Lorentz force in water: evidence that hydronium cyclotron resonance enhances polymorphism

Abstract

There is an ongoing question regarding the structure forming capabilities of water at ambient temperatures. To probe for different structures, we studied effects in pure water following magnetic field exposures corresponding to the ion cyclotron resonance of H₃O⁺. Included were measurements of conductivity and pH. We find that under ion cyclotron resonance (ICR) stimulation, water undergoes a transition to a form that is hydroxonium-like, with the subsequent emission of a transient 48.5?Hz magnetic signal, in the absence of any other measurable field. Our results indicate that hydronium resonance stimulation alters the structure of water, enhancing the concentration of EZ-water. These results are not only consistent with Del Giudice's model of electromagnetically coherent domains, but they can also be interpreted to show that these domains exist in quantized spin states.     

Allosteric regulation of pentameric ligand-gated ion channels: An emerging mechanistic perspective

Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger—a neurotransmitter—into an ion flux through the postsynaptic membrane. They are oligomeric assemblies that provide prototypical examples of allosterically regulated integral membrane proteins. Here, we present an overview of the most recent advances on the signal transduction mechanism based on the X-ray structures of both prokaryotic and invertebrate eukaryotic pLGICs and on atomistic Molecular Dynamics simulations. The present results suggest that ion gating involves a large structural reorganization of the molecule mediated by two distinct quaternary transitions, a global twisting and the blooming of the extracellular domain, which can be modulated by ligand binding at the topographically distinct orthosteric and allosteric sites. The emerging model of gating is consistent with a wealth of functional studies and will boost the development of novel pharmacological strategies.     

Role of ion channels during cell division

Ion channels are transmembrane proteins whose canonical function is the transport of ions across the plasma membrane to regulate cell membrane potential and play an essential role in neural communication, nerve conduction, and muscle contraction. However, over the last few years, non-canonical functions have been identified for many channels, having active roles in phagocytosis, invasiveness, proliferation, among others. The participation of some channels in cell proliferation has raised the question of whether they may play an active role in mitosis. There are several reports showing the participation of channels during interphase, however, the direct participation of ion channels in mitosis has received less attention. In this article, we summarize the current evidence on the participation of ion channels in mitosis. We also summarize some tools that would allow the study of ion channels and cell cycle regulatory molecules in individual cells during mitosis.     

Two recombinant α-like scorpion toxins from Mesobuthus eupeus with differential affinity toward insect and mammalian Na channels

α-Scorpion toxins are modulators of voltage-gated Na⁺ channels (Na_vs), which bind to the receptor site 3 to inhibit the fast inactivation of the channels. MeuNaTxα-12 and MeuNaTxα-13 are two new α-scorpion toxin-like peptides identified by cDNA cloning from the scorpion Mesobuthus eupeus with unknown functions. Here, we report their recombinant production, oxidative refolding, structural and functional features. By in vitro renaturation from bacterial inclusion bodies and further purification through reverse phase high-performance liquid chromatography, we obtained high purity recombinant products with a native-like conformation identified by circular dichroism analysis. Two-electrode voltage clamp recordings on five cloned mammalian Na_v subtypes (rNa_v1.1, rNa_v1.2, rNa_v1.4, rNa_v1.5, and mNa_v1.6) and the insect counterpart DmNa_v1, all expressed in Xenopus laevis oocytes, showed that these two peptides inhibited rapid inactivation of the sensitive Na⁺ channels with significant preference for DmNa_v1. The half maximal effective concentrations (EC₅₀) of MeuNaTxα-12 and MeuNaTxα-13 for this channel are 19.95 ± 2.99 nM and 65.50 ± 7.28 nM, respectively, showing 45 and 38 folds higher affinities than for rNa_v1.1, the most sensitive mammalian channel among the five isoforms. Our functional data confirms that these two peptides belong to the α-like scorpion toxin group. A combined analysis of the site 3 sequences and the pharmacological data illuminates the importance of the loop L_D4:S5–S6 of the channel in interacting with the toxins whereas affinity variations between MeuNaTxα-12 and MeuNaTxα-13 highlight a key functional role of a cationic side chain at position 28 of MeuNaTxα-12. Successful expression together with structural and functional characterization of these two new α-like scorpion toxins lays basis for further studies of their structure–function relationship.     

Extracellular Zinc Ion Regulates Transient Receptor Potential Melastatin 5 (TRPM5) Channel Activation through Its Interaction with a Pore Loop Domain

The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent cation channel activated by intracellular Ca²⁺. Expression of this channel is restricted to taste cells, the pancreas and brainstem, and is thought to be involved in controlling membrane potentials. Its endogenous ligands are not well characterized. Here, we show that extracellular application of Zn²⁺ inhibits TRPM5 activity. In whole-cell patch-clamp recordings, extracellular application of ZnCl₂ inhibited step-pulse-induced TRPM5 currents with 500 nm free intracellular Ca²⁺ in a dose-dependent manner (IC₅₀ = 4.3 μm at −80 mV). ZnSO₄ also inhibited TRPM5 activity. Extracellular application of ZnCl₂ inhibited TRPM5 activation at several temperatures. Furthermore, inhibition by 30 μm ZnCl₂ was impaired in TRPM5 mutants in which His at 896, and Glu at 926 and/or Glu at 939 in the outer pore loop were replaced with Gln. From these results, we conclude that extracellular Zn²⁺ inhibits TRPM5 channels, and the residues in the outer pore loop of TRPM5 are critically involved in the inhibition.     

