René J. M. Bindels

TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption.

Mg2+ is an essential ion involved in a multitude of physiological and biochemical processes and a major constituent of bone tissue. Mg2+ homeostasis in mammals depends on the equilibrium between intestinal Mg2+ absorption and renal Mg2+ excretion, but little is known about the molecular nature of the proteins involved in the transepithelial transport of Mg2+ in these organs. Recently, it was shown that patients with mutations in TRPM6, a member of the transient receptor potential family of cation channels, suffer from hypomagnesemia with secondary hypocalcemia (HSH) as a result of impaired renal and/or intestinal Mg2+ handling. Here, we show that TRPM6 is specifically localized along the apical membrane of the renal distal convoluted tubule and the brush-border membrane of the small intestine, epithelia particularly associated with active Mg2+ (re)absorption. In kidney, parvalbumin and calbindin-D28K, two divalent-binding proteins, are co-expressed with TRPM6 and might function as intracellular Mg2+ buffers in the distal convoluted tubule. Heterologous expression of wild-type TRPM6 but not TRPM6 mutants identified in HSH patients induces a Mg2+- and Ca2+-permeable cation channel tightly regulated by intracellular Mg2+ levels. The TRPM6-induced channel displays strong outward rectification, has a 5-fold higher affinity for Mg2+ than for Ca2+, and is blocked in a voltage-dependent manner by ruthenium red. Our data indicate that TRPM6 comprises all or part of the apical Mg2+ channel of Mg2+-absorbing epithelia.

Enhanced passive Ca2+ reabsorption and reduced Mg2+ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia.

Thiazide diuretics enhance renal Na+ excretion by blocking the Na+-Cl- cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying hypocalciuria and hypomagnesemia remain debated. Here, we show that enhanced passive Ca2+ transport in the proximal tubule rather than active Ca2+ transport in distal convolution explains thiazide-induced hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na+ and Ca2+ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca2+ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced hypocalciuria in transient receptor potential channel subfamily V, member 5-knockout (Trpv5-knockout) mice, in which active distal Ca2+ reabsorption is abolished due to inactivation of the epithelial Ca2+ channel Trpv5. Third, HCTZ upregulated the Na+/H+ exchanger, responsible for the majority of Na+ and, consequently, Ca2+ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca2+ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of hypocalciuria parallels a compensatory increase in Na+ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg2+ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation.

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

The molecular assembly of the epithelial Ca2+ 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 Cd2+ 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 Ca2+ channels that varied with respect to Ca2+‐dependent inactivation, Ba2+ selectivity and pharmacological block. Thus, Ca2+‐transporting epithelia co‐expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca2+ transport kinetics.

Permeation and Gating Properties of the Novel Epithelial Ca 2! Channel*

The recently cloned epithelial Ca2+ channel (ECaC) constitutes the Ca2+influx pathway in 1,25-dihydroxyvitamin D3-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca2+ concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca2+ gradient, demonstrating the distinctive Ca2+ permeability and constitutive activation of ECaC. Cells dialyzed with 10 mm1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding current-voltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below −40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba2+or Sr2+ replaced Ca2+ and was absent in Ca2+-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratioP Ca:P Na calculated from the reversal potential at 30 mm[Ca2+] o was larger than 100. The divalent cation selectivity profile is Ca2+ > Mn2+ > Ba2+ ∼ Sr2+. Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca2+ was replaced by Ba2+ and was virtually abolished if [Ca2+] o was lowered to 1 nm. In conclusion, ECaC is a Ca2+ selective channel, exhibiting Ca2+-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.

Impaired basolateral sorting of pro-EGF causes isolated recessive renal hypomagnesemia.

Primary hypomagnesemia constitutes a rare heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg(2+)) wasting resulting in generally shared symptoms of Mg(2+) depletion, such as tetany and generalized convulsions, and often including associated disturbances in calcium excretion. However, most of the genes involved in the physiology of Mg(2+) handling are unknown. Through the discovery of a mutation in the EGF gene in isolated autosomal recessive renal hypomagnesemia, we have, for what we believe is the first time, identified a magnesiotropic hormone crucial for total body Mg(2+) balance. The mutation leads to impaired basolateral sorting of pro-EGF. As a consequence, the renal EGFR is inadequately stimulated, resulting in insufficient activation of the epithelial Mg(2+) channel TRPM6 (transient receptor potential cation channel, subfamily M, member 6) and thereby Mg(2+) loss. Furthermore, we show that colorectal cancer patients treated with cetuximab, an antagonist of the EGFR, develop hypomagnesemia, emphasizing the significance of EGF in maintaining Mg(2+) balance.

