Epithelial Ca2+ and Mg2+ Channels in Health and Disease
Joost G.J. Hoenderop and
René J.M. Bindels
Department of Physiology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, the Netherlands
Address correspondence to: Dr. René J.M. Bindels, 160 Cell Physiology, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands. Phone: +31-24-3614211; Fax: +31-24-3616413; r.bindels{at}ncmls.ru.nl
A near constancy of the extracellular Ca2+ and Mg2+ concentrationis required for numerous physiologic functions at the organ,tissue, and cellular levels. This suggests that minor changesin the extracellular concentration of these divalents must bedetected to allow the appropriate correction by the homeostaticsystems. The maintenance of the Ca2+ and Mg2+ balance is controlledby the concerted action of intestinal absorption, renal excretion,and exchange with bone. After years of research, rapid progresswas made recently in identification and characterization ofthe Ca2+ and Mg2+ transport proteins that contribute to thedelicate balance of divalent cations. Expression-cloning approachesin combination with knockout mice models and genetic studiesin families with a disturbed Mg2+ balance revealed novel Ca2+and Mg2+ gatekeeper proteins that belong to the super familyof the transient receptor potential (TRP) channels. These epithelialCa2+ (TRPV5 and TRPV6) and Mg2+ channels (TRPM6 and TRPM7) formprime targets for hormonal control of the active Ca2+ and Mg2+flux from the urine space or intestinal lumen to the blood compartment.This review describes the characteristics of epithelial Ca2+and Mg2+ transport in general and highlights in particular thedistinctive features and the physiologic relevance of thesenew epithelial Ca2+ and Mg2+ channels in (patho)physiologicsituations.
Ca2+ and Mg2+ are of great physiologic importance by their interventionin many enzymatic systems and their function in neural excitability,muscle contraction, blood coagulation, bone formation, hormonesecretion, and cell adhesion. The human body is equipped withan efficient negative feedback system that counteracts variationsof the Ca2+ and Mg2+ balance. This system encompasses parathyroidglands, bone, intestine, and kidneys. These divalents are maintainedwithin a narrow range by the small intestine and kidney, whichboth increase their fractional (re)absorption under conditionsof deprivation (1,2). If depletion continues, then the bonestore assists to maintain appropriate serum concentrations byexchanging part of its content with the extracellular fluid.The Ca2+-sensing receptor (CaSR) represents the molecular mechanismby which parathyroid cells detect changes in the ionized Ca2+and Mg2+ concentration and modulate parathyroid hormone (PTH)secretion (3,4). In addition to the effects of these divalentson PTH secretion, this hormone in turn regulates directly theCa2+ and Mg2+ balance by modulating bone resorption, renal reabsorption,and indirectly intestinal absorption by stimulating 1-hydroxylaseactivity and consequently 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)synthesis to maintain serum Ca2+ and Mg2+ levels within a narrowphysiologic range.
The major part of the Ca2+ reabsorption takes place along theproximal tubule (PT) and thick ascending limb of Henle’sloop (TAL) through a paracellular and, therefore, passive pathway(5). Fine-tuning of the Ca2+ excretion occurs in the distalpart of the nephron, where approximately15% of the filteredload of Ca2+ is reabsorbed. This section consists of the distalconvoluted tubule (DCT), the connecting tubule (CNT), and theinitial portion of the cortical collecting duct (CCD; Figure 1).In these latter nephron segments (DCT, CNT, CCD), Ca2+ reabsorptionis active and occurs against the existing electrochemical gradient.Together with the fact that here the tight junctions are impermeablefor Ca2+ ions, this substantiates that Ca2+ is reabsorbed throughan active transcellular pathway. Active Ca2+ reabsorption isgenerally envisaged as a multistep process that consists ofpassive entry of Ca2+ across the luminal or apical membrane,cytosolic diffusion of Ca2+ bound to vitamin D3-sensitive calcium-bindingproteins (calbindin-D28K and/or calbindin-D9K), and active extrusionof Ca2+ across the opposite basolateral membrane by a Na+-Ca2+exchanger (NCX1) and/or Ca2+-ATPase (PMCA1b) (6) (Figure 1,top). This active transcellular Ca2+ transport is under hormonalcontrol of PTH (7,8), 1,25-(OH)2D3 (7,9–12,103), and calcitonin(13) but also estrogen (14,15), androgen (16), and dietary Ca2+(10) are primary regulators.
Figure 1. Transcellular Ca2+ and Mg2+ transport. Active Ca2+ and Mg2+ transport is carried out as a three-step process in the distal part of the nephron. (Top) After entry of Ca2+ in distal convoluted tubule (DCT2) and connecting tubule (CNT) through the epithelial Ca2+ channels TRPV5 and TRPV6, Ca2+ bound to calbindin diffuses to the basolateral membrane. At the basolateral membrane, Ca2+ is extruded via an ATP-dependent Ca2+-ATPase (PMCA1b) and an Na+-Ca2+–exchanger (NCX1). (Bottom) Apical Mg2+ entry in DCT via TRPM6 (and TRPM7). As extrusion mechanisms are postulated a basolateral Na+/Mg2+ exchanger and/or ATP-dependent Mg2+ pump. The Na+,K+-ATPase complex including the -subunit controls this transepithelial Mg2+ transport. In this way, there is net Ca2+ and Mg2+ reabsorption from the luminal space to the extracellular compartment.
Regulation of the total body Mg2+ balance principally resideswithin the kidney that tightly matches the intestinal absorptionof Mg2+. In the kidney, approximately 80% of the total plasmaMg2+ is ultrafiltrated across the glomerular membrane and subsequentlyreabsorbed in consecutive segments of the nephron (1). Approximately10 to 20% of Mg2+ is reabsorbed by the PT. However, the bulkamount of Mg2+ (50 to 70%) is reabsorbed by the TAL, which likelymediates Mg2+ reabsorption via paracellular transport. The finalurinary excretion of Mg2+ is mainly determined by active reabsorptionof Mg2+ in DCT, because virtually no reabsorption takes placesbeyond this segment (Figure 1) (1). Microperfusion studies haveshown that Mg2+ is reabsorbed in the superficial DCT, but littleknowledge has been gained concerning the cellular mechanismsof transcellular Mg2+ reabsorption (1,17,18). Speculatively,Mg2+ can passively enter the DCT cell across the luminal membranedriven by a favorable plasma membrane voltage (Figure 1, bottom).The molecular identity of the responsible influx protein wasunknown, however, and previous studies hypothesized that a Mg2+-specificion channel is a possible candidate (1). Subsequently, Mg2+will be transported through the cytosol and extruded at theopposing basolateral membrane by an active mechanism given theexisting electrochemical gradient. Again, the identity of responsibletransport proteins remains to be defined, and candidate mechanismsare Mg2+-binding proteins, Na+/Mg2+ exchanger and/or an ATP-dependentMg2+ pump (Figure 1, bottom).
Several genes involved in the process of transepithelial Ca2+transport have now been identified, but the Ca2+ influx mechanismremained unknown for a long time. An expression-cloning approachusing Xenopus laevis oocytes revealed the molecular identityof the Ca2+ influx systems (19,20). The first member, namedTRPV5, was cloned from primary cultures of rabbit renal distaltubules that are primarily involved in active transcellularCa2+ transport and encodes a Ca2+ channel that belongs to theTRP family (19). Likewise, a homologous member of this family,known as TRPV6, was successfully cloned from rat duodenum (20).
