Uncharged Form Research Articles

The Electrolytic Dissociation of 1,3-Cyclopentanedicarboxylic Acids

The dissociation parameters for the dilute (0.0001-0.01M) solutions of cis and trans-1,3-cyclopentanedicarboxylic acids were calculated with the aid of the original accurate and empirical equations suggested by authors for cases of electrolytic dissociation of weak multibasic organic acids with the effect of “overlapping” equilibria. The equations for determination the regions of predominance of various charged and uncharged forms of these acids were also suggested.

Investigation of the efficiency of gelation of melamine with the positional isomers of aminobenzoic acid

Novel thermoreversible gelling systems consisting of the low-molecular-weight gelators, melamine (M) and three positional isomers of aminobenzoic acid (AB) were investigated. According to the rheological measurements, the gel strength at lower temperatures was in the order of pAB/M>mAB/M>oAB/M. This sequence was consistent with a computational analysis based on the stronger hydrogen bonding and the lower energy of each cluster. When heated, the sequence of the degelation temperature was in the order of pAB/M>oAB/M>mAB/M. This degelation temperature agreed with the DSC analyses which also demonstrated the thermoreversibility of these gel systems. It was surprising that the mAB/M which is a stronger gel than the oAB/M could be more easily converted to a sol than oAB/M. FTIR revealed that during heating, the mAB from the mAB/M could change from the uncharged form to the zwitterionic form (mABz). In addition, the computational results indicated that the hydrogen bonding between the mABz and M was much weaker than between the mAB and M. This may result in the lower degelation temperature of the mAB/M system. PXRD analyses showed different patterns of the mixtures compared to the individual compounds and indicated different interactions between M and the different ABs.

Polyamines in chemiosmosis in vivo: A cunning mechanism for the regulation of ATP synthesis during growth and stress

Polyamines (PAs) are low molecular weight amines that occur in every living organism. The three main PAs (putrescine, spermidine, and spermine) are involved in several important biochemical processes covered in recent reviews. As rule of thumb, increase of the cellular titer of PAs in plants is related to cell growth and cell tolerance to abiotic and biotic stress. In the present contribution, we describe recent findings from plant bioenergetics that bring to light a previously unrecognized dynamic behavior of the PA pool. Traditionally, PAs are described by many authors as organic polycations, when in fact they are bases that can be found in a charged or uncharged form. Although uncharged forms represent less than 0.1% of the total pool, we propose that their physiological role could be crucial in chemiosmosis. This process describes the formation of a PA gradient across membranes within seconds and is difficult to be tested in vivo in plants due to the relatively small molecular weight of PAs and the speed of the process. We tested the hypothesis that PAs act as permeable buffers in intact leaves by using recent advances in vivo probing. We found that an increase of PAs increases the electric component (Δψ) and decreases the ΔpH component of the proton motive force. These findings reveal an important modulation of the energy production process and photoprotection of the chloroplast by PAs. We explain in detail the theory behind PA pumping and ion trapping in acidic compartments (such as the lumen in chloroplasts) and how this regulatory process could improve either the photochemical efficiency of the photosynthetic apparatus and increase the synthesis of ATP or fine tune antenna regulation and make the plant more tolerant to stress.

Mechanism of ion transfer in supported liquid membrane systems: electrochemical control over membrane distribution.

A polarization study carried out on a thin supported liquid membrane separating two aqueous compartments is presented. Transfer of both the ionized and uncharged form of an organic tracer dye, rhodamine B ([9-(2-carboxyphenyl)-6-diethylamino-3-xanthenylidene]-diethylammonium chloride), across supported liquid membranes composed of one of 1-octanol (octan-1-ol), 1,9-decadiene (deca-1,9-diene), 1,2-dichlorobenzene, or nitrophenyl octyl ether (1-(2-nitrophenoxy)octane) was studied using cyclic voltammetry and UV–vis absorption spectrophotometry. Concentration analysis indicates that the high membrane concentration of rhodamine B determines the ionic transfer observed via voltammetry, which is consistent with the low aqueous ionic concentration and large membrane/aqueous distribution of the molecule. The observed double-transfer voltammogram, although it has been largely neglected in previous literature, is a logical consequence of the presence of two liquid–liquid interfaces and is rationalized in terms of ion transfer across the two interfaces on either side of the membrane and supported by voltammograms obtained for a series of ions of varied lipophilicity. The bipolar nature of the voltammetric response offers an effective way of mass transport control via changing polarity of the applied voltage and finds immediate use in extraction, purification, and separation applications.

Mechanism of hERG Channel Block by the Psychoactive Indole Alkaloid Ibogaine

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go-related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells. Currents through hERG channels were blocked regardless of whether ibogaine was applied via the extracellular or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during activation of the channels and was not observed for resting channels. With increasing depolarizations, ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, whereas inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, these findings show that ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.

