008). These channels are structurally similar to other TRP channels, but are distinguished by a unique α-kinase domain at the extreme C-termini. The function of this associated kinase is currently being unravelled. Although the catalytic domain is not essential for channel function, it appears indirectly to affect TRPM6 activity (Schmitz et al. 2005; Cao et al. 2008). It is currently unclear whether TRPM6 homo-tetramers or heteromeric TRPM6/7 complexes form the Mg²⁺-permeable channels. Thus, the precise molecular organization of these channels within their native epithelia remains to be clarified. TRPM6 is expressed most abundantly in the kidney, lung and intestine, while TRPM7 shows a more ubiquitous expression pattern (Schlingmann et al. 2002; Groenestege et al. 2006).

Intestinal uptake of Ca²⁺ and Mg²⁺

In humans, dietary Ca²⁺ intake approximates 1000 mg daily, of which 400 mg is absorbed from the intestine (Wasserman et al. 1984). During steady state, Ca²⁺ absorption occurs predominantly from the small intestine (Wasserman, 2004). Dietary Ca²⁺ is absorbed either via a passive paracellular route or by an active transcellular process (Bronner, 1998). Passive Ca²⁺ efflux from the intestinal lumen is the predominant pathway for Ca²⁺ absorption when dietary Ca²⁺ intake (and thus the luminal Ca²⁺ concentration) is adequately high (Bronner & Pansu, 1999). The majority of ingested Ca²⁺ is thus absorbed from the ileum and jejunum via this passive paracellular route. Conversely, active transcellular Ca²⁺ transport occurs chiefly in the duodenum. The pathway is saturable and depends on the active transcellular movement of Ca²⁺ through the enterocyte. The exact carriers responsible for apical Ca²⁺ uptake are debated. TRPV6 appeared a likely candidate gene as Ca²⁺ homeostasis and especially intestinal Ca²⁺ absorption is disturbed in mice lacking TRPV6 (Trpv6^−/−) (Bianco et al. 2007). However, the targeting strategy used to generate the Trpv6^−/− mice also disrupted the closely adjacent Ephb6 gene, which may contribute to the observed phenotype. Trpv6^−/− mice demonstrate increased intestinal Ca²⁺ absorption during low dietary Ca²⁺ conditions, suggesting that TRPV6 is not essential for this process (Benn et al. 2008) and that another unknown entry mechanism may be rate limiting in active interstitial Ca²⁺ absorption. It is noteworthy that the increase in active duodenal transport in response to dietary Ca²⁺ restriction appeared lower in Trpv6^−/− mice than in wild-type littermates, suggesting a contribution of TRPV6 in the active absorption of Ca²⁺ (Benn et al. 2008). This is also in line with the observation that TRPV6 is regulated by 1,25-dihydroxyvitamin D₃. Transcellular diffusion, from apical to luminal membrane is mediated by high-affinity Ca²⁺ binding to calbindins (chiefly calbindin-D_9K in mouse duodenum). Secretion of Ca²⁺ occurs predominantly via an ATP-dependent basolateral pump, PMCA1b. This transcellular pathway predominates when the dietary intake of Ca²⁺ is low (Bronner & Pansu, 1999). TRPV5 colocalizes to the duodenum with TRPV6; however, mice with a genetic ablation of TRPV5 (Trpv5^−/−) are normocalcaemic in the presence of renal Ca²⁺ wasting. This is achieved by a compensatory increase in plasma 1,25-dihydroxyvitamin D₃ concentrations, which results in compensatory intestinal hyperabsorption of Ca²⁺ via both the transcellular and paracellular pathway (Wasserman & Fullmer, 1995; Hoenderop et al. 2003a). This raises the intriguing possibility that TRPV5 might also play a functional role in transcellular absorption of Ca²⁺ in the intestine. However, any potential defect in TRPV5-mediated intestinal Ca²⁺ transport is likely masked by the vitamin D induced hyperabsorption in these mice. The inability to generate TRPV5/6 double knockout mice, due to the close genomic localization of the genes relative to one another, needs to be overcome to effectively test this hypothesis.

