Cationic effect of imidazolium-based ionic liquid on the stability of myoglobin

The conformational change of myoglobin (Mb) during guanidine hydrochloride (GuHCl)-induced protein unfolding in the presence of various ionic liquids (ILs) in phosphate buffer was investigated using both the Soret band absorption and the fluorescence of tryptophan measurements. The GuHCl-induced denaturation midpoints of Mb derived from the absorption and fluorescence spectra were almost similar in the presence of 150 mM ILs with the same cation 1-butyl-3-methylimidazolium (Bmim⁺) but different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer. In addition, the denaturation midpoints of Mb in the presence of ILs were little lower than those in the absence of ILs in phosphate buffer. For the sake of clarity and comparison, we also measured the GuHCl-induced denaturation midpoints of Mb in the presence of 150 mM sodium salts with different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer and found that their corresponding denaturation midpoints of Mb were almost similar to those observed in the absence of sodium salts in phosphate buffer. These experimental data indicate that Bmim⁺ cation can promote the unfolding of Mb. Further experiments revealed that the denaturation ability of ILs increases with increasing alkyl chain length of imidazolium cation of ILs and that hydroxyl-substituted imidazolium cation could also promote the unfolding of Mb.     

Effects of phosphonium-based ionic liquids on the lipase activity evaluated by experimental results and molecular docking

In this work, the effect of several phosphonium-based ionic liquids (ILs) on the activity of lipase from Burkholderia cepacia (BCL) was evaluated by experimental assays and molecular docking. ILs comprising different cations ([P₄₄₄₄]⁺, [P_444(14)]⁺, [P_666(14)]⁺) and anions (Cl⁻, Br⁻, [Deca]⁻, [Phosp]⁻, [NTf₂]⁻) were investigated to appraise the individual roles of IL ions on the BCL activity. From the activity assays, it was found that an increase in the cation alkyl chain length leads to a decrease on the BCL enzymatic activity. ILs with the anions [Phosp]⁻ and [NTf₂]⁻ increase the BCL activity, while the remaining [P_666(14)]-based ILs with the Cl⁻, Br⁻, and [Deca]⁻ anions display a negative effect on the BCL activity. The highest activity of BCL was identified with the IL [P_666(14)][NTf₂] (increase in the enzymatic activity of BCL by 61% at 0.055 mol·L⁻¹). According to the interactions determined by molecular docking, IL cations preferentially interact with the Leu17 residue (amino acid present in the BCL oxyanion hole). The anion [Deca]⁻ has a higher binding affinity compared to Cl⁻ and Br⁻, and mainly interacts by hydrogen-bonding with Ser87, an amino acid residue which constitutes the catalytic triad of BCL. The anions [Phosp]⁻ and [NTf₂]⁻ have high binding energies (−6.2 and −5.6 kcal·mol⁻¹, respectively) with BCL, and preferentially interact with the side chain amino acids of the enzyme and not with residues of the active site. Furthermore, FTIR analysis of the protein secondary structure show that ILs that lead to a decrease on the α-helix content result in a higher BCL activity, which may be derived from an easier access of the substrate to the BCL active site.     

Fluorescence of proteins in aqueous neutral salt solutions. I. Influence of anions