During the past few years, several genes that encode proteinsthat are either directly or indirectly involved in renal Mg2+handling have been identified following a positional cloningstrategy in families with hereditary hypomagnesemia. The firstgene involved, PCLN-1 (or CLDN16), encodes the protein paracellin-1(or claudin-16) (21). This protein is specifically expressedin the TAL and shows sequence and structural homology to theclaudin family of tight junction proteins. Paracellin-1 is mutatedin patients who have hypomagnesemia, hypercalciuria, and nephrocalcinosis(HHN; MIM 248250). In this autosomal recessive disorder, thereis profound renal Mg2+ and Ca2+ wasting. The hypercalciuriaoften leads to nephrocalcinosis, resulting in progressive renalfailure (22,23). Other symptoms that have been reported in patientswith HHN include urinary tract infections, nephrolithiasis,incomplete distal tubular acidosis, and ocular abnormalities(22,24). Immunohistologic studies have shown that claudin-16co-localizes with occludin in intercellular junctions of humankidney sections, indicating that it is a tight junction protein(21). The second gene, FXYD2, encodes the -subunit of the Na+,K+-ATPasepump, which is predominantly expressed in the kidney, with thehighest expression levels in DCT and medullary TAL (25). FXYD2is mutated in patients with autosomal dominant renal hypomagnesemiaassociated with hypocalciuria (IDH; MIM 154020). Hypomagnesemiain these patients can be severe (<0.40 mM) and cause convulsions.Remarkably, in some affected individuals, there are no symptomsof Mg2+ deficiency except for chondrocalcinosis at adult age.The molecular mechanism for renal Mg2+ loss in this autosomaldominant type of primary hypomagnesemia remains to be elucidated.The third gene involved, SLC12A3, encodes the thiazide-sensitivesodium chloride co-transporter (NCC) in DCT and is mutated inpatients with Gitelman syndrome (MIM 263800) (26). This autosomalrecessive disorder is a frequent hereditary tubular disorderthat affects renal Mg2+ handling, which is characterized byhypokalemia, hypomagnesemia, and hypocalciuria. Hypomagnesemiais found in most patients with Gitelman syndrome and is assumedto be secondary to the primary defect in NCC, but the mechanismsunderlying hypomagnesemia are poorly understood.
Although these linkage analyses revealed the identificationof genes involved in Mg2+ homeostasis, the key molecules thatrepresent the mechanisms for luminal Mg2+ influx and basolateralMg2+ extrusion in the process of transcellular Mg2+ transportare still elusive. Importantly, Walder et al. (27) reportedthat hypomagnesemia associated with secondary hypocalciuria(HSH; MIM 602014) is an autosomal recessive disease that isgenetically linked to chromosome 9p22. This disease is primarilydue to defective intestinal Mg2+ absorption, and affected individualsshow neurologic symptoms of hypomagnesemic hypocalcemia, includingseizures and muscle spasms during infancy (28–30). Becausepassive Mg2+ absorption is not affected, the disease can betreated by high dietary Mg2+ intake (31). Renal Mg2+ conservationhas been reported to be normal in most patients. In some cases,however, a renal leak has been reported, suggesting impairedrenal Mg2+ reabsorption. Patients who were studied by Konradet al. and others (28,32) showed inappropriately high fractionalMg2+ excretion rates with respect to their low serum Mg2+ levels.When untreated, the disease may be fatal or may lead to severeneurologic damage. Hypocalcemia is secondary to parathyroidfailure resulting from Mg2+ deficiency. Using a positional candidategene-cloning approach, two groups headed by Konrad and Sheffield(28,29) independently identified mutations in TRPM6 in autosomalrecessive HSH, previously mapped to chromosome 9q22. The TRPM6protein is a new member of the long TRP channel (TRPM) familyand is similar to TRPM7 (also known as TRP-PLIK), a unique bifunctionalprotein known as a Mg2+-permeable cation channel propertieswith protein kinase activity (33–35). TRPM6 and TRPM7are distinct from all other ion channels in that they are composedof a channel linked to a protein kinase domain recently abbreviatedas chanzymes (36). These chanzymes are essential for Mg2+ homeostasis,which is critical for human health and cell viability (37,38).
In summary, a variety of approaches, including a genetic screenin patients with primary hypomagnesemia and expression cloningin Ca2+-transporting epithelial cells, revealed the identificationof TRP cation channels as potential gatekeepers in the maintenanceof the Ca2+ and Mg2+ balance. The TRP superfamily is a newlydiscovered family of cation-permeable ion channels (33). Thereare at least three previously recognized subfamilies of proteins—TRPC(conical), TRPV (vanilloid), and TRPM (metastatin)—thatare expressed throughout the animal kingdom (http://clapham.tch.harvard.edu/trps/).Recently, the polycystins were also included in the TRP superfamilyabbreviated as TRPP (polycystin) (39). Each of the proteinsseems to be a cation channel composed of six transmembrane-spanningdomains and a conserved pore-forming region (Figure 2) (6,33,40).Most members of the TRPC have been characterized as Ca2+-permeablecation channels playing a role in Ca2+ signaling (41). The functionalcharacterization of other TRP members, including TRPV5 and TRPV6,and TRPM6 and TRPM7, has recently been started.
Figure 2. Structural organization of TRPV5/6 and TRPM6/7. TRPV5 and TRPV6 contain a cytosolic amino- and carboxyl-terminus containing ankyrin (ANK) repeats (A). TRPM6 and TRPM7 belong to the largest TRP channels consisting of approximately 2000 amino acids, including very large cytosolic amino- and carboxyl-termini including an atypical protein kinase domain (B). The six-transmembrane unit is one of four identical or homologous subunits presumed to surround the central pore (C). The gate and selectivity filter are formed by the four two-transmembrane domains (TM5–pore loop–TM6) facing the center of the channel. Cations are selected for permeation by the extracellular-facing pore loop, held in place by the TM5 and TM6 -helices.
TRPV5 and TRPV6
TRPV5 and TRPV6 belong to the TRPV subfamily. These homologueschannel proteins are composed of approximately 730 amino acids,whereas the corresponding genes consist of 15 exons juxtaposedon chromosome 7q35 (42–44). In human embryonic kidney293 (HEK293) cells heterogeneously expressing TRPV5 or TRPV6,currents can be activated under conditions of high intracellularbuffering of Ca2+. In addition, the current is increased byhyperpolarizing voltage steps, which enhances the driving forcefor Ca2+ (45,46). Outward currents are extremely small, indicatingthat these channels are inwardly rectifying (Table 1, Figure 3).TRPV5 and TRPV6 are subject to Ca2+-dependent feedback inhibition(46,47). Both channels rapidly inactivate during hyperpolarizingvoltage steps, and this inactivation is reduced when Ba2+ orSr2+ is used as a charge carrier, confirming the Ca2+ dependence(47). Currents also diminish during repetitive activation byshort hyperpolarizing pulses (46). TRPV5 and TRPV6 are so farthe only known highly Ca2+-selective channels in the TRP superfamily.It has been demonstrated that the molecular determinants ofthe Ca2+ selectivity and permeation of TRPV5 and TRPV6 resideat a single aspartate residue (TRPV5D542 [48,49] and TRPV6D541[50], respectively) present in the predicted pore-forming region.Neutralization of these negatively charged residues affectsthe high Ca2+ selectivity of these channels. Therefore, it wassuggested that Ca2+ selectivity in TRPV5 and TRPV6 depends ona ring of four aspartate residues in the channel pore, similarto the ring of four negative residues (aspartates and/or glutamates)in the pore of voltage-gated Ca2+ channels (48,50). Recently,the substituted cysteine accessibility method was used to mapthe pore region of TRPV5 (51) and TRPV6 (50). On the basis ofthe permeability of the TRPV6 channel to organic cations, apore diameter of 5.4 Å was estimated (50). Mutating TRPV6D541,a residue involved in high-affinity Ca2+ binding, altered theapparent pore diameter, indicating that this residue indeedlines the narrowest part of the pore (50).
