Epithelial Magnesium Transport Research Articles

Intestinal hypomagnesemia in an Iranian patient with a novel TRPM6 variant: a case report and review of the literature.

TRPM6 is predominantly expressed in the kidney and colon and encodes a protein containing an ion channel domain and a protein kinase domain. It is crucial for magnesium homeostasis and plays important roles in epithelial magnesium transport and the active magnesium absorption. In this study, we present a 70-day-old Iranian female patient from consanguineous parents with hypomagnesemia and secondary hypocalcemia. She presented with seizures 19days after birth and refractory watery non-bloody diarrhea. She consequently had failure to thrive. Other features included hypotonia, wide anterior fontanel, ventriculomegaly, and pseudotumor cerebri following administration of nalidixic acid. She had severe hypomagnesemia and hypocalcemia which were treated with magnesium and calcium supplementation. Despite initial unstable response to supplemental magnesium, she eventually improved and the diarrhea discontinued. The patient was discharged by magnesium and calcium therapy. At the last follow-up at age 2.5years, the patient remained well without any recurrence or complication. Genetic testing by whole-exome sequencing revealed a novel homozygous frameshift insertion-deletion (indel) variant in exon 26 of the TRPM6 gene, c.3693-3699del GCAAGAG ins CTGCTGTTGACATCTGCT, p.L1231Ffs*36. Segregation analysis revealed the TRPM6 heterozygous variant in both parents. Patients with biallelic TRPM6 pathogenic variants typically exhibit hypomagnesemia with secondary hypocalcemia and present with neurologic manifestations including seizures. In some patients, this is also complicated by chronic diarrhea and failure to thrive. Long-term complications are rare and most of the patients show a good prognosis with supplemental magnesium therapy.

Germline De Novo Mutations in ATP1A1 Cause Renal Hypomagnesemia, Refractory Seizures, and Intellectual Disability

Over the last decades, a growing spectrum of monogenic disorders of human magnesium homeostasis has been clinically characterized, and genetic studies in affected individuals have identified important molecular components of cellular and epithelial magnesium transport. Here, we describe three infants who are from non-consanguineous families and who presented with a disease phenotype consisting of generalized seizures in infancy, severe hypomagnesemia, and renal magnesium wasting. Seizures persisted despite magnesium supplementation and were associated with significant intellectual disability. Whole-exome sequencing and conventional Sanger sequencing identified heterozygous de novo mutations in the catalytic Na+, K+-ATPase α1 subunit (ATP1A1). Functional characterization of mutant Na+, K+-ATPase α1 subunits in heterologous expression systems revealed not only a loss of Na+, K+-ATPase function but also abnormal cation permeabilities, which led to membrane depolarization and possibly aggravated the effect of the loss of physiological pump activity. These findings underline the indispensable role of the α1 isoform of the Na+, K+-ATPase for renal-tubular magnesium handling and cellular ion homeostasis, as well as maintenance of physiologic neuronal activity.

Epithelial magnesium transport by TRPM6 is essential for prenatal development and adult survival.

Mg2+ regulates many physiological processes and signalling pathways. However, little is known about the mechanisms underlying the organismal balance of Mg2+. Capitalizing on a set of newly generated mouse models, we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6. We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development. In adult mice, TRPM6 is required in the intestine to maintain organismal Mg2+ balance, but is dispensable in the kidney. Trpm6 inactivation in adult mice leads to a shortened lifespan, growth deficit and metabolic alterations indicative of impaired energy balance. Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice, but also may extend the lifespan of wildtype mice. Hence, maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood.

Insight into the molecular regulation of the epithelial magnesium channel TRPM6

Recent studies have greatly increased our knowledge concerning the molecular mechanisms of renal magnesium handling. This review highlights the functional features of the newly identified transient receptor potential channel melastatin subtype 6 (TRPM6), which forms the gatekeeper of active magnesium reabsorption in the kidney. TRPM6 confines a magnesium permeable channel of which the expression is regulated by multiple factors, including dietary magnesium, magnesiotropic hormones and drugs. TRPM6 channel activity is modulated by intracellular magnesium and pH. A recently identified point mutation in the pro-epidermal growth factor (EGF) gene, causing isolated recessive inherited renal hypomagnesemia, implicated EGF as a magnesiotropic hormone regulating TRPM6 activity. Furthermore, receptor for activated C-kinase (RACK1) was identified as the first associated protein of the TRPM6 alpha-kinase domain, which acts as a dynamic switch controlling TRPM6 activity in an auto-phosphorylation-dependent manner. Of note, the fused alpha-kinase domain functions as a sensor of the intracellular magnesium concentration and plays a feedback role in controlling TRPM6-mediated magnesium influx, preventing magnesium overload during epithelial magnesium transport. The diverse molecular regulation of TRPM6 by magnesiotropic hormones, intracellular factors and its fused alpha-kinase domain disclosed novel regulatory mechanisms of active magnesium reabsorption.

