Structural and functional comparison of magnesium transporters throughout evolution.

Magnesium (Mg2+) is the most prevalent divalent intracellular cation. As co-factor in many enzymatic reactions, Mg2+ is essential for protein synthesis, energy production, and DNA stability. Disturbances in intracellular Mg2+ concentrations, therefore, unequivocally result in delayed cell growth and metabolic defects. To maintain physiological Mg2+ levels, all organisms rely on balanced Mg2+ influx and efflux via Mg2+ channels and transporters. This review compares the structure and the function of prokaryotic Mg2+ transporters and their eukaryotic counterparts. In prokaryotes, cellular Mg2+ homeostasis is orchestrated via the CorA, MgtA/B, MgtE, and CorB/C Mg2+ transporters. For CorA, MgtE, and CorB/C, the motifs that form the selectivity pore are conserved during evolution. These findings suggest that CNNM proteins, the vertebrate orthologues of CorB/C, also have Mg2+ transport capacity. Whereas CorA and CorB/C proteins share the gross quaternary structure and functional properties with their respective orthologues, the MgtE channel only shares the selectivity pore with SLC41 Na+/Mg2+ transporters. In eukaryotes, TRPM6 and TRPM7 Mg2+ channels provide an additional Mg2+ transport mechanism, consisting of a fusion of channel with a kinase. The unique features these TRP channels allow the integration of hormonal, cellular, and transcriptional regulatory pathways that determine their Mg2+ transport capacity. Our review demonstrates that understanding the structure and function of prokaryotic magnesiotropic proteins aids in our basic understanding of Mg2+ transport.

The impact of feedback during formative testing on study behaviour and performance of (bio)medical students: a randomised controlled study.

BACKGROUND: A potential concern of formative testing using web-based applications ("apps") is provision of limited feedback. Adopting a randomised controlled trial in 463 first year (bio) medical students, we explored if providing immediate, detailed feedback during "app"-based formative testing can further improve study behaviour and study performance of (bio)medical students. METHODS: Students had access to a formative testing "app", which involved 7 formative test modules throughout the 4-week course. In a randomised order, subjects received the "app" with (n = 231, intervention) or without (n = 232, control) detailed feedback during the formative test modules. RESULTS: No differences in app-use was found between groups (P = 0.15), whereas the intervention group more frequently reviewed information compared to controls (P = 0.007). Exam scores differed between non-/moderate-/intensive- users of the "app" (P < 0.001). No differences in exam scores were found between intervention (6.6 ± 1.1) versus control (6.6 ± 1.1, P = 0.18). Time spent studying was significantly higher compared to previous courses in moderate- and intensive-users (P = 0.006 and < 0.001, respectively), but not in non-users (P = 0.55). Time spent studying did not differ between groups (P > 0.05). CONCLUSIONS: Providing detailed feedback did not further enhance the effect of a web-based application of formative testing on study behaviour or study performance in (bio)medical students, possibly because of a ceiling-effect.

Inulin significantly improves serum magnesium levels in proton pump inhibitor-induced hypomagnesaemia.

BACKGROUND: Proton pump inhibitors (PPI) are among the most widely prescribed drugs to treat gastric acid-related disorders. PPI-induced hypomagnesaemia, a defect in intestinal absorption of Mg(2+) , can be a severe side effect of chronic PPI use. AIM: To restore serum Mg(2+) concentrations in PPI-induced hypomagnesaemia patients by dietary supplementation with inulin fibres. METHODS: Eleven patients with PPI-induced hypomagnesaemia and 10 controls were treated with inulin (20 g/day). Each trial consisted of two cycles of 14-day inulin treatment followed by a washout period of 14 days. Patients continued to use their PPI. Serum Mg(2+) levels served as the primary endpoint. RESULTS: Inulin significantly enhanced serum Mg(2+) levels from 0.60 to 0.68 mmol/L in PPI-induced hypomagnesaemia patients, and from 0.84 to 0.93 mmol/L in controls. As a consequence 24 h urinary Mg(2+) excretion was significantly increased in patients with PPI-induced hypomagnesaemia (0.3-2.2 mmol/day). Symptoms related to hypomagnesaemia, including muscle cramps and paraesthesia, were reduced during intervention with inulin. CONCLUSION: Inulin increases serum Mg(2+) concentrations under PPI maintenance in patients with PPI-induced hypomagnesaemia.

TRPV4 deficiency causes sexual dimorphism in bone metabolism and osteoporotic fracture risk.

