Lourdes Millan-PerezPeña

The increase in Zinc levels and upregulation of Zinc transporters are mediated by nitric oxide in the cerebral cortex after transient ischemia in the rat

The transient occlusion of cerebral arteries causes an increase in zinc levels in the brain, which is associated with a production of nitric oxide (NO). The types of zinc transporters (ZnT) involved in zinc homeostasis in the cerebral cortex after hypoxia-ischemia are not completely known. We studied the effect of the transient occlusion (10 min) of the common carotid artery (CCA) on NO-induced zinc levels, ZnT mRNA expression, and cell-death markers in the cerebral cortex-hippocampus of the rat. Nitrites, zinc, and lipoperoxidation were quantified by colorimetric methods, ZnT expression was determined by RT-PCR, caspase-3 by ELISA and immunohistochemistry, and histopathological alterations by H&E staining. After restoration of the blood flow, the basal levels of NO and zinc increased in a biphasic manner over time, but the peaks of NO levels appeared earlier (2 h and 24 h) than those of zinc (6 h and 36 h). Upregulation of ZnT1, ZnT2, and ZnT4 mRNAs was determined after 8-h postreperfusion, but ZnT3 RNA levels were unaffected. Lipoperoxidation and caspase-3 levels were also increased, and necrosis and apoptosis were present at 24 h postreperfusion. All the effects determined were prevented by l-nitro-arginine methyl ester injected 1 h before the occlusion of the CCA. Our results suggest that the upregulation of ZnT1, ZnT2, and ZnT4 was to decrease the cytosolic zinc levels caused by NO after transient occlusion of the CCA, although this was unable to lead to physiological levels of zinc and to prevent cell damage in the cerebral cortex-hippocampus of the rat.

Identification of Navβ1 Residues Involved in the Modulation of the Sodium Channel Nav1.4

Voltage-gated sodium channels (VGSCs) are heteromeric protein complexes that initiate action potentials in excitable cells. The voltage-gated sodium channel accessory subunit, Navβ1, allosterically modulates the α subunit pore structure upon binding. To date, the molecular determinants of the interface remain unknown. We made use of sequence, knowledge and structure-based methods to identify residues critical to the association of the α and β1 Nav1.4 subunits. The Navβ1 point mutant C43A disrupted the modulation of voltage dependence of activation and inactivation and delayed the peak current decay, the recovery from inactivation, and induced a use-dependent decay upon depolarisation at 1 Hz. The Navβ1 mutant R89A selectively delayed channel inactivation and recovery from inactivation and had no effect on voltage dependence or repetitive depolarisations. Navβ1 mutants Y32A and G33M selectively modified the half voltage of inactivation without altering the kinetics. Despite low sequence identity, highly conserved structural elements were identified. Our models were consistent with published data and may help relate pathologies associated with VGSCs to the Navβ1 subunit.

An in silico approach to primaquine binding to Trp756 in the external vestibule of sodium channel Na v 1.4

The aim of our computed study was to examine the possible binding site of primaquine (PQ) using a combined homology protein modeling, automated docking, and experimental approach. The target models of wild type and mutant types of the voltage- dependent sodium channel in rat skeletal muscle (rNa v 1.4) were based on previous work by other researchers. Docking was carried out on the P-loop in the structural model of the rNa v 1.4 channel, in the open state configuration, to identify those amino acidic residues important for PQ binding. The three-dimensional models of the P-loop segment of wild types and mutant types (W402, W756C, W1239C, and W1531A at the outer tryptophan-rich lip, as well as D400C, E755C, K1237C, and A1529C of the DEKA motif) helped us to identify residues playing key roles in aminoquinoline binding. In good agreement with experimental results, a 1000-fold inhibition loss was observed. Tryptophan 756 is crucial for the reversible blocking effects of PQ. As a result, mutant-type W756C abolished the blocking effect of PQ in voltage-clamp

Mefloquine inhibits voltage dependent Nav1.4 channel by overlapping the local anaesthetic binding site

ABSTRACT Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na+ channels is unknown. To address the mechanism of action of mefloquine, we employed two‐electrode voltage clamp recordings on Xenopus laevis oocytes, site‐directed mutagenesis of the rat Na+ channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Nav1.4 currents (IC50 =60 &mgr;M), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady‐state inactivation curve to more hyperpolarized potentials. The presence of the &bgr;1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency‐independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Nav1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable &pgr;‐&pgr; interactions with this residue. We provide structure‐activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na+ channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources.