A critical role of TRPM channel‐kinase for human magnesium transport

Hereditary disorders of magnesium homeostasis comprise a heterogenous group of diseases mainly affecting the renal conservation of magnesium. In the past few years, genetic studies in affected individuals disclosed the first molecular components of epithelial magnesium transport: the tight junction protein paracellin-1 (claudin-16) was discovered as a key player in paracellular magnesium and calcium reabsorption in the thick ascending limb of Henle's loop and the gamma-subunit was identified as a component of renal Na+ -K+ -ATPase critical for transcellular magnesium reabsorption in the distal convoluted tubule. However, the molecular identity of proteins directly involved in cellular magnesium transport remained largely unknown until a series of recent studies highlighted the critical role of two members of the transient receptor potential (TRP) family, for body magnesium homeostasis. TRPM6 and TRPM7 belong to the melastatin-related TRPM subfamily of TRP channels whose eight members exhibit a significant diversity in domain structure as well as cation selectivity and activation mechanisms. Both proteins share the unique feature of an atypical kinase domain at their C-terminus for which they have been termed 'chanzymes' (channels plus enzymes). Whereas electrophysiological and biochemical analyses identified TRPM7 as an important player in cellular magnesium homeostasis, the critical role of TRPM6 for epithelial magnesium transport emerged from the discovery of loss-of-function mutations in patients with a severe form of hereditary hypomagnesaemia called primary hypomagnesaemia with secondary hypocalcaemia or HSH. The aim of this review is to summarize the data emerging from molecular genetic, biochemical and electrophysiological studies on these fascinating two new proteins combining ion channel and enzyme functions/properties.

Essential role for TRPM6 in epithelial magnesium transport and body magnesium homeostasis

Magnesium is an important cofactor for many biological processes such as protein synthesis, nucleic acid stability and neuromuscular excitability. The extracellular magnesium concentration is regulated tightly by the extent of intestinal absorption and renal excretion. Despite their critical role in magnesium handling, the molecular mechanisms mediating transepithelial transport are still not understood completely. Recently, genetic studies in patients with primary hypomagnesaemia and secondary hypocalcaemia (HSH), a combined defect of intestinal magnesium absorption and renal magnesium conservation, have identified "transient receptor potential (melastatin) 6" (TRPM6) as the first component involved directly in epithelial magnesium reabsorption. TRPM7, the closest homologue of TRPM6, has a central role in Mg(2+) uptake in vertebrate cells since TRPM7-deficient cells become Mg(2+) deficient and are not viable. TRPM7 has been characterized functionally as a constitutively active ion channel permeable for a variety of cations including calcium and magnesium and regulated by intracellular concentrations of magnesium and/or magnesium-nucleotide complexes. Both proteins share the unique feature of cation channels fused to serine/threonine kinase domains. This review summarizes recent data that has emerged from molecular genetic, biochemical and electrophysiological studies on these fascinating two new proteins and their involvement in epithelial magnesium transport.

Insights into the molecular nature of magnesium homeostasis.

Magnesium is an important cofactor for many biological processes, such as protein synthesis, nucleic acid stability, or neuromuscular excitability. Extracellular magnesium concentration is tightly regulated by the extent of intestinal absorption and renal excretion. Despite the critical role of magnesium handling, the exact mechanisms mediating transepithelial transport remained obscure. In the past few years, the genetic disclosure of inborn errors of magnesium handling revealed several new proteins along with already known molecules unexpectedly involved in renal epithelial magnesium transport, e.g., paracellin-1, a key player in paracellular magnesium and calcium reabsorption in the thick ascending limb or the gamma-subunit of the Na(+)-K(+)-ATPase in the distal convoluted tubule. In this review, we focus on TRPM6, an ion channel of the "transient receptor potential (TRP) gene family, which, when mutated, causes a combined defect of intestinal magnesium absorption and renal magnesium conservation as observed in primary hypomagnesemia with secondary hypocalcemia.

Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia.

Impaired magnesium reabsorption in patients with TRPM6 gene mutations stresses an important role of TRPM6 (melastatin-related TRP cation channel) in epithelial magnesium transport. While attempting to isolate full-length TRPM6, we found that the human TRPM6 gene encodes multiple mRNA isoforms. Full-length TRPM6 variants failed to form functional channel complexes because they were retained intracellularly on heterologous expression in HEK 293 cells and Xenopus oocytes. However, TRPM6 specifically interacted with its closest homolog, the Mg(2+)-permeable cation channel TRPM7, resulting in the assembly of functional TRPM6/TRPM7 complexes at the cell surface. The naturally occurring S141L TRPM6 missense mutation abrogated the oligomeric assembly of TRPM6, thus providing a cell biological explanation for the human disease. Together, our data suggest an important contribution of TRPM6/TRPM7 heterooligomerization for the biological role of TRPM6 in epithelial magnesium absorption.