We explored the role of transient receptor potential vanilloid 4 (TRPV4) in murine bone metabolism and association of TRPV4 gene variants with fractures in humans. Urinary and histomorphometrical analyses demonstrated reduced osteoclast activity and numbers in male Trpv4(-/-) mice, which was confirmed in bone marrow-derived osteoclast cultures. Osteoblasts and bone formation as shown by serum procollagen type 1 amino-terminal propeptide and histomorphometry, including osteoid surface, osteoblast and osteocyte numbers were not affected in vivo. Nevertheless, osteoblast differentiation was enhanced in Trpv4(-/-) bone marrow cultures. Cortical and trabecular bone mass was 20% increased in male Trpv4(-/-) mice, compared to sex-matched wild type (Trpv4(+/+)) mice. However, at the same time intracortical porosity was increased and bone matrix mineralization was reduced. Together, these lead to a maximum load, stiffness and work to failure of the femoral bone, which were not different compared to Trpv4(+/+) mice, while the bone material was less resistant to stress and less elastic. The differential impacts on these determinants of bone strength were likely responsible for the lack of any changes in whole bone strength in the Trpv4(-/-) mice. None of these skeletal parameters were affected in female Trpv4(-/-) mice. The T-allele of rs1861809 SNP in the TRPV4 locus was associated with a 30% increased risk (95% CI: 1.1-1.6; p=0.013) for non-vertebral fracture risk in men, but not in women, in the Rotterdam Study. Meta-analyses with the population-based LASA study confirmed the association with non-vertebral fractures in men. This was lost when the non-population-based studies Mr. OS and UFO were included. In conclusion, TRPV4 is a male-specific regulator of bone metabolism, a determinant of bone strength, and a potential risk predictor for fractures through regulation of bone matrix mineralization and intra-cortical porosity. This identifies TRPV4 as a unique sexually dimorphic therapeutic and/or diagnostic candidate for osteoporosis.

Omeprazole enhances the colonic expression of the Mg(2+) transporter TRPM6.

Proton pump inhibitors (PPIs) are potent blockers of gastric acid secretion, used by millions of patients suffering from gastric acid-related complaints. Although PPIs have an excellent safety profile, an increasing number of case reports describe patients with severe hypomagnesemia due to long-term PPI use. As there is no evidence of a renal Mg²âº leak, PPI-induced hypomagnesemia is hypothesized to result from intestinal malabsorption of Mg²âº. The aim of this study was to investigate the effect of PPIs on Mg ²âºhomeostasis in an in vivo mouse model. To this end, C57BL/6J mice were treated with omeprazole, under normal and low dietary Mg²âº availability. Omeprazole did not induce changes in serum Mg²âº levels (1.48 ± 0.05 and 1.54 ± 0.05 mmol/L in omeprazole-treated and control mice, respectively), urinary Mg²âº excretion (35 ± 3 µmol/24 h and 30 ± 4 µmol/24 h in omeprazole-treated and control mice, respectively), or fecal Mg²âº excretion (84 ± 4 µmol/24 h and 76 ± 4 µmol/24 h in omeprazole-treated and control mice, respectively) under any of the tested experimental conditions. However, omeprazole treatment did increase the mRNA expression level of the transient receptor potential melastatin 6 (TRPM6), the predominant intestinal Mg²âº channel, in the colon (167 ± 15 and 100 ± 7 % in omeprazole-treated and control mice, respectively, P < 0.05). In addition, the expression of the colonic H⁺,K⁺-ATPase (cHK-α), a homolog of the gastric H⁺,K⁺-ATPase that is the primary target of omeprazole, was also significantly increased (354 ± 43 and 100 ± 24 % in omeprazole-treated and control mice, respectively, P < 0.05). The expression levels of other magnesiotropic genes remained unchanged. Based on these findings, we hypothesize that omeprazole inhibits cHK-α activity, resulting in reduced extrusion of protons into the large intestine. Since TRPM6-mediated Mg²âºabsorption is stimulated by extracellular protons, this would diminish the rate of intestinal Mg²âº absorption. The increase of TRPM6 expression in the colon may compensate for the reduced TRPM6 currents, thereby normalizing intestinal Mg²âº absorption during omeprazole treatment in C57BL/6J mice, explaining unchanged serum, urine, and fecal Mg²âº levels.

Systematic review: hypomagnesaemia induced by proton pump inhibition.

