Project description:The selectivity filter of K(+) channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K(+) hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K(+). Here, we show that K(+) channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K(+) channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability pf of (6.9 ± 0.6) × 10(-13) cm(3)⋅s(-1) for both mutants. "Collapse" of the selectivity filter upon K(+) removal did not alter pf and was fully reversible, as demonstrated by current measurements through planar bilayers in a K(+)-containing medium to which K(+)-free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K(+) in the aqueous solution, which indicates an effective K(+) dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K(+) ions in the selectivity filter act to mutually destabilize binding.
Project description:Recently, we showed that synthetic anion transporters DSC4P-1 and SA-3 had activity related to cancer cell death. They were found to increase intracellular chloride and sodium ion concentrations. They were also found to induce apoptosis (DSC4P-1) and both induce apoptosis and inhibit autophagy (SA-3). However, determinants underlying these phenomenological findings were not elucidated. The absence of mechanistic understanding has limited the development of yet-improved systems. Here, we show that three synthetic anion transporters, DSC4P-1, SA-3, and 8FC4P, induce osmotic stress in cells by increasing intracellular ion concentrations. This triggers the generation of reactive oxygen species via a sequential process and promotes caspase-dependent apoptosis. In addition, two of the transporters, SA-3 and 8FC4P, induce autophagy by increasing the cytosolic calcium ion concentration promoted by osmotic stress. However, they eventually inhibit the autophagy process as a result of their ability to disrupt lysosome function through a transporter-mediated decrease in a lysosomal chloride ion concentration and an increase in the lysosomal pH.
Project description:We measured concentration changes of sodium, potassium, chloride ions, pH and the transepithelial potential difference by means of ion-selective electrodes, which were placed on both sides of a human bronchial epithelial 16HBE14? cell line grown on a porous support in the presence of ion channel blockers. We found that, in the isosmotic transepithelial concentration gradient of either sodium or chloride ions, there is an electroneutral transport of the isosmotic solution of sodium chloride in both directions across the cell monolayer. The transepithelial potential difference is below 3 mV. Potassium and pH change plays a minor role in ion transport. Based on our measurements, we hypothesize that in a healthy bronchial epithelium, there is a dynamic balance between water absorption and secretion. Water absorption is caused by the action of two exchangers, Na/H and Cl/HCO3, secreting weakly dissociated carbonic acid in exchange for well dissociated NaCl and water. The water secretion phase is triggered by an apical low volume-dependent factor opening the Cystic Fibrosis Transmembrane Regulator CFTR channel and secreting anions that are accompanied by paracellular sodium and water transport.
Project description:Ammonia is a ubiquitous, toxic by-product of cell metabolism. Its high membrane permeability and proton affinity causes ammonia to accumulate inside acidic lysosomes in its poorly membrane-permeant form: ammonium (NH4+). Ammonium buildup compromises lysosomal function, suggesting the existence of mechanisms that protect cells from ammonium toxicity. Here, we identified SLC12A9 as a lysosomal regulator of ammonium export that preserves lysosomal homeostasis. SLC12A9 knockout cells showed grossly enlarged lysosomes and elevated ammonium content. These phenotypes were reversed upon removal of the metabolic source of ammonium or dissipation of the lysosomal pH gradient. Lysosomal chloride increased in SLC12A9 knockout cells and chloride binding by SLC12A9 was required for ammonium transport. Our data indicate that SLC12A9 function is central for the handling of lysosomal ammonium and chloride, an unappreciated, fundamental mechanism of lysosomal physiology that may have special relevance in tissues with elevated ammonia, such as tumors.
