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Calcium Absorption Across Epithelia

Department of Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, Nijmegen, The Netherlands; and Department of Physiology, Campus Gasthuisberg, KU Leuven, Belgium

ABSTRACT

I. INTRODUCTION

II. PARACELLULAR TRANSPORT

A. Tight Junction

B. Regulation

III. TRANSCELLULAR TRANSPORT

A. Calcium Entry

B. Cytosolic Diffusion

C. Extrusion Mechanisms

1. The Na+/Ca2+ exchanger

2. Regulation of NCX

3. PMCA

4. Regulation of PMCA

IV. EPITHELIAL CALCIUM CHANNELS

A. Molecular Diversity

B. Ion Selectivity/Gating Mechanisms

1. Basic biophysical properties

2. Pore properties

3. Mechanism of high Ca2+ selectivity

4. Rectification

5. Gating

C. Modulation of Channel Activity

1. Intracellular Ca2+

2. pH

3. Pharmacology

D. CRAC and TRPV6

E. Molecular Structure of TRPV5 and TRPV6

V. SITES OF EPITHELIAL CALCIUM TRANSPORT

A. Kidney

1. Proximal tubule

2. Limb of Henle

3. DCT and CNT

4. Collecting duct

B. Gastrointestinal Tract

1. Stomach

C. Others

1. Placenta

2. Bone

3. Exocrine tissues

VI. REGULATION OF EPITHELIAL CALCIUM TRANSPORT

A. Regulation by 1,25-(OH)2D3

1. Calbindins

2. Basolateral extrusion mechanisms

3. Epithelial Ca2+ channels

4. Vitamin D-dependent regulation of Ca2+ transport in animal models

B. Regulation by PTH

C. Regulation by Calcitonin

D. Regulation by Stanniocalcin

E. Regulation by Estrogens

F. Regulation by Thyroid Hormone

G. Regulation by Dietary Ca2+

H. Regulation by the Ca2+-Sensing Receptor

I. Regulation by Associated Proteins

1. Calmodulin

2. S100A10-annexin 2

3. 80K-H

J. Diuretics

1. Furosemide

2. Thiazides

K. Immunosuppressants

VII. CHARACTERIZATION OF EPITHELIAL CALCIUM CHANNEL KNOCKOUT MICE

A. TRPV5 Knockout Mice

B. TRPV6 Knockout Mice

VIII. OUTLOOK

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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The maintenance of the extracellular Ca²⁺ concentration is of utmost importance for many vital functions of the body. In face of large variations in Ca²⁺ in- and output, the organism is equipped with a set of regulatory systems to keep the plasma Ca²⁺ levels around a value of 2.5 mM. To this end, the Ca²⁺ fluxes between the extracellular compartment and several organ systems are tightly controlled.

Ca²⁺ absorption occurs in epithelia, including kidney, intestine, placenta, mammary glands, and gills. In mammals, the small intestine and kidney constitute the influx pathways into the extracellular Ca²⁺ pool, while fish have an additional specialized organ for Ca²⁺ uptake, the gills (426). In addition, during reproductive events, Ca²⁺ transport in the placenta and mammary glands can seriously influence the Ca²⁺ balance (29, 345). For instance, during lactation, plasma Ca²⁺ levels can significantly drop due to Ca²⁺ excretion into the milk. Furthermore, during pregnancy Ca²⁺ transport from the mother to the fetus takes place across the placenta and challenges the plasma Ca²⁺ concentration. The Ca²⁺ demand is great in children during skeletal growth and decreases gradually with advancing age. Finally, factors as pH, extracellular Ca²⁺ concentration, and various hormones have been shown to influence the Ca²⁺ movement across epithelia (37, 129, 175).

In general, Ca²⁺ transport is mediated by a complex array of transport processes that are regulated by hormonal, developmental, and physiological factors. The distinct processes by which Ca²⁺ can be absorbed across epithelial tissues include paracellular and transcellular pathways. The paracellular pathway allows the direct exchange of Ca²⁺ between two compartments, while the transcellular route involves transport across at least two plasma membrane barriers (Fig. 1). The vitamin D metabolite 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] and parathyroid hormone (PTH), among others, are prominent hormones controlling the Ca²⁺ balance.

View larger version (53K): [in this window] [in a new window]   FIG. 1. Mechanism of epithelial Ca2+ transport. Epithelia can absorb Ca2+ by paracellular and transcellular transport. Passive and paracellular Ca2+ transport takes place across the tight junctions and is driven by the electrochemical gradient for Ca2+ (blue arrow). The active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], stimulates the individual steps of transcellular Ca2+ transport by increasing the expression levels of the luminal Ca2+ channels, calbindins, and the extrusion systems. Active and transcellular Ca2+ transport is carried out as a three-step process. Following entry of Ca2+ through the (hetero)tetrameric epithelial Ca2+ channels, TRPV5 and TRPV6, Ca2+ bound to calbindin diffuses to the basolateral membrane. At the basolateral membrane, Ca2+ is extruded via a ATP-dependent Ca2+-ATPase (PMCA1b) and a Na+/Ca2+ exchanger (NCX1). In this way, there is net Ca2+ absorption from the luminal space to the extracellular compartment.

	II. PARACELLULAR TRANSPORT

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A fundamental function of epithelia is to separate different compartments within the organism and to regulate the exchange of substances between them (151, 425). Epithelia consist of a continuous layer of individual cells, and the intercellular spaces between the epithelial cells are very narrow, but nonetheless allow the diffusion of small molecules and ions (152). This route is called the paracellular pathway, which must be regulated for the epithelium to remain selectively permeable (Fig. 1). Depending on the functional requirements of an epithelium, there may be small or large amounts of solutes flowing passively through this path. The tight junction constitutes the barrier to the passage of ions and molecules through the paracellular pathway (5). In general, the importance of this route has not been as thoroughly investigated as the role of the transepithelial pathway. However, recent studies identifying the molecular components of the paracellular transport route as causally related to particular inherited diseases, including familial hypomagnesemia (353), hypertension (429), and autosomal recessive deafness (428), confirm the importance of paracellular transport.

A. Tight Junction

The tight junction is a specialized membrane domain at the most apical region of polarized epithelial cells that not only creates a primary barrier to prevent paracellular transport of solutes, but also restricts the lateral diffusion of membrane lipids and proteins to maintain the cellular polarity. Tight junctions are intercellular structures in which the plasma membranes of adjacent epithelial cells come into very close contact. These structures consist as linear arrays of integral membrane proteins, which include occludin, claudins, and several immunoglobulin superfamily members, such as the junctional adhesion molecule (98, 152, 248). The claudin family consists of at least 20 related integral membrane proteins with four transmembrane (TM) domains and functions as major structural components of the tight junctional complex, while occludin is an accessory protein involved in the tight junction formation of which three isoforms have been described (262, 378, 384).

