Autosomal Dominant Hypomagnesemia Research Articles

A Nonredundant Role for the TRPM6 Channel in Neural Tube Closure

In humans, germline mutations in Trpm6 cause autosomal dominant hypomagnesemia with secondary hypocalcemia disorder. Loss of Trpm6 in mice also perturbs cellular magnesium homeostasis but additionally results in early embryonic lethality and neural tube closure defects. To define the mechanisms by which TRPM6 influences neural tube closure, we functionally characterized the role of TRPM6 during early embryogenesis in Xenopus laevis. The expression of Xenopus TRPM6 (XTRPM6) is elevated at the onset of gastrulation and is concentrated in the lateral mesoderm and ectoderm at the neurula stage. Loss of XTRPM6 produced gastrulation and neural tube closure defects. Unlike XTRPM6′s close homologue XTRPM7, whose loss interferes with mediolateral intercalation, depletion of XTRPM6 but not XTRPM7 disrupted radial intercalation cell movements. A zinc-influx assay demonstrated that TRPM6 has the potential to constitute functional channels in the absence of TRPM7. The results of our study indicate that XTRPM6 regulates radial intercalation with little or no contribution from XTRPM7 in the region lateral to the neural plate, whereas XTRPM7 is mainly involved in regulating mediolateral intercalation in the medial region of the neural plate. We conclude that both TRPM6 and TRPM7 channels function cooperatively but have distinct and essential roles during neural tube closure.

Hereditary hypomagnesemia

Magnesium participates in many fundamental metabolic processes and plays important roles in maintaining normal body function. Magnesium homeostasis is regulated by a fine balance between gastrointestinal absorption and renal excretion. Several hereditary disorders characterized by hypomagnesemia have been described since 1960s, including familial hypomagnesemia with hypercalciuria and nephrocalcinosis, autosomal dominant hypomagnesemia with hypocalciuria, hypomagnesemia with secondary hypocalcemia, autosomal dominant hypocalcemia and etc. Recent advances in molecular genetics and protein functions contribute to more understanding of magnesium transport. We will review clinical aspects of hereditary disorders of hypomagnesemia and summarize genetic findings related to these disorders. Key words: Magnesium; Hypomagnesemia; Magnesium deficiency; Metal metabolism,inborn errors

Functional Analysis of the Kv1.1 N255D Mutation Associated with Autosomal Dominant Hypomagnesemia

Mutations in the voltage-gated K(+) channel Kv1.1 have been linked with a mixed phenotype of episodic ataxia and/or myokymia. Recently, we presented autosomal dominant hypomagnesemia as a new phenotypic characteristic associated with a mutation in Kv1.1 (N255D) (Glaudemans, B., van der Wijst, J., Scola, R. H., Lorenzoni, P. J., Heister, A., van der Kemp, A. W., Knoers, N. V., Hoenderop, J. G., and Bindels, R. J. (2009) J. Clin. Invest. 119, 936-942). A conserved asparagine at position 255 in the third transmembrane segment was converted into an aspartic acid, resulting in a non-functional channel. In this study, we explored the functional consequence of this conserved residue by substitution with other hydrophobic, polar, or charged amino acids (N255E, N255Q, N255A, N255V, N255T, and N255H). Upon overexpression in human embryonic kidney (HEK293) cells, cell surface biotinylation revealed plasma membrane expression of all mutant channels. Next, we used the whole-cell patch clamp technique to demonstrate that the N255E and N255Q mutants were non-functional. Substitution of Asn-255 with other amino acids (N255A, N255V, N255T, and N255H) did not prevent ion conduction, and these mutant channels activated at more negative potentials when compared with wild-type channels, -41.5 +/- 1.6, -45.5 +/- 2.0, -50.5 +/- 1.9, and -33.8 +/- 1.3 mV to -29.4 +/- 1.1 mV, respectively. The time constant of activation was significantly faster for the two most hydrophobic mutations, N255A (6.2 +/- 0.2 ms) and N255V (5.2 +/- 0.3 ms), and the hydrophilic mutant N255T (9.8 +/- 0.4 ms) in comparison with wild type (13.0 +/- 0.9 ms). Furthermore, the voltage dependence of inactivation was shifted approximately 13 mV to more negative potentials in all mutant channels except for N255H. Taken together, our data showed that an asparagine at position 255 in Kv1.1 is required for normal voltage dependence and kinetics of channel gating.

