Kinase and channel activity of TRPM6 are co-ordinated by a dimerization motif and pocket interaction

Contains fulltext : 138516.pdf (publisher's version ) (Open Access)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

Gender specific effects of the calcium channel TRPV4 on osteoporotic fracture risk and osteoblast-osteoclast coupling

TRPV4 is a member of the transient receptor potential (TRP) superfamily and responds to an array of stimuli, including osmolarity, pH and pressure. Recent findings showing that TRPV4 deficiency leads to reduced sensing of mechanical stimuli led us to explore the role of TRPV4 in bone. TRPV4 mRNA was abundantly expressed in both osteoblasts and osteoclasts as assessed by qPCR. Femoral cortical and trabecular bone mass as assessed by microcomputed tomography was higher in male TRPV4 knockout mice compared to wild type mice. Despite thicker bone structures, cortical porosity was increased in the male TRPV4 knockout mice leading to reduced bone strength as assessed by 3-point bending. Osteoclast and osteoblast differentiation and function was studied, using bone marrow cultures from wildtype and TRPV4 knockout mice. Osteoclast numbers as well as the formation of resorption pits were significantly reduced in cultures of TRPV4 knockout mice compared to wildtype littermates. In contrast, osteoblast differentiation and matrix mineralization was significantly increased in TRPV4 knockout bone marrow cultures. None of these parameters were significantly different in bones and bone marrow cultures of female knock out mice. These data implicate a gender-specific osteoblast–osteoclast uncoupling and support the observed increase in bone mass in male TRPV4 deficient mice. To assess the possible impact of TRPV4 on osteoporotic outcome in humans, we extracted data from the genome-wide association study within the Rotterdam Study. Two single nucleotide polymorphisms (SNPs) in the TRPV4 gene showed strong associations with osteoporotic fracture risk fragility fracture risk and hip fracture risk in men, but not in women. This was not affected after adjusting for height, weight, age and bone mineral density (BMD). In conclusion, TRPV4 plays an important role in male but not female bone biology. Apparently, the increased periosteal bone apposition fails to overcome the increased cortical porosity, leading to reduced bone strength in TRPV4 deficient male mice. In line with the gender-specific findings in mice, variations in the TRPV4 gene are predicting fracture risk in men but not in women

Reduction of Oxidative Stress in Chronic Kidney Disease Does Not Increase Circulating alpha-Klotho Concentrations

The CKD-associated decline in soluble α-Klotho levels is considered detrimental. Some in vitro and in vivo animal studies have shown that anti-oxidant therapy can upregulate the expression of α-Klotho in the kidney. We examined the effect of anti-oxidant therapy on α-Klotho concentrations in a clinical cohort with mild tot moderate chronic kidney disease (CKD). We performed a post-hoc analysis of a prospective randomized trial involving 62 patients with mild to moderate CKD (the ATIC study), all using an angiotensin-converting enzyme inhibitor (ACEi) or angiotensin receptor blocker (ARB) for 12 months. On top of that, the intervention group received anti-oxidative therapy consisting of the combination of pravastatin (40 mg/d) and vitamin E (α-tocopherol acetate, 300 mg/d) while the placebo was not treated with anti-oxidants. α-Klotho concentrations were measured at baseline and after 12 months of anti-oxidant therapy. Data were analysed using T-tests and Generalized Estimating Equations, adjusting for potential confounders such as vitamin D, parathyroid hormone, fibroblast-growth-factor 23 (FGF23) and eGFR. The cohort existed of 62 patients with an eGFR (MDRD) of 35 ± 14 ml/min/1.72 m2, 34 were male and mean age was 53.0 ± 12.5 years old. Anti-oxidative therapy did successfully reduce oxLDL and LDL concentrations (P <0.001). α-Klotho concentrations did not change in patients receiving either anti-oxidative therapy (476.9 ± 124.3 to 492.7 ± 126.3 pg/mL, P = 0.23) nor in those receiving placebo 483.2 ± 142.5 to 489.6 ± 120.3 pg/mL, P = 0.62). Changes in α-Klotho concentrations were not different between both groups (p = 0.62). No evidence was found that anti-oxidative therapy affected α-Klotho concentrations in patients with mild-moderate CKD

Dietary Mg2+ Intake and the Na+/Mg2+ Exchanger SLC41A1 Influence Components of Mitochondrial Energetics in Murine Cardiomyocytes

Cardiomyocytes are among the most energy-intensive cell types. Interplay between the components of cellular magnesium (Mg) homeostasis and energy metabolism in cardiomyocytes is poorly understood. We have investigated the effects of dietary Mg content and presence/functionality of the Na+/Mg2+ exchanger SLC41A1 on enzymatic functions of selected constituents of the Krebs cycle and complexes of the electron transport chain (ETC). The activities of aconitate hydratase (ACON), isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (KGDH), and ETC complexes CI–CV have been determined in vitro in mitochondria isolated from hearts of wild-type (WT) and Slc41a1−/− mice fed a diet with either normal or low Mg content. Our data demonstrate that both, the type of Mg diet and the Slc41a1 genotype largely impact on the activities of enzymes of the Krebs cycle and ETC. Moreover, a compensatory effect of Slc41a1−/− genotype on the effect of low Mg diet on activities of the tested Krebs cycle enzymes has been identified. A machine-learning analysis identified activities of ICDH, CI, CIV, and CV as common predictors of the type of Mg diet and of CII as suitable predictor of Slc41a1 genotype. Thus, our data delineate the effect of dietary Mg content and of SLC41A1 functionality on the energy-production in cardiac mitochondria