It has been hypothesized that tight junctions behave similar to conventional ion channels, while others have suggested a function as water-filled channels with no ion selectivity (5, 384, 434). Recently, it was shown by applying new technology that the tight junction complex manifests biophysical properties of ion channels including ion and size selectivity, concentration-dependent ion permeability, competition between permeable molecules, anomalous mole-fraction behavior, and sensitivity to pH (378). This latter study suggests that discrete ion channels are being inserted in the tight junction to facilitate paracellular ion transport. This is further supported by the disease mutations documented in claudins (434). Mutations in claudin-16, which are associated with a renal Mg²⁺ wasting syndrome, implicate this particular claudin in paracellular resorption of Mg²⁺ and Ca²⁺, but not monovalent ions (353). Mutations in claudin-14 cause nonsyndromic recessive deafness, and this tight junction protein is essential to maintain the electrochemical gradient between the endolymph and its surrounding tissues (428).

B. Regulation

Movement of ions through the tight junctions is a passive process, which largely depends on the concentration gradient of the permeable ions and the electrical gradient across the epithelium. Hormones and factors affecting the electrochemical gradient across the epithelium, therefore, indirectly influence the passive fluxes through the tight junctions. The tight junction permeability itself is dynamically regulated under various physiological conditions (152, 299). Tight junctions undergo modulation by growth factors, cytokines, bacterial toxins, hormones, and other factors (30, 134, 153, 419). Some studies suggest that phosphorylation of tight junction proteins plays a role in tight junction assembly and function. Modulation of protein kinase C (PKC) disrupts the cell-cell junctions of epithelial cells, whose action is supposedly mediated by mitogen-activated protein kinase (419). Recently, the serine-threonine kinase WNK4 has been identified as part of the junctional complex, and mutations in this protein cause pseudoaldosteronism type II, a Mendelian trait featuring hypertension (429). Furthermore, a serine-threonine-dependent phosphorylation of tight junction proteins has been shown to enhance the paracellular permeability in rabbit nasal epithelium (294). These studies underline the essential role of serine-threonine kinase in the regulation of the paracellular route, but the precise mechanism remains to be established.

	III. TRANSCELLULAR TRANSPORT

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Transcellular Ca²⁺ transport is a multistep process, comprised of the transfer of luminal Ca²⁺ into the enterocyte or renal epithelial cell, the translocation of Ca²⁺ from point of entry to the basolateral membrane, and finally active extrusion from the cell into the circulatory system (Fig. 1).

A. Calcium Entry

Ca²⁺ is postulated to enter the epithelial cell via Ca²⁺-selective channels at the luminal membrane under the influence of a steep, inwardly directed electrochemical gradient. The molecular nature of the apical entry mechanism has remained obscure for a long time. In the past, the mechanism responsible for the luminal entry of Ca²⁺ has been extensively characterized in renal and intestinal cells, but these studies have not led to the definite identification of this Ca²⁺ transporter. A variety of studies implicated Ca²⁺ channels in mediating Ca²⁺ entry in absorptive epithelia (206, 228, 443). From these studies it is likely that several distinct Ca²⁺ channels, which resemble the classified voltage-dependent Ca²⁺ channels in part, are present in cells of the distal part of the nephron (443). Previous studies indicated the presence of the pore-forming subunit _1A or 1 of a Ca²⁺ channel expressed in the Ca²⁺-transporting renal cells. In general, voltage-sensitive Ca²⁺ channels are heteromeric, multisubunit proteins, as has been well demonstrated for the skeletal muscle (69) and brain (431) channels, and their properties are not only determined by the 1-subunit itself, but also depend on the presence of regulatory subunits. Of these, the ₃-subunit might be an interesting subunit, which can influence such fundamental channel properties as current amplitude (355), kinetics (226, 355, 400), voltage dependence (355), and regulation by cyclic nucleotide-dependent protein kinases (216). Stimulation of the Ca²⁺ influx channel in the renal distal tubule by the calciotropic hormone PTH is thought to be mediated by both protein kinase A (PKA) and PKC (124, 170). Comparable studies in rat duodenum pointed to acute effects of 1,25-(OH)₂D₃ on Ca²⁺ influx in isolated enterocytes (112–114, 144, 152). This calciotropic hormone significantly increased ⁴⁵Ca²⁺ uptake within 1–10 min in a dose-dependent manner (249). The effects of 1,25-(OH)₂D₃ were mimicked by the Ca²⁺ channel agonist BAY K 8644 and completely abolished by nifedipine and verapamil. Incubation of duodenal cells with 1,25-(OH)₂D₃ rapidly (1–5 min) increased cAMP levels. Forskolin caused a rapid increase in Ca²⁺ uptake by enterocytes that was similar to the action of the hormone. Moreover, pretreatment of cells with the specific cAMP inhibitor R_p-cAMPS suppressed the changes in ⁴⁵Ca²⁺ influx induced by 1,25-(OH)₂D₃. These results suggested the involvement of a Ca²⁺ channel activation through the cAMP/PKA-pathway by 1,25-(OH)₂D₃ in mammalian intestinal cells (249). However, the physiological significance of these postulated Ca²⁺ channels with respect to Ca²⁺ influx during Ca²⁺ (re)absorption remains unclear, since most of the studies were performed in single cell systems rather than in polarized epithelia derived from kidney or duodenum epithelia. With the use of these nonpolarized cell preparations, it was not feasible to discriminate between apical versus lateral Ca²⁺ influx. Until now, two polarized confluent epithelial cell systems representing active duodenal and renal Ca²⁺ (re)absorption have been studied. First, Caco-2 cells were employed, which represent an intestinal cell line derived from a human colorectal carcinoma that spontaneously differentiates under standard culture conditions in a tissue that exhibits functional duodenal transport processes (144). These cells become polarized columnar epithelial cells, form tight junctions and domes, and express several markers that are unique to differentiated small intestinal epithelium (e.g., high sucrase-isomaltase mRNA and protein levels) (72). Caco-2 cells exhibit saturable apical-to-basolateral Ca²⁺ transport kinetics, net transport is positive in the apical-to-basolateral direction, and the rate of transport can be increased by pretreatment with 1,25-(OH)₂D₃ (114, 143). Vitamin D-induced upregulation of Ca²⁺ transport requires transcriptional events (114) and is modulated by changes in the vitamin D receptor (VDR) content of the cell (342). Second, the use of primary cultures and immortalized cell lines originating from the renal distal tubular cells greatly facilitated our understanding of Ca²⁺ influx in these cells and how PTH and 1,25-(OH)₂D₃ regulate transepithelial Ca²⁺ transport. Two groups, Bindels and co-workers (36–39, 170, 172, 177, 320, 389–391) and Gesek and Friedman and co-workers (12, 120, 124, 126–128, 139, 138, 246, 427), have used immunodissected cell lines from rabbit and mouse kidney, respectively, to investigate PTH-stimulated Ca²⁺ transport. These studies verified that PTH increases transepithelial Ca²⁺ transport and suggested that both PKA and PKC participate in this process (124, 170). In addition, Bindels and co-workers demonstrated that the primary cultures of rabbit connecting tubule (CNT) and cortical collecting duct (CCD) cells exhibit many characteristics of the original epithelium, including 1,25-(OH)₂D₃- and vasopressin-stimulated Ca²⁺ reabsorption (36–39, 50, 170, 172, 177, 218). The stimulatory effect of cAMP/PKA on apical Ca²⁺ influx is in agreement with a report of Tan and Lau (377), describing a 25-pS, cAMP-sensitive apical Ca²⁺ channel in rabbit kidney CNT cells, that is inhibited by dihydropyridine agonists and depolarization. However, luminal administration of a variety of Ca²⁺ channel antagonists, including dihydropyridines, failed to affect Ca²⁺ absorption in primary cultures of these CNT tubules, suggesting that Tan and Lau (377) did not study the apical entry Ca²⁺ mechanism.