Micrometric segregation of fluorescent membrane lipids: relevance for endogenous lipids and biogenesis in erythrocytes

Micrometric membrane lipid segregation is controversial. We addressed this issue in attached erythrocytes and found that fluorescent boron dipyrromethene (BODIPY) analogs of glycosphingolipids (GSLs) [glucosylceramide (BODIPY-GlcCer) and monosialotetrahexosylganglioside (GM1BODIPY)], sphingomyelin (BODIPY-SM), and phosphatidylcholine (BODIPY-PC inserted into the plasma membrane spontaneously gathered into distinct submicrometric domains. GM1BODIPY domains colocalized with endogenous GM1 labeled by cholera toxin. All BODIPY-lipid domains disappeared upon erythrocyte stretching, indicating control by membrane tension. Minor cholesterol depletion suppressed BODIPY-SM and BODIPY-PC but preserved BODIPY-GlcCer domains. Each type of domain exchanged constituents but assumed fixed positions, suggesting self-clustering and anchorage to spectrin. Domains showed differential association with 4.1R versus ankyrin complexes upon antibody patching. BODIPY-lipid domains also responded differentially to uncoupling at 4.1R complexes [protein kinase C (PKC) activation] and ankyrin complexes (in spherocytosis, a membrane fragility disease). These data point to micrometric compartmentation of polar BODIPY-lipids modulated by membrane tension, cholesterol, and differential association to the two nonredundant membrane:spectrin anchorage complexes. Micrometric compartmentation might play a role in erythrocyte membrane deformability and fragility.     

The Sulfonylurea Receptor 1 (Sur1)-Transient Receptor Potential Melastatin 4 (Trpm4) Channel

The sulfonylurea receptor 1 (Sur1)-NC_Ca-ATP channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NC_Ca-ATP channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and K_ATP channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.     

The Serum- and Glucocorticoid-inducible Kinases SGK1 and SGK3 Regulate hERG Channel Expression via Ubiquitin Ligase Nedd4-2 and GTPase Rab11

The hERG (human ether-a-go-go-related gene) encodes the α subunit of the rapidly activating delayed rectifier potassium channel (I_Kr). Dysfunction of hERG channels due to mutations or certain medications causes long QT syndrome, which can lead to fatal ventricular arrhythmias or sudden death. Although the abundance of hERG in the plasma membrane is a key determinant of hERG functionality, the mechanisms underlying its regulation are not well understood. In the present study, we demonstrated that overexpression of the stress-responsive serum- and glucocorticoid-inducible kinase (SGK) isoforms SGK1 and SGK3 increased the current and expression level of the membrane-localized mature proteins of hERG channels stably expressed in HEK 293 (hERG-HEK) cells. Furthermore, the synthetic glucocorticoid, dexamethasone, increased the current and abundance of mature ERG proteins in both hERG-HEK cells and neonatal cardiac myocytes through the enhancement of SGK1 but not SGK3 expression. We have previously shown that mature hERG channels are degraded by ubiquitin ligase Nedd4-2 via enhanced channel ubiquitination. Here, we showed that SGK1 or SGK3 overexpression increased Nedd4-2 phosphorylation, which is known to inhibit Nedd4-2 activity. Nonetheless, disruption of the Nedd4-2 binding site in hERG channels did not eliminate the SGK-induced increase in hERG expression. Additional disruption of Rab11 proteins led to a complete elimination of SGK-mediated increase in hERG expression. These results show that SGK enhances the expression level of mature hERG channels by inhibiting Nedd4-2 as well as by promoting Rab11-mediated hERG recycling.     

A Pivotal Role for Tryptophan 447 in Enzymatic Coupling of Human Endothelial Nitric Oxide Synthase (eNOS): EFFECTS ON TETRAHYDROBIOPTERIN-DEPENDENT CATALYSIS AND eNOS DIMERIZATION*

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.     

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. We have recently shown that cholesterol regulates representative members of the Kir family and that in the majority of the cases, cholesterol suppresses channel function. Furthermore, recent data indicate that cholesterol regulates Kir channels by specific sterol-protein interactions, yet the location of the cholesterol binding site in Kir channels is unknown. Using a combined computational-experimental approach, we show that cholesterol may bind to two nonanular hydrophobic regions in the transmembrane domain of Kir2.1 located between adjacent subunits of the channel. The location of the binding regions suggests that cholesterol modulates channel function by affecting the hinging motion at the center of the pore-lining transmembrane helix that underlies channel gating either directly or through the interface between the N and C termini of the channel.