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Find video protocols related to scientific articles indexed in Pubmed.
Mg2+ homeostasis: the balancing act of TRPM6.
Curr. Opin. Nephrol. Hypertens.
PUBLISHED: 06-07-2014
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The tight control of blood magnesium (Mg) levels is of central importance for numerous physiological processes. A persistent low Mg 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 Mg homeostasis.
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Kinase and channel activity of TRPM6 are co-ordinated by a dimerization motif and pocket interaction.
Biochem. J.
PUBLISHED: 03-22-2014
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Mutations in the gene that encodes the atypical channel-kinase TRPM6 (transient receptor potential melastatin 6) cause HSH (hypomagnesaemia with secondary hypocalcaemia), a disorder characterized by defective intestinal Mg2+ transport and impaired renal Mg2+ reabsorption. TRPM6, together with its homologue TRPM7, are unique proteins as they combine an ion channel domain with a C-terminally fused protein kinase domain. How TRPM6 channel and kinase activity are linked is unknown. Previous structural analysis revealed that TRPM7 possesses a non-catalytic dimerization motif preceding the kinase domain. This interacts with a dimerization pocket lying within the kinase domain. In the present study, we provide evidence that the dimerization motif in TRPM6 plays a critical role in regulating kinase activity as well as ion channel activity. We identify mutations within the TRPM6 dimerization motif (Leu1718 and Leu1721) or dimerization pocket (L1743A, Q1832K, A1836N, L1840A and L1919Q) that abolish dimerization and establish that these mutations inhibit protein kinase activity. We also demonstrate that kinase activity of a dimerization motif mutant can be restored by addition of a peptide encompassing the dimerization motif. Moreover, we observe that mutations that disrupt the dimerization motif and dimerization pocket interaction greatly diminish TRPM6 ion channel activity, in a manner that is independent of kinase activity. Finally, we analyse the impact on kinase activity of ten disease-causing missense mutations that lie outwith the protein kinase domain of TRPM6. This revealed that one mutation lying nearby the dimerization motif (S1754N), found previously to inhibit channel activity, abolished kinase activity. These results provide the first evidence that there is structural co-ordination between channel and kinase activity, which is mediated by the dimerization motif and pocket interaction. We discuss that modulation of this interaction could comprise a major regulatory mechanism by which TRPM6 function is controlled.
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New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia.
Eur. J. Hum. Genet.
PUBLISHED: 06-20-2013
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Despite recent progress in our understanding of renal magnesium (Mg(2+)) handling, the molecular mechanisms accounting for transepithelial Mg(2+) transport are still poorly understood. Mutations in the TRPM6 gene, encoding the epithelial Mg(2+) channel TRPM6 (transient receptor potential melastatin 6), have been proven to be the molecular cause of hypomagnesemia with secondary hypocalcemia (HSH; OMIM 602014). HSH manifests in the newborn period being characterized by very low serum Mg(2+) levels (<0.4?mmol/l) accompanied by low serum calcium (Ca(2+)) concentrations. A proportion of previously described TRPM6 mutations lead to a truncated TRPM6 protein resulting in a complete loss-of-function of the ion channel. In addition, five-point mutations have been previously described. The aim of this study was to complement the current clinical picture by adding the molecular data from five new missense mutations found in five patients with HSH. To this end, patch-clamp analysis and cell surface measurements were performed to assess the effect of the various mutations on TRPM6 channel function. All mutant channels, expressed in HEK293 cells, showed loss-of-function, whereas no severe trafficking impairment to the plasma membrane surface was observed. We conclude that the new TRPM6 missense mutations lead to dysregulated intestinal/renal Mg(2+) (re)absorption as a consequence of loss of TRPM6 channel function.European Journal of Human Genetics advance online publication, 14 August 2013; doi:10.1038/ejhg.2013.178.
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Regulation of two renal chloride transporters, AE1 and pendrin, by electrolytes and aldosterone.