Figure 3. Current-voltage relationship of TRP channels. Representative transmembrane currents in response to a voltage ramp (I–V relation) of TRPV5/6 and TRPM6/7 channels.
The renal expression profile of TRPV5 has been studied in greatdetail (Table 1). In different species, it was demonstratedthat TRPV5 co-localizes in the kidney with the other Ca2+ transportproteins, including calbindin-D28K and the extrusion proteinsPMCA1b and NCX1 in DCT2 and CNT, with the highest immunochemicalabundance in DCT2, and a gradual decrease along CNT (52,53).A minority of cells along CNT lacked immunopositive stainingfor TRPV5 and the other Ca2+-transporting proteins. These negativecells were identified as intercalated cells (52). Taken together,these findings suggest that the major sites of transcellularCa2+ transport are DCT2 and, probably to a lesser extent, CNT.Recently, Hoenderop et al. (54) generated TRPV5 null (TRPV5–/–)mice by genetic ablation of TRPV5 to investigate the functionof TRPV5 in renal and intestinal Ca2+ (re)absorption. It isinteresting that metabolic studies demonstrated that TRPV5–/–mice exhibit a robust calciuresis compared with wild-type (TRPV5+/+)littermates. Serum analysis showed that TRPV5–/–mice have normal plasma Ca2+ concentrations but significantlyelevated 1,25-(OH)2D3 levels (54). For locating the defectivesite of the Ca2+ reabsorption along the nephron, in vivo micropuncturestudies were performed. Collections of tubular fluid revealedunaffected Ca2+ reabsorption in TRPV5–/– mice upto the last surface loop of the late proximal tubule. In contrast,Ca2+ delivery to puncturing sites within DCT and CNT were significantlyenhanced in TRPV5–/– mice. It is interesting thatpolyuria and polydipsia were consistently observed in TRPV5–/–mice compared with control littermates. Polyuria facilitatesthe excretion of large quantities of Ca2+ by reducing the potentialrisk of Ca2+ precipitations. This hypercalciuria-induced polyuriahas been observed in humans and animal models (55,56). Furthermore,TRPV5–/– mice produced urine that was significantlymore acidic compared with TRPV5+/+ littermates. Acidificationof the urine is known to prevent renal stone formation in hypercalciuria,because the formation of Ca2+ precipitates is less likely atan acidic pH (57). A significant increase in the rate of Ca2+absorption was observed in TRPV5–/– mice comparedwith wild-type littermates, indicating a compensatory role ofthe small intestine. Expression studies using quantitative real-timePCR in TRPV5–/– mice demonstrated increased TRPV6and calbindin-D9K levels in duodenum consistent with Ca2+ hyperabsorption.
Immunohistochemical studies indicated that TRPV6, originallycloned from duodenum, is localized to the brush-border membraneof the small intestine. In enterocytes, TRPV6 is co-expressedwith calbindin-D9K and PMCA1b (58,59). It is interesting thatHediger and co-workers (60) studied the functional role of TRPV6in Ca2+ absorption by inactivation of the mouse TRPV6 gene.These TRPV6 null (TRPV6–/–) mice were placed ona Ca2+-deficient diet and subsequently challenged in a 45Ca2+absorption assay. TRPV6–/– mice showed a consistentdecrease in Ca2+ absorption over time. From these initial data,it was concluded that TRPV6–/– mice show a significantCa2+ malabsorption, suggesting that TRPV6 is indeed the rate-limitingstep in 1,25-OH2D3–dependent Ca2+ absorption (60). Recently,it was found that TRPV6 is expressed in the mouse kidney alongthe apical domain of the late portion of the DCT (DCT2) throughinner medullary collecting duct (61). TRPV6 co-localizes withTRPV5 and the other Ca2+ transport proteins in DCT2, suggestinga role in Ca2+ reabsorption. In addition, the protein is detectedin the intercalated cells and the inner medullary collectingduct that are not involved in transepithelial Ca2+ transport,pointing to additional functions of TRPV6. Thus, the preciserole of this epithelial Ca2+ channel in kidney remains to beestablished, but given the widespread distribution of TRPV6throughout the nephron segments, functions of TRPV6 could involveCa2+ reabsorption, Ca2+ signaling, and others. Detailed characterizationof the TRPV6–/– mice will address these importantquestions shortly. It is interesting that quantitative PCR measurementsindicated that the mouse prostate contains high expression levelsof TRPV6 (61). Although the exact function in this organ remainsto be elucidated, previous reports have suggested that TRPV6expression correlates with prostate carcinoma tumor grade (16,62,63).Together, these findings indicate that TRPV6 expression is associatedwith prostate cancer progression and, therefore, representsa prognostic marker and a promising target for new therapeuticstrategies to treat advanced prostate cancer (63). TRPV5 andTRPV6 share several functional properties, including the permeationprofile for monovalent and divalent cations, anomalous molefraction behavior, and Ca2+-dependent inactivation (47,64).However, detailed comparison of the amino- and carboxyl-terminiof the TRPV5 and TRPV6 channels illustrates significant differences,which may account for the unique electrophysiologic propertiesof these homologous channels (65). The initial inactivationis faster in TRPV6 compared with TRPV5, and the kinetic differencesbetween Ca2+ and Ba2+ currents are more pronounced for TRPV6than for TRPV5 (66,67). It is interesting that structural determinantsof these functional dissimilarities are not located in eitherthe amino- or carboxyl-terminus but in the TM2-TM3 linker (67).It is intriguing that TRPV5 has a 100-fold higher affinity forthe potent channel blocker ruthenium red than TRPV6 (65). Physiologicconsequences of these functional differences remain to be establishedand are of interest with respect to the structural organizationof these channels (66). Cross-linking studies, co-immunoprecipitations,and molecular mass determination of TRPV5/6 complexes usingsucrose gradient sedimentation showed that TRPV5 and TRPV6 formhomo- and heterotetrameric channel complexes (Figure 2C). Hetero-oligomerizationof TRPV5 and TRPV6 might influence the functional propertiesof the formed Ca2+ channel complex. As TRPV5 and TRPV6 exhibitdifferent channel kinetics with respect to Ca2+-dependent inactivationand Ba2+ selectivity and sensitivity for the inhibitor rutheniumred, the influence of the heterotetramer composition on thesechannel properties was investigated. Concatemers that consistedof four TRPV5 and/or TRPV6 subunits that were configured ina head-to-tail manner were constructed. A different ratio ofTRPV5 and TRPV6 subunits in these concatemers showed that thephenotype resembles the mixed properties of TRPV5 and TRPV6.An increased number of TRPV5 subunits in such a concatemer displayedmore TRPV5-like properties, indicating that the stoichiometryof TRPV5/6 heterotetramers influences the channel properties(66). Consequently, regulation of the relative expression levelsof TRPV5 and TRPV6 may be a mechanism to fine-tune the Ca2+transport kinetics in TRPV5/6–co-expressing tissues (68).It is interesting that Niemeyer and colleagues (69) identifiedthe third ankyrin (ANK) repeat as being a stringent requirementfor physical assembly of TRPV6 subunits. Deletion of this repeator mutation of critical residues within this repeat rendersnonfunctional channels that do not co-immunoprecipitate or formtetramers. It was proposed that the third ANK repeat initiatesa molecular zippering process that proceeds past the fifth ANKrepeat and creates an intracellular anchor that is necessaryfor functional subunit assembly (69).