Genetics of hereditary disorders of magnesium homeostasis.

Magnesium plays an essential role in many biochemical and physiological processes. Homeostasis of magnesium is tightly regulated and depends on the balance between intestinal absorption and renal excretion. During the last decades, various hereditary disorders of magnesium handling have been clinically characterized and genetic studies in affected individuals have led to the identification of some molecular components of cellular magnesium transport. In addition to these hereditary forms of magnesium deficiency, recent studies have revealed a high prevalence of latent hypomagnesemia in the general population. This finding is of special interest in view of the association between hypomagnesemia and common chronic diseases such as diabetes, coronary heart disease, hypertension, and asthma. However, valuable methods for the diagnosis of body and tissue magnesium deficiency are still lacking. This review focuses on clinical and genetic aspects of hereditary disorders of magnesium homeostasis. We will review primary defects of epithelial magnesium transport, disorders associated with defects in Ca(2+)/ Mg(2+) sensing, as well as diseases characterized by renal salt wasting and hypokalemic alkalosis, with special emphasis on disturbed magnesium homeostasis.

Determination of epithelial magnesium transport with stable isotopes

The inability to adequately determine Mg 2+ flux rates with radiotracer studies has stymied our efforts to understand how magnesium is transported by epithelial cells. To evaluate epithelial Mg 2+ transport, a stable 25Mg isotope was used to measure magnesium uptake into normal and Mg 2+-depleted Madin–Darby canine kidney (MDCK) cells. 25Mg entry rates were significantly increased in Mg 2+-depleted cells relative to those cultured in normal magnesium media, 0.5 mM. 25Mg uptake was inhibited by external La 3+ but not Ca 2+ in both normal and Mg 2+-depleted cells suggesting a specific entry pathway. These results with 25Mg were the same as with microfluorescence determinations using mag-fura-2. We have shown that Mg 2+ entry into epithelial cells reflects transepithelial transport; accordingly, increased Mg 2+ uptake in Mg 2+-depleted cells provides an important intrinsic control of renal magnesium absorption. Furthermore, these studies indicate that cellular Mg 2+ transport may be quantitated with the use of stable isotopes that may be successfully applied to cells other than epithelia.

Epithelial magnesium transport and regulation by the kidney

Magnesium is the fourth most abundant cation in the body and the second most common cation in the intracellular fluid. It is the kidney that provides the most sensitive control for magnesium balance. About a 80% of the total serum magnesium is ultrafilterable through the glomerular membrane. In all of the mammalian species studied to date, the proximal tubule of the adult animal reabsorbs only a small fraction, 10-15%, of the filtered magnesium. Unlike the adult proximal convoluted tubule that of young rats (aged 13-15 days) reabsorbs 50-60% of filtered magnesium along the proximal tubule together with sodium, calcium, and water. Micropuncture experiments, in every species studied to date, indicates that a large part (approximately 60%) of the filtered magnesium is reabsorbed in the loop of Henle. Magnesium reabsorption in the loop occurs within the cortical thick ascending limb (cTAL) by passive means driven by the transepithelial voltage through the paracellular pathway. Micropuncture experiments have clearly showed that the superficial distal tubule reabsorbs significant amounts of magnesium. Unlike the thick ascending limb of the loop of Henle, magnesium reabsorption in the distal tubule is transcellular and active in nature. Many hormones and nonhormonal factors influence renal magnesium reabsorption to variable extent in the cTAL and distal tubule. Moreover, nonhormonal factors may have important implications on hormonal controls of renal magnesium conservation. Dietary magnesium restriction leads to renal magnesium conservation with diminished urinary magnesium excretion. Adaptation of magnesium transport with dietary magnesium restriction occurs in both the cTAL and distal tubule. Elevation of plasma magnesium or calcium concentration inhibits magnesium and calcium reabsorption leading to hypermagnesiuria and hypercalciuria. The identification of an extracellular Ca2+/Mg2+ -sensing receptor located on the peritubular side of cTAL and distal tubule cells explains this phenomenon. Loop diuretics, such as furosemide and bumetanide, diminish salt absorption in the cTAL whereas the distally acting diuretics, amiloride and chlorothiazide stimulate magnesium reabsorption within the distal convoluted tubule. Finally, metabolic acidosis, potassium depletion or phosphate restriction can diminish magnesium reabsorption within the loop and distal tubule. Research in the 90's have greatly contributed to our understanding of renal magnesium handling.

Epithelial magnesium transport and regulation by the kidney

Epithelial magnesium transport and regulation by the kidney