TRPM7 Provides an Ion Channel Mechanism for Cellular Entry of Trace Metal Ions

Trace metal ions such as Zn2+, Fe2+, Cu2+, Mn2+, and Co2+ are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca2+- and Mg2+-permeable cation channel, whose activity is regulated by intracellular Mg2+ and Mg2+·ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide–regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn2+ and Ni2+, which both permeate TRPM7 up to four times better than Ca2+. Similarly, native MagNuM currents are also able to support Zn2+ entry. Furthermore, TRPM7 allows other essential metals such as Mn2+ and Co2+ to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd2+, Ba2+, and Sr2+. Equimolar replacement studies substituting 10 mM Ca2+ with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn2+ ≈ Ni2+ >> Ba2+ > Co2+ > Mg2+ ≥ Mn2+ ≥ Sr2+ ≥ Cd2+ ≥ Ca2+, while trivalent ions such as La3+ and Gd3+ are not measurably permeable. With the exception of Mg2+, which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn2+, Co2+, or Ni2+ suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca2+ and Mg2+, suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells

Maternal and offspring intelligence in relation to BMI across childhood and adolescence

Objective: The present study tested the association between both mothers’ and offspring’s intelligence and offspring’s body mass index (BMI) in youth. Method: Participants were members of the National Longitudinal Survey of Youth 1979 (NLSY-79) Children and Young Adults cohort (n = 11,512) and their biological mothers who were members of the NLSY-79 (n = 4932). Offspring’s IQ was measured with the Peabody Individual Achievement Test (PIAT). Mothers’ IQ was measured with the Armed Forces Qualification Test (AFQT). A series of regression analyses tested the association between IQ and offspring’s BMI by age group, while adjusting for pre-pregnancy BMI and family SES. The analyses were stratified by sex and ethnicity (non-Black and non-Hispanic, Black, and Hispanic). Results: The following associations were observed in the fully adjusted analyses. For the non-Blacks and non-Hispanics, a SD increment in mothers’ IQ was negatively associated with daughters’ BMI across all age-groups, ranging from β = −0.12 (95% CI −0.22 to −0.02, p = 0.021) in late childhood, to β = −0.17 (95% C.I. −0.27 to −0.07, p = 0001), in early adolescence and a SD increment in boys’ IQ was positively associated with their BMI in early adolescence β = 0.09 (95% CI 0.01–0.18, p = 0.031). For Blacks, there was a non-linear relationship between mothers’ IQ and daughters’ BMI across childhood and between girls’ IQ and BMI across adolescence. There was a positive association between mothers’ IQ and sons’ BMI in early adolescence (β = 0.17, 95% CI 0.02–0.32, p = 0.030). For Hispanic boys, there was a positive IQ-BMI association in late childhood (β = 0.19, 95% CI 0.05–0.33, p = 0.008) and early adolescence (β = 0.17, 95% CI 0.04–0.31, p = 0.014). Conclusion: Mothers’ IQ and offspring’s IQ were associated with offspring’s BMI. The relationships varied in direction and strength across ethnicity, age group and sex. Obesity interventions may benefit from acknowledging the heterogeneous influence that intelligence has on childhood BMI

The Stature of Boys Is Inversely Correlated to the Levels of Their Sertoli Cell Hormones: Do the Testes Restrain the Maturation of Boys?