Previous studies in animal and cell models demonstrated thatTRPV5 and TRPV6 channels are tightly regulated at the transcriptionallevel by various hormones, including 1,25-(OH)2D3, estradiol,androgen, and also dietary Ca2+ intake, which are describedin detail (6,70). Recently, the effect of PTH was studied onthe renal expression of TRPV5 (71). The parathyroid glands playa key role in maintaining the extracellular Ca2+ concentrationthrough their capacity to sense minute changes in the levelof blood Ca2+ (4). Early studies using micropuncture and cellpreparations demonstrated that PTH stimulates active Ca2+ reabsorptionin the distal part of the nephron via a dual signaling mechanisminvolving protein kinase A–and protein kinase C–dependentprocesses (7,8,72,73). Preliminary studies demonstrated thatparathyroidectomy in rats resulted in decreased serum PTH levelsand hypocalcemia, which were accompanied by decreased levelsof TRPV5, calbindin-D28K, and NCX1. Supplementation with PTHrestored serum Ca2+ concentrations and abundance of these Ca2+transporters in kidney. These data suggest that long-term treatmentwith PTH affects renal Ca2+ handling through the regulationof the expression of the Ca2+ transport proteins, includingTRPV5 (71). Promoter analysis should reveal the molecular mechanismof this PTH-mediated increase in TRPV5 expression. In addition,several regulatory proteins that interact with TRPV5 and/orTRPV6 have been identified, including calmodulin (74–76),S100A10-annexin 2 (58), and 80K-H (77) (Table 1). These newlyidentified associated proteins have facilitated the elucidationof important molecular pathways modulating transport activity.Calmodulin and 80K-H both have been implicated as Ca2+ sensors.Disturbance of the EF-hand structures in these proteins directlyaffects TRPV5/6 channel activity. Interaction of TRPV5/6 withthe S100A10-annexin 2 complex is critical for trafficking ofthese epithelial Ca2+ channels toward the plasma membrane.
TRPM6 and TRPM7
TRPM6 is a protein of approximately 2000 amino acids encodedby a large gene that contains 39 exons (28,29,33). TRPM6 showsapproximately 50% sequence homology with TRPM7, which formsa Ca2+- and Mg2+-permeable cation channel (38). Unlike othermembers of the TRP family, TRPM6 and TRPM7 contain long carboxyl-terminaldomains with similarity to the -kinases (Figure 2B) (35). Thecombination of channel and enzyme domains in TRPM6 and TRPM7is unique among known ion channels and raises intriguing questionsconcerning the function of the enzymatic domains and physiologicrole of these chanzymes. The identification of TRPM6 as thegene mutated in HSH represents the first case in which a humandisorder has been attributed to a channel kinase. However, theprecise function of this kinase domain remains to be established.To date, TRPM7 regulation has received most of the attention.TRPM7 is ubiquitously expressed and implicated in cellular Mg2+homeostasis, whereas TRPM6 has a more restricted expressionpattern predominantly present in absorbing epithelia (28,29,38).Although in HSH the defect at the level of the intestine isestablished, there is also evidence for impaired renal Mg2+reabsorption (28,32). The renal expression of TRPM6, in additionto the renal leak in patients with HSH, stresses the potentialimportant role of TRPM6 in renal Mg2+ reabsorption. In kidney,TRPM6 is expressed in DCT, known as the main site of activetranscellular Mg2+ reabsorption along the nephron (38). In linewith the expected function of being the gatekeeper of Mg2+ influx,TRPM6 was predominantly localized along the apical membraneof these immunopositive tubules. Immunohistochemical studiesof TRPM6 and NCC, which were used as specific markers for DCT,indicated a complete co-localization of these transport proteinsin the kidney (38). Until now, specific Mg2+-binding proteinshave not been identified, but it is interesting to mention thatthe Ca2+-binding proteins parvalbumin and calbindins also bindMg2+ (78). Importantly, TRPM6 co-localized with parvalbuminin DCT1 and with calbindin-D28K in DCT2 (38). In addition tothe DCT segment, Schlingmann et al. (28,38) reported the presenceof TRPM6 mRNA by nephron segment–specific PCR analysisin the proximal tubule, which was not confirmed by immunohistochemistry.In small intestine, absorptive epithelial cells stained positivelyfor TRPM6 detected by in situ hybridization and immunohistochemistry(28,38). In these cells, TRPM6 was localized along the brush-bordermembrane (38).
To functionally characterize TRPM6, the protein was heterogeneouslyexpressed in HEK293 cells. TRPM6-transfected HEK293 cells perfusedwith an extracellular solution that contained 1 mM Mg2+ or Ca2+exhibited characteristic outwardly rectifying currents uponestablishment of the whole-cell configuration as was demonstratedfor TRPM7 (Figure 3) (37,38,79). It is intriguing that at physiologicmembrane potentials of the DCT cell (–80 mV), small butsignificant inward currents were observed in TRPM6-expressingHEK293 cells with all tested divalent cations as the sole chargecarrier. However, mutations in TRPM6 are linked directly toHSH, emphasizing that this channel is an essential componentof the epithelial Mg2+ uptake machinery. It is possible thatthe TRPM6-mediated Mg2+ inward current is more pronounced innative DCT and intestinal cells as a result of specific co-factors,such as intracellular Mg2+ buffers, that are missing in overexpressionsystems such as HEK293 cells.
The unique permeation rank order determined from the inwardcurrent amplitude at –80 mV was comparable to TRPM7 (Ba2+ Ni2+ > Mg2+ > Ca2+) (35,38). Experiments using the Mg2+-sensitiveradiometric fluorescent dye Magfura-2 demonstrated a coherentrelationship between the applied extracellular Mg2+ concentrationand the measured intracellular Mg2+ level in TRPM6-expressingcells. Intracellular Mg2+ was elevated further when the plasmamembrane was hyperpolarized to the physiologic level of –80mV, consistent with influx through the TRPM6 channel. For evaluatingthe effect of intracellular Mg2+ on TRPM6 activity, the Mg2+concentration was altered directly in a spatially uniform mannerusing flash photolysis of the photolabile Mg2+ chelator DM-nitrophen.Elevation of intracellular Mg2+ by a flash of ultraviolet lightreduced the TRPM6-induced current, indicating that the channelis regulated by the intracellular concentration of this ion.Likewise, TRPM7 channel activity is strongly suppressed by Mg2+-ATPconcentrations in the millimolar range (37,80). Kozak and Cahalan(81) demonstrated that internal Mg2+ rather than ATP inhibitschannel activity.
Micropuncture studies have demonstrated that the luminal concentrationof free Mg2+ in DCT ranges from 0.2 to 0.7 mM (1). Because theCa2+ concentration is in the millimolar range, the apical Mg2+influx pathway should exhibit a higher affinity for Mg2+ thanfor Ca2+. It is interesting that dose-response curves for theNa+ current block at –80 mV indicated four times higherKD values for Ca2+ compared with Mg2+ (38). These data suggestthat the pore of the TRPM6 has a higher affinity for Mg2+ thanfor Ca2+. In this way, TRPM6 comprises a unique channel becauseall known Ca2+-permeable channels, including members of theTRP superfamily, generally display a 10 to 1000 times loweraffinity for Mg2+ than for Ca2+. It is interesting that HEK293cells transfected with the TRPM6 mutants identified in HSH patients(TRPM6Ser590X and TRPM6Arg736fsX737) displayed currents withsimilar amplitude and activation kinetics as nontransfectedHEK293 cells, indicating that these mutant proteins are nonfunctional,in line with the postulated function of TRPM6 being Mg2+ influxstep in epithelial Mg2+ transport (38). The observation thatTRPM7 conducts Mg2+ and is required for cell viability suggestedthat the TRPM7-mediated Mg2+ influx is essential for cellularMg2+ homeostasis rather than the extracellular Mg2+ homeostasis(37). It is interesting that Schmitz et al. (82) demonstratedthat Mg2+ supplementation of cells that lack TRPM7 expressionrescued growth arrest and cell lethality that was caused byTRPM7 inactivation (Table 1). Although TRPM7 is permeable forCa2+, as well as trace divalents such as Zn2+, Ni2+, Ba2+, andCo2+, supplementation with these cations was ineffective, indicatingthe specific effect of Mg2+ on these cellular processes.
