Cellular Magnesium Research Articles

823: Magnesium decreases inflammatory cytokine production: a novel innate immunomodulatory mechanism

ObjectiveTo determine if magnesium sulfate (MgSO4) influences inflammatory cytokine production.Study DesignMgSO4 exposure before preterm birth reduces the risk of cerebral palsy and major motor dysfunction. As neonatal inflammatory cytokine levels strongly correlate with neurologic outcome, we assessed the effect of MgSO4 on cytokine production within monocytic cell lines, cord blood mononuclear cells and maternal blood from women receiving parenteral magnesium sulfate for clinical indications.ResultsIn vivo MgSO4 treatment substantially reduced maternal TNF-α and IL-6 production. THP-1 cells exposed to TLR4 ligand in the presence of a clinically-effective MgSO4 concentration (6mg/dl) demonstrated reduced IL-1β, IL-6, IL-8 and TNF-α production. We next exposed term and preterm cord blood mononuclear cells to MgSO4 in vitro and found a significantly reduced frequency of neonatal monocytes producing cytokines under constitutive and TLR-stimulated conditions, as measured by intracellular cytokine staining. Total cellular magnesium content rapidly increased upon MgSO4 exposure, and the immunomodulatory effect was mediated by magnesium and not the sulfate moiety of the compound. Decreased cytokine production was linked to lower cytokine and IκBα gene expression and diminished NF-κB activation. Reduced cytokine production was seen following exposure to different TLR ligands, suggesting that magnesium acts intracellularly, directly influencing NF-κB or IκBα activity.ConclusionOur findings potentially elucidate the mechanism by which magnesium sulfate reduces the risk of cerebral palsy and establishes a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis. ObjectiveTo determine if magnesium sulfate (MgSO4) influences inflammatory cytokine production. To determine if magnesium sulfate (MgSO4) influences inflammatory cytokine production. Study DesignMgSO4 exposure before preterm birth reduces the risk of cerebral palsy and major motor dysfunction. As neonatal inflammatory cytokine levels strongly correlate with neurologic outcome, we assessed the effect of MgSO4 on cytokine production within monocytic cell lines, cord blood mononuclear cells and maternal blood from women receiving parenteral magnesium sulfate for clinical indications. MgSO4 exposure before preterm birth reduces the risk of cerebral palsy and major motor dysfunction. As neonatal inflammatory cytokine levels strongly correlate with neurologic outcome, we assessed the effect of MgSO4 on cytokine production within monocytic cell lines, cord blood mononuclear cells and maternal blood from women receiving parenteral magnesium sulfate for clinical indications. ResultsIn vivo MgSO4 treatment substantially reduced maternal TNF-α and IL-6 production. THP-1 cells exposed to TLR4 ligand in the presence of a clinically-effective MgSO4 concentration (6mg/dl) demonstrated reduced IL-1β, IL-6, IL-8 and TNF-α production. We next exposed term and preterm cord blood mononuclear cells to MgSO4 in vitro and found a significantly reduced frequency of neonatal monocytes producing cytokines under constitutive and TLR-stimulated conditions, as measured by intracellular cytokine staining. Total cellular magnesium content rapidly increased upon MgSO4 exposure, and the immunomodulatory effect was mediated by magnesium and not the sulfate moiety of the compound. Decreased cytokine production was linked to lower cytokine and IκBα gene expression and diminished NF-κB activation. Reduced cytokine production was seen following exposure to different TLR ligands, suggesting that magnesium acts intracellularly, directly influencing NF-κB or IκBα activity. In vivo MgSO4 treatment substantially reduced maternal TNF-α and IL-6 production. THP-1 cells exposed to TLR4 ligand in the presence of a clinically-effective MgSO4 concentration (6mg/dl) demonstrated reduced IL-1β, IL-6, IL-8 and TNF-α production. We next exposed term and preterm cord blood mononuclear cells to MgSO4 in vitro and found a significantly reduced frequency of neonatal monocytes producing cytokines under constitutive and TLR-stimulated conditions, as measured by intracellular cytokine staining. Total cellular magnesium content rapidly increased upon MgSO4 exposure, and the immunomodulatory effect was mediated by magnesium and not the sulfate moiety of the compound. Decreased cytokine production was linked to lower cytokine and IκBα gene expression and diminished NF-κB activation. Reduced cytokine production was seen following exposure to different TLR ligands, suggesting that magnesium acts intracellularly, directly influencing NF-κB or IκBα activity. ConclusionOur findings potentially elucidate the mechanism by which magnesium sulfate reduces the risk of cerebral palsy and establishes a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis. Our findings potentially elucidate the mechanism by which magnesium sulfate reduces the risk of cerebral palsy and establishes a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis.