Serum Calcium Levels Are Associated with Novel Cardiometabolic Risk Factors in the Population-Based CoLaus Study

BACKGROUND: Associations of serum calcium levels with the metabolic syndrome and other novel cardio-metabolic risk factors not classically included in the metabolic syndrome, such as those involved in oxidative stress, are largely unexplored. We analyzed the association of albumin-corrected serum calcium levels with conventional and non-conventional cardio-metabolic risk factors in a general adult population. METHODOLOGY/PRINCIPAL FINDINGS: The CoLaus study is a population-based study including Caucasians from Lausanne, Switzerland. The metabolic syndrome was defined using the Adult Treatment Panel III criteria. Non-conventional cardio-metabolic risk factors considered included: fat mass, leptin, LDL particle size, apolipoprotein B, fasting insulin, adiponectin, ultrasensitive CRP, serum uric acid, homocysteine, and gamma-glutamyltransferase. We used adjusted standardized multivariable regression to compare the association of each cardio-metabolic risk factor with albumin-corrected serum calcium. We assessed associations of albumin-corrected serum calcium with the cumulative number of non-conventional cardio-metabolic risk factors. We analyzed 4,231 subjects aged 35 to 75 years. Corrected serum calcium increased with both the number of the metabolic syndrome components and the number of non-conventional cardio-metabolic risk factors, independently of the metabolic syndrome and BMI. Among conventional and non-conventional cardio-metabolic risk factors, the strongest positive associations were found for factors related to oxidative stress (uric acid, homocysteine and gamma-glutamyltransferase). Adiponectin had the strongest negative association with corrected serum calcium. CONCLUSIONS/SIGNIFICANCE: Serum calcium was associated with the metabolic syndrome and with non-conventional cardio-metabolic risk factors independently of the metabolic syndrome. Associations with uric acid, homocysteine and gamma-glutamyltransferase were the strongest. These novel findings suggest that serum calcium levels may be associated with cardiovascular risk via oxidative stress

Magnesium reduces calcification in bovine vascular smooth muscle cells in a dose-dependent manner

WOS: 000300421300010PubMed ID: 21750166Vascular calcification (VC), mainly due to elevated phosphate levels, is one major problem in patients suffering from chronic kidney disease. In clinical studies, an inverse relationship between serum magnesium and VC has been reported. However, there is only few information about the influence of magnesium on calcification on a cellular level available. Therefore, we investigated the effect of magnesium on calcification induced by beta-glycerophosphate (BGP) in bovine vascular smooth muscle cells (BVSMCs). BVSMCs were incubated with calcification media for 14 days while simultaneously increasing the magnesium concentration. Calcium deposition, transdifferentiation of cells and apoptosis were measured applying quantification of calcium, von Kossa and Alizarin red staining, real-time reverse transcription-polymerase chain reaction and annexin V staining, respectively. Calcium deposition in the cells dramatically increased with addition of BGP and could be mostly prevented by co-incubation with magnesium. Higher magnesium levels led to inhibition of BGP-induced alkaline phosphatase activity as well as to a decreased expression of genes associated with the process of transdifferentiation of BVSMCs into osteoblast-like cells. Furthermore, estimated calcium entry into the cells decreased with increasing magnesium concentrations in the media. In addition, higher magnesium concentrations prevented cell damage (apoptosis) induced by BGP as well as progression of already established calcification. Higher magnesium levels prevented BVSMC calcification, inhibited expression of osteogenic proteins, apoptosis and further progression of already established calcification. Thus, magnesium is influencing molecular processes associated with VC and may have the potential to play a role for VC also in clinical situations.Fresenius Medical Care Deutschland GmbH, GermanyThis study was supported by Fresenius Medical Care Deutschland GmbH, Germany

Influence of 1α, 25-dihydroxyvitamin D3 [1, 25(OH)2D3] on the expression of Sox 9 and the transient receptor potential vanilloid 5/6 ion channels in equine articular chondrocytes

Background Sox 9 is a major marker of chondrocyte differentiation. When chondrocytes are cultured in vitro they progressively de-differentiate and this is associated with a decline in Sox 9 expression. The active form of vitamin D, 1, 25 (OH)2D3 has been shown to be protective of cartilage in both humans and animals. In this study equine articular chondrocytes were grown in culture and the effects of 1, 25 (OH)2D3 upon Sox 9 expression examined. The expression of the transient receptor potential vanilloid (TRPV) ion channels 5 and 6 in equine chondrocytes in vitro, we have previously shown, is inversely correlated with de-differentiation. The expression of these channels in response to 1, 25 (OH)2D3 administration was therefore also examined. Results The active form of vitamin D (1, 25 (OH)2D3) when administered to cultured equine chondrocytes at two different concentrations significantly increased the expression of Sox 9 at both. In contrast 1, 25 (OH)2D3 had no significant effect upon the expression of either TRPV 5 or 6 at either the protein or the mRNA level. Conclusions The increased expression of Sox 9, in equine articular chondrocytes in vitro, in response to the active form of vitamin D suggests that this compound could be utilized to inhibit the progressive de-differentiation that is normally observed in these cells. It is also supportive of previous studies indicating that 1α, 25-dihydroxyvitamin D3 can have a protective effect upon cartilage in animals in vivo. The previously observed correlation between the degree of differentiation and the expression levels of TRPV 5/6 had suggested that these ion channels may have a direct involvement in, or be modulated by, the differentiation process in vitro. The data in the present study do not support this