Previous studies indicated that over a wide range of transepithelial Ca²⁺ transport rates, the Ca²⁺ influx at the apical membrane is correlated in a 1:1 fashion with the apical to basolateral ⁴⁵Ca²⁺ flux (320). The mechanism underlying this tight coupling could in principle be located at three distinct Ca²⁺ transport positions, namely, influx, cytosolic diffusion, or efflux (175, 320). Ca²⁺ influx across the apical or luminal membrane could be the rate-limiting step for transcellular Ca²⁺ transport. This would imply that the availability of cytosolic Ca²⁺ for the basolateral Ca²⁺ efflux pumps controls the rate of Ca²⁺ efflux. This is in line with the finding that apical H⁺ directly inhibits the Ca²⁺ influx pathway and consequently decreases the intracellular Ca²⁺ concentration, which in turn limits the Ca²⁺ extrusion (37). In addition, extracellular H⁺ are known to inhibit voltage-gated Ca²⁺ channels in excitable tissues by changing their conformations and modifying their gating properties.

To unravel the molecular identity of the apical Ca²⁺ influx protein in Ca²⁺-transporting epithelia, several different experimental strategies have been employed. The classic approach of purification of the Ca²⁺ transporter followed by amino acid sequencing of the isolated peptide has been hindered by the lack of a rich source of channel protein. In another strategy, a calbindin-D_28K affinity column has been applied to purify associated proteins potentially involved in transepithelial Ca²⁺ (re)absorption (191). Previous studies indicated that the intracellular Ca²⁺ concentration is a critical determinant for Ca²⁺ influx. In theory, the Ca²⁺ buffer calbindin, which reaches submillimolar concentrations in these epithelial cells, might interact with this putative influx channel to buffer and bind Ca²⁺ in a rapid way to facilitate an efficient Ca²⁺ transport. This attempt, however, did not unveil the molecular identify of the Ca²⁺ influx mechanism. Alternatively, investigators have focused on homology-based cloning strategies using sequences of previously described voltage-gated Ca²⁺ channels (21, 22, 397, 443), but also this approach did not result in the identification of the apical Ca²⁺ entry transporter.

Subsequently, functional expression cloning using a rabbit primary CNT/CCD cDNA library in Xenopus laevis oocytes was applied by Hoenderop et al. (178). This technique has previously been successful for the cloning of several epithelial transporters (66). The rationale to use the functional expression cloning approach was threefold: 1) mRNA isolated from primary cultures of rabbit CNT tubules, when injected in oocytes, induced a ⁴⁵Ca²⁺ uptake two to three times above background; 2) transcellular Ca²⁺ transport in primary cultures of rabbit CNT tubules was not affected by voltage-gated Ca²⁺ channel blockers, which allowed us to distinguish between voltage-gated Ca²⁺ channels and the Ca²⁺ transporter involved in transepithelial Ca²⁺ transport; and 3) the absence of a substantial endogenous Ca²⁺ influx in Xenopus laevis oocytes. Based on these characteristics, an expression cloning strategy was established to identify the apical Ca²⁺ entry channel. First, a cDNA library from poly(A)⁺ RNA isolated from primary cultures of rabbit kidney CNT and CCD was generated and subsequently screened for Ca²⁺ uptake activity in Xenopus laevis oocytes in the presence of a cocktail of known voltage-gated Ca²⁺ channel antagonists, including nifedipine, verapamil, and Ba²⁺. After an extensive screening procedure, a single transcript was isolated encoding for a novel epithelial Ca²⁺ channel, named ECaC1 and recently renamed as the transient receptor potential channel TRPV5 (178, 257).

To study the functional characteristics of this new Ca²⁺ channel, TRPV5 was initially heterogeneously expressed in Xenopus laevis oocytes and later in human embryonic kidney (HEK293) cells. Functional data on TRPV5 include ⁴⁵Ca²⁺ uptake, electrophysiology using voltage-clamp and patch-clamp experiments, and fluorimetric measurements (see sect. IV). In short, the channel was inhibited in order of potency by La³⁺ > Cd²⁺ > Mn²⁺. Permeability to Na⁺ was negligible in the situation where Ca²⁺ and Na⁺ were both present, whereas Ba²⁺ and Sr²⁺ did not affect the Ca²⁺ influx. These experiments unequivocally demonstrated that TRPV5 exhibits the defining properties of Ca²⁺ influx in Ca²⁺-transporting epithelia. Subsequently, Hediger and co-workers (257, 304) applied the functional expression cloning technique and identified the Ca²⁺ transporter 1 (CaT1 or recently renamed as TRPV6) from rat intestine, which shares 80% amino acid identity with TRPV5 (note: TRPV5 is also known in the literature as ECaC, ECaC1, and CaT2, whereas TRPV6 has been named previously CaT1, ECaC2, and CaT-like). Electrophysiological studies demonstrated that the characteristics of TRPV6 are comparable to those measured for TRPV5, but its expression pattern is more ubiquitous (see sect. IV). The functional properties of TRPV5 and TRPV6 are in line with those of epithelial Ca²⁺ (re)absorption, providing the first evidence that these transporters are the anticipated Ca²⁺ influx proteins initiating the process of transcellular Ca²⁺ transport, which is described in detail in section IV (Fig. 1) (175, 302).