Silencing TRPM7 mimics the effects of magnesium deficiency in human microvascular endothelial cells

Evidence has accumulated to suggest that magnesium might play a role in controlling angiogenesis. Since microvascular endothelial cells are protagonists in this process, we investigated the behavior of these cells cultured in low extracellular magnesium or silenced for its transporter Transient Receptor Potential Melastatin (TRPM)7, essential for cellular magnesium homeostasis. In particular, we focused on some crucial steps of the angiogenic process, i.e. proliferation, migration, protease production and organization in tridimensional structures. Silencing TRPM7 mimics the effects of low extracellular magnesium on human microvascular endothelial cells (HMEC). Indeed, while no effects were observed on the production of metalloproteases and on tridimensional organization on matrigel, both magnesium deficiency and silencing of TRPM7 impair cell migration and inhibit growth by arresting the cells in the G0/G1 and G2/M phases of the cell cycle. Since low extracellular magnesium markedly decreases TRPM7 in HMEC, we suggest that TRPM7 downregulation might mediate low magnesium-induced inhibition of cell growth and migration. Human endothelial cells from the umbilical vein are growth inhibited by low magnesium and growth stimulated after TRPM7 silencing. An impairment of ERK phosphorylation in HMEC silencing TRPM7 is responsible, in part, for the different proliferative behavior of these two cell types. We broadened our studies also to endothelial colony-forming cells and found that they are sensitive to fluctuations of the concentrations of extracellular magnesium, while their proliferation rate is not modulated by TRPM7 silencing. Our results point to magnesium and TRPM7 as a modulators of the angiogenic phenotype of microvascular endothelial cells.

MagT1: a highly specific magnesium channel with important roles beyond cellular magnesium homeostasis

Over recent years, the study of magnesium homeostasis has greatly benefited from molecular genetic approaches that identified several new classes of magnesium transporters. These proteins demonstrate a diversity of structural properties and biophysical functions that often translate into a wide range of tissue-specific cellular activities. Among these novel channels, MagT1 has gained most of the attention, given its high selectivity for Mg(2+) and its possible involvement in cellular functions reaching far beyond magnesium homeostasis, as the latest findings seem to imply. Indeed, a signaling role for MagT1 has been proven in T lymphocytes, where Mg(2+) functions as a second messenger, coupling TCR activation to intracellular effectors. We herein review these intriguing results and discuss their potential implications for magnesium research, and ultimately for therapeutic opportunities. As our knowledge of magnesium advances, it becomes increasingly clear that a deeper understanding of magnesium homeostasis is the key for a deeper insight into relevant pathophysiological conditions, and their treatment.

Decreased Intracellular Phosphate and Magnesium Concentrations in Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats.