Dietary Mg²⁺ intake averages 300 mg daily (Quamme & de Rouffignac, 2000). However, depending on the dietary Mg²⁺ load, absorption varies between 11 and 65% (Fine et al. 1991). As with Ca²⁺, Mg²⁺ can be absorbed passively or it can be actively transported, with the majority of Mg²⁺ being absorbed from the small intestine and to a lesser extent from the colon (Fine et al. 1991). Again, the paracellular route predominates during conditions of high dietary Mg²⁺ intake while the active transcellular route prevails when the intestinal Mg²⁺ concentration is low (Milla et al. 1979). Active absorption of Mg²⁺ occurs predominantly from the colon (Karbach, 1989), but may also occur in the small intestine (Juttner & Ebel, 1998). Although not fully delineated, transcellular influx of Mg²⁺ is probably mediated via TRPM6, the putative Mg²⁺ channel situated within the apical membrane of the enterocyte. TRPM6 was originally identified as the underlying cause of the autosomal recessive disorder hypomagnesaemia with secondary hypocalcaemia (HSH) (Schlingmann et al. 2002; Walder et al. 2002). Patients affected by this disease present early in life with symptoms of neuromuscular instability such as muscle spasms, tetany and generalized convulsions due to severe hypomagnesaemia and hypocalcaemia (Paunier et al. 1968). Treatment with high dose Mg²⁺ by supplementation effectively reduces several of the symptoms, but plasma Mg²⁺ levels remain below normal (Shalev et al. 1998). Milla et al. demonstrated that individuals with HSH fail to effectively absorb Mg²⁺ when the intraluminal intestinal concentration of Mg²⁺ is low (Milla et al. 1979). Furthermore, Ca²⁺ balance remained positive, thereby inferring that the absorptive defect exclusively affects Mg²⁺ transport. Thus, these data suggest that TRPM6 plays an integral role in Mg²⁺ entry into the enterocyte, as a component of the saturable transcellular Mg²⁺ transport pathway. Consequently, hypomagnesaemia in HSH patients results from intestinal malabsorption of Mg²⁺. As will be discussed later, an additional defect due to the renal loss of TRPM6 probably exaggerates the hypomagnesaemia observed in these patients. The transport machinery that buffers intracellular Mg²⁺ in the enterocyte and secretes these ions at the basolateral membrane remains undefined.

Storage and exchange minerals in bone

Both Ca²⁺ and Mg²⁺ are to a great extent stored in the bone, as elements of the hydroxyapatite crystal. Osseous tissue, the mineralized connective tissue of bone, is partly composed of these apatite crystals, which are embedded within a collagen matrix. Ninety-nine per cent of bodily Ca²⁺ is stored in bone, leaving 1% distributed between soft tissues and the extracellular fluid. Ca²⁺ can be exchanged between extracellular fluid and bone by osteoblasts (specialized cells involved in bone formation) and osteoclasts (specialized cells that remove the mineralized bone matrix via a process known as resorption). Bone remodelling occurs via coordinated interplay between these cell types and occurs slowly over time (Parfitt, 2003).

TRPV5 localizes to the ruffled border membrane of osteoclasts, the cellular domain that facilitates removal of bone matrix (van der Eerden et al. 2005) (Fig. 1). Genetic ablation of the channel in mice causes dysfunctional osteoclasts with an impaired ability to sufficiently reabsorb Ca²⁺ from bone, suggesting that TRPV5 could play an important role in osteoclastic Ca²⁺ resorption (van der Eerden et al. 2005). However, Trpv5^−/− mice exhibit reduced bone mineralization probably due to excessive renal Ca²⁺ wasting (Hoenderop et al. 2003a). This observation is curious since impaired bone resorption would normally promote osteopetrosis. Furthermore, treatment with the bisphosphonate bone resorption inhibitor, alendronate, normalizes reduced bone thickness in Trpv5^−/− mice (Nijenhuis et al. 2008). Thus, it is likely that a significant amount of resorption still occurs in these animals, indicating that perhaps an alternative transport pathway exists for the vectorial movement of Ca²⁺ through the osteoclast. Whether vectorial transport of Ca²⁺ through the osteoclast is essential for bone resorption is also not clear (Berger et al. 2001).

Figure 1. Bone resorption and the potential role of TRPV channels in osteoclast function.

Schematic drawing of the osteoclast, expressing TRPV5 at its resorptive surface near the bone matrix. Vectorial Ca²⁺ transport may be facilitated by the driving force of the PMCA. TRPV4 is also found in the basolateral membrane, driving NFATc1 activation and controlling differentiation of the osteoclast. TRPV5, transient receptor potential vanilloid 5; TRPV4, transient receptor potential vanilloid 4; NFATc1, nuclear factor-activated T cells c1; PMCA, plasma membrane Ca²⁺-ATPase.

TRPV4 is expressed in both osteoblasts and osteoclasts (Masuyama et al. 2008). Interestingly, mice with a targeted deletion of the Trpv4 gene (Trpv4^−/−) have osteopetrosis. This feature is not due to a defect in osteoblast function, as bone formation is unaffected in Trpv4^−/− mice. However, the Trpv4^−/− mice show a marked decrease in osteoclast surface area and number, suggesting that the channel is important for osteoclast development or maintenance. In the mature osteoclast, TRPV4 localizes to the basolateral side of the cell. Subsequent experiments demonstrated that Ca²⁺ influx via TRPV4 is important for maintaining basal intracellular Ca²⁺ levels, which control the activation of the nuclear factor-activated T cells c1 (NFATc1) in large osteoclasts (Fig. 1). Ca²⁺-dependent activation of NFATc1 causes translocation of the protein to the nucleus, which induces NFATc1-regulated gene transcription leading to terminal differentiation of the osteoclasts. Thus, in Trpv4^−/− mice, osteoclast differentiation is affected, disturbing the resorption process and ultimately increasing bone mass (Masuyama et al. 2008).