B. Cytosolic Diffusion

Epithelial cells involved in transcellular Ca²⁺ transport are continuously challenged by substantial Ca²⁺ traffic through the cytosol, while simultaneously maintaining low levels of cytosolic Ca²⁺. To date, two models have been proposed to explain transport of Ca²⁺ across these cells. First, the facilitated diffusion model in which the basal rate of Ca²⁺ uptake and Ca²⁺ extrusion from the absorptive epithelial cell is proposed to be sufficient to accommodate the elevated rate of transport observed after vitamin D stimulation. In contrast, mathematical modeling predicts that intracellular diffusion is the rate-limiting step (58, 357). There are two major subclasses of vitamin D-dependent Ca²⁺-binding proteins, calbindin-D_9K and calbindin-D_28K. These cytosolic proteins have been proposed as shuttles that can bind Ca²⁺ and facilitate the Ca²⁺ diffusion between the apical and basolateral surfaces of the cell. Calbindin-D_28K is highly conserved during evolution and present in kidney, small intestine (only birds), pancreas, placenta, bone, and brain, and calbindin-D_9K is present in highest concentrations in small intestine as well as in kidney (only mouse). The expression level of these calbindins in kidney and intestine is closely correlated with the efficiency of Ca²⁺ (re)absorption and, therefore, these proteins play a central role in the facilitated diffusion model. Second, a vesicular model was proposed in which the absorptive cells use lysosomes to sequester Ca²⁺ and facilitate its movement to the basolateral membrane (230). Formation of Ca²⁺-enriched vesicles is initiated by influx of Ca²⁺ through Ca²⁺ channels in the apical or luminal membrane. The rapid increase in Ca²⁺ concentrations in close vicinity to the apical membrane disrupts the actin filaments near the Ca²⁺ channels and initiates the formation of endocytic vesicles. Ca²⁺ bind to calmodulin (CaM) associated with myosin I or alternatively CaM-associated with the Ca²⁺ channels, which leads to inactivation of the channels. Inactivation of the Ca²⁺ channels causes a decrease in the free Ca²⁺ levels close to the apical membrane, and the actin filament network can be restored. The formed Ca²⁺-containing vesicles are transported by microtubules and fuse with lysosomes (230). While calbindins have been found to associate with lysosomes, the role of these Ca²⁺-binding proteins in this latter model is less clear. Experimental evidence suggests, however, an important role in epithelial Ca²⁺ transport, which is best described by the first model in which calbindin-D_9K and calbindin-D_28K facilitate the cytosolic diffusion of Ca²⁺ from the apical influx to the basolateral efflux sites and acts as cytosolic Ca²⁺ buffer to maintain low intracellular Ca²⁺ levels during changes in transcellular Ca²⁺ transport (35, 107, 108). Interestingly, calbindin-D_9K may directly enhance plasma membrane Ca²⁺-ATPase (PMCA) activity (417). Due to the relatively slow binding kinetics of these Ca²⁺-binding proteins, Ca²⁺ signaling can occur independently of transcellular Ca²⁺ movement mediated by calbindin-D_9K and calbindin-D_28K (218). Depending on the vitamin D state, the cytosolic calbindin-D concentration can reach values in the submillimolar range, which is indeed sufficient to fulfill the above-mentioned functions (see sect. VIA) (38, 106).

Previously, a homozygous mutant of calbindin-D_28K gene-knockout mice was generated by gene targeting which developed normally (3, 23, 361, 362). These animals exhibited a two times higher urinary Ca²⁺ excretion compared with wild-type littermates, but no significant differences in serum Ca²⁺, PTH, or in serum and urinary Mg²⁺ and phosphate were observed (361). This suggests that the hypercalciuria induced by calbindin-D_28K deficiency is compensated by, for instance, increased intestinal absorption of Ca²⁺. Renal calbindin-D_9K expression was not affected in these knockout mice. The cytosolic Ca²⁺ transport function of calbindin-D_9K remains to be confirmed, since calbindin-D_9K knockout mice have not been generated until now.

The calbindins, like CaM, belong to a group of intracellular proteins that bind Ca²⁺ with high affinity and undergo structural changes upon binding (33). Each calbindin is encoded by a separate gene, and there is no direct association between the two genes. In fact, nature has produced a wide variety of EF-hand Ca²⁺-binding proteins (i.e., calbindin-D_9K, calbindin-D_28K, CaM, parvalbumin, S100, troponin C) that display minor overall sequence homology (307). An important functional feature of CaM and troponin is their ability to interact with and regulate the function of voltage-gated Ca²⁺ channels in a Ca²⁺-dependent fashion. The ubiquitously expressed CaM directly interacts with an IQ motif present in the carboxy termini of these channels where it functions as a Ca²⁺ sensor (358). This IQ motif is, however, not present in TRPV5 and TRPV6. At present, it is unknown whether calbindin-D_9K or calbindin-D_28K could fulfill a similar Ca²⁺ sensor function for which a specific interaction with TRPV5 and/or TRPV6 would be required. Freud and Christakos (118) reported an interaction of calbindin-D_28K with microsomal membranes in kidney (118), whereas Shimura and Wasserman (351) showed that calbindin-D_28K is associated with purified chicken intestinal brush-border membranes. Together with the striking colocalization of calbindin-D_9K and/or calbindin-D_28K in all TRPV5/6-expressing tissues, this suggests a functional interaction between these two proteins. Future experiments are needed to delineate whether the function of calbindin is restricted to its buffer capacity maintaining low Ca²⁺ concentrations in close vicinity of the channel mouth or whether a physical interaction between calbindin and TRPV5/6 is needed to exert a direct regulatory function.

C. Extrusion Mechanisms

The efflux of Ca²⁺ occurs against a considerable electrochemical gradient, and two Ca²⁺ transporters have been located in the basolateral membrane of absorptive cells to extrude Ca²⁺, i.e., a Na⁺/Ca²⁺ exchange mechanism (NCX) and a Ca²⁺-ATPase (PMCA).