A residue W756 in the P-loop segment of the sodium channel is critical for primaquine binding.

Our study on the wild-type and mutants of the voltage-dependent sodium channel in the rat skeletal muscle Na(v) 1.4 was to examine the possible binding site of primaquine PQ by using an experimental approach. We used a standard voltage-clamp in oocytes. Previously, we had demonstrated that PQ blocks the voltage-dependent sodium current in rat myocytes and that this blocking is concentration-dependent and voltage-independent. The direct-site mutagenesis in the P-loop segment W402C, W756C, W1239C, W1531A at the outer tryptophan-rich lip, and D400C, E758C, K1237C, A1529C of the DEKA locus helped us to identify residues playing a key role in aminoquinoline binding. In full agreement with our computed results, where a 1000-fold reduction of inhibition was measured, the tryptophan 756 is crucial for the reversible modulating effects of PQ. The W756C decreased the blocking effect of PQ in voltage-clamp assays. This new binding site may be important to the development of new drugs that modulate sodium inward currents.

Characterization of specific allosteric effects of the Na+ channel β1 subunit on the Nav1.4 isoform

The mechanism of inactivation of mammalian voltage-gated Na+ channels involves transient interactions between intracellular domains resulting in direct pore occlusion by the IFM motif and concomitant extracellular interactions with the β1 subunit. Navβ1 subunits constitute single-pass transmembrane proteins that form protein–protein associations with pore-forming α subunits to allosterically modulate the Na+ influx into the cell during the action potential of every excitable cell in vertebrates. Here, we explored the role of the intracellular IFM motif of rNav1.4 (skeletal muscle isoform of the rat Na+ channel) on the α-β1 functional interaction and showed for the first time that the modulation of β1 is independent of the IFM motif. We found that: (1) Nav1.4 channels that lack the IFM inactivation particle can undergo a “C-type-like inactivation” albeit in an ultraslow gating mode; (2) β1 can significantly accelerate the inactivation of Nav1.4 channels in the absence of the IFM motif. Previously, we identified two residues (T109 and N110) on the β1 subunit that disrupt the α-β1 allosteric modulation. We further characterized the electrophysiological effects of the double alanine substitution of these residues demonstrating that it decelerates inactivation and recovery from inactivation, abolishes the modulation of steady-state inactivation and induces a current rundown upon repetitive stimulation, thus causing a general loss of function. Our results contribute to delineating the process of the mammalian Na+ channel inactivation. These findings may be relevant to the design of pharmacological strategies, targeting β subunits to treat pathologies associated to Na+ current dysfunction.

Study of Cellular Processes in Higher Eukaryotes Using the Yeast Schizosaccharomyces pombe as a Model

Schizosaccharomyces pombe (Sz. pombe), or fission yeast, is an ascomycete unicellular fungus that has been used as a model system for studying diverse biological processes of higher eukaryotic cells, such as the cell cycle and the maintenance of cell shape, apoptosis, and ageing. Sz. pombe is a rod-shaped cell that grows by apical extension; it divides along the long axis by medial fission and septation. The fission yeast has a doubling time of 2–4 hours, it is easy and inexpensive to grow in simple culture conditions, and can be maintained in the haploid or the diploid state. Sz. pombe can be genetically manipulated using methods such as mutagenesis or gene disruption by homologous recombination. Fission yeast was defined as a micro-mammal because it shares many molecular, genetic, and biochemical features with cells of higher eukaryotes in mRNA splicing, post-translational modifications as N-glycosylation protein, cell-cycle regulation, nutrient-sensing pathways as the target of rapamycin (TOR) network, cAMP-PKA pathway, and autophagy. This chapter uses Sz. pombe as a useful model for studying important cellular processes that support life such as autophagy, apoptosis, and the ageing process. Therefore, the molecular analysis of these processes in fission yeast has the potential to generate new knowledge that could be applied to higher eukaryotes.