Rare but Relevant Kidney Disorders

Occurrence of hypomagnesemia (serum Mg2+ levels <0.7 mmol/L) in the general population has been estimated to be approximately 2%. The kidney is central for the maintenance of the Mg+ balance. A large body of knowledge has been generated from the study of inherited defects of renal Mg2+ handling. The majority of filtered Mg+ is reabsorbed along the proximal tubule and thick ascending limb of Henle's loop via a passive paracellular pathway. Claudin 16, formerly paracellin 1, and claudin 19 are renal tight junction proteins that are important for paracellular reabsorption of calcium and magnesium in thick ascending limb. Mutations of the corresponding genes, CLDN 16 and 19, result in a rare autosomal recessive tubular disorder that is characterized by familial hypomagnesemia, hypercalciuria, and nephrocalcinosis (FHHNC). This disease is frequently associated with progressive renal failure as a result of both intrarenal calcium deposition and additional roles of claudins in the maintenance of tight junction integrity. Children with a complete loss of function of CLDN16 have a more rapid progression of renal failure than those with residual function (1). The thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule (DCT) is mutated in Gitelman syndrome. Hypokalemic metabolic alkalosis and hypocalciuria are associated with hypomagnesemia. At the apical membrane of DCT cells is also present the Mg2+-permeable transient receptor potential cation channel, subfamily M (TRPM6). Mg2+ uptake from the tubular fluid via TRPM6 is primarily driven by the negative potential across the luminal membrane. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6. Mutations in FXYD2, encoding the γ-subunit of the basolateral Na+-K+-ATPase, is responsible for dominant isolated renal Mg2+ wasting, as a result of misrouting of this subunit. This defect has been described so far in a single family (but read this summary to the end). Finally, the recently discovered magnesiotropic hormone EGF causes isolated recessive renal hypomagnesemia (see references in Glaudemans et al.). Glaudemans et al. identified a new mechanism for autosomal dominant hypomagnesemia. A large Brazilian family was studied. Twenty-one affected members had serum Mg2+ levels <0.4 mmol/L, which was responsible for muscle symptoms and tetany from infancy. The locus was identified on chromosome 12. The mutation was found in KCNA1, a gene encoding the voltage-gated K+ channel, Kv1.1. This channel co-localized with TRPM6 along the luminal membrane of DCT. Upon overexpression in a human kidney cell line, patch-clamp analysis revealed that the mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. The negative potential across the apical membrane is normally maintained by an apical K+ efflux via Kv1.1 energized by the action of Na+-K+-ATPase. When Kv1.1 function is lost, Mg2+ reabsorption in DCT is impaired. Recently, an unexpected mechanism of renal magnesium wasting and hypomagnesemia was discovered by Adalat et al. (2). They first identified a teenager who had hepatocyte nuclear factor 1B (HNF 1B) mutation and presented with tetany and hypomagnesemia (0.620 mmol/L). Then they retrospectively reviewed 91 pediatric cases of renal malformation screened for HNF 1B mutation. Twenty-one had heterozygous mutations. Eight (44%) of 18 mutation carriers had hypomagnesemia (<0.65 mmol/L) compared with one (2%) of 48 of those without mutations. HNF 1B is a transcription factor that is expressed in developing kidneys, whereas, postnatally, proximal and distal tubules express the gene. Hnf 1b in mouse upregulates transcription of various genes whose human homologues are involved in inherited cystic kidney diseases. Adalat et al. (2) detected a highly conserved HNF 1B recognition site in FXYD2. They demonstrated HNF 1B–mediated transactivation of FXYD2. Thus, HNF 1B regulates transcription of FXYD2, which participates in the tubular handling of Mg2+. These results extend the phenotype of HNF 1B mutations to include hypomagnesemia. Serum Mg2+ levels should be measured in patients with HNF 1B mutations (2).

HNF1B mutations associate with hypomagnesemia and renal magnesium wasting.