1. The Na⁺/Ca²⁺ exchanger

To date, three genes for NCX, designated NCX1, NCX2, and NCX3, have been identified in mammals. Similarities between these proteins include a homology of 70% sequence identity, the presence of an amino-terminal signal sequence, two sets of multiple transmembrane -helices near the ends of the protein, and a large intracellular loop (46, 340). Splicing of RNA transcripts is a general characteristic of the NCX genes in mammals to generate diversity. Reilly and Shugrue (325) published the sequence of the rabbit kidney NCX1, and in kidney the expression of this transporter is restricted to the distal part of the nephron where it is predominantly localized along the basolateral membrane (40, 171, 240). NCX1 is widely distributed in many different mammalian tissues, whereas NCX2 and NCX3 are only expressed in brain and skeletal muscle (236, 275). Unfortunately, specific inhibitors of NCX1 are not available to substantiate the relative importance of this exchanger for overall Ca²⁺ reabsorption. Bindels and co-workers (39, 391) demonstrated that NCX1 is the primary extrusion mechanism, whereas only a minor amount of Ca²⁺ in the distal tubular cells is extruded by the plasma Ca²⁺ pump. NCX1 is also expressed in the basolateral membrane of the enterocytes (164, 210, 387). In fish enterocytes, NCX appears to be the main mechanism by which transcellular Ca²⁺ fluxes are extruded from the cells at the basolateral surface, whereas in mammals PMCA is the predominant extrusion mechanism (115, 164, 337, 387). Together these functional studies suggest that in kidney, basolateral Ca²⁺ extrusion is mainly carried out via NCX1, whereas Na⁺/Ca²⁺ exchange seems of minor importance in the small intestine. Recently, it was demonstrated that targeted deletion of NCX1 results in NCX1-null embryos that do not have a spontaneously beating heart and die in utero (219, 327). Therefore, this animal model is, unfortunately, not suitable to verify the importance of NCX1 in renal epithelial Ca²⁺ transport.

In addition, several K⁺-dependent Na⁺-Ca²⁺ exchangers (NCKX) have been described (46, 308). Northern blot analysis demonstrated that some isoforms [i.e., NCKX4 (235) and NCKX6 (65)] of this family are expressed in epithelia including small intestine and kidney. Ubiquitous expression of these exchangers in various tissues suggests a key role in regulating intracellular Ca²⁺ homeostasis in mammalian cells. It remains to be established whether these transporters play a role in epithelial Ca²⁺ transport.

2. Regulation of NCX

Kimura et al. (212) were the first to directly measure a NCX current. Their experiments were carried out in guinea pig cardiac myocytes and provided evidence that the NCX exchanger actually generates a measurable membrane current in which the exchanger can translocate a net positive charge across the plasma membrane (100). The stoichiometry of NCX has been investigated by several groups, and calculations varied from 3 Na⁺:1 Ca²⁺ (15) to 4 Na⁺:1 Ca²⁺ (266, 267), indicating that this transporter is electrogenic. Comprehensive functional studies in oocytes and mammalian cell systems indicated that NCX is regulated by several factors including the membrane potential, PKC activation, protons, nucleotides, and calciotropic hormones (46).

Interestingly, it has been shown that NCX1 is regulated by PTH. Functional data demonstrated that PTH markedly stimulates Ca²⁺ reabsorption in the distal part of the nephron primarily by augmenting NCX1 activity via a cAMP-mediated mechanism.

However, the exact mechanism by which PTH activates the exchanger remains controversial. Initial experiments suggested that the kidney isoform of NCX1 is poorly activated by PKA (161). In another study, it was found that PTH does not affect the intracellular Ca²⁺ concentration (312), indicating that the activation of NCX1 cannot be explained by increased substrate availability. Moreover, it was postulated that PTH increases Cl^– conductance in DCT cells leading to decreased intracellular Cl^– activity and membrane hyperpolarization (137). Hyperpolarization of the basolateral membrane will increase the Na⁺/Ca²⁺ exchange rate. Therefore, coactivation of apical Ca²⁺ entry through TRPV5 occurs under conditions that are favorable for basolateral Ca²⁺ extrusion (see also sect. IV). Other studies on isolated basolateral membrane vesicles implied that the PTH-induced rise in the intracellular Ca²⁺ concentration is not required for the stimulatory effect of PTH (195). Whether PTH stimulation affects expression of the NCX1 mRNA or the protein itself is not known. In addition to PTH, the calciotropic hormone 1,25-(OH)₂D₃ also regulates the renal expression of NCX1. Studies in vitamin D-deficient knockout models showed an impressive downregulation of NCX1 mRNA that could be normalized by 1,25-(OH)₂D₃ supplementation (sect. VI) (169). In these animal models there was no significant downregulation of PMCA in line with a primary role of NCX in Ca²⁺ extrusion. These findings point to a supportive role of the exchanger in vitamin D-stimulated Ca²⁺ reabsorption.

3. PMCA

PMCAs are high-affinity Ca²⁺ efflux pumps present in virtually all eukaryotic cells, wherein they are responsible for the maintenance and resetting of the resting intracellular Ca²⁺ levels (45). Four genes encode separate isoforms designated PMCA1–4. In addition, alternative splicing of the transcripts yields a large variety of splice variants differing mainly in their carboxy-terminal amino acid sequence (365, 369). PMCAs are a universal system for the extrusion of Ca²⁺ in cells. In kidney, PMCA is, in contrast to NCX, present in all nephron segments with highest expression in the basolateral membrane of cells lining the distal part of the nephron. Compared with other nephron segments, the distal convoluted tubule (DCT) possesses the highest Ca²⁺-ATPase activity (95) and exhibits the strongest immunocytochemical reactivity for PMCA protein expression (48, 49, 245). Studies at the transcript level using RT-PCR and advanced tissue microdissection techniques indicated that all four PMCA isoforms are distinctively expressed in the kidney, and variable abundance of the individual isoforms along the different regions of the nephron has been documented (369). Other studies using RT-PCR on whole kidney RNA as well as studies at the protein level have been controversial but suggested that PMCA1 and PMCA4 are the major isoforms expressed in the kidney, while PMCA2 and PMCA3 may be minor components (246). Based on the fact that PMCA1 and PMCA4 are widespread, while PMCA2 and PMCA3 are more tissue specific, it has been suggested that PMCA1 and PMCA4 are housekeeping isoforms involved in the maintenance of cellular Ca²⁺ homeostasis (365). At variance with this conclusion is the observation that PMCA1b transcripts were definitely observed in rabbit CNT and CCD, whereas expression of the PMCA2 isoform was not (171, 213). In addition, Kip and Strehler (213, 214) demonstrated in Madin-Darby canine kidney (MDCK) cells that PMCA4b plays a significant role in basolateral Ca²⁺ extrusion. Furthermore, PMCA1b is the predominant isoform and abundantly expressed in the small intestine, where NCX1 is expressed at a low level. These data suggest indirectly that PMCA1b is the principal Ca²⁺ extrusion mechanism in intestinal Ca²⁺ absorption.