Recently, it was postulated that TRPM6 requires assembly withTRPM7 to form functional channel complexes in the plasma membraneand that disruption of multimer formation by a mutated TRPM6variant, TRPM6S141L, results in HSH (83). In this study, TRPM6S141Lwas not directed to the cell surface by TRPM7 and failed tointeract with the coexpressed TRPM7. Remarkably, in contrastto TRPM7, Gudermann and co-workers (83) found that TRPM6 expressionin Xenopus oocytes and HEK293 cells did not entail significantion currents. In contrast, Voets et al. (38) measured significantlylarger currents in TRPM6-transfected HEK293 cells compared withcontrol cells. An explanation for this discrepancy might bethe existence of specific TRPM6 splice variants with differentfunctional properties. Chubanov et al. (83) demonstrated that5' rapid amplification of cDNA ends revealed three short alternative5' exons, called 1A, 1B, and 1C, that were found to be individuallyspliced onto exon 2, suggesting that the TRPM6 gene harborsa promoter with alternative transcription start sites. ThesecDNA have been named accordingly TRPM6a, TRPM6b, and TRPM6c,and additional functional measurements are needed to explainpossible biophysical differences.
A key question concerns the nature of mechanisms underlyingthe activation and regulation of TRPM6 and TRPM7. In particular,what is the function of the atypical protein -kinase domainlocated in the carboxyl terminus? -Kinases are a recently discoveredfamily of proteins that have no detectable sequence homologyto conventional protein kinases (84). To characterize the TRPM7kinase activity in vitro, we performed studies in which thecatalytic domain was expressed in bacteria (85). This kinaseis able to undergo autophosphorylation and to phosphorylatesubstrates such as myelin basic protein and histone H3 on serineand threonine residues. The kinase is specific for ATP and requiresMg2+ or Mn2+ for optimal activity. Clapham and co-workers (35)found that kinase activity is necessary for TRPM7 channel function.Although kinases have long been known to modulate ion channels,TRPM7 is unusual in that the channel has its own kinase. Futurestudies will address the question of whether the kinase, presentin TRPM6 and TRPM7, has specific cellular targets that mightmodulate ion channel activity and, therefore, the Mg2+ balance.
A tight coupling of the Ca2+ and Mg2+ balance is frequentlyobserved in patients and animal models (86,87). In hypomagnesemiawith secondary hypocalcemia, Simon et al. (21) proposed thatparacellin-1 is involved in controlling both the Ca2+ and Mg2+permeability of the paracellular pathway in TAL. Immunolocalizationstudies demonstrated that this tight junction protein is expressedin TAL. Defective paracellular Ca2+ and Mg2+ absorption by inactiveparacellin-1 explains the observed hypomagnesemia and hypercalciuriain patients with HHN (Table 2).
Table 2. Inherited disorders with mutual disturbance in Ca2+ and Mg2+ balancea
Mutations in the Ca2+-sensing receptor (CaSR) are also associatedwith disturbed Mg2+ handling (Table 2). Mutations in this receptorare identified in autosomal dominant hypoparathyroidism, whichis characterized by hypercalcemia and hypocalciuria (88). Hypomagnesemiais observed in up to half of the patients (89). Mutations inthe parathyroid and kidney CaSR result in a lower set pointfor plasma Ca2+ and Mg2+ on PTH secretion (86). Consequently,renal Ca2+ and Mg2+ reabsorption is suppressed, and the diseaseis characterized by inappropriately low serum PTH and increasedCa2+ and Mg2+ excretion. Furthermore, mutations in CaSR wereidentified in patients with hypercalcemic disorders of familialbenign (hypocalciuric) hypercalcemia and neonatal severe primaryhyperparathyroidism (90). Inactivation of the CaSR likely leadsto inappropriate reabsorption of Ca2+ and Mg2+ in the TAL (91)and Mg2+ transport in DCT (92). Therefore, renal excretion ofCa2+ and Mg2+ is reduced, which leads to hypercalcemia and insome cases hypermagnesemia (93). It should be noted that theCaSR plays a role in controlling renal Ca2+ and Mg2+ secretionindependent of its role in regulating PTH release. Recent studiesusing double knockout mice for CaSR and PTH showed a much widerrange of values for serum Ca2+ and renal excretion of Ca2+ thanthose in control (PTH–/–) littermates, despite theabsence of any circulating PTH (94,95).
Patients with mutations in TRPM6, the -subunit of the Na+-K+-ATPase,or NCC exhibit besides hypomagnesemia also hypocalciuria (Table 2).Expression of these affected genes is restricted to theDCT. However, an interaction between transcellular Ca2+ andMg2+ pathways in the distal part of the nephron is still unclear.There is limited overlap in expression between the Ca2+ transportproteins and TRPM6, -subunit, or NCC (38,53,96). It is interestingthat hypocalcemia in HSH patients can be corrected only by administrationof high dietary Mg2+ content. Several studies reported thatnormalization of the hypomagnesemia by dietary supplementationresulted in a prompt release of PTH and subsequent correctionof the hypocalcemia (97–99). These findings suggest thathypocalcemia in HSH is caused by a disturbance in PTH-mediatedCa2+ reabsorption. The factors that determine whether Mg2+ deficiencywill result in inhibition of PTH release, a lack of responseof the bone to PTH, or both remain to be clarified.
Gitelman syndrome in adults is characterized by consistent hypomagnesemia,hypocalciuria, and hypokalemic metabolic alkalosis (Table 2).The affected NCC gene results in loss of normal thiazide function,and the phenotype is identical to patients with chronic useof thiazide diuretics. Likewise, hypocalciuria is observed whenanimals receive long-term treatment with thiazides (100). Recently,it was postulated that thiazides induce hypovolemia, which stimulatesproximal electrolyte reabsorption, explaining the observed hypocalciuria(100). This finding is in line with the observation that thiazideexposure leads to structural degeneration of DCT, resultingin downregulation of Ca2+ transport proteins, arguing an increasedtranscellular Ca2+ transport in this segment (100,101). Thisincreased rate of apoptosis might reduce the absorptive surfacearea of the DCT in general and, therefore, could explain theobserved hypomagnesemia.
Furthermore, mutations in the -subunit of Na+-K+-ATPase arethe cause of IDH (Table 2). A cell model that hypothesized thatthe mutated -subunit manipulates the activity of the Na+,K+-ATPaseby disturbing routing of the total protein complex to the plasmamembrane has been proposed (25). As a consequence, the reducedintracellular K+ concentration, increased intracellular Na+concentration, or depolarization of the membrane may subsequentlylead to reduced Mg2+ influx through the apical TRPM6 channel,resulting in Mg2+ wasting (25). However, the exact molecularmechanism of decreased Mg2+ reabsorption and the associatedhypocalciuria remains to be elucidated.