The effects of anions of neutral salts on the fluorescence emission of six proteins as well as on tryptophan and tyrosine were studied in relation to the structure of proteins. Most anions are good quenchers of tryptophyl and tyrosyl fluorescence, free or in proteins. The results with tryptophan and tyrosine indicate involvement of a collisional quenching mechanism due to agreement with Stern–Volmer law. The deactivation of fluorescence probably occurs because of the transition from singlet state to triplet state. Lehrer's modification of Stern–Volmer law was applied to proteins. The effective quenching constants ([K_Q]eff) and the fraction of fluorescence available ([f_a]eff) to the quencher are also calculated. In contrast to its effect on tryptophan, CH₃COO^? quenches tyrosyl fluorescence and ClO₄^? does not. The effects on fluorescence of ribonuclease and free tyrosine are similar and without any changes in emission maximum. The anions are divided into three groups based on the effect they have on tryptophan-containing proteins. (1) NO₃^?, NO₂^?, Br^?, and I^? have high [K_Q]eff values and readily quench tryptophyl fluorescence of proteins causing a shift of emission maximum to a shorter wavelength. This change is due to the specific quenching of “exposed” tryptophan residues which are accessible to quenchers and the observed residual fluorescence is from the “buried” tryptophyls. (2) ClO₄^? and SCN^? also quench fluorescence of tryptophan in proteins and have lower ([K_Q]eff) values. In their presence the fluorescence maximum is shifted to a longer wavelength, which indicates the unfolding of a protein with [(f_a)eff] = 1. (3) Cl^?, CH₃COO^?, and SO₄? do not have a direct effect on the fluorescence of tryptophan. Besides the “direct” effects, “indirect” effects on fluorophors in protein are also seen, pointing out that the neutral salts can interact in more than one manner with proteins. The effectiveness of anions in quenching fluorescence of proteins follows similar sequences which almost resemble the Hofmeister series, viz., SO₄⁼, CH₃COO^? ? Cl^? < ClO₄^? < SCN^? < Br^? < I^? < NO₃^? < NO₂^?.     

Solvation structures of sodium halides in dimethyl sulfoxide (DMSO)–methanol (MeOH) mixtures

A constrained molecular dynamics technique has been used to study the structures and dynamics of the solvation shells of three sodium halides, namely sodium chloride (Na⁺–Cl^?), sodium bromide (Na⁺–Br^?) and sodium iodide (Na⁺–I^?) in DMSO–MeOH mixtures. In the case of Na⁺–Cl^? and Na⁺–Br^?, Na⁺ is preferentially solvated by DMSO and Cl^? and Br^? are preferentially solvated by methanol in the contact ion pair (CIP) state. In the solvent-assisted ion pair (SAIP) configuration, Na⁺ ions of Na⁺–Cl^? and Na⁺–Br^? are preferentially solvated by methanol and Cl^? and Br^? also show preferential solvation by methanol over DMSO. In the case of Na⁺–I^?, the only preferential solvation is in the SAIP state for I^? ion by methanol. These observations are supported by the calculated excess coordination numbers and spatial density maps. The heights of the transition states barriers for CIPs and SAIPs/solvent-shared ion pairs (SSHIPs) are significantly affected when the mole fraction of methanol (x_MeOH) changes from 0.0 to 0.25 because of a significant increase in the methanol density around halides. From the analysis of angular distribution functions of DMSO and methanol around the cations and anions, it is seen that DMSO and methanol molecules are present in parallel dipolar orientations (with respect to cation–solvent vector) in the first coordination shell of these three ion pairs at the CIP and SAIP states. Methanol molecules are nearly in an antiparallel (with respect to ion–solvent vector) orientation around the three halide ions.     

Permeant Anions Control Gating of Calcium-dependent Chloride Channels

The effects of external anions (SCN⁻, NO₃⁻, I⁻, Br⁻, F⁻, glutamate, and aspartate) on gating of Ca²⁺-dependent Cl⁻ channels from rat parotid acinar cells were studied using the whole-cell configuration of the patch-clamp technique. Shifts in the reversal potential of the current induced by replacement of external Cl⁻ with foreign anions, gave the following selectivity sequence based on permeability ratios (P_x/P_Cl): SCN⁻>I⁻>NO₃⁻>Br⁻>Cl⁻>F⁻>aspartate>glutamate. Using a continuum electrostatic model we calculated that this lyotropic sequence resulted from the interaction between anions and a polarizable tunnel with an effective dielectric constant of ∼23. Our data revealed that anions with P_x/P_Cl > 1 accelerated activation kinetics in a voltage-independent manner and slowed deactivation kinetics. Moreover, permeant anions enhanced whole-cell conductance (g, an index of the apparent open probability) in a voltage-dependent manner, and shifted leftward the membrane potential-g curves. All of these effects were produced by the anions with an effectiveness that followed the selectivity sequence. To explain the effects of permeant anions on activation kinetics and g_Cl we propose that there are 2 different anion-binding sites in the channel. One site is located outside the electrical field and controls channel activation kinetics, while a second site is located within the pore and controls whole-cell conductance. Thus, interactions of permeant anions with these two sites hinder the closing mechanism and stabilize the channel in the open state.This revised version was published online in August 2005 with a corrected cover date.     