Sample conditions to avoid pH distortion in RP‐LC

Band deformations might take place for acids and bases in preparative applications and adsorption studies where it is necessary to use overloaded injections. In this study, we focus on how deformations can be prevented without using highly concentrated buffers that may precipitate in the eluent. We have systematically investigated how the elution zones depend on which protolytic form the analyte has when it is dissolved. Basic and acidic model compounds are investigated using eluents with different pH values and the resulting elution profiles are compared when the analytes are dissolved in their protonated and deprotonated form, i.e., in uncharged form or as different kinds of salts. Depending on the analyte's protolytic form, a sample zone is created at the column inlet whose pH deviates, more or less, from the bulk eluent's. If the local adsorption strength in this sample zone is greater than the bulk eluent's, the elution profiles are compressed. Under opposite conditions, the eluted bands are more or less deformed and may even be split; completely different deformations can even take place for different kinds of salt combinations. Explanations of these, and other, effects, together with detailed guidelines for proper sample preparation to avoid peak deformations, are given.

The Electrolytic Dissociation of 1,2-Cyclopentanedicarboxylic Acids

The dissociation parameters for the dilute (0.0001-0.01M) solutions of cis and trans-1,2-cyclopentanedicarboxylic acids were calculated with the aid of the original accurate and empirical equations suggested by authors for cases of electrolytic dissociation of weak multibasic organic acids with the effect of “overlapping” equilibria. The equations for determination the regions of predominance of various charged and uncharged forms of these acids were also suggested.

The Electrolytic Dissociation of 1,3-Cyclobutanedicarboxylic Acids

The dissociation parameters for the dilute (0.0001-0.01M) solutions of cis and trans-1,3-cyclobutanedicarboxylic acids were determined with the aid of the original method for analysis the regularities of electrolytic dissociation of weak multibasic organic acids with the close values of the dissociation constants of separate steps (the effect of “overlapping” equilibria). The equations for determination the regions of predominance of various charged and uncharged forms of these acids were also described.

The Ammonia Transport, Retention and Futile Cycling Problem in Cyanobacteria

Ammonia is the preferred nitrogen source for many algae including the cyanobacterium Synechococcus elongatis (Synechococcus R-2; PCC 7942). Modelling ammonia uptake by cells is not straightforward because it exists in solution as NH(3) and NH (4) (+) . NH(3) is readily diffusible not only via the lipid bilayer but also through aquaporins and other more specific porins. On the other hand, NH (4) (+) requires cationic transporters to cross a membrane. Significant intracellular ammonia pools (≈1-10mol m(-3)) are essential for the synthesis of amino acids from ammonia. The most common model envisaged for how cells take up ammonia and use it as a nitrogen source is the "pump-leak model" where uptake occurs through a simple diffusion of NH(3) or through an energy-driven NH (4) (+) pump balancing a leak of NH(3) out of the cell. The flaw in such models is that cells maintain intracellular pools of ammonia much higher than predicted by such models. With caution, [(14)C]-methylamine can be used as an analogue tracer for ammonia and has been used to test various models of ammonia transport and metabolism. In this study, simple "proton trapping" accumulation by the diffusion of uncharged CH(3)NH(2) has been compared to systems where CH(3)NH (3) (+) is taken up through channels, driven by the membrane potential (ΔU (i,o)) or the electrochemical potential for Na(+) (ΔμNa (i,o) (+) ). No model can be reconciled with experimental data unless the permeability of CH(3)NH(2) across the cell membrane is asymmetric: permeability into the cell is very high through gated porins, whereas permeability out of the cell is very low (≈40nm s(-1)) and independent of the extracellular pH. The best model is a Na (in) (+) /CH(3)NH (3) (+) (in) co-porter driven by ΔμNa (i,o) (+) balancing synthesis of methylglutamine and a slow leak governed by Ficks law, and so there is significant futile cycling of methylamine across the cell membrane to maintain intracellular methylamine pools high enough for fixation by glutamine synthetase. The modified pump-leak model with asymmetric permeability of the uncharged form is a viable model for understanding ammonia uptake and retention in plants, free-living microbes and organisms in symbiotic relationships.

The Regularities of Electrolytic Dissociation of 1,2-Cyclobutanedicarboxylic Acids

The dissociation parameters for the dilute (0.0001-0.01M) solutions of cis and trans-1,2-cyclobutanedicarboxylic acids were calculated with the aid of the new original method for analysis the regularities of electrolytic dissociation of weak multibasic organic acids with the effect of "overlapping" equilibria. The equations for determination the regions of predominance of various charged and uncharged forms of these acids were also described.

The Pivotal Twin-Histidine Element of the Escherichia Coli Ammonium Channel AmtB Functions as a Substrate Selectivity Filter

Ammonium functions as both a primary nutrient and waste product and, thus, its transport across biological membranes is of fundamental importance. Because the uncharged form, NH3, readily traverses phospholipid bilayers by simple diffusion the role of protein-catalyzed transport of the protonated species, NH4+, is unusually interesting. The Amt family of channels mediates the transport of NH4+ and is required for microbial growth when diffusion of NH3 becomes limiting for nitrogen uptake. Whereas all other characterized channels facilitate downhill substrate movement, Amt proteins are active channels - hybrids between passive channels and active transporters - and concentrate NH4+ against a gradient. Amt family members function as homotrimers, with each monomeric unit carrying a pore for substrate conduction. Each pore is lined entirely with hydrophobic residues, save for a pair of conserved hydrogen-bonded histidines postulated to play a critical role in mediating NH4+ transport. We examined the impact that changes to this histidine pairing had on the function of one of the best-characterized members of the Amt family, the AmtB protein of Escherichia coli. Our initial analysis indicated that AmtB can accommodate, by either direct substitution or suppressor generation, acidic residues at one or both positions of the twin-histidine site while retaining good-to-excellent transport activity. Subsequent work shows that a number of mutant AmtB proteins carrying such alterations leak K+ ions and that this leakage is energetically costly. These findings lead us to conclude that whereas the twin-histidine element is not required to conduct NH4+ it serves as a filter to prevent AmtB-mediated K+ transport.

Antidepressants inhibit proton currents and tumor necrosis factor-α production in BV2 microglial cells

Proton channels are gated by voltage and pH gradients, and play an important role in the microglial production of pro-inflammatory cytokines, which are known to be suppressed by antidepressants. In the present study we tested the hypothesis that cytokine inhibition by antidepressants is due to an inhibitory action on proton currents by comparing their effects on tumor necrosis factor-α production with the effects on the proton currents in BV2 murine microglial cells. Imipramine, amitriptyline, desipramine and fluoxetine potently and reversibly inhibited proton currents at micromolar concentrations at an intracellular/extracellular pH gradient of 5.5/7.3. Raising extracellular pH to 8.3 sped up the rate and enhanced the extent of block whereas raising intracellular pH to 6.3 reduced the blocking potency of imipramine. These results support a mechanism where the uncharged drug form penetrates the cell membrane, and the charged form blocks the proton channel from the internal side of membrane. This mode of action was corroborated by an experiment with imipraminium, a permanently charged quaternary derivative, which showed far less block compared to imipramine. The lipopolysaccharide-induced release of tumor necrosis factor-α was inhibited by imipramine at concentrations comparable to those inhibiting the proton current. These results support the hypothesis that tumor necrosis factor-α inhibition by imipramine is related to its inhibitory effects on proton channels.

The Regularities of Electrolytic Dissociation of 1,2-Cyclopropanedicarboxylic Acids

The dissociation parameters for the dilute (0.0001-0.01M) solutions of cis and trans-1,2-cyclopropanedicarboxylic acids were calculated with the aid of the new original method for analysis the regularities of electrolytic dissociation of weak multibasic organic acids with the effect of "overlapping" equilibria. The equations for determination the regions of predominance of various charged and uncharged forms of these acids were also described.

Photooxidation of Alkaloids: Considerable Quantum Yield Enhancement by Rose Bengal‐sensitized Singlet Molecular Oxygen Generation

The photooxidation of sanguinarine, coralyne and berberine was studied in oxygenated alkaline methanol solutions. Rose bengal as photosensitizer significantly accelerates the process, indicating the importance of singlet molecular oxygen in the reaction mechanism. The quantum yield of sensitized oxidation was found to increase significantly with pH and reaches 0.4 for berberine at pH 13.8. The direct oxidation of alkaloids is less efficient, the quantum yield does not exceed 0.01 even in oxygen-saturated solutions. The photoinduced electron ejection does not play a role in the oxidation. The uncharged pseudobase forms, which are present in alkaline medium, are oxidized much more easily than the alkaloid cations.

Permeation through the Cell Membrane of a Boron-Based β-Lactamase Inhibitor

Bacteria express beta-lactamases to counteract the beneficial action of antibiotics. Benzo[b]-thiophene-2-boronic acid (BZB) derivatives are β-lactamase inhibitors and, as such, promising compounds to be associated with β-lactam antibacterial therapies. The uncharged form of BZB, in particular, is suggested to diffuse through the outer membrane of Gram negative bacteria. In this study, through the combination of electrophysiological experiments across reconstituted PC/n-decane bilayers and metadynamics-based free energy calculations, we investigate the permeation mechanism of boronic compounds. Our experimental data establish that BZB passes through the membrane, while computer simulations provide hints for the existence of an aqueous, water-filled monomolecular channel. These findings provide new perspectives for the design of boronic acid derivatives with high membrane permeability.

Interaction of local anaesthetics with model phospholipid membranes. The effect of pH and phospholipid composition studied by quenching of an intramembrane fluorescent probe.

The interaction of local anaesthetics, tetracaine and procaine, with model phospholipid membranes has been examined by measurement of drug-induced quenching of the fluorescence of a membrane incorporated probe, 12-(9-anthroyl)stearic acid. The pH dependence of quenching curves obtained for neutral phosphatidylcholine and acidic phosphatidylserine bilayers indicates that, contrary to previous suggestions, both charged and uncharged forms of amine local anaesthetics may be incorporated into lipid membranes.

A polymeric film responding in diffusion properties to environmental pH stimuli: a model for a self-regulating drug delivery system.

A new polymeric film, ethylene-vinyl-NN-diethylglycinate random copolymer, has been studied to evaluate possible applications of such a material to produce controlled release dosage forms. Protonation of the substituted amino groups took place to different extents according to environmental pH conditions. The polymeric film proved to be permeable only to uncharged species, the rate of diffusion of which increased with the increasing charge of the membrane. This effect might be related to strong polymer-proton interactions affecting structure and transport properties of the film and enhancing the solvation process. The charged form of a number of barbituric acid derivatives did not diffuse through the membrane. The uncharged forms permeated the polymeric sheeting at rates consistent with their partition coefficients and which increased with the increasing charge of the polymer.

Engineered pH-Dependence at the Kir6.2 Helix Bundle Crossing

The hallmark functional property of KATP (ATP-sensitive potassium) channels is inhibition by intracellular ATP, which binds to a well-defined binding site on Kir6.x subunits and stabilizes the closed conformation of a gate in the channel pore. Numerous inwardly-rectifying potassium (Kir) channels possess an aromatic residue in the ‘helix bundle crossing’ region, forming the narrowest pore constriction in crystal structures of Kir channels, indicating an important role in channel gating. We have identified a remarkable phenotype of mutant channels carrying a glutamate at this position (F168E). Despite the structural prediction of four glutamates in close proximity, F168E channels are predominantly closed at physiological pH. However, intracellular alkalinization causes rapid and reversible channel activation. These findings suggest that F168E glutamates are uncharged at physiological pH but become deprotonated with a pKa∼9, resulting in opening due to mutual repulsion of multiple nearby glutamate sidechains. The K-channel pore scaffold likely brings these glutamates into close proximity, stabilizing the protonated (uncharged) form of the glutamate sidechain, and resulting in a dramatic pKa shift relative to free glutamate. Only at more alkaline pH do the glutamates deprotonate, with their mutual repulsion driving channel opening. Consistent with a role in ATP-mediated channel closure, alkalinization also affects channel sensitivity to ATP. Taken together, these findings demonstrate an engineered (not intrinsic) mechanism of channel gating by pH, and suggest that ATP-mediated gating of Kir6.2 involves conformational rearrangement of the bundle crossing region.

The permanently charged lidocaine analogue QX222 acts as a blocker from the intracellular side and as an inactivation modulator from the extracellular side in a mutant NaV1.4 channel

Background QX222 is a quaternary amine analogue of lidocaine, which, unlike lidocaine, is permanently charged. Lidocaine has its binding site in the internal vestibule of the voltage-gated sodium channel. Due to the hydrophobic nature of its uncharged form, lidocaine reaches the binding site by passing through the membrane, QX222 can reach this binding site only by a hydrophilic pathway, presumably through the channel protein. However, such a pathway has been reported only in the heart-type sodium channel (NaV1.5) and some mutants of other sodium channels. Notably, mutations at site 1575 in the skeletal muscle-type sodium channel (NaV1.4) open an access pathway from the external side. In this study we tested the properties of QX222 block on the mutant I1575E.

Molecular dynamics simulations of local anesthetic articaine in a lipid bilayer

In order to investigate structural and dynamical properties of local anesthetic articaine in a model lipid bilayer, a series of molecular dynamics simulations have been performed. Simulations were carried out for neutral and charged (protonated) forms of articaine inserted in fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer. For comparison purpose, a fully hydrated DMPC bilayer without articaine was also simulated. The length of each simulation was 200 ns. Various properties of the lipid bilayer systems in the presence of both charged and uncharged forms of articaine taken at two different concentrations have been examined: membrane area per lipid, mass density distributions, order parameters, radial distribution functions, head group tilt, diffusion coefficients, electrostatic potential, etc, and compared with results of previous simulations of DMPC bilayer in the presence of lidocaine. It was shown that addition of both charged and neutral forms of articaine causes increase of the dipole electrostatic potential in the membrane interior.