The testes of preadolescent boys appear to be dormant, as they produce only trace levels of testosterone [1]. However, they release supra-adult levels of Müllerian Inhibiting Substance (MIS, anti-Müllerian hormone) and lesser levels of inhibin B (InhB), for unknown reasons [2], [3]. Boys have a variable rate of maturation, which on average is slower than girls. The height of children relative to their parents is an index of their maturity [4], [5]. We report here that a boy's level of MIS and InhB is stable over time and negatively correlates with his height and his height relative to his parent's height. This suggests that boy's with high levels of MIS and InhB are short because they are immature, rather than because they are destined to be short men. The levels of MIS and InhB in the boys did not correlate with known hormonal modulators of growth, and were additive with age and the growth hormone/IGF1 axis as predictors of a boy's height. If MIS and InhB were causal regulators of maturity, then the inter-boy differences in the levels of these hormone produces variation in maturation equivalent to 18-months of development. MIS and InhB may thus account for most of the variation in the rate of male development. If boys lacked these hormones, then an average 5-year-old boy would be over 5 cm taller than age-matched girls, making boys almost as dimorphic as men, for height. This indicates that boys have a high growth potential that is initially suppressed by their testes. The concept of the childhood testes suppressing an adult male feature appears paradoxical. However, the growth of children requires intergenerational transfer of nutrients. Consequently, the MIS/InhB slowing of male growth may have been historically advantageous, as it would minimizes any sex bias in the maternal cost of early child rearing

Human Tumor Cell Proliferation Evaluated Using Manganese-Enhanced MRI

Tumor cell proliferation can depend on calcium entry across the cell membrane. As a first step toward the development of a non-invasive test of the extent of tumor cell proliferation in vivo, we tested the hypothesis that tumor cell uptake of a calcium surrogate, Mn(2+) [measured with manganese-enhanced MRI (MEMRI)], is linked to proliferation rate in vitro.Proliferation rates were determined in vitro in three different human tumor cell lines: C918 and OCM-1 human uveal melanomas and PC-3 prostate carcinoma. Cells growing at different average proliferation rates were exposed to 1 mM MnCl(2) for one hour and then thoroughly washed. MEMRI R(1) values (longitudinal relaxation rates), which have a positive linear relationship with Mn(2+) concentration, were then determined from cell pellets. Cell cycle distributions were determined using propidium iodide staining and flow cytometry. All three lines showed Mn(2+)-induced increases in R(1) compared to cells not exposed to Mn(2+). C918 and PC-3 cells each showed a significant, positive correlation between MEMRI R(1) values and proliferation rate (p≤0.005), while OCM-1 cells showed no significant correlation. Preliminary, general modeling of these positive relationships suggested that pellet R(1) for the PC-3 cells, but not for the C918 cells, could be adequately described by simply accounting for changes in the distribution of the cell cycle-dependent subpopulations in the pellet.These data clearly demonstrate the tumor-cell dependent nature of the relationship between proliferation and calcium influx, and underscore the usefulness of MEMRI as a non-invasive method for investigating this link. MEMRI is applicable to study tumors in vivo, and the present results raise the possibility of evaluating proliferation parameters of some tumor types in vivo using MEMRI

Blockade of TRPM7 Channel Activity and Cell Death by Inhibitors of 5-Lipoxygenase

TRPM7 is a ubiquitous divalent-selective ion channel with its own kinase domain. Recent studies have shown that suppression of TRPM7 protein expression by RNA interference increases resistance to ischemia-induced neuronal cell death in vivo and in vitro, making the channel a potentially attractive pharmacological target for molecular intervention. Here, we report the identification of the 5-lipoxygenase inhibitors, NDGA, AA861, and MK886, as potent blockers of the TRPM7 channel. Using a cell-based assay, application of these compounds prevented cell rounding caused by overexpression of TRPM7 in HEK-293 cells, whereas inhibitors of 12-lipoxygenase and 15-lipoxygenase did not prevent the change in cell morphology. Application of the 5-lipoxygenase inhibitors blocked heterologously expressed TRPM7 whole-cell currents without affecting the protein's expression level or its cell surface concentration. All three inhibitors were also effective in blocking the native TRPM7 current in HEK-293 cells. However, two other 5-lipoxygenase specific inhibitors, 5,6-dehydro-arachidonic acid and zileuton, were ineffective in suppressing TRPM7 channel activity. Targeted knockdown of 5-lipoxygenase did not reduce TRPM7 whole-cell currents. In addition, application of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), the product of 5-lipoxygenase, or 5-HPETE's downstream metabolites, leukotriene B4 and leukotriene D4, did not stimulate TRPM7 channel activity. These data suggested that NDGA, AA861, and MK886 reduced the TRPM7 channel activity independent of their effect on 5-lipoxygenase activity. Application of AA861 and NDGA reduced cell death for cells overexpressing TRPM7 cultured in low extracellular divalent cations. Moreover, treatment of HEK-293 cells with AA861 increased cell resistance to apoptotic stimuli to a level similar to that obtained for cells in which TRPM7 was knocked down by RNA interference. In conclusion, NDGA, AA861, and MK886 are potent blockers of the TRPM7 channel capable of attenuating TRPM7's function during cell stress, making them effective tools for the biophysical characterization and suppression of TRPM7 channel conductance in vivo