New Screening Method for the Rapid Identification of Blocked Mutants and Their Products: Application to Erythromycin and Oleandomycin Producing Strains

γ-Glutamyltransferase from fruiting bodies of Lentinus edodes was further tested for its activation by chaotropic ions such as SCN^?, NO₃^?, Cl^?, Br^?, I^?, F^? and C1O₄^?. The thiocyanate ion increased the Km value for γ-glutamyl-p-nitroanilide without affecting the V_max value of the reaction, whereas other anions as represented by NO₃^? and Br^? increased the V_max without affecting the Km. Jhe inactivation of the enzyme by the SH group-orienting reagents, iodoacetamide and hydrogen peroxide, was stimulated by SCN^? but not by the other anions.

The activator anions protected the enzyme against its inactivation by chemical modification with 2,3-butanedione in borate. Their efficiency was parallel to the activator potency of the respective anions, except for SCN^? which provided less protection than expected from its activation potency. These dissociable effects of activator anions might be explained by two different mechanisms; binding of SCN^? to a basic group to bring about a significant change in protein conformation and binding of other anions by electrostatic and hydrophobic forces to an arginyl residue located near the active site of the enzyme.     

Structural evidence for the order of preference of inorganic substrates in mammalian heme peroxidases: crystal structure of the complex of lactoperoxidase with four inorganic substrates,SCN-, I-, Br– and Cl-

Lactoperoxidase (LPO) is a member of the family of mammalian heme peroxidases. It catalyzes the oxidation of halides and pseudohalides in presence of hydrogen peroxide. LPO has been co-crystallized with inorganic substrates, SCN^-, I^-, Br^- and Cl^-. The structure determination of the complex of LPO with above four substrates showed that all of them occupied distinct positions in the substrate binding site on the distal heme side. The bound substrate ions were separated from each other by one or more water molecules. The heme iron is coordinated to His-351 N^ϵ2 on the proximal side while it is coordinated to conserved water molecule W-1 on the distal heme side. W-1 is hydrogen bonded to Br^- ion which is followed by Cl^- ion with a hydrogen bonded water molecule W-5′ between them. Next to Cl^- ion is a hydrogen bonded water molecule W-7′ which in turn is hydrogen bonded to W-8′ and N atom of SCN^-. W-80 is hydrogen bonded to W-9′ which is hydrogen bonded to I^-. SCN^- ion also interacts directly with Asn-230 and through water molecules with Ser-235 and Phe-254. Therefore, according to the locations of four substrate anions, the order of preference for binding to lactoperoxidase is observed as Br^- > Cl^- > SCN^- > I^-. The positions of anions are further defined in terms of subsites where Br^- is located in subsite 1, Cl^- in subsite 2, SCN^- in subsite 3 and I^- in subsite 4.     

Effect of Single-Charged Anions of Different Nature on the DNA Molecule Conformation in Water–Salt Solutions

Methods of intrinsic viscosity () and beam flow birefringence were used to study the effects of some single-charged ions (F^–, Cl^–, Br^–, I^–, NO^– ₂, NO^– ₃, ClO^– ₄, SCN^–, CH₃COO^–) on the size and thermodynamic rigidity of a DNA molecule in aqueous solutions of sodium salts in a broad interval of ionic strength when temperature T is changed. It has been shown that the close interactions in a macromolecule and the resulting DNA persistent length a are independent of the type of the salt anion over the whole interval of . On the contrary, the specific volume of the DNA molecule in solution, proportional to the value, is quite sensitive to the anionic composition of the solvent, which is due to the effect of anions and their hydration on the long-range interactions in the macromolecule. The presence of polyatomic and halide anions is manifested differently in the value of DNA. Possible factors responsible for the observed effect and the role of structural alterations of water upon anion hydration are discussed.     

