Theoretical investigation of hydroxylated analogues of valinomycin as potassium transporter.

Valinomycin is a potent ionophore known for its ability to transport potassium ions across biological membranes. The study focuses on the hydroxylated analogues of valinomycin (HyVLMs) and compares their energy profiles and capabilities for transporting potassium ions across phospholipid membranes. Using metadynamics, we investigated the energy profiles of wildtype valinomycin (VLM_1) and its three hydroxylated analogues (VLM_2, VLM_3, and VLM_4). We observed that all analogues exhibited energy maxima in the centre of the membrane and preferred positions below the phospholipid heads. Furthermore, the entry barriers for membrane penetration were similar among the analogues, suggesting that the hydroxyl group did not significantly affect their passage through the membrane. Transition state calculations provided insights into the ability of valinomycin analogues to capture potassium ions, with VLM_4 showing the lowest activation energy and VLM_2 displaying the highest. Our findings contribute to understanding the mechanisms of potassium transport by valinomycin analogues and highlight their potential as ionophores. The presence of the hydroxyl group is of particular importance because it paves the way for subsequent chemical modifications and the synthesis of new antiviral agents with reduced intrinsic toxicity.

Ionophore constructed from non-covalent assembly of a G-quadruplex and liponucleoside transports K+-ion across biological membranes.

The selective transport of ions across cell membranes, controlled by membrane proteins, is critical for a living organism. DNA-based systems have emerged as promising artificial ion transporters. However, the development of stable and selective artificial ion transporters remains a formidable task. We herein delineate the construction of an artificial ionophore using a telomeric DNA G-quadruplex (h-TELO) and a lipophilic guanosine (MG). MG stabilizes h-TELO by non-covalent interactions and, along with the lipophilic side chain, promotes the insertion of h-TELO within the hydrophobic lipid membrane. Fluorescence assays, electrophysiology measurements and molecular dynamics simulations reveal that MG/h-TELO preferentially transports K+-ions in a stimuli-responsive manner. The preferential K+-ion transport is presumably due to conformational changes of the ionophore in response to different ions. Moreover, the ionophore transports K+-ions across CHO and K-562 cell membranes. This study may serve as a design principle to generate selective DNA-based artificial transporters for therapeutic applications.

Glibenclamide inhibits NLRP3 inflammasome-mediated IL-1ß secretion in human trophoblasts.

Infection-associated pregnancy complications cause premature delivery. Caspase-1 is involved in the maturation of interleukin (IL)-1ß, which is activated by the NLRP3 inflammasome. To characterize the significance of the NLRP3 inflammasome pathway in the placenta, the effects of activators and inhibitors on NLRP3-related molecules were examined using isolated primary trophoblasts. Caspase-1 and IL-1ß mRNA expression was markedly increased in response to lipopolysaccharide (LPS), a toll-like receptor (TLR)4 ligand. Treatment with the potassium ionophore nigericin significantly increased the level of activated caspase-1. Treatment with either LPS or nigericin stimulated IL-1ß secretion, whereas pretreatment with the ATP-sensitive K+ channel inhibitor glibenclamide, the Rho-associated coiled-coil kinase inhibitor Y-27632, or a caspase-1 inhibitor significantly decreased nigericin-induced IL-1ß secretion. In addition, dibutyryl-cAMP, which induces trophoblast differentiation, decreased expression of NLRP3, caspase-1, and IL-1ß. These findings suggest that trophoblasts can secrete IL-1ß through the NLRP3/caspase-1 pathway, which is suppressed by glibenclamide, and that the TLR4-mediated NLRP3 inflammasome pathway is more likely to be stimulated in undifferentiated than differentiated trophoblasts. Our data support the hypothesis that inhibition of the NLRP3 inflammasome can suppress placental inflammation-associated disorders.

Role of potassium and pH on the initiation of sperm motility in the European eel.

The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing K+ from this media, and by testing the use of K+ channel inhibitors and a K-ionophore. Sperm motility was measured using a CASA system, intracellular K+ and pH were measured by flow cytometry, and sperm head area was measured by ASMA: Automated Sperm Morphometry Analyses. Sperm motility was notably inhibited by the removal of K+ from the seminal plasma and by treatment with the K+ ionophore valinomycin. This therefore indicates that a reduction of K+ levels in the quiescent stage inhibits further motility. The normal decrease in sperm head area induced by seawater activation was altered by the removal of K+ from the seminal plasma, and an increase in the pHi in the quiescent stage was also induced. Intracellular pH (pHi) was quantitatively measured for the first time in European eel spermatozoa, being 7.2 in the quiescent stage and 7.1 post-activation. Intracellular and external pH levels influenced sperm motility both in the quiescent stage and at activation. The alkalinization of the pHi (by NH4Cl) inhibited sperm motility activation, while acidification (by Na-acetate) did not have any effect. Our results indicate that a pH gradient between the sperm cell and the seminal plasma is necessary for sperm motility activation. The presence of the ion K+ in the seminal plasma (or in the extender medium) is necessary in order to maintain sperm volume, intracellular pH and sperm motility.

Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.

K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.

Mdm31 protein mediates sensitivity to potassium ionophores but does not regulate mitochondrial morphology or phospholipid trafficking in Schizosaccharomyces pombe.

Mdm31p is an inner mitochondrial membrane (IMM) protein with unknown function in Saccharomyces cerevisiae. Mutants lacking Mdm31p contain only a few giant spherical mitochondria with disorganized internal structure, altered phospholipid composition and disturbed ion homeostasis, accompanied by increased resistance to the electroneutral K+ /H+ ionophore nigericin. These phenotypes are interpreted as resulting from diverse roles of Mdm31p, presumably in linking mitochondrial DNA (mtDNA) to the machinery involved in segregation of mitochondria, in mediating cation transport across IMM and in phospholipid shuttling between mitochondrial membranes. To investigate which of the roles of Mdm31p are conserved in ascomycetous yeasts, we analysed the Mdm31p orthologue in Schizosaccharomyces pombe. Our results demonstrate that, similarly to its S. cerevisiae counterpart, SpMdm31 is a mitochondrial protein and its absence results in increased resistance to nigericin. However, in contrast to S. cerevisiae, Sz. pombe cells lacking SpMdm31 are also less sensitive to the electrogenic K+ ionophore valinomycin. Moreover, mitochondria of the fission yeast mdm31Δ mutant display no changes in morphology or phospholipid composition. Therefore, in terms of function, the two orthologous proteins appear to have considerably diverged between these two evolutionarily distant yeast species, possibly sharing only their participation in ion homeostasis.

Calibration of potentiometric sensor arrays with a reduced number of standards.

In this paper a novel calibration procedure for the parameter determination of ion-selective electrodes used in an array is described. Commonly used procedures require a large number of standards to determine the parameters based on the Nicolsky-Eisenman model. The elaborated procedure reduces the number of standards to a minimum by using a standard containing a mixture of ions instead of a couple of pure standards. This paper presents a complete calibration procedure, which consists of designing the composition of the standards, parameter determination and verification of the calibration results. Comparison of the results obtained by the procedure presented with results obtained by the Two-Point Calibration and Separate Solution methods proves that the accuracies of both procedures are comparable. The outlined procedure can be applied in multicomponent analysers.

Development of a potassium-selective optode for hydroponic nutrient solution monitoring.

Highly efficient and reliable plant growth such as that required in biological life support systems for future space-based missions can be better achieved with knowledge of ion concentrations within the hydroponic nutrient solution. This paper reports on the development and application of ion-selective bulk optodes to plant growth systems. Membranes for potassium-selective sensing are reported that have been tailored so that their dynamic range is centred on potassium activities within typical nutrient solution recipes. The developed sensors have been shown to exhibit a potassium activity measuring range from 0.134 to 117 mM at pH 6.0. These bulk optodes show full scale response on the order of several minutes. They show minimal interference to other cations and meet worst-case selectivity requirements for potassium monitoring in the considered half strength Hoagland solution. When continuously immersed in nutrient solution, these sensors demonstrated predicable lifetimes on the order of 50h. The developed instrument for absorption-based measurements including light source, mini-spectrometer and optode probe is presented. Custom instrument control and monitoring software including a spectral normalization procedure, use of a dual-wavelength absorbance ratio technique and automatic adjustment for pH variation result in an instrument that is self-calibrating and one that can account for effects such as light source fluctuations, membrane thickness variations and a variety of other factors. The low mass, low volume nature of bulk optode sensing systems, make them a promising technology for future space-based plant production systems. Their low-cost and technology transfer potential suggest that they could provide terrestrial growers a new and reliable mechanism to obtain ion-selective knowledge of their nutrient solution, improving yields, reducing costs and aiding in compliance to continually more stringent environmental regulation.

Transportation behavior of alkali ions through a cell membrane ion channel. A quantum chemical description of a simplified isolated model.

Quantum chemical model calculations were carried out for modeling the ion transport through an isolated ion channel of a cell membrane. An isolated part of a natural ion channel was modeled. The model channel was a calixarene derivative, hydrated sodium and potassium ions were the models of the transported ion. The electrostatic potential of the channel and the energy of the channel-ion system were calculated as a function of the alkali ion position. Both attractive and repulsive ion-channel interactions were found. The calculations - namely the dependence of the system energy and the atomic charges of the water molecules with respect to the position of the alkali ion in the channel - revealed the molecular-structural background of the potassium selectivity of this artificial ion channel. It was concluded that the studied ion channel mimics real biological ion channel quite well.