Mg2+ homeostasis: the balancing act of TRPM6.

REVIEW URRENT C OPINION

Mg2þ homeostasis: the balancing act of TRPM6 Jenny van der Wijst, Rene´ J.M. Bindels, and Joost G.J. Hoenderop

Purpose of review The tight control of blood magnesium (Mg2þ) levels is of central importance for numerous physiological processes. A persistent low Mg2þ status (hypomagnesemia) is associated with severe health risks and is involved in the pathogenesis of type 2 diabetes mellitus, osteoporosis, asthma, and heart and vascular diseases. The current view has expanded significantly as a result of the identification of novel genes and regulatory pathways involved in hypomagnesemic disorders. This review aims to give an up-to-date overview of transient receptor potential melastatin 6 (TRPM6) regulation and its role in the maintenance of Mg2þ homeostasis. Recent findings The epithelial Mg2þ channel TRPM6 is considered to be the Mg2þ entry pathway in the distal convoluted tubule of the kidney, where it functions as gatekeeper for controlling the body’s Mg2þ balance. Various factors and hormones contribute not only to the function, but also to the dysregulation of TRPM6, which has a substantial impact on renal Mg2þ handling. Recent genetic and molecular studies have further elucidated the signaling processes of epithelial Mg2þ transport, including their effect on the expression and function of TRPM6. Summary Knowledge of TRPM6 functioning is of vital importance to decipher its role in Mg2þ handling and will, in particular, provide a molecular basis for achieving a better understanding of Mg2þ mal(re)absorption and hence systemic Mg2þ balance. Keywords distal convoluted tubule, hypomagnesemia, magnesium channel, TRPM

INTRODUCTION 2þ

The tight control of plasma magnesium (Mg ) levels (0.7–1.1 mmol/l) occurs through the interplay between the intestine, which determines Mg2þ uptake, bones that store Mg2þ and the kidney, which regulates Mg2þ excretion. This is of central importance for various physiological processes, as Mg2þ functions as a cofactor in multiple reactions involved in protein synthesis, nucleic acid stability, neuromuscular excitability, and oxidative phosphorylation. Clinical manifestations of an altered Mg2þ balance are multifold. Hypermagnesemia can cause lethargy, confusion, and coma and extreme cases can result in cardiac arrest [1]. Symptoms of hypomagnesemia are cramps, tetany, seizures, and cardiac arrhythmias [1,2]. The occurrence of hypomagnesemia is linked to several conditions. First, it is observed in patients using drugs such as the immunosuppressants cyclosporine and tacrolimus [3], proton pump inhibitors such as omeprazole [4], and the anticancer drugs cetuximab and cisplatin [5,6]. Second, disturbances in the Mg2þ balance have been associated with diabetes mellitus, osteoporosis,

asthma, and heart and vascular disease [7–11]. Importantly, numerous inherited Mg2þ-wasting disorders have been published and genetic analysis of families with monogenetic forms of hypomagnesemia resulted in the identification of several genes. Next to the transient receptor potential melastatin 6 (TRPM6), these include SLC12A3 encoding the thiazide-sensitive Naþ/Cl – co-transporter NCC [12], the genes CLDN16 and CLDN19 that code for the tight-junction proteins claudin 16 and 19 [13,14], the g-subunit of the basolateral Naþ/KþATPase, encoded by the FXYD2 gene [15], EGF that codes for pro-epidermal growth factor (EGF) [16], CNNM2 encoding the basolateral cyclin M2 Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Correspondence to Joost G.J. Hoenderop, Department of Physiology (286), Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel: +31 24 3610580; e-mail: Joost. [email protected] Curr Opin Nephrol Hypertens 2014, 23:361–369 DOI:10.1097/01.mnh.0000447023.59346.ab

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Mineral metabolism

(CNNM2) [17], the apical voltage-gated Kþ channel Kv1.1, encoded by the KCNA1 gene [18], KCNJ10 encoding the basolateral inward rectifier Kþ channel Kir4.1 [19], HNF1B coding for the transcription factor hepatocyte nuclear factor 1B (HNF1B) [20], and recently mutations have been described in PCBD1, encoding hepatocyte nuclear factor 1 homeobox A (PCBD1) [21] (Fig. 1). Taken together, identification of these disease genes and the expression patterns of their encoded proteins support the vital role of the kidney in maintaining the Mg2þ balance. The major part of Mg2þ reabsorption in the kidney depends on passive, paracellular transport in the proximal tubule (10%) and the thick ascending limb of Henle’s loop (70%). The fine-tuning of the filtered Mg2þ occurs through an active, transcellular mechanism in the

KEY POINTS Control of TRPM6 function is key in the maintenance of the body’s Mg2þ balance. Screening of families with inherited forms of hypomagnesemia has led to the identification of new Mg2þ-related genes and their role in renal Mg2þ handling. Identification of the Mg2þ buffering and extrusion proteins will greatly improve the knowledge of the control of Mg2þ homeostasis. Clarifying the mechanisms causing drug-induced TRPM6 transcriptional regulation needs further investigation.