Effects of Potentiators of Muscular Contraction on Binding of Cations by Sarcoplasmic Reticulum

Anionic (NO₃^-, Br^-, I^-, and SCN^-) and cationic (Zn⁺⁺ and Cd⁺⁺) potentiators of the twitch output of skeletal muscle depress the active binding of Ca by sarcoplasmic reticulum isolated from rabbit skeletal muscle. Zinc and Cd exchange for Ca and Mg at the binding sites of the reticular membranes, whereas the anions effectively induce a replacement by Mg of Ca bound actively in the presence of ATP. In the absence of ATP, the passive binding of both Ca and Mg is increased by the anions tested. Furthermore, the anions increase the total capacity of the membrane fragments for passive cation binding. The Ca-stimulated ATPase activity of the membranes is inhibited by Zn and Cd, but not by the anions. Shifts in cations bound to muscle membrane systems caused by agents that increase the force of contraction developed during the twitch are considered to be the primary event modifying excitation-contraction coupling, and thus leading to potentiation.     

Theoretical study on the interactions between methanol and imidazolium-based ionic liquids

To better understand the property of the binary systems composing of imidazolium salt, [emim]⁺Aˉ (A=Clˉ, Brˉ, BF₄ˉ, and PF₆ˉ) and methanol, we have investigated in detail the interactions of methanol molecule with anions Aˉ, cation [emim]⁺, and ion pair [emim]⁺Aˉ of several ionic liquids (ILs) based on 1-ethyl-3-methylimidazolium cation by performing density functional theory calculations. It is found that H-bonds are universally involved in these systems, which may play an important role for the miscibility of methanol with imidazolium-based ILs. The interaction mechanisms of methanol molecule with anion and cation are found to be different in nature: the former mainly involves LP_X-s_{O - H}^* \sigma_{{O - H}}^{*} interaction, while the latter relates with the decisive orbital overlap of the type of LP_O-s_{C - H}^* \sigma_{{C - H}}^{*} . Based on the present calculations, we have provided some reasonable interpretations for properties of the binary mixtures of ILs and alcohol and revealed valuable information for the interaction details between ILs and alcohols, which is expected to be useful for the design of more efficient ILs to form superior solvent system with alcohol.     

A theoretical investigation of the interactions between hydroxyl-functionalized ionic liquid and water/methanol/dimethyl sulfoxide

Density functional calculations have been used to investigate the interactions of 1-(2-hydroxyethyl)-3-methylimidazolium ([C₂OHmim]⁺)-based ionic liquids (hydroxyl ILs) with water (H₂O), methanol (CH₃OH), and dimethyl sulfoxide (DMSO). It was found that the cosolvent molecules interact with the anion and cation of each ionic liquid through different atoms, i.e., H and O atoms, respectively. The interactions between the cosolvent molecules and 1-ethyl-3-methylimizolium ([C₂mim]⁺)-based ionic liquids (nonhydroxyl ILs) were also studied for comparison. In the cosolvent–[nonhydroxyl ILs] systems, a furcated H-bond was formed between the O atom of the cosolvent molecule and the C2-H and C6-H, while there were always H-bonds involving the OH group of the cation in the cosolvent–[hydroxyl ILs] systems. Introducing an OH group on the ethyl side of the imidazolium ring may change the order of solubility of the molecular liquids.     

Synthesis, characterisation of a complex of Cu(II) with a multidentate macrocyclic ligand and its interactions with anions

A macrocyclic ligand possessing a donor set of {N₃S₂} synthesised via Cs⁺-templation, 4-(pyridin-2-ylmethyl)-1,7-dithia-4,10-diazacyclododecane (L) and its Cu(II) complex, [CuL(NCMe)]²⁺ (6), are described. This Cu(II) complex interacts with a range of anions, F⁻, Cl⁻, Br⁻, I⁻, HCOO⁻, AcO⁻, CO₃²⁻, NO₃⁻, C₂O₄²⁻, H₂PO₄⁻, SCN⁻, CN⁻, BF₄⁻. Of the investigated anions, I⁻, SCN⁻, and CN⁻, show strong interaction with the Cu(II) centre as indicated by their spectral variations. The iodide particularly demonstrates distinct change in colour. This change originates from a newly appeared band at 471 nm upon iodide binding, which arises from the ligand (I⁻) to Cu(II) charge transfer (LMCT) in the iodide-substituted Cu(II) complex, [CuLI]⁺ (7). All organic compounds are characterised by NMR spectroscopy and/or microanalysis. The identities of the two Cu(II) complexes are confirmed by using microanalysis and the complex 6 is crystallographically analysed.     