Taken together, many diseases in which Ca2+ and Mg2+ disturbancesare linked have been reported (Table 2). In some cases, thereis an explanation for the mutual disorder in Ca2+ and Mg2+ handling,but in the majority of the diseases, the origin of this couplingis still unclear. Particularly, the limited overlap betweenthe Ca2+ transport (DCT2-CNT) and Mg2+ transport (DCT1-DCT2)machinery indicated that additional mechanisms might be involved.
This review has focused on the identification, function, andregulation of the Ca2+ and Mg2+ transport proteins. In the pastfew years, significant advances were achieved in our knowledgeabout the maintenance of the Ca2+ and Mg2+ balance. The identificationof the epithelial Ca2+ (TRPV5 and TRPV6) and Mg2+ (TRPM6 andTRPM7) channels provided insight in a new molecular conceptof Ca2+ and Mg2+ influx in specialized epithelia and other cellsystems in which these channels facilitate Ca2+ and Mg2+ transport.There are striking similarities between the characteristicsof the TRPV5/6 and TRPM6/7 channel pairs, such as expressionprofiling, structural organization, and function with respectto the maintenance the Ca2+ and Mg2+ balance (Table 1). To date,several studies have focused on the regulation of TRPV5 andTRPV6, whereas many questions remain to be investigated forTRPM6 and TRPM7. For instance, the hormonal regulation of theseMg2+ channels has not been studied yet. The next step is toclarify the cellular events in epithelial Ca2+ and Mg2+ transport.For instance, the mechanisms by the DCT cells to sense the extracellularCa2+ and Mg2+ concentration and appropriately adapt the transportrates are fertile areas for future research. The continued useof molecular and cell physiologic techniques to probe the constitutiveand congenital disturbances of Ca2+ and Mg2+ metabolism willincrease further our understanding of renal electrolyte transportand provide new insights into the way in which renal diseasesare diagnosed and managed.
Acknowledgments
This work was supported by the Dutch Organization of ScientificResearch (Zon-Mw 016.006.001, Zon-Mw 902.18.298, NWO-ALW 810.38.004,NWO-ALW 805-09.042, NWO-ALW 814-02.001, NWO 812-08.002), theStomach Liver Intestine Foundation (MWO 03-19), Human FrontiersScience Program (RGP32/2004), and the Dutch Kidney Foundation(C10.1881 and C03.6017).
Dai LJ, Ritchie G, Kerstan D, Kang HS, Cole DE, Quamme GA: Magnesium transport in the renal distal convoluted tubule. Physiol Rev 81: 51–84, 2001[Abstract/Free Full Text]
Hoenderop JG, Nilius B, Bindels RJ: Molecular mechanism of active Ca2+ reabsorption in the distal nephron. Annu Rev Physiol 64: 529–549, 2002[CrossRef][Medline]
Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC: Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 366: 575–580, 1993[CrossRef][Medline]
Brown EM, MacLeod RJ: Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 81: 239–297, 2001[Abstract/Free Full Text]
Suki WN: Calcium transport in the nephron. Am J Physiol (Lond) 237: F1–F6, 1979
Hoenderop JG, Nilius B, Bindels RJ: Molecular mechanisms of active Ca2+ reabsorption in the distal nephron. Annu Rev Physiol 64: 529–549, 2002
Bindels RJ, Hartog A, Timmermans J, Van Os CH: Active Ca2+ transport in primary cultures of rabbit kidney CCD: Stimulation by 1,25-dihydroxyvitamin D3 and PTH. Am J Physiol (Lond) 261: F799–F807, 1991
Friedman PA, Coutermarsh BA, Kennedy SM, Gesek FA: Parathyroid hormone stimulation of calcium transport is mediated by dual signaling mechanisms involving protein kinase A and protein kinase C. Endocrinology 137: 13–20, 1996[Abstract]
Van Cromphaut SJ, Dewerchin M, Hoenderop JG, Stockmans I, Van Herck E, Kato S, Bindels RJ, Collen D, Carmeliet P, Bouillon R, Carmeliet G: Duodenal calcium absorption in vitamin D receptor-knockout mice: Functional and molecular aspects. Proc Natl Acad Sci U S A 98: 13324–13329, 2001[Abstract/Free Full Text]
Hoenderop JG, Dardenne O, van Abel M, van der Kemp AW, Van Os CH, St-Arnaud R, Bindels RJ: Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3-1-hydroxylase knockout mice. FASEB J 16: 1398–1406, 2002[Abstract/Free Full Text]
Hoenderop JG, Muller D, Van Der Kemp AW, Hartog A, Suzuki M, Ishibashi K, Imai M, Sweep F, Willems PH, Van Os CH, Bindels RJ: Calcitriol controls the epithelial calcium channel in kidney. J Am Soc Nephrol 12: 1342–1349, 2001[Abstract/Free Full Text]
Friedman PA, Gesek FA: Vitamin D3 accelerates PTH-dependent calcium transport in distal convoluted tubule cells. Am J Physiol (Lond) 265: F300–F308, 1993
Gesek FA, Friedman PA: Calcitonin stimulates calcium transport in distal convoluted tubule cells. Am J Physiol (Lond) 264: F744–F751, 1993
Van Abel M, Hoenderop JG, Dardenne O, St Arnaud R, Van Os CH, Van Leeuwen HJ, Bindels RJ: 1,25-dihydroxyvitamin D3-independent stimulatory effect of estrogen on the expression of ECaC1 in the kidney. J Am Soc Nephrol 13: 2102–2109, 2002[Abstract/Free Full Text]
Van Cromphaut SJ, Rummens K, Stockmans I, Van Herck E, Dijcks FA, Ederveen AG, Carmeliet P, Verhaeghe J, Bouillon R, Carmeliet G: Intestinal calcium transporter genes are upregulated by estrogens and the reproductive cycle through vitamin D receptor-independent mechanisms. J Bone Miner Res 18: 1725–1736, 2003[CrossRef][Medline]
Peng JB, Zhuang L, Berger UV, Adam RM, Williams BJ, Brown EM, Hediger MA, Freeman MR: CaT1 expression correlates with tumor grade in prostate cancer. Biochem Biophys Res Commun 282: 729–734, 2001[CrossRef][Medline]
Quamme GA, Dirks JH: Magnesium transport in the nephron. Am J Physiol (Lond) 239: F393–F401, 1980
Quamme GA, Dirks JH: Intraluminal and contraluminal magnesium on magnesium and calcium transfer in the rat nephron. Am J Physiol (Lond) 238: F187–F198, 1980
Hoenderop JG, van der Kemp AW, Hartog A, van de Graaf SF, van Os CH, Willems PH, Bindels RJ: Molecular identification of the apical Ca2+ channel in 1, 25-dihydroxyvitamin D3-responsive epithelia. J Biol Chem 274: 8375–8378, 1999[Abstract/Free Full Text]
Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H, Brown EM, Hediger MA: Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J Biol Chem 274: 22739–22746, 1999[Abstract/Free Full Text]
Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP: Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 285: 103–106, 1999[Abstract/Free Full Text]
Nicholson JC, Jones CL, Powell HR, Walker RG, McCredie DA: Familial hypomagnesaemia—hypercalciuria leading to end-stage renal failure. Pediatr Nephrol 9: 74–76, 1995[CrossRef][Medline]
Torralbo A, Pina E, Portoles J, Sanchez-Fructuoso A, Barrientos A: Renal magnesium wasting with hypercalciuria, nephrocalcinosis and ocular disorders. Nephron 69: 472–475, 1995[Medline]