Na+

Mg2+

Cl+

NCC

Apical

TRPM6 TRPM7

TRPM6 PIP3

K+

Mg2+

Kv1.1

Mg2+

PIP2

–70 mV

ATP

Rac1 EGF

Cyclosporine: EGF Cisplatin: EGF Furosemide:

Akt

EGFR

TRPM6 TRPM6 TRPM6 HNF1B

FXYD2

PCBD1

PI3K IR CDK5 Insulin Na+

Mg2+ Mg2+

K+

?

ATP Na – K – ATPase ?

?

Na+

Kir4.1 Basolateral

CNNM2

K+

FIGURE 1. Overview of the regulatory pathways in the distal convoluted tubule (DCT) cell. Transient receptor potential melastatin 6 (TRPM6) channels, located in the luminal membrane, facilitate transport of Mg2þ from the pro-urine into the cell, which is primarily driven by the luminal membrane potential established by the voltage-gated Kþ channel Kv1.1. Epidermal growth factor (EGF) and insulin function as magnesiotropic hormones stimulating TRPM6 activity, through activation of the PI3K-AKT pathway. Insulin can also act on TRPM6 via CDK5 phosphorylation. Furthermore, TRPM6 channel activity can be modulated by PIP2 and by Mg-ATP levels. The expression of TRPM6 in the DCT is affected by treatment with furosemide, cyclosporine A, and cisplatin; the latter two were shown to also downregulate EGF levels. The Mg2þ buffering and extrusion systems are not yet known. CNNM2 is suggested as playing a role in the Mg2þ extrusion and can bind Mg-ATP, which might play a role in this process. Transcription factor HNF1B, together with its regulator PCBD1, are proposed to regulate the expression of FXYD2, encoding the g-subunit of the Naþ/Kþ-ATPase. 362


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Mg2R homeostasis: the balancing act of TRPM6 van der Wijst et al.

distal convoluted tubule (DCT), which therefore determines the final serum Mg2þ concentration as no Mg2þ is reabsorbed beyond this point. In the DCT, TRPM6 functions as the gatekeeper of apical Mg2þ entry, and regulation here has been the main focus of research in the past decade. This review provides an update on the regulatory mechanisms of TRPM6 involved in Mg2þ handling.

BASIS OF TRPM6 (PATHO)PHYSIOLOGY Mutations in the TRPM6 gene cause the autosomal recessive disorder hypomagnesemia with secondary hypocalcemia (HSH, OMIM: 602014) [22,23], which is characterized by extremely low serum Mg2þ levels (0.1–0.3 mmol/l) accompanied by low serum Ca2þ levels. This often results in severe muscular and neurologic complications from early infancy that can lead to neurologic damage or cardiac arrest if left untreated. Although most identified mutations are nonsense–splice site and frame shift mutations leading to a truncated and nonfunctional form of TRPM6–in the last decade several missense mutations have also been identified (Table 1) [23–33]. These mutations, located throughout TRPM6, can alter channel function (Table 1, Fig. 2). TRPM6 is specifically expressed in the colon and in the DCTs of the kidney where it is responsible for transcellular Mg2þ transport [30,34]. Hence, mutations result in impaired intestinal uptake and renal Mg2þ wasting. The significance of TRPM6 is supported by recent studies that demonstrate embryonic lethality in TRPM6 knockout mice because of an inability to control Mg2þ levels [35,36]. In addition, both groups showed that the heterozygous TRPM6 mice display a mild hypomagnesemia [35,36]. The limited number of TRPM6 knockout mice that were viable in Walder’s study had neural tube defects pointing toward a role of TRPM6 in embryonic development. Further investigation by generating conditional knockout mice with specific TRPM6 deletion in the kidney or other organs will be highly interesting in clarifying the physiological function of TRPM6 in adult mice.

THE STRUCTURE-FUNCTION BOND OF TRANSIENT RECEPTOR POTENTIAL MELASTATIN 6 The TRPM6 gene holds 39 exons that code for a protein of 2022 amino acids [22,23]. A variety of splice variants have been identified including three alternative first exons [24]. TRPM6 is predicted to share structural homology to other TRP channels, and shows highest homology to TRPM7. They are