Mg²⁺ is also an integral part of bone, with approximately 50% of bodily Mg²⁺ stored there (Elin, 1987). Dietary reduction in Mg²⁺ decreases bone Mg²⁺ content in rats (Alfrey et al. 1974). Evaluation of the chemical and mechanical properties of bone during hypomagnesaemia in the rat also revealed evidence of accelerated bone turnover, decreased bone volume, and decreased bone strength in Mg²⁺-depleted animals (Boskey et al. 1992; Kenney et al. 1994). Hence chronic hypomagnesaemia leads to severe osteopenia (Lai et al. 1975; Burnell et al. 1986). It is therefore likely that Mg²⁺ is stored and exchanged with bone in a similar manner to Ca²⁺, and that chronic Mg²⁺ depletion can affect bone resorption and formation. However, a regulatory system to mobilize Mg²⁺ from bone has not been identified. In this respect, the localization of channels and transporters permitting Mg²⁺ to permeate bone cells needs to be established. Additionally, it is not clear how Mg²⁺ deficiency contributes to osteoporosis in humans (as discussed in Rude & Gruber, 2004).

Ca²⁺ and Mg²⁺ reabsorption by the kidney

The kidney controls the excretion of Ca²⁺ and Mg²⁺ in response to changes in the systemic concentration of these ions. Transport of divalent cations across various nephron segments occurs either paracellularly in the proximal tubule and thick ascending limb, or actively in the distal convolutions (this anatomical structure comprises the distal convoluted tubule (DCT), connecting tubule (CNT), and the initial collecting duct (CD) (Kriz & Bankir, 1988).

Only 1–2% of the Ca²⁺ that is filtered by the glomerulus is excreted in the urine, the majority being reabsorbed along the course of the nephron. The paracellular pathway is responsible for reclaiming the majority of Ca²⁺ from the proximal tubule and thick ascending limb. Micropuncture studies estimate that the distal convolution reabsorbs 3–7% of filtered Ca²⁺ via an active process (Dimke et al. 2010a). TRPV5 allows the entry of Ca²⁺ across the apical membrane in the late part of the distal convolution (mainly DCT2 and CNT), thus providing the basis for active Ca²⁺ reabsorption in the kidney (Hoenderop et al. 2001; Loffing & Kaissling, 2003). The remainder of the machinery involved in active Ca²⁺ transport within these cells is similar to that of the intestine. The Ca²⁺-binding protein calbindin-D_28K acts to buffer, shuttling Ca²⁺ across the cell, while the basolateral extrusion proteins – the plasma membrane ATPase 1b (PMCA1b) and the Na⁺–Ca²⁺ exchanger type 1 (NCX1) – act to facilitate the efflux of Ca²⁺. The importance of TRPV5 in active Ca²⁺ transport in the distal convolution is highlighted by the phenotypic characteristics of the Trpv5^−/− mice. These animals show an almost sixfold increase in their urinary excretion of Ca²⁺. Moreover, detailed micropuncture studies reveal that Ca²⁺ wasting in the Trpv5^−/− mice originates in the distal convolution (Hoenderop et al. 2003a).

TRPV5 is subject to regulation by a variety of factors as outlined in Fig. 2, thereby controlling the final urinary excretion of Ca²⁺ by the kidney. Calciotropic hormones including 1,25-dihydroxyvitamin D₃ and parathyroid hormone increase the expression of the channel (Boros et al. 2009). Other systemic factors such as dietary Ca²⁺, acid/base balance and the sex hormones oestrogen and testosterone also affect expression of TRPV5 (Boros et al. 2009; Hsu et al. 2010). In addition to the transcriptional effects of parathyroid hormone (PTH), this hormone stimulates TRPV5-dependent Ca²⁺ transport via activation of the protein kinase A (PKA) pathway. Initiation of this signalling cascade leads to PTH-dependent phosphorylation of TRPV5, which directly increases the open probability of TRPV5 (de Groot et al. 2009). In addition, PTH also inhibits caveolae-mediated endocytosis of TRPV5, thereby increasing cell surface abundance of the channel. This process occurs via a protein kinase C-dependent pathway (Cha et al. 2008b) (Fig. 2). Novel hormones, such as Klotho have also been shown to regulate channel abundance at the plasma membrane, by hydrolysing oligosaccharide chains from the N-glycan tree on TRPV5 (Chang et al. 2005; Cha et al. 2008a) (Fig. 2). This is in line with the phenotypic characteristics of the Klotho^−/− mice which show renal Ca²⁺ wasting, secondary hypervitaminosis D, osteopenia, and nephrocalcinosis (Alexander et al. 2009). Furthermore, a range of associated proteins regulate TRPV5 activity (Boros et al. 2009).