Project description:Bilayer ion-exchange membranes are mainly used for separating single and multiply charged ions. It is well known that in membranes in which the layers have different charges of the ionogenic groups of the matrix, the limiting current decreases, and the water splitting reaction accelerates in comparison with monolayer (isotropic) ion-exchange membranes. We study samples of bilayer ion-exchange membranes with very thin cation-exchange layers deposited on an anion-exchange membrane-substrate in this work. It was revealed that in bilayer membranes, the limiting current's value is determined by the properties of a thin surface film (modifying layer). A linear regularity of the dependence of the non-equilibrium effective rate constant of the water-splitting reaction on the resistance of the bipolar region, which is valid for both bilayer and bipolar membranes, has been revealed. It is shown that the introduction of the catalyst significantly reduces the water-splitting voltage, but reduces the selectivity of the membrane. It is possible to regulate the fluxes of salt ions and water splitting products (hydrogen and hydroxyl ions) by changing the current density. Such an ability makes it possible to conduct a controlled process of desalting electrolytes with simultaneous pH adjustment.
Project description:Salt stress defense mechanisms in plant roots, such as active Na+ efflux and storage, require energy in the form of ATP. Understanding the energy required for these transport mechanisms is an important step toward achieving an understanding of salt tolerance. However, accurate measurements of the fluxes required to estimate these energy costs are difficult to achieve by experimental means. As a result, the magnitude of the energy costs of ion transport in salt-stressed roots relative to the available energy is unclear, as are the relative contributions of different defense mechanisms to the total cost. We used mathematical modeling to address three key questions about the energy costs of ion transport in salt-stressed Arabidopsis roots: are the energy requirements calculated on the basis of flux data feasible; which transport steps are the main contributors to the total energy costs; and which transport processes could be altered to minimize the total energy costs? Using our biophysical model of ion and water transport we calculated the energy expended in the trans-plasma membrane and trans-tonoplast transport of Na+, K+, Cl-, and H+ in different regions of a salt-stressed model Arabidopsis root. Our calculated energy costs exceeded experimental estimates of the energy supplied by root respiration for high external NaCl concentrations. We found that Na+ exclusion from, and Cl- uptake into, the outer root were the major contributors to the total energy expended. Reducing the leakage of Na+ and the active uptake of Cl- across outer root plasma membranes would lower energy costs while enhancing exclusion of these ions. The high energy cost of ion transport in roots demonstrates that the energetic consequences of altering ion transport processes should be considered when attempting to improve salt tolerance.
Project description:Ion-specific effects in aqueous solution, known as the Hofmeister effect, are prevalent in diverse systems ranging from pure ionic to complex protein solutions. The objective of this paper is to explicitly demonstrate how complex ion-ion and ion-water interactions manifest themselves in the Hofmeister effect based on a series of recent experimental observations. These effects are not considered in the classical descriptions of ion effects, such as the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, and therefore they fail to describe the origin of the phenomenological Hofmeister effect. However, given that models considering the basic forces of electrostatic and van der Waals interactions can offer rationalization for the core experimental observations, a universal interaction model stands a chance of being developed. In this perspective, we separately derive the contribution from ion-ion electrostatic interactions and ion-water interactions from second harmonic generation (SHG) data at the air-ion solution interface, which yields an estimate of the ion-water interactions in solution. The Hofmeister ion effect observed for biological solutes in solution should be similarly influenced by contributions from ion-ion and ion-water interactions, where the same ion-water interaction parameters derived from SHG data at the air-ion solution interface could be applicable. A key experimental data set available from solution systems to probe ion-water interactions is the modulation of water diffusion dynamics near ions in a bulk ion solution, as well as near biological liposome surfaces. This is obtained from Overhauser dynamic nuclear polarization (ODNP), a nuclear magnetic resonance (NMR) relaxometry technique. The surface water diffusivity is influenced by the contribution from ion-water interactions, both from localized surface charges and adsorbed ions, although the relative contribution of the former is larger on liposome surfaces. In this perspective, ion-water interaction energy values derived from experimental data for various ions are compared with theoretical values in the literature. Ultimately, quantifying ion-induced changes in the surface energy for the purpose of developing valid theoretical models for ion-water interactions will be critical to rationalizing the Hofmeister effect.