composed of six membrane-spanning domains that make up the channel pore, and large intracellular amino-terminal and carboxy-terminal domains (Fig. 2) [37,38]. The unique feature is the protein kinase domain fused to the carboxy terminus [39,40], and regulation of this is discussed in the next paragraph. TRPM6 is a cation-selective channel with strong outward rectification. It is found to be constitutively open, but strongly regulated by intracellular Mg2þ levels [30]. In addition, the channel activity is potentiated by an acidic extracellular pH, and 2-aminoethoxydeiphenyl borate is found to increase TRPM6 currents [25,41]. A study by Xie et al. [42] indicated that PIP2 is required for TRPM6 channel function dependent on basic residues in the TRP domain and it has been recently shown that the phospholipid sphingosine inhibits TRPM6 activity by affecting the open probability of the channel [43]. The functional unit is thought to be a homotetramer or heterotetramer with TRPM7 [24,25], but the exact composition is unclear. Most importantly, the tetrameric structure seems to influence channel characteristics and pharmacology [25,44 ]. The combination of a channel moiety and a protein kinase domain has given TRPM6 the name ‘chanzyme’ [40]. A critical point of interest is the interrelationship between these domains and how the kinase domain is involved in controlling channel activity. To date, no major functional link has been identified, as truncation of the kinase domain or kinase inactivating mutations in TRPM6 or TRPM7 does not disrupt channel activity [45,46]. However, later studies revealed an indirect regulatory role in channel function. First, several interacting proteins of the TRPM6 kinase, including receptor for activated C-kinase 1 (RACK1) and prohibitin2 (PHB2/REA), have been identified that inhibit channel activity [46,47]. Second, ATP was found to regulate the kinase domain and thereby function as a modulator of TRPM6 and TRPM7 channels [44 ,48–51]. On one hand, Mg-ATP is suggested as a regulator of free Mg2þ that inhibits channel activity [52], while on the other hand, it has also been shown to act directly via binding to the kinase domain [49,50]. Interestingly, a recent study demonstrates that Mg-ATP sensitivity is dependent on the homomerization or heteromerization, in which TRPM6 kinase activity is critical in determining the functionality of a heteromeric TRPM6/7 complex [44 ]. Furthermore, Yu et al. [53] found that halide ions (chloride, bromide, iodide) inhibit TRPM7 in synergy with intracellular Mg2þ, and established that the Mg-ATP binding site in the kinase domain facilitates this inhibition. This mechanism remains to be investigated for TRPM6. Further


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Mineral metabolism Table 1. Table summarizing all mutations in the TRPM6 gene that were identified in patients suffering from hypomagnesemia with secondary hypocalcemia No

Mutation

Functional effect

Nucleotide

Amino acid

Channel

Kinase

References

X

–

[22,24,25]

–

[26]

1

166C > T

Arg56X

2

422C > T

Ser141Leu

[23]

3

469G > T

Glu157X

4

521T > G

Ile174Arg

[27]

5

668delA

Asp233fsX263

[26]

6

1010 þ 5G > C

Splicing

7

1060A > C

Thr354Pro

8

1134 þ 5G > C

Splicing

[27]

9

1196delC

Ala399fsX401

[28]

[23] –

[27]

10

1208-1G > A

Splicing

11

1280delA

His427fsX429

[26]

12

1308 þ 1G > A

Splicing

13

1437C > A

Tyr479X

[27]

14

1444-1G > T

Splicing

[29]

15

1450C > T

Arg484X

16

1769C > G

Ser590X

17

Del1796-1797

Pro599fsX609

18

2009 þ 1G > A

Splicing

19

2120G > A

Cys707Tyr

20

2123T > C

Leu708Pro

X

21

2207delG

Arg736fsX737

X

22

2391 þ 2T > G

Splicing

23

2537 – 2A > T

Splicing

24

2615A > G

Glu872Gly

25

2667 þ 1G > A

Splicing

26

2782C > T

Arg928X

27

del Ex 21

28

Del2831_2832insG

Ile944fsX959

29

Ins2999T

Ser1000fsX1016

30

3050C > G

Pro1017Arg

X

31

3158A > G

Ty1053Cys

X

32

3209-68A > G

Splicing

33

del Ex 22 þ 23

34

3428T > C

X

[22,26] [26]

[23] X

[22,30] [26] [23] – –

[27] [31] [22,30] [31] [26]

X

–

[31] [22]

X

[26] [26] [26] [32] [33] –

[31] [23] [26]

Leu1143Pro

X

–

[31]

35

3537-1G > A

Splicing

36

3556C > T

Gln1186X

[22] [29]

37

3779-91del

Glu1260fsX1283

[22]

38

4577G > A

Trp1526X

[28]

39

del Ex 25–27

40

del Ex 26

Arg1533X

41

4988A > G

Gln1663Arg

–

42

5017-18delT

Leu1673fsX1675

X

43

del Ex 31 þ 32

44

5057 þ 2T > G

Splicing

45

5084-2A > G

Splicing

46

5261G > A

Ser1754Asn

47

5775A > G

Splicing

[26] X

[26] –

[31] [26] [26] [26] [31]

X

X

[31] [26]

X means that the function of the encoded protein is disrupted. – indicates that there is no effect of the mutation on protein function. Other mutations have not been investigated.

364





31 32

(a)

1

2

21

3 25

4

31 32 5

6

27 35 37

20

13 15

Extracellular

Intracellular

17

7

42

41 39

47

4

Kina se

2 3 N 1

dom ain

C

(b)

Extracellular

1 62 5 4 3

Intracellular

N

C N

N

C

N

C

C

FIGURE 2. (a) Schematic representation of a TRPM6 subunit, which comprises six transmembrane segments and intracellular amino (N)-terminal and carboxy (C)-terminal domains. The missense mutations are depicted in dark grey, according to the table numbering. The mutations leading to a stop codon resulting in a truncated protein product are shown in light grey. (b) Overview of a tetrameric structure of TRPM6 consisting of four subunits that construct a functional channel. The pore region is formed by transmembrane segments 5 and 6.

work is also required to understand how TRPM6 is expected to function in a physiological setting.

