Structure of the mammalian TRPM7, a magnesium channel required during embryonic development.

The transient receptor potential ion channel subfamily M, member 7 (TRPM7), is a ubiquitously expressed protein that is required for mouse embryonic development. TRPM7 contains both an ion channel and an α-kinase. The channel domain comprises a nonselective cation channel with notable permeability to Mg2+ and Zn2+ Here, we report the closed state structures of the mouse TRPM7 channel domain in three different ionic conditions to overall resolutions of 3.3, 3.7, and 4.1 Å. The structures reveal key residues for an ion binding site in the selectivity filter, with proposed partially hydrated Mg2+ ions occupying the center of the conduction pore. In high [Mg2+], a prominent external disulfide bond is found in the pore helix, which is essential for ion channel function. Our results provide a structural framework for understanding the TRPM1/3/6/7 subfamily and extend the knowledge base upon which to study the diversity and evolution of TRP channels.

Structure of full-length human TRPM4.

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na+-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4-S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π-π bonds and cation-π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.

Histone phosphorylation by TRPM6's cleaved kinase attenuates adjacent arginine methylation to regulate gene expression.

TRPM6 and TRPM7 are members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels. Deletion of either gene in mice is embryonically lethal. TRPM6/7 are the only known examples of single polypeptides containing both an ion channel pore and a serine/threonine kinase (chanzyme). Here we show that the C-terminal kinase domain of TRPM6 is cleaved from the channel domain in a cell type-specific fashion and is active. Cleavage requires that the channel conductance is functional. The cleaved kinase translocates to the nucleus, where it is strictly localized and phosphorylates specific histone serine and threonine (S/T) residues. TRPM6-cleaved kinases (M6CKs) bind subunits of the protein arginine methyltransferase 5 (PRMT5) molecular complex that make important epigenetic modifications by methylating histone arginine residues. Histone phosphorylation by M6CK results in a dramatic decrease in methylation of arginines adjacent to M6CK-phosphorylated amino acids. Knockout of TRPM6 or inactivation of its kinase results in global changes in histone S/T phosphorylation and changes the transcription of hundreds of genes. We hypothesize that M6CK associates with the PRMT5 molecular complex in the nucleus, directing M6CK to a specific genomic location and providing site-specific histone phosphorylation. M6CK histone phosphorylation, in turn, regulates transcription by attenuating the effect of local arginine methylation.

TRPM7 senses oxidative stress to release Zn2+ from unique intracellular vesicles.

TRPM7 (transient receptor potential cation channel subfamily M member 7) regulates gene expression and stress-induced cytotoxicity and is required in early embryogenesis through organ development. Here, we show that the majority of TRPM7 is localized in abundant intracellular vesicles. These vesicles (M7Vs) are distinct from endosomes, lysosomes, and other familiar vesicles or organelles. M7Vs accumulate Zn2+ in a glutathione-enriched, reduced lumen when cytosolic Zn2+ concentrations are elevated. Treatments that increase reactive oxygen species (ROS) trigger TRPM7-dependent Zn2+ release from the vesicles, whereas reduced glutathione prevents TRPM7-dependent cytosolic Zn2+ influx. These observations strongly support the notion that ROS-mediated TRPM7 activation releases Zn2+ from intracellular vesicles after Zn2+ overload. Like the endoplasmic reticulum, these vesicles are a distributed system for divalent cation uptake and release, but in this case the primary divalent ion is Zn2+ rather than Ca2.

Altered conductance and permeability of Cx40 mutations associated with atrial fibrillation.

Gap junctions ensure the rapid propagation of the action potential throughout the myocardium. Three mutant forms of connexin40 (Cx40; A96S, M163V, and G38D), the primary component of the atrial gap junction channel, are associated with atrial fibrillation and retain the ability to form functional channels. We determined the biophysical properties of these mutant gap junctions in transiently transfected HeLa and N2A cells. All three mutants showed macroscopic junctional conductances over the range of 0.5 to 40 nS, and voltage dependences comparable to those of wild-type (WT) Cx40. However, the unitary conductance of G38D channels was ∼1.6-fold higher than that of WT Cx40 channels (∼220 vs. ∼135 pS), whereas the unitary conductances of the A96S and M163V mutants were similar to that of WT Cx40. Furthermore, the M163V and G38D channels exhibited approximately two- and approximately fivefold higher permeability to the anionic dye Lucifer yellow (LY) relative to K+ (LY/K+) compared with that of WT Cx40, whereas A96S LY transfer was similar to that of WT (G38D > M163V > A96S ≈ Cx40WT). In contrast, G38D channels were almost impermeable to cationic ethidium bromide (EtBr), suggesting that G38D alters channel selectivity. Conversely, A96S and M163V channels showed enhanced EtBr permeability relative to WT Cx40, with the following permeability order: M163V > A96S > Cx40WT > G38D. Altered conductive and permeability properties of mutant channels suggest an essential role for Cx40-mediated biochemical and electrical coupling in cardiac tissues. The altered properties of the three single-base substitution mutants may play a role in mechanisms of reentry arrhythmias.

Synthesis and nematocide activity of S-glycopyranosyl-6,7-diarylthiolumazines.

