Functional Characterization of Homo- and Heteromeric Channel Kinases TRPM6 and TRPM7

TRPM6 and TRPM7 are two known channel kinases that play important roles in various physiological processes, including Mg2+ homeostasis. Mutations in TRPM6 cause hereditary hypomagnesemia and secondary hypocalcemia (HSH). However, whether TRPM6 encodes functional channels is controversial. Here we demonstrate several signature features of TRPM6 that distinguish TRPM6 from TRPM7 and TRPM6/7 channels. We show that heterologous expression of TRPM6 but not the mutant TRPM6S141L produces functional channels with divalent cation permeability profile and pH sensitivity distinctive from those of TRPM7 channels and TRPM6/7 complexes. TRPM6 exhibits unique unitary conductance that is 2- and 1.5-fold bigger than that of TRPM7 and TRPM6/7. Moreover, micromolar levels of 2-aminoethoxydiphenyl borate (2-APB) maximally increase TRPM6 but significantly inhibit TRPM7 channel activities; whereas millimolar concentrations of 2-APB potentiate TRPM6/7 and TRPM7 channel activities. Furthermore, Mg2+ and Ca2+ entry through TRPM6 is enhanced three- to fourfold by 2-APB. Collectively, these results indicate that TRPM6 forms functional homomeric channels as well as heteromeric TRPM6/7 complexes. The unique characteristics of these three channel types, TRPM6, TRPM7, and TRPM6/7, suggest that they may play different roles in vivo

Potentiation of TRPM7 Inward Currents by Protons

TRPM7 is unique in being both an ion channel and a protein kinase. It conducts a large outward current at +100 mV but a small inward current at voltages ranging from −100 to −40 mV under physiological ionic conditions. Here we show that the small inward current of TRPM7 was dramatically enhanced by a decrease in extracellular pH, with an ∼10-fold increase at pH 4.0 and 1–2-fold increase at pH 6.0. Several lines of evidence suggest that protons enhance TRPM7 inward currents by competing with Ca2+ and Mg2+ for binding sites, thereby releasing blockade of divalent cations on inward monovalent currents. First, extracellular protons significantly increased monovalent cation permeability. Second, higher proton concentrations were required to induce 50% of maximal increase in TRPM7 currents when the external Ca2+ and Mg2+ concentrations were increased. Third, the apparent affinity for Ca2+ and Mg2+ was significantly diminished at elevated external H+ concentrations. Fourth, the anomalous-mole fraction behavior of H+ permeation further suggests that protons compete with divalent cations for binding sites in the TRPM7 pore. Taken together, it appears that at physiological pH (7.4), Ca2+ and Mg2+ bind to TRPM7 and inhibit the monovalent cationic currents; whereas at high H+ concentrations, the affinity of TRPM7 for Ca2+ and Mg2+ is decreased, thereby allowing monovalent cations to pass through TRPM7. Furthermore, we showed that the endogenous TRPM7-like current, which is known as Mg2+-inhibitable cation current (MIC) or Mg nucleotide–regulated metal ion current (MagNuM) in rat basophilic leukemia (RBL) cells was also significantly potentiated by acidic pH, suggesting that MIC/MagNuM is encoded by TRPM7. The pH sensitivity represents a novel feature of TRPM7 and implies that TRPM7 may play a role under acidic pathological conditions

The Cation Selectivity Filter of the Bacterial Sodium Channel, NaChBac

The Bacillus halodurans voltage-gated sodium-selective channel (NaChBac) (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001b. Science. 294:2372–2375), is an ideal candidate for high resolution structural studies because it can be expressed in mammalian cells and its functional properties studied in detail. It has the added advantage of being a single six transmembrane (6TM) orthologue of a single repeat of mammalian voltage-gated Ca2+ (CaV) and Na+ (NaV) channels. Here we report that six amino acids in the pore domain (LESWAS) participate in the selectivity filter. Replacing the amino acid residues adjacent to glutamatic acid (E) by a negatively charged aspartate (D; LEDWAS) converted the Na+-selective NaChBac to a Ca2+- and Na+-permeant channel. When additional aspartates were incorporated (LDDWAD), the mutant channel resulted in a highly expressing voltage-gated Ca2+-selective conductance

Hydroxychloroquine enhances anticancer effect of DOX/folate-phytosterol-carboxymethyl cellulose nanoparticles in A549 lung cancer cells