TRANSIENT RECEPTOR POTENTIAL MELASTATIN 6 AND ITS REGULATORY AFFAIRS Renal Mg2þ excretion is correlated to dietary Mg2þ intake; mice fed a Mg2þ-restricted diet display renal Mg2þ conservation, whereas a Mg2þ-enriched diet has the opposite effect [54]. The Mg2þ-deficient diet resulted in significantly increased renal TRPM6 abundance, an observation confirmed by a recent study of van Angelen et al. [55]. They also demonstrate that dietary Mg2þ restriction in mice affects renal Ca2þ handling, in line with the hypocalcemia observed in patients suffering from HSH. The Ca2þ-related proteins transient receptor potential

vanilloid 5 (TRPV5) and calbindin-D28k were significantly downregulated [55]. In the past decade, several hormones have been implicated in renal Mg2þ handling including EGF, estrogen, and insulin. Importantly, EGF and estrogen were termed magnesiotropic hormones considering their effect on both TRPM6 expression and channel function [16,47,54,56,57]. The effect of EGF is supported by studies demonstrating that patients treated with the anticancer drug cetuximab develop hypomagnesemia, owing to antagonizing the EGF-stimulated TRPM6 channel activity [16,58,59]. Regarding insulin, there is a growing body of evidence correlating a disturbed Mg2þ balance to insulin resistance and type 2 diabetes mellitus [60–62], also reviewed in [63]. In addition, an increased TRPM6 expression has been observed in diabetic rats, a phenomenon that can be reversed by insulin



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Mineral metabolism

administration [64]. A recent study investigated the role of two nonsynonymous polymorphisms in TRPM6 (Ile1393Val and Lys1584Glu) that were identified in relation to the risk of gestational type 2 diabetes mellitus in women with low Mg2þ intake [65,66]. They demonstrated that insulin stimulates TRPM6 activity via increased plasma membrane abundance, which is mediated by the same signaling pathway as EGF stimulation (Fig. 1). This is hampered in the genetic variants of TRPM6 [66]. Furthermore, the authors showed an association of the TRPM6 polymorphisms with the total glycosylated hemoglobin level, a measure of insulin resistance, in pregnant women [66]. A recent study provided evidence for additional receptor-activated signaling of TRPM6. Here, an inhibitory effect of the P2X4 purinergic receptor on TRPM6 channel function was demonstrated. Interestingly, the ATP-insensitive P2X4 mutant did not affect TRPM6, indicating that the inhibition of TRPM6 is dependent on P2X4 downstream signaling [67]. In addition to hormonal regulation of TRPM6, studies from the last few years have given more insights into mechanisms of various drug-induced Mg2þ deficiencies (Fig. 1). First, furosemide has been associated with renal Mg2þ wasting, but this may vary upon the patient’s Mg2þ intake and Mg2þ balance [68–70]. A recent study showed that mice do not develop hypomagnesemia upon furosemide treatment, hypothesizing that the observed increased TRPM6 expression in DCT compensates for potential diminished Mg2þ reabsorption in earlier segments of the kidney [71]. This would highlight the immense capacity of the DCT and its importance in the maintenance of Mg2þ balance. Second, several case reports have linked the use of proton pump inhibitors to hypomagnesemia (reviewed in [72]), suggested as resulting from intestinal malabsorption of Mg2þ. Recently, Lameris et al. [73] revealed that mice treated with the proton pump inhibitor omeprazole display increased expression of TRPM6 in the colon. The current model proposes this is a compensatory mechanism for the reduced TRPM6 channel activity, which results from decreased proton extrusion by the omeprazole-inhibited colonic Hþ,Kþ-ATPase [73]. Third, renal Mg2þ wasting is a known side-effect of the use of immunosuppressant drugs such as cyclosporine A and tacrolimus [74,75]. A previous study in tacrolimus-treated rats demonstrated increased Mg2þ excretion together with significantly diminished levels of TRPM6 mRNA in the kidney [76]. Furthermore, two recent studies link the cyclosporineinduced hypomagnesemia with hampered EGF signaling toward TRPM6 [77,78 ]. In addition, &