4. Regulation of PMCA

In general, there is only limited data available regarding the regulation of PMCA by hormones or signaling mechanisms. Several studies indicated that PMCA is positively regulated by 1,25-(OH)₂D₃ in the intestine to increase Ca²⁺ absorption. Cai et al. (64) addressed the effect of vitamin D on the synthesis of the chicken intestinal PMCA mRNA. Northern blot analysis indicated that repletion of vitamin D-deficient chickens with vitamin D increases PMCA mRNAs in the duodenum, jejunum, ileum, and colon. After injection of 1,25-(OH)₂D₃ intravenously in these deficient chickens, duodenal PMCA mRNA tended to increase by 2 h, reached a maximum at 16 h, and returned to baseline levels at 48 h (64). These results were confirmed by Johnson and Kumar (199), who demonstrated that 1,25-(OH)₂D₃ causes an increase in abundance of the PMCA and stimulates Ca²⁺-pumping activity. Kip and Strehler (214) showed that 1,25-(OH)₂D₃ upregulates the expression of the PMCAs (mainly PMCA4b) in MDCK cell lysates in a time- and dose-dependent manner. Interestingly, 1,25-(OH)₂D₃ caused a decrease of the PMCAs in the apical plasma membrane fraction and a concomitant increase of the number of pumps in the basolateral membrane. Functional transport assays demonstrated that transcellular ⁴⁵Ca²⁺ flux from the apical-to-basolateral compartment was significantly enhanced by 1,25-(OH)₂D₃. These findings confirm that 1,25-(OH)₂D₃ is a positive regulator of the PMCAs in MDCK cells.

Prince and colleagues (90) demonstrated a stimulatory effect of estrogen and dihydrotestosterone on PMCA activity measured in isolated vesicles from Madin-Darby bovine kidney cells (MDBK) with a magnitude comparable to that of 1,25-(OH)₂D₃. Unlike 1,25-(OH)₂D₃, which stimulates PMCA protein expression in MDBK cells, neither estrogen nor dihydrotestosterone increased PMCA protein expression (90). This suggests that these latter hormones regulate Ca²⁺ transport by increasing PMCA activity, rather than by increasing PMCA protein abundance. Furthermore, PMCA activation is dependent on CaM, and inhibition of CaM is in turn known to prevent PMCA stimulation. In MDCK cells, trifluoperazine and calmidazolium, two inhibitors of CaM, decreased the Ca²⁺ transport by 45 and 33%, respectively (213). Kip et al. (213) used a variety of agents known to inhibit the PMCAs in these cells to determine the contribution of the Ca²⁺ pump to transcellular Ca²⁺ flux. Interestingly, 30% of this flux was due to PMCA and the remaining flux depended on a Na⁺/Ca²⁺ exchange process. These results are in line with the dominant role of NCX1 in transcellular Ca²⁺ flux studies in cultured rabbit kidney cells isolated from CNT and CCD (39).

	IV. EPITHELIAL CALCIUM CHANNELS

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A. Molecular Diversity

TRP channel proteins constitute a large and diverse family of proteins that are expressed in many tissues and cell types (74, 256). The large functional diversity of TRPs is reflected in their diverse permeability to ions, activation mechanisms, and involvement in biological processes ranging from pain perception to male aggression. Mammalian homologs of the Drosophila TRP gene encode a family of at least 20 ion channel proteins (Fig. 2). They are widely distributed in mammalian tissues, but most of their specific physiological functions are largely unknown. The molecular structure that is conserved among all members of the TRP family is a channel subunit, containing six TM spanning domains and a pore region loop, that most probably assemble into tetramers to form unique ion channels allowing the influx of cations into cells (Fig. 3A) (182). The TRP channels can be divided by sequence homology into at least six subfamilies, designated TRPC (canonical or classical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystins, PKD-type), TRPA (for ankyrin), TRPML (for mucolipin), and the more distant subfamily TRPN (N for "nomp" no mechano-receptor potential). ANKTM1, a new member of the TRP ion channel family recently implicated in the detection of noxious cold, is sufficiently different from members of the other TRP subfamilies to warrant assigning this protein to a new subfamily, referred to as TRPA (201, 368). While there is only one mammalian TRPA member, there are four in flies and two in worms (74, 77). To maintain consistency with the rest of the TRP nomenclature, it is recommended to refer to ANKTM1 as TRPA1 in future studies as suggested and approved by the HUGO nomenclature committee. Polycystic kidney disease is an autosomal-dominantly inherited disease causing progressive development of cysts in the kidney and liver (268, 269). Nearly all cases result from mutations in either PKD1 or PKD2 genes, both of which encode proteins of the TRP family. PKD1 and PKD2 are usually lumped together in the TRPP branch, but the two groups have a markedly different molecular architecture and represent distinct phylogenetic branches of the superfamily. PKD1 is a very large protein of 12 TM segments, whereas PKD2 has the predicted topology of the TRP channel consisting of 6 TM spanning domains and clearly conduct ions (156). Importantly, TRPP channels are mainly expressed in ciliated epithelial cells (270).

View larger version (59K): [in this window] [in a new window]   FIG. 2. Mammalian TRP family tree. The evolutionary distance between the TRP channels is shown by the total branch lengths in point accepted mutations (PAM) units, which is the mean number of substitutions per 100 residues. The tree was calculated using the neighbor-joining method for human, rat, and mouse sequences. [From Clapham (74).]

View larger version (33K): [in this window] [in a new window]   FIG. 3. Structural organization of TRPV5 and TRPV6. A: the epithelial Ca2+ channels are 730 amino acids long with a predicted molecular mass of 83 kDa. TRPV5 and TRPV6 contain a core domain consisting of 6 transmembrane (TM) segments. In addition, a large cytosolic amino and carboxy terminus are present containing ankyrin repeats. Between TM5 and TM6 there is a short hydrophobic stretch predicted to be the pore-forming region of these channels. Inner and outer side of the membrane is indicated. B: potential regulatory sites in the amino and carboxy tail of TRPV5 and TRPV6 including ankyrin repeats and PDZ motifs and conserved PKC phosphorylation sites.