BACKGROUND: Proton pump inhibitors (PPIs) are a mainstay therapy for all gastric acid-related diseases. Clinical concerns arise from a small but growing number of case reports presenting PPI-induced hypomagnesaemia (PPIH) as a consequence of long-term PPI use. Current opinion is that reduced intestinal magnesium absorption might be involved, but nothing is known on the molecular mechanism underlying PPIH. AIM: To investigate whether or not PPIH is a true, long-term drug-class effect of all PPIs and to scrutinise a possible role of comorbidity in its aetiology. Therefore, the primary objective in particular was to investigate serum magnesium dynamics in trials drug withdrawal and re-challenge. The secondary objective was to profile the 'patient at risk'. METHODS: We reviewed systematically all currently available case reports on the subject and performed a statistical analysis on extracted data. RESULTS: Proton pump inhibitor-induced hypomagnesaemia PPIH is a drug-class effect and occurred after 5.5 years (median) of PPI use, onset was broad and ranged from 14 days to 13 years. Discontinuation of PPIs resulted in fast recovery from PPIH in 4 days and re-challenge led to reoccurrence within 4 days. Histamine-2-receptor antagonists were the preferable replacement therapy in PPIH and prevented reoccurrence of hypomagnesaemia. In PPIH no specific risk profile was identified that was linked to the hypomagnesaemia. CONCLUSIONS: The cases of PPIH show severe symptoms of magnesium depletion and identification of its causation was only possible through withdrawal of the PPI. Clinical awareness of PPIH is key to avoid putting patients at risk.

Vascular calcification in chronic kidney disease: new developments in drug therapy.

Controlling the development of vascular calcification in chronic kidney disease is essential, because it is associated with increased cardiovascular pathology. However, the precise mechanism of vascular calcification has not been completely elucidated. In the literature, the involvement of passive calcium and phosphate deposition as well as an active process stimulating the transformation of vascular smooth muscle cells into an osteoblastic phenotype is suggested. New promising insights into the etiology could lead to better treatment strategies, as Mizobuchi et al. now report.

Physiology of epithelial Ca2+ and Mg2+ transport.

Ca2+ and Mg2+ are essential ions in a wide variety of cellular processes and form a major constituent of bone. It is, therefore, essential that the balance of these ions is strictly maintained. In the last decade, major breakthrough discoveries have vastly expanded our knowledge of the mechanisms underlying epithelial Ca2+ and Mg2+ transport. The genetic defects underlying various disorders with altered Ca2+ and/or Mg2+ handling have been determined. Recently, this yielded the molecular identification of TRPM6 as the gatekeeper of epithelial Mg2+ transport. Furthermore, expression cloning strategies have elucidated two novel members of the transient receptor potential family, TRPV5 and TRPV6, as pivotal ion channels determining transcellular Ca2+ transport. These two channels are regulated by a variety of factors, some historically strongly linked to Ca2+ homeostasis, others identified in a more serendipitous manner. Herein we review the processes of epithelial Ca2+ and Mg2+ transport, the molecular mechanisms involved, and the various forms of regulation.

TRPV5, the gateway to Ca2+ homeostasis.

Ca2+ homeostasis in the body is tightly controlled, and is a balance between absorption in the intestine, excretion via the urine, and exchange from bone. Recently, the epithelial Ca2+ channel (TRPV5) has been identified as the gene responsible for the Ca2+ influx in epithelial cells of the renal distal convoluted tubule. TRPV5 is unique within the family of transient receptor potential (TRP) channels due to its high Ca2+ selectivity. Ca2+ flux through TRPV5 is controlled in three ways. First, TRPV5 gene expression is regulated by calciotropic hormones such as vitamin D3 and parathyroid hormone. Second, Ca2+ transport through TRPV5 is controlled by modulating channel activity. Intracellular Ca2+, for example, regulates channel activity by feedback inhibition. Third, TRPV5 is controlled by mobilization of the channel through trafficking toward the plasma membrane. The newly identified anti-aging hormone Klotho regulates TRPV5 by cleaving off sugar residues from the extracellular domain of the protein, resulting in a prolonged expression of TRPV5 at the plasma membrane. Inactivation of TRPV5 in mice leads to severe hypercalciuria, which is compensated by increased intestinal Ca2+ absorption due to augmented vitamin D3 levels. Furthermore, TRPV5 deficiency in mice is associated with polyuria, urine acidification, and reduced bone thickness. Some pharmaceutical compounds, such as the immunosuppressant FK506, affect the Ca2+ balance by modulating TRPV5 gene expression. This underlines the importance of elucidating the role of TRPV5 in Ca(2+)-related disorders, thereby enhancing the possibilities for pharmacological intervention. This chapter describes a unique TRP channel and highlights its regulation and function in renal Ca2+ reabsorption and overall Ca2+ homeostasis.

Concerted action of associated proteins in the regulation of TRPV5 and TRPV6.