Purpose: To study the in vitro anticancer effect of doxorubicin-loaded folate-phytosterol-carboxymethyl cellulose nanoparticles (DOX/FPCMC NPs), alone and in combination with the antimalarial drug hydroxychloroquine (HCQ) on human lung cancer cells (A549 cells). Methods: Human lung adenocarcinoma A549 cell line was treated with blank FPCMC NPs, HCQ, free DOX, DOX/FPCMC NPs, free DOX + HCQ or DOX/FPCMC NPs + HCQ. The concentrations of HCQ, DOX and FPCMC NPs varied within the ranges of 20-120 μmol/L, 2-12 mg/L and 50-500 mg/L, respectively. Cell viability and free folate competitive inhibition were determined using MTT assay. Cell proliferation and migration were investigated with wound healing assay, while confocal laser scanning microscopy (CLSM) was used to determine cellular uptake of drugs. Results: In all formulations, the DOX/FPCMC NPs + HCQ produced the highest cytotoxicity in A549 cells due to high cytotoxicity arising from folate-receptor-mediated endocytosis and HCQ-induced inhibition of autophagy. Free folate competitively inhibited the cytotoxicity of DOX/FPCMC NPs on A549 cells. Wound healing assay showed that A549 cells treated with DOX/FPCMC NPs + HCQ had the lowest cell levels of proliferation and migration capacity. The cellular uptake of DOX/FPCMC NPs by A549 cells was higher than that of free DOX. Conclusion: The combination of DOX/FPCMC NPs and HCQ produced the best antitumor effect and had a promising potential for reversal of MDR Keywords: Folate-phytosterol-carboxymethyl cellulose, Doxorubicin, Hydroxychloroquine, Anticancer, Lung cance

Brazil and China in Mozambican Agriculture: Emerging Insights from the Field

Mozambique, a country undergoing rapid transformations driven by the recent discovery of mineral resources, is one of the top destinations for Chinese and Brazilian cooperation and investment in Africa. This article provides an account of the policies, narratives, operational modalities and underlying motivations of Brazilian and Chinese development cooperation in Mozambique. It is particularly interested in understanding how the engagements are perceived and talked about, what drives them and what formal and informal relations are emerging at the level of particular exchanges. The article draws on three cases (1) ProSavana, Brazil's current flagship programme in Mozambique, which aims to transform the country's savanna, spreading along the Nacala corridor, drawing on Brazil's own experience in the Cerrado ; (2) the Chinese Agricultural Technology Demonstration Centre (ATDC); and (3) a private Chinese rice investment project in the Xai?Xai irrigation scheme, which builds on a technical cooperation initiative. Commonalities and differences between the Brazilian and Chinese approaches are discussed

Phosphatidylinositol 4,5-bisphosphate (PIP2) controls magnesium gatekeeper TRPM6 activity

TRPM6 is crucial for human Mg2+ homeostasis as patients carrying TRPM6 mutations develop hypomagnesemia and secondary hypocalcemia (HSH). However, the activation mechanism of TRPM6 has remained unknown. Here we demonstrate that phosphatidylinositol-4,5-bisphophate (PIP2) controls TRPM6 activation and Mg2+ influx. Stimulation of PLC-coupled M1-receptors to deplete PIP2 potently inactivates TRPM6. Translocation of over-expressed 5-phosphatase to cell membrane to specifically hydrolyze PIP2 also completely inhibits TRPM6. Moreover, depolarization-induced-activation of the voltage-sensitive-phosphatase (Ci-VSP) simultaneously depletes PIP2 and inhibits TRPM6. PLC-activation induced PIP2-depletion not only inhibits TRPM6, but also abolishes TRPM6-mediated Mg2+ influx. Furthermore, neutralization of basic residues in the TRP domain leads to nonfunctional or dysfunctional mutants with reduced activity by PIP2, suggesting that they are likely to participate in interactions with PIP2. Our data indicate that PIP2 is required for TRPM6 channel function; hydrolysis of PIP2 by PLC-coupled hormones/agonists may constitute an important pathway for TRPM6 gating, and perhaps Mg2+ homeostasis

Genetic deletion of Rnd3 results in aqueductal stenosis leading to hydrocephalus through up-regulation of Notch signaling

Rho family guanosine triphosphatase (GTPase) 3 (Rnd3), a member of the small Rho GTPase family, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration, and proliferation through the Rho kinase-dependent signaling pathway. We report a role of Rnd3 in the pathogenesis of hydrocephalus disorder. Mice with Rnd3 genetic deletion developed severe obstructive hydrocephalus with enlargement of the lateral and third ventricles, but not of the fourth ventricles. The cerebral aqueducts in Rnd3-null mice were partially or completely blocked by the overgrowth of ependymal epithelia. We examined the molecular mechanism contributing to this Rnd3-deficiency–mediated hydrocephalus and found that Rnd3 is a regulator of Notch signaling. Rnd3 deficiency, through either gene deletion or siRNA knockdown, resulted in a decrease in Notch intracellular domain (NICD) protein degradation. However, there was no correlated change in mRNA change, which in turn led to an increase in NICD protein levels. Immunoprecipitation analysis demonstrated that Rnd3 and NICD physically interacted, and that down-regulation of Rnd3 attenuated NICD protein ubiquitination. This eventually enhanced Notch signaling activity and promoted aberrant growth of aqueduct ependymal cells, resulting in aqueduct stenosis and the development of congenital hydrocephalus. Inhibition of Notch activity rescued the hydrocephalus disorder in the mutant animals