Mechanism of Ion Permeation in Skeletal Muscle Chloride Channels

Voltage-gated Cl⁻ channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl⁻ channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I⁻ and related anions. Extracellular and intracellular I⁻ exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br⁻) and polyatomic (SCN⁻, NO₃ ⁻, CH₃SO₃ ⁻) anions. In addition, I⁻ block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I⁻ causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I⁻ slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined K _D values for I⁻ block in three distinct kinetic states and found that binding of I⁻ to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I⁻ binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.     

Synthesis,characterization, and evaluation of (E)-methyl 2-((2-oxonaphthalen-1(2H)-ylidene)methylamino)acetate as a biological agent and an anion sensor

An amino acid based and bidentate Schiff base, (E)-methyl 2-((2-oxonaphthalen-1(2H)-ylidene)methylamino)acetate (ligand), was synthesized from the reaction of glycine-methyl ester hydrochloride with 2-hydroxy-1-naphthaldehyde. Characterization of the ligand was carried out using theoretical quantum–mechanical calculations and experimental spectroscopic methods. The molecular structure of the compound was confirmed using X-ray single-crystal data, NMR, FTIR and UV–Visible spectroscopy, which were in good agreement with the structure predicted by the theoretical calculations using density functional theory (DFT). Antimicrobial activity of the ligand was investigated for its minimum inhibitory concentration (MIC) to several bacteria and yeast cultures. UV–Visible spectroscopy studies also shown that the ligand can bind calf thymus DNA (CT-DNA) electrostatic binding. In addition, DNA cleavage study showed that the ligand cleaved DNA without the need for external agents. Energetically most favorable docked structures were obtained from the rigid molecular docking of the compound with DNA. The compound binds at the active site of the DNA proteins by weak non-covalent interactions. The colorimetric response of the ligand in DMSO to the addition of equivalent amount of anions (F⁻, Br⁻, I⁻, CN⁻, SCN⁻, ClO₄⁻, HSO₄⁻, AcO⁻, H₂PO₄⁻, N₃⁻ and OH⁻) was investigated and the ligand was shown to be sensitive to CN⁻ anion.     

Reaction Product Variability and Biological Activity of the Lactoperoxidase System Depending on Medium Ionic Strength and pH,and on Substrate Relative Concentration

The potential of ions produced in water by the lactoperoxidase system against plant pests has shown promising results. We tested the bioactivity of ions produced by the lactoperoxidase oxidation of I⁻ and SCN⁻ in several buffers or in tap water and characterized the ions produced. In vitro biological activity was tested against Penicillium expansum, the causal agent of mold in fruits, and the major cause of patulin contamination of fruit juices and compotes. In buffers, the ionic concentration was increased 3‐fold, and pathogen inhibition was obtained down to the 1:15 dilution. In tap water, the ionic concentration was weaker, and pathogen inhibition was obtained only down to the 1:3 dilution. Acidic buffer increased ion concentrations as compared to less acidic (pH 5.6 or 6.2) or neutral buffers, as do increased ionic strength. ¹³C‐labelled SCN⁻ and MS showed that different ions were produced in water and in buffers. In specific conditions the ion solution turned yellow and a product was formed, probably diiodothiocyanate (I₂SCN⁻), giving an intense signal at 49.7 ppm in ¹³C‐NMR. The formation of the signal was unambiguously favored in acidic media and disadvantaged or inhibited in neutral or basic conditions. It was enhanced at a specific SCN⁻: I⁻ ratio of 1:4.5, but decreased when the ratio was 1:2, and was inhibited at ratio SCN⁻>I⁻. We demonstrated that the formation of the signal required the interaction between I₂ and SCN⁻, and MS showed the presence of I₂SCN⁻.     