Mutations in hepatocyte nuclear factor 1B (HNF1B), which is a transcription factor expressed in tissues including renal epithelia, associate with abnormal renal development. While studying renal phenotypes of children with HNF1B mutations, we identified a teenager who presented with tetany and hypomagnesemia. We retrospectively reviewed radiographic and laboratory data for all patients from a single center who had been screened for an HNF1B mutation. We found heterozygous mutations in 21 (23%) of 91 cases of renal malformation. All mutation carriers had abnormal fetal renal ultrasonography. Plasma magnesium levels were available for 66 patients with chronic kidney disease (stages 1 to 3). Striking, 44% (eight of 18) of mutation carriers had hypomagnesemia (<1.58 mg/dl) compared with 2% (one of 48) of those without mutations (P < 0.0001). The median plasma magnesium was significantly lower among mutation carriers than those without mutations (1.68 versus 2.02 mg/dl; P < 0.0001). Because hypermagnesuria and hypocalciuria accompanied the hypomagnesemia, we analyzed genes associated with hypermagnesuria and detected highly conserved HNF1 recognition sites in FXYD2, a gene that can cause autosomal dominant hypomagnesemia and hypocalciuria when mutated. Using a luciferase reporter assay, we demonstrated HNF1B-mediated transactivation of FXYD2. These results extend the phenotype of HNF1B mutations to include hypomagnesemia. HNF1B regulates transcription of FXYD2, which participates in the tubular handling of Mg(2+), thus describing a role for HNF1B not only in nephrogenesis but also in the maintenance of tubular function.

Inherited forms of renal hypomagnesemia: an update

The kidney plays an important role in ion homeostasis in the human body. Several hereditary disorders characterized by perturbations in renal magnesium reabsorption leading to hypomagnesemia have been described over the past 50 years, with the most important of these being Gitelman syndrome, familial hypomagnesemia with hypercalciuria and nephrocalcinosis, familial hypomagnesemia with secondary hypocalcemia, autosomal dominant hypomagnesemia with hypocalciuria, and autosomal recessive hypomagnesemia. Only recently, following positional cloning strategies in affected families, have mutations in renal ion channels and transporters been identified in these diseases. In this short review, I give an update on these hypomagnesemic disorders. Elucidation of the genetic etiology and, for most of these disorders, also the underlying pathophysiology of the disease, has greatly increased our understanding of the normal physiology of renal magnesium handling. This is yet another example of the importance of studying rare disorders in order to unravel physiological and pathophysiological processes in the human body.

The voltage-gated K+ channel subunit Kv1.1 links kidney and brain

Analysis of Mendelian Mg2+ wasting disorders helps us to unravel the mechanisms of Mg2+ homeostasis. In this issue of the JCI, Glaudemans andcolleagues show that mutations in voltage-gated K+ channel subtype 1.1(Kv1.1) cause autosomal dominant hypomagnesemia in humans (see the related article beginning on page 936). Interestingly, other mutations in the same protein cause the neurological disease episodic ataxia type 1. The authors show, using cells with heterologous expression of the wild-type and mutant channels, that the mutant channel is dysfunctional and speculate that Mg2+ wasting results from changes in apical membrane voltage along the nephron. Mechanisms by which the apical voltage is generated and howKv1.1 fits within this context are discussed herein.

A missense mutation in the Kv1.1 voltage-gated potassium channel–encoding gene KCNA1 is linked to human autosomal dominant hypomagnesemia

Primary hypomagnesemia is a heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg2+) wasting, resulting in tetany, cardiac arrhythmias, and seizures. The kidney plays an essential role in maintaining blood Mg2+ levels, with a prominent function for the Mg2+-transporting channel transient receptor potential cation channel, subfamily M, member 6 (TRPM6) in the distal convoluted tubule (DCT). In the DCT, Mg2+ reabsorption is an active transport process primarily driven by the negative potential across the luminal membrane. Here, we studied a family with isolated autosomal dominant hypomagnesemia and used a positional cloning approach to identify an N255D mutation in KCNA1, a gene encoding the voltage-gated potassium (K+) channel Kv1.1. Kv1.1 was found to be expressed in the kidney, where it colocalized with TRPM6 along the luminal membrane of the DCT. Upon overexpression in a human kidney cell line, patch clamp analysis revealed that the KCNA1 N255D mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. These data suggest that Kv1.1 is a renal K+ channel that establishes a favorable luminal membrane potential in DCT cells to control TRPM6-mediated Mg2+ reabsorption.