Meij IC, Koenderink JB, van Bokhoven H, Assink KF, Groenestege WT, de Pont JJ, Bindels RJ, Monnens LA, van den Heuvel LP, Knoers NV: Dominant isolated renal magnesium loss is caused by misrouting of the Na+,K+-ATPase gamma-subunit. Nat Genet 26: 265–266, 2000[CrossRef][Medline]
Simon DB, Nelson-Williams C, Bia MJ, Ellison D, Karet FE, Molina AM, Vaara I, Iwata F, Cushner HM, Koolen M, Gainza FJ, Gitleman HJ, Lifton RP: Gitelman’s variant of Bartter’s syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nat Genet 12: 24–30, 1996[CrossRef][Medline]
Walder RY, Shalev H, Brennan TM, Carmi R, Elbedour K, Scott DA, Hanauer A, Mark AL, Patil S, Stone EM, Sheffield VC: Familial hypomagnesemia maps to chromosome 9q, not to the X chromosome: Genetic linkage mapping and analysis of a balanced translocation breakpoint. Hum Mol Genet 6: 1491–1497, 1997[Abstract/Free Full Text]
Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H, Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S, Sassen M, Waldegger S, Seyberth HW, Konrad M: Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet 31: 166–170, 2002[CrossRef][Medline]
Paunier L, Radde IC, Kooh SW, Conen PE, Fraser D: Primary hypomagnesemia with secondary hypocalcemia in an infant. Pediatrics 41: 385–402, 1968[Abstract/Free Full Text]
Shalev H, Phillip M, Galil A, Carmi R, Landau D: Clinical presentation and outcome in primary familial hypomagnesaemia. Arch Intern Med 78: 127–130, 1998
Matzkin H, Lotan D, Boichis H: Primary hypomagnesemia with a probable double magnesium transport defect. Nephron 52: 83–86, 1989[Medline]
Clapham DE, Runnels LW, Strubing C: The TRP ion channel family. Nat Rev Neurosci 2: 387–396, 2001[Medline]
Montell C, Birnbaumer L, Flockerzi V, Bindels RJ, Bruford EA, Caterina MJ, Clapham D, Harteneck C, Heller S, Julius D, Kojima I, Mori Y, Penner R, Prawitt D, Scharenberg AM, Schultz G, Shimizu S, Zhu MX: A unified nomenclature for the superfamily of TRP cation channels. Mol Cell 9: 229–231, 2002[CrossRef][Medline]
Runnels LW, Yue L, Clapham DE: TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science 291: 1043–1047, 2001[Abstract/Free Full Text]
Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, Fleig A: LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature 411: 590–595, 2001[CrossRef][Medline]
Voets T, Nilius B, van der Kemp AW, Droogmans G, Bindels RJ, Hoenderop JG: TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. J Biol Chem 279: 19–25, 2004[Abstract/Free Full Text]
Montell C: Physiology, phylogeny, and functions of the TRP superfamily of cation channels. Sci STKE 2001: RE1, 2001
Clapham DE: TRP channels as cellular sensors. Nature 426: 517–524, 2003[CrossRef][Medline]
Minke B, Cook B: TRP channel proteins and signal transduction. Physiol Rev 82: 429–472, 2002[Abstract/Free Full Text]
Muller D, Hoenderop JG, Merkx GF, van Os CH, Bindels RJ: Gene structure and chromosomal mapping of human epithelial calcium channel. Biochem Biophys Res Commun 275: 47–52, 2000[CrossRef][Medline]
Peng JB, Brown EM, Hediger MA: Structural conservation of the genes encoding CaT1, CaT2, and related cation channels. Genomics 76: 99–109, 2001[CrossRef][Medline]
Weber K, Erben RG, Rump A, Adamski J: Gene structure and regulation of the murine epithelial calcium channels ECaC1 and 2. Biochem Biophys Res Commun 289: 1287–1294, 2001[CrossRef][Medline]
Hoenderop JG, Vennekens R, Muller D, Prenen J, Droogmans G, Bindels RJ, Nilius B: Function and expression of the epithelial Ca2+ channel family: Comparison of the mammalian epithelial Ca2+ channel 1 and 2. J Physiol (Lond) 537: 747–761, 2001[Abstract/Free Full Text]
Vennekens R, Hoenderop JG, Prenen J, Stuiver M, Willems PH, Droogmans G, Nilius B, Bindels RJ: Permeation and gating properties of the novel epithelial Ca2+ channel. J Biol Chem 275: 3963–3969, 2000[Abstract/Free Full Text]
Nilius B, Prenen J, Vennekens R, Hoenderop JG, Bindels RJ, Droogmans G: Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+. Cell Calcium 29: 417–428, 2001[CrossRef][Medline]
Nilius B, Vennekens R, Prenen J, Hoenderop JG, Droogmans G, Bindels RJ: The single pore residue D542 determines Ca2+ permeation and Mg2+ block of the epithelial Ca2+ channel. J Biol Chem 276: 1020–1025, 2001[Abstract/Free Full Text]
Jean K, Bernatchez G, Klein H, Garneau L, Sauvé R, Parent L: The role of aspartate residues in Ca2+ affinity and permeation of the distal ECaC1 channel. Am J Physiol (Lond) 282: C665–C672, 2002
Voets T, Janssens A, Droogmans G, Nilius B: Outer pore architecture of a Ca2+-selective TRP channel. J Biol Chem 279: 15223–15230, 2004[Abstract/Free Full Text]
Dodier Y, Banderali U, Klein H, Topalak O, Dafi O, Simoes M, Bernatchez G, Sauve R, Parent L: Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis. J Biol Chem 279: 6853–6862, 2004[Abstract/Free Full Text]
Hoenderop JG, Hartog A, Stuiver M, Doucet A, Willems PH, Bindels RJ: Localization of the epithelial Ca2+ channel in rabbit kidney and intestine. J Am Soc Nephrol 11: 1171–1178, 2000[Abstract/Free Full Text]
Loffing J, Loffing-Cueni D, Valderrabano V, Klausli L, Hebert SC, Rossier BC, Hoenderop JG, Bindels RJ, Kaissling B: Organization of the mouse distal nephron: Distributions of transcellular calcium and sodium transport pathways. Am J Physiol Renal Physiol 281: F1021–F1027, 2001[Abstract/Free Full Text]
Hoenderop JG, van Leeuwen JP, van der Eerden B, Kersten F, van der Kemp AW, Mérrliat A, Waarsing E, Rossier B, Vallon V, Hummler E, Bindels RJ: Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5. J Clin Invest 112: 1906–1914, 2003[CrossRef][Medline]
Frick KK, Bushinsky DA: Molecular mechanisms of primary hypercalciuria. J Am Soc Nephrol 14: 1082–1095, 2003[Free Full Text]
Miller LA, Stapleton FB: Urinary volume in children with urolithiasis. J Urol 141: 918–920, 1989[Medline]
Baumann JM: Stone prevention: Why so little progress? Urol Res 26: 77–81, 1998[CrossRef][Medline]
van de Graaf SF, Hoenderop JG, Gkika D, Lamers D, Prenen J, Rescher U, Gerke V, Staub O, Nilius B, Bindels RJ: Functional expression of the epithelial Ca2+ channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex. EMBO J 22: 1478–1487, 2003[CrossRef][Medline]
Zhuang L, Peng JB, Tou L, Takanaga H, Adam RM, Hediger MA, Freeman MR: Calcium-selective ion channel, CaT1, is apically localized in gastrointestinal tract epithelia and is aberrantly expressed in human malignancies. Lab Invest 82: 1755–1764, 2002[Medline]
Bianco S, Peng JB, Takanaga H, Kos CH, Crescenzi A, Brown EM, Hediger MA: Mice lacking the epithelial calcium channel CaT1 (TRPV6) show a deficiency in intestinal calcium absorption despite high plasma levels of 1,25-dihydroxy vitamin D. FASEB J 18: A706, 2004
Nijenhuis T, Hoenderop JG, van der Kemp AW, Bindels RJ: Localization and regulation of the epithelial Ca2+ channel TRPV6 in the kidney. J Am Soc Nephrol 14: 2731–2740, 2003[Abstract/Free Full Text]
Wissenbach U, Niemeyer BA, Fixemer T, Schneidewind A, Trost C, Cavalie A, Reus K, Meese E, Bonkhoff H, Flockerzi V: Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer. J Biol Chem 276: 19461–19468, 2001[Abstract/Free Full Text]
Fixemer T, Wissenbach U, Flockerzi V, Bonkhoff H: Expression of the Ca2+-selective cation channel TRPV6 in human prostate cancer: A novel prognostic marker for tumor progression. Oncogene 22: 7858–7861, 2003[CrossRef][Medline]
Vennekens R, Prenen J, Hoenderop JG, Bindels RJ, Droogmans G, Nilius B: Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells. J Physiol (Lond) 530: 183–191, 2001[Abstract/Free Full Text]
Hoenderop JGJ, Vennekens R, Müller D, Prenen J, Droogmans G, Bindels RJM, Nilius B: Function and expression of the epithelial Ca2+ channel family: Comparison of the epithelial Ca2+ channel 1 and 2. J Physiol (Lond) 537: 747–761, 2001
Hoenderop JG, Voets T, Hoefs S, Weidema AF, Prenen J, Nilius B, Bindels RJ: Homo- and heterotetrameric architecture of the epithelial Ca2+ channels, TRPV5 and TRPV6. EMBO J 22: 776–785, 2003[CrossRef][Medline]
Nilius B, Prenen J, Hoenderop JG, Vennekens R, Hoefs S, Weidema AF, Droogmans G, Bindels RJ: Fast and slow inactivation kinetics of the Ca2+ channels ECaC1 and ECaC2 (TRPV5 and 6): Role of the intracellular loop located between transmembrane segment 2 and 3. J Biol Chem 277: 30852–30858, 2002[Abstract/Free Full Text]
Song Y, Peng X, Porta A, Takanaga H, Peng JB, Hediger MA, Fleet JC, Christakos S: Calcium transporter 1 and epithelial calcium channel messenger ribonucleic acid are differentially regulated by 1,25 dihydroxyvitamin D3 in the intestine and kidney of mice. Endocrinology 144: 3885–3894, 2003[Abstract/Free Full Text]
Erler I, Hirnet D, Wissenbach U, Flockerzi V, Niemeyer BA: Ca2+-selective TRPV channel architecture and function require a specific ankyrin repeat. J Biol Chem 279: 34456–34463, 2004[Abstract/Free Full Text]
Peng JB, Brown EM, Hediger MA: Epithelial Ca2+ entry channels: Transcellular Ca2+ transport and beyond. J Physiol (Lond) 551[Suppl]: 729–740, 2003
van Abel M, Hoenderop JG, Van Leeuwen HJ, Bindels RJ: Down-regulation of calcium transporters in kidney and duodenum by the calcimimetic compound NPS R-467 [Abstract]. J Am Soc Nephrol 14: 459A–34463, 2003
Greger R, Lang F, Oberleithner H: Distal site of calcium reabsorption in the rat nephron. Pflugers Arch 374: 153–157, 1978[CrossRef][Medline]
Hoenderop JG, De Pont JJ, Bindels RJ, Willems PH: Hormone-stimulated Ca2+ reabsorption in rabbit kidney cortical collecting system is cAMP-independent and involves a phorbol ester-insensitive PKC isotype. Kidney Int 55: 225–233, 1999[CrossRef][Medline]
Niemeyer BA, Bergs C, Wissenbach U, Flockerzi V, Trost C: Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin. Proc Natl Acad Sci U S A 98: 3600–3605, 2001[Abstract/Free Full Text]
Lambers TT, Weidema AF, Nilius B, Hoenderop JG, Bindels RJ: Regulation of the mouse epithelial Ca2+ channel TRPV6, by the Ca2+-sensor calmodulin. J Biol Chem 279: 28855–28861, 2004[Abstract/Free Full Text]
Hirnet D, Olausson J, Fecher-Trost C, Bodding M, Nastainczyk W, Wissenbach U, Flockerzi V, Freichel M: The TRPV6 gene, cDNA and protein. Cell Calcium 33: 509–518, 2003[CrossRef][Medline]
Gkika D, Mahieu F, Nilius B, Hoenderop JG, Bindels RJ: 80K-H as a new Ca2+ sensor regulating the activity of the epithelial Ca2+ channel transient receptor potential cation channel V5 (TRPV5). J Biol Chem 279: 26351–26357, 2004[Abstract/Free Full Text]
Yang W, Lee HW, Hellinga H, Yang JJ: Structural analysis, identification, and design of calcium-binding sites in proteins. Proteins 47: 344–356, 2002[CrossRef][Medline]
Runnels LW, Yue L, Clapham DE: The TRPM7 channel is inactivated by PIP2 hydrolysis. Nat Cell Biol 4: 329–336, 2002
Top
Abstract
Introduction
-hydroxylase activity and consequently 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) synthesis to maintain serum Ca2+ and Mg2+ levels within a narrow physiologic range. 
-subunit of the Na+,K+-ATPase pump, which is predominantly expressed in the kidney, with the highest expression levels in DCT and medullary TAL (25). FXYD2 is mutated in patients with autosomal dominant renal hypomagnesemia associated with hypocalciuria (IDH; MIM 154020). Hypomagnesemia in these patients can be severe (<0.40 mM) and cause convulsions. Remarkably, in some affected individuals, there are no symptoms of Mg2+ deficiency except for chondrocalcinosis at adult age. The molecular mechanism for renal Mg2+ loss in this autosomal dominant type of primary hypomagnesemia remains to be elucidated. The third gene involved, SLC12A3, encodes the thiazide-sensitive sodium chloride co-transporter (NCC) in DCT and is mutated in patients with Gitelman syndrome (MIM 263800) (26). This autosomal recessive disorder is a frequent hereditary tubular disorder that affects renal Mg2+ handling, which is characterized by hypokalemia, hypomagnesemia, and hypocalciuria. Hypomagnesemia is found in most patients with Gitelman syndrome and is assumed to be secondary to the primary defect in NCC, but the mechanisms underlying hypomagnesemia are poorly understood. 
Ni2+ > Mg2+ > Ca2+) (35,38). Experiments using the Mg2+-sensitive radiometric fluorescent dye Magfura-2 demonstrated a coherent relationship between the applied extracellular Mg2+ concentration and the measured intracellular Mg2+ level in TRPM6-expressing cells. Intracellular Mg2+ was elevated further when the plasma membrane was hyperpolarized to the physiologic level of –80 mV, consistent with influx through the TRPM6 channel. For evaluating the effect of intracellular Mg2+ on TRPM6 activity, the Mg2+ concentration was altered directly in a spatially uniform manner using flash photolysis of the photolabile Mg2+ chelator DM-nitrophen. Elevation of intracellular Mg2+ by a flash of ultraviolet light reduced the TRPM6-induced current, indicating that the channel is regulated by the intracellular concentration of this ion. Likewise, TRPM7 channel activity is strongly suppressed by Mg2+-ATP concentrations in the millimolar range (37,80). Kozak and Cahalan (81) demonstrated that internal Mg2+ rather than ATP inhibits channel activity. 