Figure 2. Schematic model of the cellular composition of renal Ca²⁺ and Mg²⁺ proteins within the distal convolution.

The Mg²⁺ transport machinery and regulators are listed in the upper cell, while the Ca²⁺ transporters and regulators can be found in the cell below. DCT, distal convoluted tubule; CNT, connecting tubule; TRPM6, transient receptor potential melastin 6 Mg²⁺ channel; EGFR, epidermal growth factor receptor; FXYD2, γ-subunit of the Na⁺–K⁺-ATPase; HNF1B, the hepatocyte nuclear factor 1B; Kir4.1, ATP-sensitive inward rectifier potassium channel 10; Kv1.1, Shaker-related voltage-gated K⁺ channel. PTHR, parathyroid hormone receptor; TRPV5, transient receptor potential vanilloid 5 Ca²⁺ channel; PMCA1b, plasma membrane Ca²⁺-ATPase 1b; NCX1, Na⁺–Ca²⁺ exchanger 1; 28K, calbindin-D_28K.

The kidney excretes 3–5% of the Mg²⁺ ions appearing in the ultrafiltrate (Dimke et al. 2010a). The majority of the filtered Mg²⁺ is reabsorbed along the nephron; this occurs by a paracellular mechanism in segments prior to the distal convolution. Micropuncture studies estimate that the distal convolution reabsorbs approximately 5–6% of the Mg²⁺ in the ultrafiltrate (Le Grimellec et al. 1973; Brunette et al. 1974). These studies also suggest that active Mg²⁺ reabsorption occurs predominantly in the early distal tubule (Bailly et al. 1985). The immunohistochemical localization of TRPM6 is constrained to the apical membrane of the DCT and the channel probably provides the apical entry step for Mg²⁺ in this segment (Voets et al. 2004). Consistent with this is the observation that HSH patients with TRPM6 mutations have a noticeable renal Mg²⁺ leak, which is particularly visible when these individuals are given a Mg²⁺ load (Walder et al. 2002). The intracellular Mg²⁺ buffer proteins as well as the basolateral excursion machinery remain to be identified.

TRPM6 expression is regulated by systemic factors such as dietary Mg²⁺ content, oestrogens, and acid/base balance. In addition, epidermal growth factor (EGF) was discovered as the first magnesotropic hormone, regulating the activity of TRPM6 (Fig. 2). This is evidenced by the fact that EGFR inhibitors promote hypomagnesaemia, which is the result of renal wasting (Groenestege et al. 2007; Dimke et al. 2010b). Similarly, patients with mutations in the pro-EGF gene display isolated renal hypomagnesaemia (Groenestege et al. 2007). Transport of Mg²⁺ across the apical membrane is predicted to occur independently of the chemical driving force, since the concentrations of Mg²⁺ in the lumen and inside the cell are similar. As a consequence, the voltage difference across the apical membrane is probably the major driving force for Mg²⁺ reabsorption via TRPM6 in the kidney. This is supported by the observation that mutations in channels and regulators implicated in stabilizing the membrane voltage in the DCT cell cause clinical hypomagnesaemia. These include the γ-subunit of the Na⁺–K⁺-ATPase (FXYD2) (Meij et al. 2000), the hepatocyte nuclear factor 1B (HNF1B), which regulates transcription of the FXYD2 protein (Adalat et al. 2009), the ATP-sensitive inward rectifier potassium channel 10 (Kir4.1) (Bockenhauer et al. 2009; Scholl et al. 2009), and the Shaker-related voltage-gated K⁺ channel (Kv1.1) (Glaudemans et al. 2009) (Fig. 2).

Conclusion

It is essential for the body to maintain circulating concentrations of Ca²⁺ and Mg²⁺ within a narrow physiological window. Failure to adequately maintain extracellular concentrations of these minerals results in the instability of neuronal and cardiac systems. Maintenance of systemic concentrations of divalent cations is achieved by the coordinated interplay between intestine, bone and the kidneys. Multiple studies have delineated the important contribution of the TRPV and TRPM subfamilies in active transcellular transport of Ca²⁺ and Mg²⁺, respectively. Consequently, defects in the genes encoding these channels can greatly impact divalent cation homeostasis and lead to phenotypic characteristics of divalent cation deficiency including changes in bone morphology, altered intestinal absorption and renal wasting.