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Gouadon et al. [79] used Caco2 cells to determine differential effects of cyclosporine and tacrolimus on Mg2þ influx without altering expression of TRPM6 or other Mg2þ transporters. Fourth, in 1979, hypomagnesemia was established as a serious side-effect in patients receiving cisplatin chemotherapy [6]. Previous studies mainly highlighted the nephrotoxicity as a consequence of cisplatin accumulation in the kidney, but the mono-symptotic Mg2þ loss is interesting and points toward a direct effect on the Mg2þ-reabsorbing epithelia. Last year, two animal studies reported on the mechanism explaining cisplatin-induced hypomagnesemia [78 ,80]. On one hand, the study in mice showed a significant decrease in TRPM6 expression, but also other DCT markers (NCC and parvalbumin) were markedly reduced, thereby suggesting that cisplatin induces cell death in the DCT or particularly affects this nephron segment [80]. On the other hand, Ledeganck et al. [77] present a specific downregulation of EGF and TRPM6 in the rat kidney upon cisplatin treatment. They indicate EGF as the key component of Mg2þ loss through its influence on expression and activity of TRPM6. &

BEYOND TRANSIENT RECEPTOR POTENTIAL MELASTATIN 6 IN THE DISTAL CONVOLUTED TUBULE As is evident from the aforementioned sections, TRPM6 constitutes the Mg2þ entry pathway in the DCT. However, the mechanism of basolateral Mg2þ extrusion is unknown, although it is speculated to occur through exchange with Naþ [81]. However, extrusion via Naþ-independent mechanisms has also been suggested. Romani et al. [82] provide a list of putative Mg2þ extrusion proteins in different cell types. Two recently studied examples are the solute carrier family 41 member 1 (SLC41A1) and the mitochondrial RNA splicing 2 (MRS2), which could be involved in subcellular Mg2þ transport [83–86]. However, future work should reveal whether these function as extrusion proteins in the DCT. Furthermore, mutations found in CNNM2 related to hypomagnesemia, together with its observed Mg2þ sensitivity and the basolateral localization in the DCT, imply an involvement in basolateral Mg2þ extrusion [17,87 ] (Fig. 1). In addition, a zebrafish model has recently been used to knockdown CCNM2, which resulted in a phenotype related to disturbances in brain development, neurological behavior, and Mg2þ management that has also been observed in patients suffering from hypomagnesemia [87 ]. Besides Mg2þ extrusion, it is unknown whether intracellular Mg2þ is buffered in the renal cells and &&

&&




possible chaperones are also speculative. Even though the intracellular Mg2þ concentration is not known to fluctuate, a chaperone could be beneficial in increasing cellular Mg2þ diffusion; it can increase the rate of diffusion from apical to basolateral, as well as relieve TRPM6 from its Mg2þ-inhibited state. So far, ATP constitutes the most abundant cellular moiety [88], but other phosphonucleotides, and possibly Ca2þ-binding proteins such as S100 [89] and calmodulin [90], can add to the cellular Mg2þ homeostasis. Another interesting candidate is parvalbumin, as it conspicuously colocalizes with TRPM6 in the DCT and is characterized by its binding affinities for Ca2þ and Mg2þ [30,91]. Although the parvalbumin knockout mice do not display hypomagnesemia, a recent animal study by van Angelen et al. [55] demonstrated a marked increase in parvalbumin levels as a result of dietary Mg2þ restriction. This is confirmed in a microarray study of isolated DCT cells, which aimed to identify Mg2þ-sensitive genes by comparing high-Mgþ versus low-Mg2þ diets [92 ]. Furthermore, this latter study underlines the opportunities of the newly established COPAS (Complex Parametric Analyser and Sorter), which allows sorting and subsequent culture of primary DCT cells. A large number of new genes were identified that are potentially involved in epithelial Mg2þ transport, with a main focus on SLC41A3, TBC1D4, and Umod [92 ]. Future studies should clarify their role in renal Mg2þ handling. &

&

CONCLUSION Recent advances have increased our knowledge of the molecular regulation of TRPM6. Numerous factors and hormones control the channel at the level of transcription, membrane expression, and function. However, it is clear that several proteins involved in DCT-mediated Mg2þ reabsorption remain to be identified. Fortunately, the expression profile of this nephron segment, termed DCT transcriptome, which has recently been published [92 ], will advance the discovery of new Mg2þ-related proteins. This will ultimately provide further understanding of transepithelial Mg2þ transport, and contribute to the diagnosis and treatment of hypomagnesemia and associated diseases. &

Acknowledgements This work was supported by grants from the Netherlands Organization for Scientific Research (ZonMw 9120.8026, NWO ALW 818.02.001), a NWO Veni grant for J. van der Wijst (863.13.010), a NWO Vici grant for J.G.J. Hoenderop (016.130.668), and EURenOmics funding from the European Union seventh

Framework Program (FP7/2007–2013, agreement no. 305608). Conflicts of interest There are no conflicts of interest.