A decrease in total magnesium content is not a direct proof of a decreased magnesium ion concentration. It could reflect a phosphate alteration or an ATP metabolism disorder. Plasma phosphate levels are lower in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY), and defects in membrane regulation or mitochondrial ATP synthase occur. Only sparse data exist concerning cellular magnesium and phosphate concentrations in hypertensive cells. In aortic smooth muscle cells from 10 SHR of the Münster strain and 10 age-matched normotensive WKY, the intracellular phosphate and magnesium content was measured by electron probe X-ray microanalysis (Camscan CS 24 apparatus, Cambridge, U.K.). The Mg(++) content was 0.90 +/- 0.15 g/kg dry weight in SHR versus 1.15 +/- 0.10 g/kg dry weight in WKY (p < 0.05). Vascular smooth muscle phosphate content was 23.6 +/- 0.79 g/kg dry weight in WKY versus 15.81 +/- 1.22 g/kg dry weight in SHR (p < 0.01). Aortic smooth muscle cells from SHR are characterized by markedly lowered cellular phosphate and magnesium concentrations and an altered ATP metabolism. </hea

Electron-probe X-ray Microanalysis of Magnesium and Sodium Ion Content in Vascular Smooth Muscle Cells from Spontaneously Hypertensive and Normotensive Rats.

Whereas in blood cells decreased magnesium concentrations and increased sodium concentrations in essential hypertension have often been described, only sparse data exist on cellular magnesium or sodium content in vascular smooth muscle cells. Therefore, in aortic smooth muscle cells from seven spontaneously hypertensive rats (SHR) of the Münster strain and seven normotensive Wistar-Kyoto rats (WKY), the intracellular magnesium and sodium content were measured by electron-probe X-ray microanalysis. Measurements were performed in aortic cryosections 3 µm thick. The magnesium ion content was 0.93 +/- 0.17 g/kg dry weight in SHR vs 1.14 +/- 0.12 g/kg dry weight in WKY (p < 0.05). Vascular smooth muscle sodium ion content was measured at 6.85 +/- 0.59 g/kg dry weight in WKY and 12.47 +/- 1.62 g/kg dry weight in SHR (p < 0.05). In conclusion, aortic smooth muscle cells from SHR are characterized by a markedly lowered intracellular magnesium ion content and increased sodium ion concentrations compared with normotensive cells. The results may be due to genetically determined disturbances in transmembrane magnesium and sodium ion transport. Cellular magnesium and sodium handling may be disturbed in SHR aortic smooth muscle as it is in hypertensive blood cells.

Lowered Total Intracellular Magnesium Status in a Subgroup of Hypertensives.

A new method to determine total Mg++ content in lymphocytes was developed, offering advantages for routine measurements as compared to fluorescence methods. Intracellular total Mg++ measurements were performed in lymphocytes of 18 healthy subjects and 19 untreated essential hypertensive patients. Mg++ content was referred to lymphocytic and membrane protein, which was determined according to Bradford's method. Mg++ measurements were performed by atomic absorption spectroscopy using a Video 12 apparatus of Thermo Electron Instrumentation Laboratory, Andover, USA. The results show that in patients with essential hypertension total intralymphocytic Mg++ content is significantly lower (0.07 +/- 0.05 mmol/g lymphocytic protein, mean +/- s.d.) as compared to controls (0.11 +/- 0.04 mmol/g lymphocytic protein, mean +/- s.d., p < 0.05). Free intracellular Mg++ content was measured in lymphocytes by the fluorescent indicator mag-fura-II, showing no significant differences in normotensives and hypertensives (0.30 +/- 0.16 versus 0.38 +/- 0.17 mmol/l). Additionally, in platelets free intracellular Mg++ concentrations were not found of significant difference in normotensives and hypertensives (0.52 +/- 0.23 versus 0.47 +/- 0.27 mmol/l) using mag-fura-II. In plasma Mg++ concentrations there was no significant difference in the normotensive and hypertensive group (0.92 +/- 0.07 versus 0.88 +/- 0.07 mmol/l). There was no correlation between plasma or free or total cellular magnesium concentrations in both groups. Furthermore this method seems also suitable for routine measurements of total intracellular Mg++ concentrations in even larger measurements like mag-fur-II. Lowered total intracellular Mg++ concentrations in a subgroup of primary hypertensives may contribute to the development of this disorder, perhaps due to different buffering systems.http://link.springer-ny.com/link/service/journals/00547/bibs/8n3p154.html