The epithelial Ca²⁺ channel family is restricted to two members, and genomic cloning demonstrated that these Ca²⁺ channels are transcribed from distinct genes rather than being splice variants (181, 301). TRPV5 and TRPV6 are juxtaposed on the human chromosome 7q35 with a distance of only 22 kb, which suggests an evolutionary gene duplication event. The same situation was observed in the mouse genome in which TRPV5 and TRPV6 are localized on chromosome 6 in a region that is syntenic to human chromosome 7q33–35 (180, 301). There is one other example found as a direct repeat of two homologs TRP channels. Comparable to the TRPV5/6 couple, TRPV3 is located juxtaposed the TRPV1 gene at a distance of 7.5 kb (41). To date, TRPV5 and TRPV6 have been cloned from many species including rabbit, rat, mouse, and human (181). The identified sequences exhibit an overall homology of 75%. Strikingly, several domains in TRPV5/6 are completely conserved within these species including the core structure of the protein consisting of 6 TM segments and the pore region, ankyrin repeats, PDZ motifs, and putative PKC phosphorylation sites, of which three are conserved among all identified TRPV5/6 channels (Fig. 3A). Detailed sequence analysis of TRPV5 and TRPV6 revealed the identification of several putative phosphorylation sites including PKC, PKA, and cGMP-dependent kinase (89). For instance, in rabbit TRPV5, two combined putative phosphorylation sites for PKA and cGMP-dependent kinase (S669 and T709) were originally identified (178). However, these predicted phosphorylation sites are not conserved in other species or in TRPV6. This is in contrast to the putative PKC phosphorylation sites of which three are conserved within the complete TRPV5/6 subfamily (Fig. 3A) (181). However, the physiological relevance of the conserved PKC sites is not clear, since mutations of these putative regions did not affect channel activity in HEK293 cells (Hoenderop and Bindels, unpublished data). To date, no information is available about the phosphorylation of TRPV5 and TRPV6. In addition, TRPV5 and TRPV6 contain PDZ motifs and ankyrin repeat domains in the amino-terminal region, which are also present in a diverse range of receptors and ion channels including the TRP superfamily. PDZ motifs are recognized by PDZ domains that are modular protein interaction domains playing a role in protein targeting and protein complex assembly (292). There is evidence that they can regulate the functions of their ligands in addition to serving as scaffolds. Although binding to carboxy-terminal motifs appears to be the typical mode of interaction, PDZ domains could also interact with internal motifs that are present in TRPV5/6. In general, ankyrins link transporters and cell adhesion molecules to the spectrin-based cytoskeletal elements in specialized membrane domains (197). Neural-specific isoforms of ankyrin have been demonstrated to participate in the maintenance and targeting of ion channels to subcellular regions in cells (217). So far, no PDZ domain- or ankyrin domain-interacting proteins have been identified.

B. Ion Selectivity/Gating Mechanisms

1. Basic biophysical properties

The conspicuous biophysical hallmarks of TRPV5, which are also representative for TRPV6, are shown in Figure 4 (181, 444). In HEK293 cells heterogeneously expressing TRPV5, currents through TRPV5 can be activated under conditions of high intracellular buffering of Ca²⁺ by hyperpolarizing voltage steps. In the absence of divalent cations, large inward currents are observed, which show a typical time-dependent increase ("gating") and are constant over more than 1 min (Fig. 4A). Outward currents are extremely small, indicating that the channel is nearly completely inwardly rectifying. In the presence of Mg²⁺, but the absence of Ca²⁺, the initial currents are large and rapidly inactivate due to block by extracellular Mg²⁺, which is driven into the open pore by the hyperpolarizing voltage. In the presence of Ca²⁺, but in a nominally Mg²⁺-free solution, currents through TRPV5 increase again when the extracellular Ca²⁺ concentration is elevated. If the extracellular Ca²⁺ concentration is changed from 0 to 30 mM, then 1) increasing extracellular Ca²⁺ up to 100 µM reduces the current amplitudes through TRPV5/6, and 2) an increase from an extracellular Ca²⁺ concentration of 100 µM up to 30 mM or higher enhances the current again. This finding is reminiscent of the anomalous mole fraction behavior described previously for L-type voltage-gated Ca²⁺ channels (4, 85, 163). In analogy to L-type Ca²⁺ channels, permeation through TRPV5/6 can by described by "repulsion" pore models considering a pore consisting of two high-affinity binding sites, whereby the double occupation of the two binding sites by either Na⁺ or Ca²⁺ provides the "drive" for Ca²⁺ conduction due to mutual repulsion of the two cations (403, 406) or by a three binding-site model in which two low-affinity sites flank a high-affinity binding site for Ca²⁺ (181, 403, 406). The current through TRPV5/6 is carried exclusively by Ca²⁺ at extracellular Ca²⁺ concentrations exceeding 10 µM.

View larger version (27K): [in this window] [in a new window]   FIG. 4. Hallmarks of currents through TRPV5. A: currents are activated in HEK293 cells transfected with TRPV5. Holding potential is +20 mV; test steps were applied from +140 to –100 mV in 40-mV increments. Extracellular ionic conditions are changed from left to right: nominally Ca2+- and Mg2+-free extracellular solution, nominally Ca2+-free solution with 1 mM free Mg2+, 1 mM Ca2+ but nominally free Mg2+, 30 mM Ca2+ in a nominally Mg2+-free solution. Note the increase in current due to elevation of the extracellular Ca2+ concentration and the fast inhibition (block) in the presence of Mg2+. Same calibration was used for all current protocols. B: current-voltage (I-V) relationships from the same cell as shown in A. Ionic conditions are indicated at the left of each I-V curve. Note the rightward shift of the reversal potential for increased extracellular [Ca2+]. The fast current decay in 1 mM Mg2+ reflects the fast inactivation seen in the step protocol. [Modified from Vennekens (403).]

View larger version (38K): [in this window] [in a new window]   FIG. 5. Single-channel currents through TRPV5 channels. A: Na+ currents through TRPV5 channels in cell-attached patches. Cells were zeroed in an isotonic extracellular K+ solution. Pipette solution contains 140 mM Na+ in the absence of Ca2+ and Mg2+, 5 mM EDTA. Two consecutive sweeps are shown for potentials. From amplitude histograms, single-channel current-voltage relationships were constructed (I-V). Conductance was measured from the I-V slope between –200 and –100 mV and –100 and +20 mV. Estimation of the single-channel conductance is 75 and 35 pS, respectively. Note that also at the single-channel level inward rectification occurs. B: ensemble-averaged current from 82 sweeps for a voltage step from +20 mV to –100 mV. Note the similarities between ensemble-averaged current and whole cell currents. Increasing the time resolutions (right) unmasks a slow gating behavior at the single-channel level as shown in the whole cell experiment step. (From B. Nilius, unpublished data.)