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Mineral metabolism 26. Schlingmann KP, Sassen MC, Weber S, et al. Novel TRPM6 mutations in 21 families with primary hypomagnesemia and secondary hypocalcemia. J Am Soc Nephrol 2005; 16:3061–3069. 27. Jalkanen R, Pronicka E, Tyynismaa H, et al. Genetic background of HSH in three Polish families and a patient with an X;9 translocation. Eur J Hum Genet 2006; 14:55–62. 28. Zhao Z, Pei Y, Huang X, et al. Novel TRPM6 mutations in familial hypomagnesemia with secondary hypocalcemia. Am J Nephrol 2013; 37:541–548. 29. Guran T, Akcay T, Bereket A, et al. Clinical and molecular characterization of Turkish patients with familial hypomagnesaemia: novel mutations in TRPM6 and CLDN16 genes. Nephrol Dial Transplant 2012; 27:667–673. 30. Voets T, Nilius B, Hoefs S, et al. TRPM6 forms the Mg2þ influx channel involved in intestinal and renal Mg2þ absorption. J Biol Chem 2004; 279:19– 25. 31. Lainez S, Schlingmann KP, van der Wijst J, et al. New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia. Eur J Hum Genet 2014; 22:497–504. 32. Habeb AM, Al-Harbi H, Schlingmann KP. Resolving basal ganglia calcification in hereditary hypomagnesemia with secondary hypocalcemia due to a novel TRPM6 gene mutation. Saudi J Kidney Dis Transpl 2012; 23:1038–1042. 33. Chubanov V, Schlingmann KP, Wa¨ring J, et al. Hypomagnesemia with secondary hypocalcemia due to a missense mutation in the putative poreforming region of TRPM6. J Biol Chem 2007; 282:7656–7667. 34. Dai LJ, Ritchie G, Kerstan D, et al. Magnesium transport in the renal distal convoluted tubule. Physiol Rev 2001; 81:51–84. 35. Walder RY, Yang B, Stokes JB, et al. Mice defective in Trpm6 show embryonic mortality and neural tube defects. Hum Mol Genet 2009; 18:4367–4375. 36. Woudenberg-Vrenken TE, Sukinta A, van der Kemp AW, et al. Transient receptor potential melastatin 6 knockout mice are lethal whereas heterozygous deletion results in mild hypomagnesemia. Nephron Physiol 2011; 117:11–19. 37. Schaefer M. Homo- and heteromeric assembly of TRP channel subunits. Pflugers Arch 2005; 451:35–42. 38. Ramsey IS, Delling M, Clapham DE. An introduction to TRP channels. Annu Rev Physiol 2006; 68:619–647. 39. Runnels LW, Yue L, Clapham DE. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science 2001; 291:1043–1047. 40. Montell C. Mg2þ homeostasis: the Mg2þnificent TRPM chanzymes. Curr Biol 2003; 13:R799–R801. 41. Li M, Du J, Jiang J, et al. Molecular determinants of Mg2þ and Ca2þ permeability and pH sensitivity in TRPM6 and TRPM7. J Biol Chem 2007; 282:25817–25830. 42. Xie J, Sun B, Du J, et al. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) controls magnesium gatekeeper TRPM6 activity. Sci Rep 2011; 1:146. 43. Qin X, Yue Z, Sun B, et al. Sphingosine and FTY720 are potent inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels. Br J Pharmacol 2013; 168:1294–1312. 44. Zhang Z, Yu H, Huang J, et al. The TRPM6 kinase domain determines the Mg && ATP-sensitivity of TRPM7/M6 heteromeric ion channels. J Biol Chem 2014; 289:5217–5227. doi:10.1074/jbc.M113.512285. [Epub ahead of print] TRPM6 can form heteromeric complexes with TRPM7, which gives rise to altered channel characteristics. This study now includes the role of the kinase domains in modulating the homomeric or heteromeric channel complexes. Their findings suggest that the TRPM6 kinase directs the phenotype of the complex, likely by cross-phosphorylation of TRPM7. 45. Matsushita M, Kozak JA, Shimizu Y, et al. Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPM7/ChaK1. J Biol Chem 2005; 280:20793–20803. 46. Cao G, Thebault S, van der Wijst J, et al. RACK1 inhibits TRPM6 activity via phosphorylation of the fused alpha-kinase domain. Curr Biol 2008; 18:168– 176. 47. Cao G, van der Wijst J, van der Kemp A, et al. Regulation of the epithelial Mg2þ channel TRPM6 by estrogen and the associated repressor protein of estrogen receptor activity (REA). J Biol Chem 2009; 284:14788–14795. 48. Nadler MJ, Hermosura MC, Inabe K, et al. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature 2001; 411:590–595. 49. Demeuse P, Penner R, Fleig A. TRPM7 channel is regulated by magnesium nucleotides via its kinase domain. J Gen Physiol 2006; 127:421–434. 50. Thebault S, Cao G, Venselaar H, et al. Role of the alpha-kinase domain in transient receptor potential melastatin 6 channel and regulation by intracellular ATP. J Biol Chem 2008; 283:19999–20007. 51. Gwanyanya A, Sipido KR, Vereecke J, Mubagwa K. ATP and PIP2 dependence of the magnesium-inhibited, TRPM7-like cation channel in cardiac myocytes. Am J Physiol Cell Physiol 2006; 291:C627–C635. 52. Kozak JA, Cahalan MD. MIC channels are inhibited by internal divalent cations but not ATP. Biophys J 2003; 84:922–927. 53. Yu H, Zhang Z, Lis A, et al. TRPM7 is regulated by halides through its kinase domain. Cell Mol Life Sci 2013; 70:2757–2771. 54. Groenestege WMT, Hoenderop JG, van den Heuvel L, et al. The epithelial Mg2þ channel transient receptor potential melastatin 6 is regulated by dietary Mg2þ content and estrogens. J Am Soc Nephrol 2006; 17:1035–1043. 55. van Angelen AA, San-Cristobal P, Pulskens WP, et al. The impact of dietary magnesium restriction on magnesiotropic and calciotropic genes. Nephrol Dial Transplant 2013; 28:2983–2993.