Intracellular magnesium detection: imaging a brighter future

A wide variety of biochemical reactions and physiological functions are known to require magnesium; nonetheless, its regulatory mechanisms (both at the cellular and systemic level) are still poorly characterised. Derangement of magnesium homeostasis is associated with several relevant human pathologies, e.g. diabetes, neuromuscular disorders, hypertension and other cardiovascular diseases. The study of the regulation of magnesium has gained particular interest in the last decades thanks to the molecular characterisation of specific magnesium transporters and the exploitation of molecular biology techniques to clarify their cellular and physiological function(s). In contrast, experimental tools to trace cellular magnesium and to define its homeostasis in living cells have not witnessed a corresponding progress. It was not until recently that efforts were paid to design more appropriate fluorescent indicators that could translate the advances of live imaging techniques into the field of magnesium research. Herein we critically summarise the state of the art in intracellular magnesium detection by fluorescent probes and focus on the need for improving methods and techniques in this area. We highlight the advantages of last-generation fluorescent indicators and discuss a number of challenges and opportunities that the development of novel and better sensors for magnesium still faces.

Mammalian MagT1 and TUSC3 are required for cellular magnesium uptake and vertebrate embryonic development

Magnesium (Mg(2+)) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg(2+) transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3, as major mechanisms of Mg(2+) influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg(2+). Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg(2+) concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg(2+) or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg(2+) transport system.

Huntingtin-interacting Proteins, HIP14 and HIP14L, Mediate Dual Functions, Palmitoyl Acyltransferase and Mg2+ Transport

Polyglutamine expansions of huntingtin protein are responsible for the Huntington neurological disorder. HIP14 protein has been shown to interact with huntingtin. HIP14 and a HIP14-like protein, HIP14L, with a 69% similarity reside in the Golgi and possess palmitoyl acyltransferase activity through innate cysteine-rich domains, DHHC. Here, we used microarray analysis to show that reduced extracellular magnesium concentration increases HIP14L mRNA suggesting a role in cellular magnesium metabolism. Because HIP14 was not on the microarray platform, we used real-time reverse transcriptase-PCR to show that HIP14 and HIP14L transcripts were up-regulated 3-fold with low magnesium. Western analysis with a specific HIP14 antibody also showed that endogenous HIP14 protein increased with diminished magnesium. Furthermore, we demonstrate that when expressed in Xenopus oocytes, HIP14 and HIP14L mediate Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with Michaelis constants of 0.87 +/- 0.02 and 0.74 +/- 0.07 mm, respectively. Diminished magnesium leads to an apparent increase in HIP14-green fluorescent protein and HIP14L-green fluorescent fusion proteins in the Golgi complex and subplasma membrane post-Golgi vesicles of transfected epithelial cells. We also show that inhibition of palmitoylation with 2-bromopalmitate, or deletion of the DHHC motif HIP14DeltaDHHC, diminishes HIP14-mediated Mg2+ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14DeltaDHHC mutant restored Mg2+ transport to values observed with wild-type HIP14. Although we did not directly measure palmitoylation of HIP14 in these studies, the data are consistent with a regulatory role of autopalmitoylation in HIP14-mediated Mg2+ transport. We conclude that the huntingtin interacting protein genes, HIP14 and HIP14L, encode Mg2+ transport proteins that are regulated by their innate palmitoyl acyltransferases thus fulfilling the characteristics of "chanzymes."