Molecular Diagnostics as an Indispensable Tool for the Diagnosis of Infectious Diseases of Viral Origin and Global Impact

Infectious diseases are responsible for a considerable number of deaths in infectious in entire world. Infectious diseases are human diseases caused by viruses, bacteria, parasites, fungi and other microorganisms. Most of them have been controlled by vaccines or antimicrobials. However, some of them still represent global public health problems and are being monitored by the WHO and Center for Disease Control and Prevention. This chapter provides an overview of the applications of molecular methods for infectious diseases caused by viruses (intracellular obligate parasites) of global impact such as Dengue virus, Hepatitis B virus or influenza A virus. The infectious diseases not only represent a potential danger to the life of all human beings but also a significant investment in its detection, treatment and control of their spread. The increase in opportunities of infection by globalization, high rates of mobility among most countries around the world, the patient susceptibility to diseases due to genetic variation in populations [1], the ability of the microorganisms to evade the host immune response has forced the World Health Organization (WHO) to establish better methods of detection, prevention and control of infectious diseases caused by viruses as influenza A virus, coronaviruses, dengue virus, among others [2]. On the other hand, some types of cancer are the result of chronic viral infections caused by human papillomavirus, hepatitis B and C virus. Other infectious diseases are related to the development of neurological disorders caused by the measles virus, or human immunodeficiency virus [3]. In the determination of the etiology

Chlorthalidone inhibits the KvLQT1 potassium current in guinea-pig ventricular myocytes and oocytes from Xenopus laevis

Chlorthalidone is used for the treatment of hypertension as it produces a lengthening of the cardiac action potential. However, there is no experimental evidence that chlorthalidone has electrophysiological effects on the potassium currents involved in cardiac repolarization.

Prophylactic Zinc and Therapeutic Selenium Administration Increases the Antioxidant Enzyme Activity in the Rat Temporoparietal Cortex and Improves Memory after a Transient Hypoxia-Ischemia

In the cerebral hypoxia-ischemia rat model, the prophylactic administration of zinc can cause either cytotoxicity or preconditioning effect, whereas the therapeutic administration of selenium decreases the ischemic damage. Herein, we aimed to explore whether supplementation of low doses of prophylactic zinc and therapeutic selenium could protect from a transient hypoxic-ischemic event. We administrated zinc (0.2 mg/kg of body weight; ip) daily for 14 days before a 10 min common carotid artery occlusion (CCAO). After CCAO, we administrated sodium selenite (6 μg/kg of body weight; ip) daily for 7 days. In the temporoparietal cerebral cortex, we determined nitrites by the Griess method and lipid peroxidation by the Gerard-Monnier assay. qPCR was used to measure mRNA of nitric oxide synthases, antioxidant enzymes, chemokines, and their receptors. We measured the enzymatic activity of SOD and GPx and protein levels of chemokines and their receptors by ELISA. We evaluated long-term memory using the Morris-Water maze test. Our results showed that prophylactic administration of zinc caused a preconditioning effect, decreasing nitrosative/oxidative stress and increasing GPx and SOD expression and activity, as well as eNOS expression. The therapeutic administration of selenium maintained this preconditioning effect up to the late phase of hypoxia-ischemia. Ccl2, Ccr2, Cxcl12, and Cxcr4 were upregulated, and long-term memory was improved. Pyknotic cells were decreased suggesting prevention of neuronal cell death. Our results show that the prophylactic zinc and therapeutic selenium administration induces effective neuroprotection in the early and late phases after CCAO.