Anions induced structural variety of macrocycles containing imidazolium cations

Six compounds including macrocyclic imidazolium cations ([H₂L^I]²⁺, [H₂L^II]²⁺ or [H₂L^III]²⁺) and anions such as Cl⁻, SCN⁻ and have been synthesized and characterized, in which different anions were introduced to the macrocyclic skeleton and the interactions of anions with the macrocycle have been investigated. Single-crystal X-ray analyses indicated that the macrocyclic configuration changed from a boat-like configuration to a chair-like configuration with the geometric variety of anions from spherical (Cl⁻) to linear (SCN⁻) or trigonal planner . Meanwhile, hydrogen bonding interactions between the macrocycle and anions were also studied.     

Effects of monovalent cations and anions on ADP-induced aggregation of bovine platelets, and mechanism thereof

The effects of monovalent cations - inorganic alakali metal cations and organic quanternary ammonium cations - and monovalent inorganic anions on ADP-induced aggregation of bovine platelets were investigated. In the presence of K⁺, Rb⁺, Cs⁺, choline or tetramethylammonium, aggeregation proceeded. However, aggregation was markedly restricted in media containing Li⁺, Na⁺, tetrabutylammonium or dimethyldibenzylammonium. With anions, aggregation proceeded in the order Cl⁻ > Br⁻ > I⁻ > Clo₄⁻ > SCN⁻. The effects of cations significantly depended on Ca²⁺ concentration, whereas those of the anions depended little of Ca²⁺. Anions such as SCN⁻ and ClO₄⁻ markedly decreased the fluorescence of the surface charge probe 2-p-tuluidinylnaphthalene-6-sulfonate, whereas cations had less pronouced effects. The relative effects of the anions on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that inhibition of platelet aggregation by the anions is due to a change in the surface change of the platelet plasma membrane. On the other hand, kinetic analysis suggests that the effects of monovalent cations on platelet aggregation are due to their competition with Ca²⁺ during the process of aggregation.     

Permeation and Block of the Skeletal Muscle Chloride Channel,ClC-1, by Foreign Anions

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl⁻ as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO₃ ⁻, BrO₃ ⁻); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl⁻ at the regulatory binding site but impair Cl⁻ passage through the channel pore (Br⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻, SCN⁻). The permeability sequence for rClC-1, SCN⁻ ∼ ClO₄ ⁻ > Cl⁻ > Br⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ > I⁻ >> BrO₃ ⁻ > HCO₃ ⁻ >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P _open curve, SCN⁻ ∼ ClO₄ ⁻ > I⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ ∼ methanesulfonate > Br⁻ > cyclamate > BrO₃ ⁻ > HCO₃ ⁻ > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl⁻ and SCN⁻ or ClO₄ ⁻ suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.     

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, GdnH⁺, and Ca²⁺), identity. The Q* decreased in the order F⁻ > Cl⁻ > Br⁻ > NO₃⁻ ∼ I⁻ > SCN⁻ > ClO₄⁻ ≫ SO₄²⁻, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO₄²⁻ anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.     

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca²⁺-activated Cl⁻ channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl⁻ with other anions (P_X/P_Cl) was SCN⁻ > I⁻ > NO₃⁻ > Br⁻ > Cl⁻ > F⁻ > gluconate. When external Cl⁻ was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca²⁺ were modified according to the sequence of permeability ratios, with anions more permeant than Cl⁻ slowing both activation and deactivation and anions less permeant than Cl⁻ accelerating them. Moreover, replacement of external Cl⁻ with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl⁻ with SCN⁻ shifted G-V to more negative potentials. Dose–response relationships for Ca²⁺ in the presence of different extracellular anions indicated that the apparent affinity for Ca²⁺ at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca²⁺ in the presence of intracellular SCN⁻ also increased compared with that in Cl⁻. Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating.