Antonio Alcaraz

Electric field-assisted proton transfer and water dissociation at the junction of a fixed-charge bipolar membrane

Abstract Electric field-enhanced (EFE) water dissociation can occur at the interfacial space charge junction of both biological and synthetic fixed-charge bipolar membranes. This dissociation has so far been analysed on an electrochemical basis using modified second Wien effect and absolute rate theories. We propose a statistical thermodynamics model to describe the cooperative orientation of the water molecules by the electric field at the bipolar junction. The approach is simple and retains some of the essential aspects of the phenomenon. In particular, the EFE water dissociation can now be rationalised on the basis of a field-assisted proton transfer mechanism involving the membrane fixed charge and the water molecules at the space charge region of the bipolar junction.

Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis.

Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence.

Coronavirus E protein forms ion channels with functionally and structurally-involved membrane lipids

Abstract Coronavirus (CoV) envelope (E) protein ion channel activity was determined in channels formed in planar lipid bilayers by peptides representing either the transmembrane domain of severe acute respiratory syndrome CoV (SARS-CoV) E protein, or the full-length E protein. Both of them formed a voltage independent ion conductive pore with symmetric ion transport properties. Mutations N15A and V25F located in the transmembrane domain prevented the ion conductivity. E protein derived channels showed no cation preference in non-charged lipid membranes, whereas they behaved as pores with mild cation selectivity in negatively-charged lipid membranes. The ion conductance was also controlled by the lipid composition of the membrane. Lipid charge also regulated the selectivity of a HCoV-229E E protein derived peptide. These results suggested that the lipids are functionally involved in E protein ion channel activity, forming a protein–lipid pore, a novel concept for CoV E protein ion channel entity.

Ion selectivity and water dissociation in polymer bipolar membranes studied by membrane potential and current-voltage measurements

Abstract A polymer bipolar ion-exchange membrane consists of a layered structure involving one cation and one anion ion-exchange layer joined together in series. In this study, the ionic selectivity and water dissociation rate of six commercial bipolar membranes was evaluated from the measurements of the membrane potential in a concentration cell and the current–voltage curve in a four-point measuring cell. Bipolar membrane technology requires polymer membranes presenting high ion selectivities and water dissociation rates, and in this paper we have addressed the basic physico-chemical phenomena involved, both theoretically and experimentally. We have shown that the effects of the bipolar junction and the membrane fixed charge concentrations on the ion transport rates observed can be understood on the basis of simple concepts.

Hydrophobic Pulmonary Surfactant Proteins SP-B and SP-C Induce Pore Formation in Planar Lipid Membranes: Evidence for Proteolipid Pores

Pulmonary surfactant is a complex mixture of lipids and specific surfactant proteins, including the hydrophobic proteins SP-B and SP-C, in charge of stabilizing the respiratory surface of mammalian lungs. The combined action of both proteins is responsible for the proper structure and dynamics of membrane arrays in the pulmonary surfactant network that covers the respiratory surface. In this study, we explore the possibility that proteins SP-B and SP-C induce the permeabilization of phospholipid membranes via pore formation. To this end, electrophysiological measurements have been carried out in planar lipid membranes prepared with different lipid/protein mixtures. Our main result is that channel-like structures are detected in the presence of SP-B, SP-C, or the native mixture of both proteins. Current traces show a high variety of conductance states (from pS to nS) that are dependent both on the lipid composition and the applied potential. We also show that the type of host lipid crucially determines the ionic selectivity of the observed pores: the anionic selectivity observed in zwitterionic membranes is inverted to cationic selectivity in the presence of negatively charged lipids. All those results suggest that SP-B and SP-C proteins promote the formation of proteolipid channels in which lipid molecules are functionally involved. We propose that proteolipidic membrane-permeabilizing structures may have an important role to tune ionic and lipidic flows through the pulmonary surfactant membrane network at the alveolar surfaces.

AC impedance spectra of bipolar membranes : an experimental study

A bipolar membrane (BM) is composed of one cation and one anion ion-exchange layers joined together in series. In order to obtain the AC electrical impedance of a BM, a small sinusoidal current perturbation was superimposed to the DC current, and the resulting frequency-dependent impedance spectra were recorded under different conditions of electrical polarisation and temperature for five BMs. The experimental spectra were measured in three current ranges: below the limiting current region, at the onset of the overlimiting region and in the electric field enhanced water dissociation region. This allows for a better understanding of the contributions of the salt and water ions to the measured impedance spectra. Measurements of the impedance of the forward biased membrane were also carried out. Although the experimental impedance spectra appear to be in qualitative agreement with previous theoretical models incorporating the effect of the electric field enhanced water dissociation, a quantitative analysis of the results is not still possible due to the high number of parameters involved.

pH and supporting electrolyte concentration effects on the passive transport of cationic and anionic drugs through fixed charge membranes

The effects of pH and supporting electrolyte concentration on the passive transport of an ionized (cationic or anionic) drug through a thick fixed charge membrane have been theoretically studied. This system constitutes a simplified model for the pH controlled ion transport and drug delivery through membranes of biological and pharmaceutical interest. Calculations were carried out for different values of the membrane fixed charge, supporting electrolyte and drug concentrations covering a broad range of the conditions usually found in experiments. The theoretical approach employed is based on the Nernst‐Planck flux equations, and all of the species present in the system (the neutral and ionized forms of the drug, the two supporting electrolyte ions and the hydrogen and hydroxide ions) have been taken into account without any additional assumption. It has been shown that the Goldman constant field assumption together with the total co-ion exclusion assumption provide good approximated solutions for high membrane fixed charge concentrations. The model predictions show that the internal pH within the membrane, the total drug flux and the membrane potential are very sensitive to the external pH values. Comparison of our results with available experimental data confirms the potential utility of the calculations for the analysis and design of experiments involving the pH dependent passive transport of ionized drugs through a fixed charge membrane, especially in the cases of thick biomembranes, biochemical sensors and pH-controlled drug delivery systems. # 1999 Elsevier Science B.V. All rights reserved.

Membrane potential bistability in nonexcitable cells as described by inward and outward voltage-gated ion channels.

The membrane potential of nonexcitable cells, defined as the electrical potential difference between the cell cytoplasm and the extracellular environment when the current is zero, is controlled by the individual electrical conductance of different ion channels. In particular, inward- and outward-rectifying voltage-gated channels are crucial for cell hyperpolarization/depolarization processes, being amenable to direct physical study. High (in absolute value) negative membrane potentials are characteristic of terminally differentiated cells, while low membrane potentials are found in relatively depolarized, more plastic cells (e.g., stem, embryonic, and cancer cells). We study theoretically the hyperpolarized and depolarized values of the membrane potential, as well as the possibility to obtain a bistability behavior, using simplified models for the ion channels that regulate this potential. The bistability regions, which are defined in the multidimensional state space determining the cell state, can be relevant for the understanding of the different model cell states and the transitions between them, which are triggered by changes in the external environment.

Analysis of SARS-CoV E protein ion channel activity by tuning the protein and lipid charge

Abstract A partial characterization of the ion channels formed by the SARS coronavirus (CoV) envelope (E) protein was previously reported (C. Verdiá-Báguena et al., 2012 [12]). Here, we provide new significant insights on the involvement of lipids in the structure and function of the CoV E protein channel on the basis of three series of experiments. First, reversal potential measurements over a wide range of pH allow the dissection of the contributions to channel selectivity coming from ionizable residues of the protein transmembrane domain and also from the negatively charged groups of diphytanoyl phosphatidylserine (DPhPS) lipid. The corresponding effective pKas are consistent with the model pKas of the acidic residue candidates for titration. Second, the change of channel conductance with salt concentration reveals two distinct regimes (Donnan-controlled electrodiffusion and bulk-like electrodiffusion) fully compatible with the outcomes of selectivity experiments. Third, by measuring channel conductance in mixtures of neutral diphytanoyl phosphatidylcholine (DPhPC) lipids and negatively charged DPhPS lipids in low and high salt concentrations we conclude that the protein–lipid conformation in the channel is likely the same in charged and neutral lipids. Overall, the whole set of experiments supports the proteolipidic structure of SARS-CoV E channels and explains the large difference in channel conductance observed between neutral and charged membranes.

Effects of pH on ion transport in weak amphoteric membranes

Abstract We have studied theoretically the effect of pH on the ion transport through amphoteric polymer membranes composed of weak polyelectrolytes where the charged groups are randomly distributed along the axial direction of the membrane. This system serves as a simplified model for the pH controlled ion transport and drug delivery through membranes of biological interest. The theoretical approach employed is based on the Nernst-Planck equations. The complete system of electrical charges formed by: (i) the pH dependent, amphoretic membrane fixed charge, and (ii) the four mobile charges (the salt ions and the hydrogen and hydroxide ions) have been taken into account without any additional assumption. The model predictions show that the ionic fluxes and the membrane potential are very sensitive to the external pH, and the potential utility of these predictions for the analysis of experiments involving pH dependent passive transport through membranes is emphasized.