Ca(2+) is an essential ion in all organisms and many physiological functions in the body rely on the exact maintenance of the Ca(2+) balance. The epithelial Ca(2+) channels TRPV5 [TRP (transient receptor potential) vanilloid 5] and TRPV6 are the most Ca(2+)-selective members of the TRP superfamily and are generally considered as the gatekeepers of Ca(2+) entry across epithelia. TRPV5 is involved in Ca(2+) reabsorption from pro-urine, while TRPV6 has an essential role in intestinal Ca(2+) uptake. These channels are the prime targets of calciotropic hormonal regulation, including vitamin D and parathyroid hormone. In addition, extra- and intra-cellular signalling by associated proteins and Ca(2+) itself play key roles in TRPV5 and TRPV6 regulation. In this paper, we describe the present understanding of the concerted action of calbindin-D(28k), klotho and BSPRY (B-box and SPRY-domain-containing protein) at different levels throughout the epithelial cell to control Ca(2+) influx at the luminal entry gate.

Coordinated control of renal Ca2+ handling.

Ca2+ homeostasis is an important factor, which is underlined by the numerous clinical symptoms that involve Ca2+ deficiencies. The overall Ca2+ balance is maintained by the concerted action of Ca2+ absorption in the intestine, reabsorption in the kidney, and exchange from bone, which are all under the control of the calciotropic hormones that are released upon a demand for Ca2+. In the kidney, these calciotropic hormones affect active Ca2+ reabsorption, which consists of TRPV5 as the apical entry gate for Ca2+ influx, calbindin-D28K as an intracellular ferry for Ca2+ and, NCX1 and PMCA1b for extrusion of Ca2+ across the basolateral membrane. This review highlights the action of hormones on renal Ca2+ handling and focuses on the coordinated control of the renal Ca2+ transport proteins. Parathyroid hormone stimulates renal Ca2+ handling by regulating active Ca2+ reabsorption on both the genomic and non-genomic level. Estrogens harbor calciotropic hormone characteristics positively regulating the expression of TRPV5, independently of vitamin D. Besides having a strong regulatory effect on the expression of the intestinal Ca2+ transport proteins, vitamin D contributes to the overall Ca2+ balance by enhancing the expression of the Ca2+ transport machinery in the kidney. Dietary Ca2+ is involved in regulating its own handling by controlling the expression of the renal Ca2+ transport proteins. Thus, the magnitude of Ca2+ entry via TRPV5 controls the expression of the other Ca2+ transport proteins underlining the gatekeeper function of this Ca2+ channel in the renal Ca2+ handling.

[From gene to disease; mutations in the SLC12A3 gene as the cause of Gitelman's syndrome]. / Van gen naar ziekte; mutaties in het SLC12A3-gen als oorzaak van het syndroom van Gitelman.

Gitelman's syndrome is characterised by persistent hypokalaemia, hypomagnesaemia and hypocalciuria (OMIM 263800). This rare autosomal recessive disorder is caused by renal Na+, Cl-, K+ and Mg2+ wasting. Other typical features include hypocalciuria and an intact renal concentrating ability. Gitelman's syndrome is caused by mutations in the SLC12A3 gene, encoding the thiazide-sensitive sodium-chloride co-transporter (NCC). NCC is located in the distal convoluted tubule of the kidney, a segment known to play an important role in active magnesium reabsorption in the nephron. The exact mechanisms underlying hypomagnesaemia and hypocalciuria in Gitelman's syndrome are still poorly understood, but point to enhanced proximal Na+ and Ca2+ reabsorption and apoptosis of distal convoluted tubule cells.

Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6.

The molecular assembly of the epithelial Ca(2+) channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immuno blotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd(2+) sensitivity and voltage-dependent gating. Immuno precipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca(2+) channels that varied with respect to Ca(2+)-dependent inactivation, Ba(2+) selectivity and pharmacological block. Thus, Ca(2+)-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca(2+) transport kinetics.

Current understanding of mammalian TRP homologues.

Calcium influx into the cell from the extracellular medium is crucial for important processes including muscle contraction, secretion and gene expression. This calcium influx is mainly mediated through calcium influx channels, which on the basis of their activation mechanism can be subdivided in voltage-gated calcium channels, which have already been thoroughly characterized and non-voltage-gated calcium permeable channels. This latter group includes ion channels activated by binding of extra and intracellular messengers, mechanical stress or depletion of intracellular calcium stores. Currently little molecular data is available concerning this class of calcium influx channels. However, recent studies have indicated that members of the transient receptor potential (TRP) family of ion channels can function as calcium influx channels both in excitable and non-excitable tissues. On the basis of structural information the TRP family is subdivided in three main subfamilies: the TRPC (canonical) group, the TRPV (vanilloid) group and the TRPM (melastatin) group. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of tissues and species, including mammals, insects and yeast. This review summarizes the currently available information concerning members of the TRP family expressed in mammalian tissues.