SLC41A1 Is a Novel Mammalian Mg2+ Carrier

The molecular biology of mammalian magnesium transporters and their interrelations in cellular magnesium homeostasis are largely unknown. Recently, the mouse SLC41A1 protein was suggested to be a candidate magnesium transporter with channel-like properties when overexpressed in Xenopus laevis oocytes. Here, we demonstrate that human SLC41A1 overexpressed in HEK293 cells forms protein complexes and locates to the plasma membrane without, however, giving rise to any detectable magnesium currents during whole cell patch clamp experiments. Nevertheless, in a strain of Salmonella enterica exhibiting disruption of all three distinct magnesium transport systems (CorA, MgtA, and MgtB), overexpression of human SLC41A1 functionally substitutes these transporters and restores the growth of the mutant bacteria at magnesium concentrations otherwise non-permissive for growth. Thus, we have identified human SLC41A1 as being a bona fide magnesium transporter. Most importantly, overexpressed SLC41A1 provide HEK293 cells with an increased magnesium efflux capacity. With outwardly directed Mg(2+) gradients, a SLC41A1-dependent reduction of the free intracellular magnesium concentration accompanied by a significant net decrease of the total cellular magnesium concentration could be observed in such cells. SLC41A1 activity is temperature-sensitive but not sensitive to the only known magnesium channel blocker, cobalt(III) hexaammine. Taken together, these data functionally identify SLC41A1 as a mammalian carrier mediating magnesium efflux.

Recent developments in intestinal magnesium absorption

Recent identification and characterization of novel Mg transporters have clarified our understanding of intestinal magnesium absorption. The predominant Mg transporters include TRPM6 and TRPM7, members of the transient receptor potential melastatin family of cation channels. Mutations of TRPM6 result in a primary disorder termed hypomagnesemia with secondary hypocalcemia. Both TRPM6 and TRPM7 channels possess an atypical alpha-kinase domain. Recent studies have shown that TRPM7 channel activity is regulated by intracellular Mg and magnesium-nucleotides and modulated via this phosphotransferase kinase. TRPM6 channel function and intestinal magnesium absorption is altered by a variety of hormones and factors. Although it is apparent that TRPM6 and TRPM7 form heteromeric ion channels, controversy surrounds the nature of this interaction. Some studies show that TRPM6 may function on its own whereas other research concludes that TRPM7 is required for effective trafficking of TRPM6 to the plasma membrane. Finally, a number of other Mg transporters have been identified in intestinal epithelial cells but the role of these proteins is unclear. The recent developments in intestinal magnesium absorption and cellular magnesium homeostasis provide a basis for understanding magnesium deficiency disorders and provide a platform for future investigations.

Transient receptor potential melastatin 6 and 7 channels, magnesium transport, and vascular biology: implications in hypertension

Magnesium, an essential intracellular cation, is critically involved in many biochemical reactions involved in the regulation of vascular tone and integrity. Decreased magnesium concentration has been implicated in altered vascular reactivity, endothelial dysfunction, vascular inflammation, and structural remodeling, processes important in vascular changes and target organ damage associated with hypertension. Until recently, very little was known about mechanisms regulating cellular magnesium homeostasis, and processes controlling transmembrane magnesium transport had been demonstrated only at the functional level. Two cation channels of the transient receptor potential melastatin (TRPM) cation channel family have now been identified as magnesium transporters, TRPM6 and TRPM7. These unique proteins, termed chanzymes because they possess a channel and a kinase domain, are differentially expressed, with TRPM6 being found primarily in epithelial cells and TRPM7 occurring ubiquitously. Vascular TRPM7 is modulated by vasoactive agents, pressure, stretch, and osmotic changes and may be a novel mechanotransducer. In addition to its magnesium transporter function, TRPM7 has been implicated as a signaling kinase involved in vascular smooth muscle cell growth, apoptosis, adhesion, contraction, cytoskeletal organization, and migration, important processes involved in vascular remodeling associated with hypertension and other vascular diseases. Emerging evidence suggests that vascular TRPM7 function may be altered in hypertension. This review discusses the importance of magnesium in vascular biology and implications in hypertension and highlights the transport systems, particularly TRPM6 and TRPM7, which may play a role in the control of vascular magnesium homeostasis. Since the recent identification and characterization of Mg2+-selective transporters, there has been enormous interest in the field. However, there is still a paucity of information, and much research is needed to clarify the exact mechanisms of magnesium regulation in the cardiovascular system and the implications of aberrant transmembrane magnesium transport in the pathogenesis of hypertension and other vascular diseases.