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56. Thebault S, Alexander RT, Tiel Groenestege WM, et al. EGF increases TRPM6 activity and surface expression. J Am Soc Nephrol 2009; 20:78–85. 57. Ikari A, Sanada A, Okude C, et al. Up-regulation of TRPM6 transcriptional activity by AP-1 in renal epithelial cells. J Cell Physiol 2010; 222:481–487. 58. Tejpar S, Piessevaux H, Claes K, et al. Magnesium wasting associated with epidermal-growth-factor receptor-targeting antibodies in colorectal cancer: a prospective study. Lancet Oncol 2007; 8:387–394. 59. Fakih MG, Wilding G, Lombardo J. Cetuximab-induced hypomagnesemia in patients with colorectal cancer. Clin Colorectal Cancer 2006; 6:152–156. 60. Barbagallo M, Dominguez LJ, Resnick LM. Magnesium metabolism in hypertension and type 2 diabetes mellitus. Am J Ther 2007; 14:375–385. 61. Barbagallo M, Dominguez L J, Galioto A, et al. Role of magnesium in insulin action, diabetes and cardio-metabolic syndrome X. Mol Aspects Med 2003; 24:39–52. 62. Song Y, Manson JE, Buring JE, Liu S. Dietary magnesium intake in relation to plasma insulin levels and risk of type 2 diabetes in women. Diabetes Care 2004; 27:59–65. 63. Hruby A, McKeown NM, Song Y, Djousse´ L. Dietary magnesium and genetic interactions in diabetes and related risk factors: a brief overview of current knowledge. Nutrients 2013; 5:4990–5011. 64. Lee C-T, Lien Y-HH, Lai L-W, et al. Increased renal calcium and magnesium transporter abundance in streptozotocin-induced diabetes mellitus. Kidney Int 2006; 69:1786–1791. 65. Song Y, Hsu Y-H, Niu T, et al. Common genetic variants of the ion channel transient receptor potential membrane melastatin 6 and 7 (TRPM6 and TRPM7), magnesium intake, and risk of type 2 diabetes in women. BMC Med Genet 2009; 10:4. 66. Nair AV, Hocher B, Verkaart S, et al. Loss of insulin-induced activation of TRPM6 magnesium channels results in impaired glucose tolerance during pregnancy. Proc Natl Acad Sci U S A 2012; 109:11324–11329. 67. de Baaij JHF, Blanchard MG, Lavrijsen M, et al. P2X4 receptor regulation of transient receptor potential melastatin type 6 (TRPM6) Mg(2þ) channels. Pflugers Arch 2014. doi:10.1007/s00424-014-1440-3. [Epub ahead of print] 68. Duarte CG. Effects of ethacrynic acid and furosemide on urinary calcium, phosphate and magnesium. Metab Clin Exp 1968; 17:867–876. 69. Cohen N, Almoznino-Sarafian D, Zaidenstein R, et al. Serum magnesium aberrations in furosemide (frusemide) treated patients with congestive heart failure: pathophysiological correlates and prognostic evaluation. Heart 2003; 89:411–416. 70. Davies DL, Fraser R. Do diuretics cause magnesium deficiency? Br J Clin Pharmacol 1993; 36:1–10. 71. van Angelen AA, van der Kemp AW, Hoenderop JG, Bindels RJ. Increased expression of renal TRPM6 compensates for Mg2þ wasting during furosemide treatment. Clin Kidney J 2012; 5:535–544. 72. Hess MW, Hoenderop JGJ, Bindels RJM, Drenth JPH. Systematic review: hypomagnesaemia induced by proton pump inhibition. Aliment Pharmacol Ther 2012; 36:405–413. 73. Lameris ALL, Hess MW, van Kruijsbergen I, et al. Omeprazole enhances the colonic expression of the Mg(2þ) transporter TRPM6. Pflugers Arch 2013; 465:1613–1620. 74. Barton CH, Vaziri ND, Martin DC, et al. Hypomagnesemia and renal magnesium wasting in renal transplant recipients receiving cyclosporine. Am J Med 1987; 83:693–699. 75. McDiarmid SV, Colonna JO, Shaked A, et al. A comparison of renal function in cyclosporine- and FK-506-treated patients after primary orthotopic liver transplantation. Transplantation 1993; 56:847–853. 76. Nijenhuis T, Hoenderop JGJ, Bindels RJM. Downregulation of Ca(2þ) and Mg(2þ) transport proteins in the kidney explains tacrolimus (FK506)-induced hypercalciuria and hypomagnesemia. J Am Soc Nephrol 2004; 15:549–557. 77. Ledeganck KJ, Boulet GA, Horvath CA, et al. Expression of renal distal tubule transporters TRPM6 and NCC in a rat model of cyclosporine nephrotoxicity and effect of EGF treatment. 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Mg2R homeostasis: the balancing act of TRPM6 van der Wijst et al. 83. Hurd TW, Otto EA, Mishima E, et al. Mutation of the Mg2þ transporter SLC41A1 results in a nephronophthisis-like phenotype. J Am Soc Nephrol 2013; 24:967–977. 84. Kolisek M, Nestler A, Vormann J, Schweigel-Ro¨ntgen M. Human gene SLC41A1 encodes for the Naþ/Mg2þ exchanger. Am J Physiol Cell Physiol 2012; 302:C318–C326. 85. Piskacek M, Zotova L, Zsurka G, Schweyen RJ. Conditional knockdown of hMRS2 results in loss of mitochondrial Mg(2þ) uptake and cell death. J Cell Mol Med 2009; 13:693–700. 86. Shindo Y, Fujii T, Komatsu H, et al. Newly developed Mg2þ-selective fluorescent probe enables visualization of Mg2þ dynamics in mitochondria. PLoS ONE 2011; 6:e23684. 87. Arjona FJ, de Baaij JHF, Schlingmann KP, et al. CNNM2 mutations cause && impaired brain development and seizures in patients with hypomagnesemia. PLoS Genet 2014; 10:e1004267. This study describes the identification of new mutations in the CNNM2 gene associated with hypomagnesemia, mental retardation, and seizures. They established a zebrafish knockdown model in which CNNM2 loss-of-function resulted in impaired brain development and neurological disorders, together with a disturbed Mg2þ balance. Furthermore, stable Mg2þ isotopes were used to measure increased Mg2þ uptake in cells expressing wild type CNNM2, compared with nontransfected or mutant CNNM2-transfected cells.