PLoS ONE
PUBLISHED: 01-31-2013
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The renal handling of salt and protons and bicarbonate are intricately linked through shared transport mechanisms for sodium, chloride, protons, and bicarbonate. In the collecting duct, the regulated fine-tuning of salt and acid-base homeostasis is achieved by a series of transport proteins located in different cell types, intercalated and principal cells. Intercalated cells are considered to be of less importance for salt handling but recent evidence has suggested that the anion exchanger pendrin may participate in salt reabsorption and blood pressure regulation. Here, we examined the regulated expression of two functionally related but differentially expressed anion exchangers, AE1 and pendrin, by dietary electrolyte intake and aldosterone. Cortical expression of pendrin was regulated on mRNA and protein level. The combination of NaHCO? and DOCA enhanced pendrin mRNA and protein levels, whereas DOCA or NaHCO? alone had no effect. NaCl or KHCO? increased pendrin mRNA, KCl decreased its mRNA abundance. On protein level, NH?Cl, NaCl, and KCl reduced pendrin expression, the other treatments were without effect. In contrast, AE1 mRNA or protein expression in kidney cortex was regulated by none of these treatments. In kidney medulla, NaHCO?/DOCA or NaHCO? alone enhanced AE1 mRNA levels. AE1 protein abundance was increased by NH?Cl, NaHCO?/DOCA, and NaCl. Immunolocalization showed that during NH?Cl treatment the relative number of AE1 positive cells was increased and pendrin expressing cells reduced. Thus, pendrin and AE1 are differentially regulated with distinct mechanisms that separately affect mRNA and protein levels. Pendrin is regulated by acidosis and chloride intake, whereas AE1 is enhanced by acidosis, NaCl, and the combination of DOCA and NaHCO?.
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Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.
Am. J. Hum. Genet.
PUBLISHED: 08-09-2010
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Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.
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Effects of the EGFR Inhibitor Erlotinib on Magnesium Handling.
J. Am. Soc. Nephrol.
PUBLISHED: 07-01-2010
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A mutation in pro-EGF causes isolated hypomagnesemia, and monoclonal antibodies targeting the extracellular domain of the EGF receptor (EGFR) affect epithelial Mg(2+) transport. The effect of the EGFR tyrosine kinase inhibitor erlotinib on Mg(2+) homeostasis, however, remains unknown. Here, we injected C57BL/6 mice with erlotinib for 23 days and observed a small but significant decrease in serum Mg(2+) concentrations at days 16 and 23, but the fractional excretion of Mg(2+) remained unchanged after 23 days. Semiquantitative immunohistochemical evaluation did not reveal detectable changes in renal expression of transient receptor potential melastatin 6 (TRPM6) protein, the channel that mediates Mg(2+) reabsorption. Patch clamp analysis in TRPM6-expressing cells demonstrated that 30 muM erlotinib inhibited EGF-induced changes in TRPM6 current density and tyrosine phosphorylation of EGFR; 0.3 muM erlotinib did not have these effects. Furthermore, 30 muM erlotinib inhibited EGF-stimulated increases in the mobile fraction of endomembrane TRPM6 channels. In summary, erlotinib can influence Mg(2+) handling but its effect on the systemic Mg(2+) concentration seems less potent than that observed with antibody-based EGFR inhibitors. These data suggest that typical human dosages of erlotinib are unlikely to severely affect serum Mg(2+) concentrations.
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Methionine sulfoxide reductase B1 (MsrB1) recovers TRPM6 channel activity during oxidative stress.
J. Biol. Chem.
PUBLISHED: 06-28-2010
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Mg(2+) is an essential ion for many cellular processes, including protein synthesis, nucleic acid stability, and numerous enzymatic reactions. Mg(2+) homeostasis in mammals depends on the equilibrium between intestinal absorption, renal excretion, and exchange with bone. The transient receptor potential melastatin type 6 (TRPM6) is an epithelial Mg(2+) channel, which is abundantly expressed in the luminal membrane of the renal and intestinal cells. It functions as the gatekeeper of transepithelial Mg(2+) transport. Remarkably, TRPM6 combines a Mg(2+)-permeable channel with an alpha-kinase domain. Here, by the Ras recruitment system, we identified methionine sulfoxide reductase B1 (MsrB1) as an interacting protein of the TRPM6 alpha-kinase domain. Importantly, MsrB1 and TRPM6 are both present in the renal Mg(2+)-transporting distal convoluted tubules. MsrB1 has no effect on TRPM6 channel activity in the normoxic conditions. However, hydrogen peroxide (H(2)O(2)) decreased TRPM6 channel activity. Co-expression of MsrB1 with TRPM6 attenuated the inhibitory effect of H(2)O(2) (TRPM6, 67 +/- 5% of control; TRPM6 + MsrB1, 81 +/- 5% of control). Cell surface biotinylation assays showed that H(2)O(2) treatment does not affect the expression of TRPM6 at the plasma membrane. Next, mutation of Met(1755) to Ala in TRPM6 reduced the inhibitory effect of H(2)O(2) on TRPM6 channel activity (TRPM6 M1755A: 84 +/- 10% of control), thereby mimicking the action of MsrB1. Thus, these data suggest that MsrB1 recovers TRPM6 channel activity by reducing the oxidation of Met(1755) and could, thereby, function as a modulator of TRPM6 during oxidative stress.