6,7-Diaryl derivatives of mono and di-S-glycopyranosylthiolumazine derivatives 5-8 were prepared to test their nematocide activity. In vitro tests against Caenorhabditis elegans were performed and it was found that monosubstituted derivatives 5-7 showed higher activity than the corresponding unsubstituted 2-thiolumazines 1-3, whilst 2-S,4-S-di-glycopyranosylpteridine derivative 8 was inactive in contrast to unsubstituted derivative 4. In order to check whether the lack of activity of 8 was due to the two bulky substituents of the pteridine nucleus, 2-S,4-S-dimethyl derivative 9 was synthesized and assayed showing also lack of activity. A theoretical study on the stability of the different possible tautomers of compound 4 was carried out in an attempt to explain some, in appearance, anomalous (13)C NMR data of this compound.

Evaluación de los aportes de la biología molecular al diagnóstico del cancer mamario humano / Evaluation of the contributions of molecular biology to diagnosis of the human mammary cancer

En el presente trabajo hemos expuesto los hallazgos más relevantes de la biología molecular, que contribuyen a una mejor comprensión del mecanismo de carcinogénesis, posibilitando el desarrollo de nuevos métodos para la detección del cáncer mamario humano. Se ha revisado exhaustivamente la literatura internacional relacionada con la detección, aislamiento y aplicación de marcadores biológicos orientados al diagnóstico del cáncer mamario humano. Es así que la expresión de genes embrionarios (antígeno carcinoembrionario, alfafetoproteína, isoenzima de Regan), en las manifestaciones neoplásicas mamarias, y en otros desórdenes, constituye uno de los rasgos más singulares de las alteraciones a nivel de expresión genética en células transformadas. Se han desarrollado diversos métodos para su detección en especímenes tumorales y en fluídos biológicos, resultando de enorme ayuda para el diagnóstico de estadíos preneoplásicos. Sin embargo, su mayor aplicación radica en el seguimiento de la enfermedad durante el curso de su tratamiento y remisión. La desventaja en común de estos marcadores es su especificidad relativa, hecho que no invalida su aplicación. Probablemente, la mayor contribución resultante de estos marcadores clásicos es el haber estimulado la búsqueda de nuevos marcadores que exhiban mayor especificadad para el diagnóstico anatomo-patológico y precoz del cáncer de mama. Las técnicas de hibridización somática han permitido el desarrollo de hibridomas secretantes, cuyos clones debidamente seleccionados, poseen la propiedad de sintetizar anticuerpos dirigidos hacia un determinante antigénico de una proteína tumor específica. Los anticuerpos monoclonales como H59,B6.2 y otros, forman parte de una batería molecular de inapreciables facultades. El cáncer mamario es una entidad nosológica con carcterísticas particulares. Su dependencia o su antomía hormonal es en gran parte debida a la presencia de receptores específicos para cada hormona o grupo de ellas. La síntesis de nuevas macromoléculas, manifestación biológica de la interacción hormona-recptor, es considerada como un excelente modelo para determinar la funcionalidad hormonal. Nuestra orientación en el campo de la biología molecular es la descripción y purificación de nuevos marcadores que puedan integrarse a una precisa tecnología para el diagnóstico de neoplasias mamarias humanas... (TRUNCADO)

Evaluación de los aportes de la biología molecular al diagnóstico del cancer mamario humano / Evaluation of the contributions of molecular biology to diagnosis of the human mammary cancer

En el presente trabajo hemos expuesto los hallazgos más relevantes de la biología molecular, que contribuyen a una mejor comprensión del mecanismo de carcinogénesis, posibilitando el desarrollo de nuevos métodos para la detección del cáncer mamario humano. Se ha revisado exhaustivamente la literatura internacional relacionada con la detección, aislamiento y aplicación de marcadores biológicos orientados al diagnóstico del cáncer mamario humano. Es así que la expresión de genes embrionarios (antígeno carcinoembrionario, alfafetoproteína, isoenzima de Regan), en las manifestaciones neoplásicas mamarias, y en otros desórdenes, constituye uno de los rasgos más singulares de las alteraciones a nivel de expresión genética en células transformadas. Se han desarrollado diversos métodos para su detección en especímenes tumorales y en fluídos biológicos, resultando de enorme ayuda para el diagnóstico de estadíos preneoplásicos. Sin embargo, su mayor aplicación radica en el seguimiento de la enfermedad durante el curso de su tratamiento y remisión. La desventaja en común de estos marcadores es su especificidad relativa, hecho que no invalida su aplicación. Probablemente, la mayor contribución resultante de estos marcadores clásicos es el haber estimulado la búsqueda de nuevos marcadores que exhiban mayor especificadad para el diagnóstico anatomo-patológico y precoz del cáncer de mama. Las técnicas de hibridización somática han permitido el desarrollo de hibridomas secretantes, cuyos clones debidamente seleccionados, poseen la propiedad de sintetizar anticuerpos dirigidos hacia un determinante antigénico de una proteína tumor específica. Los anticuerpos monoclonales como H59,B6.2 y otros, forman parte de una batería molecular de inapreciables facultades. El cáncer mamario es una entidad nosológica con carcterísticas particulares. Su dependencia o su antomía hormonal es en gran parte debida a la presencia de receptores específicos para cada hormona o grupo de ellas. La síntesis de nuevas macromoléculas, manifestación biológica de la interacción hormona-recptor, es considerada como un excelente modelo para determinar la funcionalidad hormonal. Nuestra orientación en el campo de la biología molecular es la descripción y purificación de nuevos marcadores que puedan integrarse a una precisa tecnología para el diagnóstico de neoplasias mamarias humanas... (TRUNCADO)(AU)