Sensing cellular magnesium with RNA

A recently characterized 'M-box' genetic switch from bacteria is proposed to directly sense cellular Mg2+ levels through the conformation of its newly synthesized RNA. This Mg2+-sensing riboswitch controls transcription termination in front of a Mg2+ transporter gene, thus introducing a new and direct level of genetic regulation to metal ion homeostasis.

Measuring Metals with RNA

In a recent issue of Cell, Winkler and coworkers (Dann et al., 2007) describe in atomic detail the structural changes that allow a specific RNA aptamer (the M box) to regulate transcription in response to changing cellular magnesium levels.

Cellular magnesium acquisition: An anomaly in embryonic cation homeostasis

The intracellular dominance of magnesium ion makes clinical assessment difficult despite the critical role of Mg ++ in many key functions of cells and enzymes. There is general consensus that serum Mg ++ levels are not representative of the growing number of conditions for which magnesium is known to be important. There is no consensus method or sample source for testing for clinical purposes. High intracellular Mg ++ in vertebrate embryos results in part from interactions of cations which influence cell membrane transport systems. These are functionally competent from the earliest stages, at least transiently held over from the unfertilized ovum. Kinetic studies with radiotracer cations, osmolar variations, media lacking one or more of the four biological cations, Na +, Mg ++, K +, and Ca ++, and metabolic poison 0.05 mEq/L NaF, demonstrated that: (1) all four cations influence the behavior of the others, and (2) energy is required for uptake and efflux on different time scales, some against gradient. Na + uptake is energy dependent against an efflux gradient. The rate of K + loss is equal with or without fluoride, suggesting a lack of an energy requirement at these stages. Ca ++ efflux took twice as long in the presence of fluoride, likely due in part to intracellular binding. Mg ++ is anomalous in that early teleost vertebrate embryos have an intracellular content exceeding the surrounding sea water, an isolated unaffected yolk compartment, and a clear requirement for energy for both uptake and efflux. The physiological, pathological, and therapeutic roles of magnesium are poorly understood. This will change: (1) when 28Mg is once again generally available at a reasonable cost for both basic research and clinical assessment, and (2) when serum or plasma levels are determined simultaneously with intracellular values, preferably as part of complete four cation profiles. Atomic absorption spectrophotometry, energy-dispersive x-ray analysis, and inductively coupled plasma emission spectroscopy on sublingual mucosal and peripheral blood samples are potential methods of value for coordinated assessments.

Spironolactone Reduced Arrhythmia and Maintained Magnesium Homeostasis in Patients With Congestive Heart Failure

Patients with congestive heart failure (CHF) often have increased aldosterone activity that leads to hypomagnesemia. Hypomagnesemia can induce arrhythmias, an important cause of death in patients with CHF. We determined whether the aldosterone receptor antagonist spironolactone improved magnesium homeostasis and reduced arrhythmias in patients with CHF. We randomized 116 consecutive patients with CHF into placebo control group (n = 58) and spironolactone group (20 mg daily, n = 58) in addition to conventional therapy. Plasma magnesium concentration (PMC), erythrocyte magnesium concentration (EMC), and erythrocyte magnesium efflux were not different between the 2 groups of patients before treatment. Compared with control patients, patients treated with spironolactone for 6 months had increased PMC and EMC and decreased erythrocyte magnesium efflux. Patients on spironolactone therapy also had a marked decrease of 24-hour mean heart rate, ventricular and atrial premature beats, and the risk of atrial fibrillation/flutter. Pooled data from the 116 patients showed that patients with a higher EMC or a lower sodium-dependent erythrocyte magnesium efflux had a slower heart rate, fewer ventricular premature beats, and a lower risk of atrial fibrillation/flutter. Our results suggest that reducing cellular magnesium efflux and loss may contribute to the spironolactone-reduced arrhythmias in patients with CHF.