88. Cittadini A, Scarpa A. Intracellular Mg2þ homeostasis of Ehrlich ascites tumor cells. Arch Biochem Biophys 1983; 227:202–209. 89. Ogoma Y, Kobayashi H, Fujii T, et al. Binding study of metal ions to S100 protein: 43Ca, 25Mg, 67Zn and 39K n.m.r. Int J Biol Macromol 1992; 14:279–286. 90. Ohki S, Ikura M, Zhang M. Identification of Mg2þ-binding sites and the role of Mg2þ on target recognition by calmodulin. Biochemistry 1997; 36:4309– 4316. 91. Schwaller B. Cytosolic Ca2þ buffers. Cold Spring Harb Perspect Biol 2010; 2:a004051–a14051. 92. de Baaij JHF, Groot Koerkamp MJ, Lavrijsen M, et al. Elucidation of the distal & convoluted tubule transcriptome identifies new candidate genes involved in renal Mg(2þ) handling. Am J Physiol Renal Physiol 2013; 305:F1563– F1573. Parvalbumin-GFP (green fluorescent protein) transgenic mice were subjected to a high-Mg2þ and low-Mg2þ diet, and the use of the COPAS allows specific sampling of the GFP-positive DCT cells that were subsequently prepared for microarray analysis. This resulted in the identification of genes that are regulated by the Mg2þ diet and therefore could play a role in renal Mg2þ handling. Future research on these genes in the so-called DCT transcriptome should establish their role in the Mg2þ reabsorption process.



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The balancing act.

The balancing act.

NAD(+) Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus.

Thrombosis: Balancing act.

7 channels from inhibition by cytosolic Mg2+ and Mg·ATP.

The balancing act of GABAergic synapse organizers.

A balancing act times two: sensing and regulating cell envelope homeostasis in Bacillus subtilis.

Balancing act of undergraduate clinical teaching.

Survival or Safety: Balancing act with Colistin.

Platelet signaling--blood's great balancing act.

P2X4 receptor regulation of transient receptor potential melastatin type 6 (TRPM6) Mg2+ channels.

Multisensory Processes: A Balancing Act across the Lifespan.

[It's a balancing act being on the safe side].

Balancing act: optimal care in the real world.

Vitamin A and bone health: the balancing act.

Forword. Regulation of RhoGTPases in motility: A fine balancing act.

Effect of Alcohol Administration on Mg2+ Homeostasis in H9C2 Cells.

A Balancing Act: Stability versus Reactivity of Mn(O) Complexes.

Early dislodgement of Indwelling Pleural Catheter (IPC): a balancing act.

ATP-dependent modulation of MgtE in Mg2+ homeostasis.

Intraprotein signal transduction by HAMP domains: a balancing act.

A balancing act: achieving glycemic control in hospitalized patients.

Between liberal aspirations and market forces: Obamacare's precarious balancing act.

Dengue virus and host antibody: a dangerous balancing act.