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.

Calcium and phosphate homeostasis: concerted interplay of new regulators.

Calcium (Ca2+) and phosphate (Pi) are essential to many vital physiological processes. Consequently the maintenance of Ca2+ and Pi homeostasis is essential to a healthy existence. This occurs through the concerted action of intestinal, renal, and skeletal regulatory mechanisms. Ca2+ and Pi handling by these organs is under tight hormonal control. Disturbances in their homeostasis have been linked to pathophysiological disorders including chronic renal insufficiency, kidney stone formation, and bone abnormalities. Importantly, the kidneys fine‐tune the amount of Ca2+ and Pi retained in the body by altering their (re)absorption from the glomerular filtrate. The ion transport proteins involved in this process have been studied extensively. Recently, new key players have been identified in the regulation of the Ca2+ and Pi balance. Novel regulatory mechanisms and their implications were introduced for the antiaging hormone klotho and fibroblast growth factor member 23 (FGF23). Importantly, transgenic mouse models, exhibiting disturbances in Ca2+ and Pi balance, have been of great value in the elucidation of klotho and FGF23 functioning. This review highlights the current knowledge and ongoing research into Ca2+ and Pi homeostasis, emphasizing findings from several relevant knockout mouse models.

Angiotensin II contributes to podocyte injury by increasing TRPC6 expression via an NFAT-mediated positive feedback signaling pathway.

The transient receptor potential channel C6 (TRPC6) is a slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Gain-of-function mutations in TRPC6 cause hereditary focal segmental glomerulosclerosis (FSGS), and several human acquired proteinuric diseases show increased glomerular TRPC6 expression. Angiotensin II (AngII) is a key contributor to glomerular disease and may regulate TRPC6 expression in nonrenal cells. We demonstrate that AngII regulates TRPC6 mRNA and protein levels in cultured podocytes and that AngII infusion enhances glomerular TRPC6 expression in vivo. In animal models for human FSGS (doxorubicin nephropathy) and increased renin-angiotensin system activity (Ren2 transgenic rats), glomerular TRPC6 expression was increased in an AngII-dependent manner. TRPC6 expression correlated with glomerular damage markers and glomerulosclerosis. We show that the regulation of TRPC6 expression by AngII and doxorubicin requires TRPC6-mediated Ca(2+) influx and the activation of the Ca(2+)-dependent protein phosphatase calcineurin and its substrate nuclear factor of activated T cells (NFAT). Accordingly, calcineurin inhibition by cyclosporine decreased TRPC6 expression and reduced proteinuria in doxorubicin nephropathy, whereas podocyte-specific inducible expression of a constitutively active NFAT mutant increased TRPC6 expression and induced severe proteinuria. Our findings demonstrate that the deleterious effects of AngII on podocytes and its pathogenic role in glomerular disease involve enhanced TRPC6 expression via a calcineurin/NFAT positive feedback signaling pathway.

EGF Increases TRPM6 Activity and Surface Expression

Recent identification of a mutation in the EGF gene that causes isolated recessive hypomagnesemia led to the finding that EGF increases the activity of the epithelial magnesium (Mg2+) channel transient receptor potential M6 (TRPM6). To investigate the molecular mechanism mediating this effect, we performed whole-cell patch-clamp recordings of TRPM6 expressed in human embryonic kidney 293 (HEK293) cells. Stimulation of the EGF receptor increased current through TRPM6 but not TRPM7. The carboxy-terminal alpha-kinase domain of TRPM6 did not participate in the EGF receptor-mediated increase in channel activity. This activation relied on both the Src family of tyrosine kinases and the downstream effector Rac1. Activation of Rac1 increased the mobility of TRPM6, assessed by fluorescence recovery after photobleaching, and a constitutively active mutant of Rac1 mimicked the stimulatory effect of EGF on TRPM6 mobility and activity. Ultimately, TRPM6 activation resulted from increased cell surface abundance. In contrast, dominant negative Rac1 decreased TRPM6 mobility, abrogated current development, and prevented the EGF-mediated increase in channel activity. In summary, EGF-mediated stimulation of TRPM6 occurs via signaling through Src kinases and Rac1, thereby redistributing endomembrane TRPM6 to the plasma membrane. These results describe a regulatory mechanism for transepithelial Mg2+ transport and consequently whole-body Mg2+ homeostasis.

CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia

Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.