NIPA1(SPG6), the Basis for Autosomal Dominant Form of Hereditary Spastic Paraplegia, Encodes a Functional Mg2+ Transporter

Mutations in the NIPA1(SPG6) gene, named for "nonimprinted in Prader-Willi/Angelman" has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression of NIPA1 suggesting a role in cellular magnesium metabolism. Indeed NIPA1 mediates Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with a Michaelis constant of 0.69+/-0.21 mM when expressed in Xenopus oocytes. Subcellular localization with immunofluorescence showed that endogenous NIPA1 protein associates with early endosomes and the cell surface in a variety of neuronal and epithelial cells. As expected of a magnesium-responsive gene, we find that altered magnesium concentration leads to a redistribution between the endosomal compartment and the plasma membrane; high magnesium results in diminished cell surface NIPA1 whereas low magnesium leads to accumulation in early endosomes and recruitment to the plasma membrane. The mouse NIPA1 mutants, T39R and G100R, corresponding to the respective human mutants showed a loss-of-function when expressed in oocytes and altered trafficking in transfected COS7 cells. We conclude that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to abnormal trafficking of the mutated protein provides the basis of the HSP phenotype.

The reference range of serum, plasma and erythrocyte magnesium

The interest in the clinical importance of serum magnesium level has just recently begun with the analysis and findings of abnormal magnesium level in cardiovascular, metabolic and neuromuscular disorder. Although the serum level does not reflect the body magnesium level, but currently, only serum magnesium determination is widely used. Erythrocyte magnesium is considered more sensitive than serum magnesium as it reflects intracellular magnesium status. According to NCCLS (National Committee for Clinical Laboratory Standards) every laboratory is recommended to have its own reference range for the tests it performs, including magnesium determination. The reference range obtained is appropriate for the population and affected by the method and technique. This study aimed to find the reference range of serum and plasma magnesium and also intracellular magnesium i.e. erythrocyte magnesium by direct method, and compare the results of serum and plasma magnesium. Blood was taken from 114-blood donor from Unit Transfusi Darah Daerah (UTDD) Budhyarto Palang Merah Indonesia (PMI) DKI Jakarta, consisted of 57 male and 57 female, aged 17 – 65 years, clinically healthy according to PMI donor criteria. Blood was taken from blood set, collected into 4 ml vacuum tube without anticoagulant for serum magnesium determination and 3 ml vacuum tube with lithium heparin for determination of erythrocyte and plasma magnesium Determination of magnesium level was performed with clinical chemistry auto analyzer Hitachi 912 by Xylidil Blue method colorimetrically. This study showed no significant difference between serum and heparinized plasma extra cellular magnesium. The reference range for serum or plasma magnesium was 1.30 – 2.00 mEq/L and for erythrocyte magnesium was 4.46 - 7.10 mEq/L. (Med J Indones 2006; 15:229-35) Keywords: Reference range, extracellular magnesium, intracellular magnesium

Total cellular calcium and magnesium content of cultured human peripheral blood mononuclear cells following exposure to antidiabetic drugs

Drugs modulating the ATP-sensitive potassium (K(ATP)) channel activity are widely used for the treatment of diabetes mellitus, the target being pancreatic beta-cells. However, any cell type possessing K(ATP) channels might be concomitantly affected. We investigated the metabolic effect of glibenclamide, a K(ATP) channel closer, and/or diazoxide, a K(ATP) channel opener, on total intracellular content of calcium (Ca) and magnesium (Mg) of cultured peripheral blood mononuclear cells (PBMC). Metformin and rosiglitazone, acting via cellular mechanisms other than K(ATP) channels, were also tested. Ca and Mg were assessed in PBMC from healthy subjects following 72 h in vitro treatment with the respective drugs. Ca content increased significantly in PBMC treated with glibenclamide or rosiglitazone, however apparently via different intracellular pathways. Mg increased only following treatment with rosiglitazone. Metformin had no effect on intracellular Ca or Mg. Pretreatment with diazoxide resulted in a significant intracellular Ca and Mg loss in each experimental situation. If verified clinically, rosiglitazone-induced increase in Mg content of PBMC might prove beneficial beyond hypoglycaemic control. On the other hand, loss of intracellular Ca/Mg content following K(ATP) channel opening by diazoxide might eventually result in significant intracellular Ca and/or Mg depletion.

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.