Using C. elegans to decipher the cellular and molecular mechanisms underlying neurodevelopmental disorders

Prova tipográfica (uncorrected proof)Neurodevelopmental disorders such as epilepsy, intellectual disability (ID), and autism spectrum disorders (ASDs) occur in over 2 % of the population, as the result of genetic mutations, environmental factors, or combination of both. In the last years, use of large-scale genomic techniques allowed important advances in the identification of genes/loci associated with these disorders. Nevertheless, following association of novel genes with a given disease, interpretation of findings is often difficult due to lack of information on gene function and effect of a given mutation in the corresponding protein. This brings the need to validate genetic associations from a functional perspective in model systems in a relatively fast but effective manner. In this context, the small nematode, Caenorhabditis elegans, presents a good compromise between the simplicity of cell models and the complexity of rodent nervous systems. In this article, we review the features that make C. elegans a good model for the study of neurodevelopmental diseases. We discuss its nervous system architecture and function as well as the molecular basis of behaviors that seem important in the context of different neurodevelopmental disorders. We review methodologies used to assess memory, learning, and social behavior as well as susceptibility to seizures in this organism. We will also discuss technological progresses applied in C. elegans neurobiology research, such as use of microfluidics and optogenetic tools. Finally, we will present some interesting examples of the functional analysis of genes associated with human neurodevelopmental disorders and how we can move from genes to therapies using this simple model organism.The authors would like to acknowledge Fundação para a Ciência e Tecnologia (FCT) (PTDC/SAU-GMG/112577/2009). AJR and CB are recipients of FCT fellowships: SFRH/BPD/33611/2009 and SFRH/BPD/74452/2010, respectively

Dissociation of the store-operated calcium current ICRAC and the Mg-nucleotide-regulated metal ion current MagNuM

Rat basophilic leukaemia cells (RBL-2H3-M1) were used to study the characteristics of the store-operated Ca2+ release-activated Ca2+ current (ICRAC) and the magnesium-nucleotide-regulated metal cation current (MagNuM) (which is conducted by the LTRPC7 channel). Pipette solutions containing 10 mm BAPTA and no added ATP induced both currents in the same cell, but the time to half-maximal activation for MagNuM was about two to three times slower than that of ICRAC. Differential suppression of ICRAC was achieved by buffering free [Ca2+]i to 90 nm and selective inhibition of MagNuM was accomplished by intracellular solutions containing 6 mm Mg.ATP, 1.2 mm free [Mg2+]i or 100 μm GTP-γ-S, allowing investigations on these currents in relative isolation. Removal of extracellular Ca2+ and Mg2+ caused both currents to be carried significantly by monovalent ions. In the absence or presence of free [Mg2+]i, ICRAC carried by monovalent ions inactivated more rapidly and more completely than MagNuM carried by monovalent ions. Since several studies have used divalent-free solutions on either side of the membrane to study selectivity and single-channel behaviour of ICRAC, these experimental conditions would have favoured the contribution of MagNuM to monovalent conductance and call for caution in interpreting results where both ICRAC and MagNuM are activated