View larger version (23K): [in this window] [in a new window]   FIG. 6. Ca2+ and Ba2+ currents through TRPV5 and recovery from inactivation. A: during 3-s hyperpolarizing pulses from +70 to –100 mV, large currents are activated in the presence of 30 mM Ca2+ and Ba2+ as the only charge carrier. Note that Ca2+ currents inactivate much faster than Ba2+ currents. B: inactivation can also by monitored by application of 400-ms voltage ramps from –100 to +100 mV, with 5-s intervals between the ramps. Currents were measured at –100 mV. If Ba2+ and Ca2+ are the only charge carriers (all monovalent cations substituted by NMDG+), currents decay rapidly and much faster in the presence of Ca2+. However, recovery from inactivation monitored by the monovalent currents in the absence of divalent cations is much slower when Ca2+ was the charge carrier than with Ba2+. C: I-V curve obtained from the protocol shown in B measured at the times indicated in B. Charge carrier is Ba2+. Note the large monovalent currents and rightward shift of the reversal potential when Ba2+ is the charge carrier. D: same protocol as in B and C. However, Ca2+ is the charge carrier. Although I-V curves are very similar in C and D, the recovery from inactivation is strikingly different (Nilius et al., unpublished data). Letters refer to the time course shown in B and indicated by the same letters.

View larger version (24K): [in this window] [in a new window]   FIG. 7. Determinants of the fast component of inactivation of TRPV6. Functional differences between TRPV5 and TRPV6 include Ca2+-dependent inactivation, Ba2+ selectivity (A), and ruthenium red block (C). Shown is the critical region responsible for the fast Ca2+-dependent inactivation of TRPV6. Alignment depicts the distinctive amino acids within this intracellular loop (B).

View larger version (29K): [in this window] [in a new window]   FIG. 8. Comparison of currents through TRPV5 and TRPV6. A and B: currents through TRPV5 and TRPV6 during a voltage step from +70 to –100 mV. Charge carrier is either Ca2+ (30 mM) or Ba2+ (30 mM). All of the other cations are substituted by 150 mM NMDG+. Cells were loaded with 10 mM BAPTA. Insets show currents normalized to the peak. C: data compare the time to 10% inactivation (t10%) during a voltage step as in A and B for currents through TRPV5 and TRPV6. Data compare the times to 10% inactivation when Ca2+ or Ba2+ is the charge carrier through TRPV5 (left) and TRPV6 (right). [Modified from Nilius et al. (281).] D: the time to 10% decay for Ba2+ and Ca2+ currents through TRPV5 (left) and TRPV6 (right).

TRPV5 and TRPV6 are so far the only known highly Ca²⁺-selective channels in the TRP superfamily. This unique permeation property is also not conserved in the TRPV subfamily, which shares the highest homology with TRPV5/6. TRPV1–4 are, however, all Ca²⁺ and Mg²⁺ permeable, but discriminate much less between divalent and monovalent cations, e.g., the relative selectivity for Ca²⁺ and Mg²⁺ over Na⁺ is between P_Ca/P_Na = 1–10 (TRPV1-TRPV4) and P_Mg/P_Na = 2–3 (only measured for TRPV4) (32, 155, 287, 411). TRPV5/6 display P_Ca/P_Na values of >100. Permeation of monovalent cations is Na⁺ > Li⁺ > K⁺ > Cs⁺ (181, 285, 286, 404, 406). This refers to an Eisenman sequence X for a strong field-strength binding site (181, 285, 286, 406). For TRPV6, a sequence of K⁺ > Na⁺ > Li⁺, e.g., Eisenman V or VI (304), has been reported which, however, seems to be unlikely given the identical pores of TRPV5/6. For divalent cations, a permeation sequence of Ca²⁺ > Ba²⁺ > Sr²⁺ > Mn²⁺ has been reported (181, 305, 404, 406).

A striking and important feature of TRPV5/6 channels is the open pore blockage by intracellular Mg²⁺ (Fig. 9) (409, 410). In the absence of extracellular Ca²⁺, hyperpolarizing voltage steps (prestep, Fig. 9A) activate inward currents with a slowly rising phase ("gating") (Fig. 9B). If after complete opening of the channels at negative potentials steps are applied to different test potentials, inward, but not outward, currents can be observed, indicating that TRPV5/6 channels nearly completely rectify. The typical current-voltage relationships, shown in Figure 9D, indicate that at negative potentials the intracellular Mg²⁺ concentration is ineffective and also an intrinsic rectification remains in the absence of Mg²⁺. The plot of the instantaneous initial current after the negative test potential against the preceding variable prepotentials defines the fraction of open channels (or the apparent open probability, P_o) at the end of the prepotentials. Stepping back from the test potential to the prestep potential unmasks clear voltage dependence: at less negative potentials, partially blocked channels open time dependently due to unblock (voltage dependence). The number of available channels is decreased in a Boltzmann-type voltage dependence, which completely disappears in Mg²⁺-free intracellular solutions (Fig. 9E). These three features (i.e., gating, rectification, and voltage dependence) only appear in the presence of intracellular Mg²⁺. In the absence of intracellular Mg²⁺, gating and voltage dependence disappear, whereas rectification is still present, but diminished (Fig. 9C). An open pore blockage by intracellular Mg²⁺ explains the following findings: at depolarizing potentials, Mg²⁺ moves towards the pore, thereby plugging the permeation pathway for monovalent ions. Unblock occurs at hyperpolarizing voltages. At very large depolarization, Mg²⁺ is pushed through the pore, which results in a partial unblock of the channels (increased apparent P_o, Fig. 9E). These results are crucial to understand the pore properties of TRPV5/6.

View larger version (25K): [in this window] [in a new window]   FIG. 9. Block by intracellular Mg2+ of monovalent currents through TRPV6. A: voltage protocol to activate monovalent currents through TRPV6. Holding potential is 0 mV; a 100-ms prestep to –100 mV is followed by 100-ms test steps to –160 to +100 mV (increment 20 mV). The arrows mark the time for measuring of the apparent open probability of TRPV6 dependent on the preceding test potential. B: voltage-dependent gating occurs in the presence of intracellular Mg2+ (1 mM). Note the strong inward rectification, e.g., almost no outward currents appear. The step back to the prestep potential as indicated in A shows a clear voltage dependence, e.g., currents are smaller at depolarized potential in time-dependently increase to the stationary level. C: same protocol as in B; however, pipette solution contains 10 mM EDTA and no Mg2+. Note that gating and voltage dependence disappear. Rectification is less pronounced. D: average I-V relations obtained from this step protocol and normalized to the current at –100 mV. Shown are steady-state currents measured at the end of the test pulse (arrow in A). E: voltage dependence of the apparent open probability (Po) is shown in the absence and presence of 1 mM intracellular Mg2+. Apparent Po of TRPV6 was determined from the inward current immediately after the 100-ms test pulses (arrow in A) and was normalized to the maximal inward current. Voltage dependence completely disappeared in the absence of intracellular Mg2+. The solid line represent a Boltzmann fit through the first part of the V-Po relationship (–160 to 0 mV, half-maximal inactivation at –52 mV, slope 9.5 mV, fraction open channels at +20 mV, 0.23). [Modified from Voets et al. (409).]