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Functional analysis of the Kv1.1 N255D mutation associated with autosomal dominant hypomagnesemia.
J. Biol. Chem.
PUBLISHED: 11-10-2009
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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.
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Epithelial Mg2+ channel TRPM6: insight into the molecular regulation.
Magnes Res
PUBLISHED: 09-29-2009
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Our understanding of the molecular mechanisms of renal magnesium (Mg2+) handling has greatly enhanced over recent years. This review highlights the regulatory factors controlling Mg2+ homeostasis through its effects on the epithelial Mg2+ channel TRPM6 (Transient Receptor Potential Melastatin subtype 6), the gatekeeper of the bodys Mg2+ balance. Drug treatment, acid-base status, and several hormones have been shown to regulate TRPM6 expression, while its channel activity is modified by intracellular Mg2+, pH, and ATP. Recently, epidermal growth factor (EGF) and estrogen have been implicated as magnesiotropic hormones. The stimulation of the EGF receptor (EGFR) leads to an intracellular cascade involving Rac1 that promotes trafficking of TRPM6 to the plasma membrane. Furthermore, long-term EGF treatment upregulates the expression of TRPM6. Estrogen has also been shown to stimulate TRPM6 activity upon short-term treatment, next to its long-term regulatory effect on TRPM6 transcription. TRPM6, and its closest homologue TRPM7, are composed of a Mg2+ -permeable channel fused to an alpha-kinase domain. In the intracellular compartment, the receptor for activated C-kinase (RACK1), the repressor for estrogen receptor activity (REA), and ATP were identified as negative modulators of TRPM6 activity through its alpha-kinase domain. Therefore, the a-kinase domain acts as an indirect player involved in Mg2+ homeostasis by its feedback function in the TRPM6-mediated Mg2+ influx.
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Regulation of the epithelial Mg2+ channel TRPM6 by estrogen and the associated repressor protein of estrogen receptor activity (REA).
J. Biol. Chem.
PUBLISHED: 03-26-2009
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The maintenance of the Mg(2+) balance of the body is essential for neuromuscular excitability, protein synthesis, nucleic acid stability, and numerous enzymatic systems. The Transient Receptor Potential Melastatin 6 (TRPM6) functions as the gatekeeper of transepithelial Mg(2+) transport. However, the molecular regulation of TRPM6 channel activity remains elusive. Here, we identified the repressor of estrogen receptor activity (REA) as an interacting protein of TRPM6 that binds to the 6(th), 7(th), and 8(th) beta-sheets in its alpha-kinase domain. Importantly, REA and TRPM6 are coexpressed in renal Mg(2+)-transporting distal convoluted tubules (DCT). We demonstrated that REA significantly inhibits TRPM6, but not its closest homologue TRPM7, channel activity. This inhibition occurs in a phosphorylation-dependent manner, since REA has no effect on the TRPM6 phosphotransferase-deficient mutant (K1804R), while it still binds to this mutant. Moreover, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA potentiated the inhibitory effect of REA on TRPM6 channel activity. Finally, we showed that the interaction between REA and TRPM6 is a dynamic process, as short-term 17beta-estradiol treatment disassociates the binding between these proteins. In agreement with this, 17beta-estradiol treatment significantly stimulates the TRPM6-mediated current in HEK293 cells. These results suggest a rapid pathway for the effect of estrogen on Mg(2+) homeostasis in addition to its transcriptional effect. Together, these data indicate that REA operates as a negative feedback modulator of TRPM6 in the regulation of active Mg(2+) (re)absorption and provides new insight into the molecular mechanism of renal transepithelial Mg(2+) transport.
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A missense mutation in the Kv1.1 voltage-gated potassium channel-encoding gene KCNA1 is linked to human autosomal dominant hypomagnesemia.
J. Clin. Invest.
PUBLISHED: 01-07-2009
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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.
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