Ion transport across transmembrane pores

To study the pore-mediated transport of ionic species across a lipid membrane, a series of molecular dynamics simulations have been performed of a dipalmitoyl-phosphatidyl-choline bilayer containing a preformed water pore in the presence of sodium and chloride ions. It is found that the stability of the transient water pores is greatly reduced in the presence of the ions. Specifically, the binding of sodium cations at the lipid/water interface increases the pore line tension, resulting in a destabilization of the pore. However, the application of mechanical stress opposes this effect. The flux of ions through these mechanically stabilized pores has been analyzed. Simulations indicate that the transport of the ions through the pores depends strongly on the size of the water channel. In the presence of small pores (radius <1.5 nm) permeation is slow, with both sodium and chloride permeating at similar rates. In the case in which the pores are larger (radius >1.5 nm), a crossover is observed to a regime where the anion flux is greatly enhanced. Based on these observations, a mechanism for the basal membrane permeability of ions is discussed.     

Diffusion of cations in salt solutions between ice walls

It is important for food, medical and energy engineering to control ice growth during cooling processes. Adding salts and antifreeze protein to water is a promising method for doing this. However, the effects of ions in solution on ice–water interface in narrow spaces have not yet been clarified. Therefore, we carried out molecular dynamics simulations of sodium chloride, potassium chloride and calcium chloride in water between ice walls. It was found that the peaks of the radial distribution function for calcium ions are highest among the cations. In addition, the diffusion coefficient of calcium ions was lower than those of the other ions. A further finding was that one water molecule on the interface and two water molecules adjacent to the interface in liquid phase were positioned in the first or second hydration shell for the calcium ion, which was located at the closest point to the interface. Such hydration shells caused the reductions in motion and diffusion coefficient of the specific calcium ion.     

Effects of impermeant medium ions on the composition of rabbit renal cortical slices

When incubated in isosmotic oxygenated medium in which chloride was completely replaced by gluconate, rabbit renal cortical slices lost chloride with sodium, potassium and water before reaching a new steady-state composition after 15-30 min. When corrected for extracellular space, there was an electroneutral loss of alkali metal cations (Na + K) with chloride, accompanied by isosmotic loss of water from the cells. The losses of chloride and water were independent of medium pH over the range of 6.4-8.2, and were the same with potassium rather than sodium as the dominant medium cation. Incubation in isosmotic sodium chloride medium restored tissue composition of slices transferred from gluconate medium. This recovery was not dependent specifically upon medium chloride, for slice water content also recovered when nitrate rather than chloride was substituted for medium gluconate. With sodium completely replaced by n-methyl d-glucamine (nmdG+), cells in slices lost far more sodium and potassium than chloride before reaching a new steady-state composition after some 30 min. However, the loss of water was as predicted from the total losses of measured inorganic ions. With sodium and chloride completely replaced by nmdG+ and gluconate, there was a greater loss of water than found with unilateral substitutions. Again, the combined loss of diffusible inorganic cations exceeded the loss of chloride but the water loss was that expected for isosmotic loss accompanying the measured losses of ions. These results reveal that both gluconate and nmdG+ behave as impermeant ions in this tissue preparation. It is suggested that, in the absence of medium sodium, sodium-hydrogen exchange is inhibited. Retained hydrogen ions are buffered on charged cellular non-diffusible solutes and the associated hydroxyl (or bicarbonate) ions are lost from the cells accompanied by the inorganic univalent cations lost in excess of chloride in nmdG+ medium.     

The Origin of the Short-Circuit Current in the Isolated Skin of the South American Frog Leptodactylus ocellatus

In isolated skins of Leptodactylus ocellatus the short-circuit current is smaller than the sodium net flux and this difference disappears when the skins are bathed in solutions in which the chloride ions have been replaced by sulfate or methylsulfate ions. There is a net movement of chloride ions from outside to inside of the skins in the short-circuit condition with chloride Ringer's solutions bathing the skins. The addition of ouabain to the inside solution markedly reduced not only sodium net flux but also the chloride net influx found. Copper ions added to the outside solutions produced a rise in short-circuit current, as well as the known increase in potential difference. In sodium-free Ringer's (sodium replaced by choline) the orientation of the potential difference across the skins was reversed, the inside being negative instead of positive. The results are interpreted as direct or indirect indications of the presence of a net transfer of chloride ions from outside to inside of these frog skins.     

Purification of chicken brain choline acetyltransferase

Chicken brain choline acetyltransferase was purified to homogeneity using ammonium sulfate fractionation, followed by chromatography on DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150, immunoabsorption and Sepharose-CoA columns. A purification of 3500-fold was achieved and the final preparation had a specific activity of 2:32 μmol acetylcholine formed per minute per milligram protein. The purified chicken choline acetyltransferase migrated as a single band on polyacrylamide gel electrophoresis in the presence and absence of sodium deodecyl sulfate. The native enzyme, with a molecular weight of 67,000 daltons, consists of two subunits of identical molecular weight. Chicken choline acetyltransferase has a sharp pH optimum of 7.4. It is activated by sodium chloride and potassium chloride but inhibited by cupric ion and N-ethylmaleimide.     

Study on the diuretic activity of Strychnos potatorum Linn. seed extract in albino rats.

Methanol extract of Strychnos potatorum Linn. seeds (SPSE) was evaluated for its diuretic activity in Wistar albino rats. The SPSE was administered at the graded doses of 200, 400, and 600 mg/kg body weight. The parameters which were taken into account during the experimental on each rat were: total urine volume (corrected for water intake during the test period), body weight before and after the experiment, and the concentration of sodium, potassium, and chloride ions in urine. The total urine volumes of the SPSE (600 mg/kg)-treated rats were evaluated nearly two and half fold then compared with the control (saline treated) group. Excretion of cations (sodium and potassium ions) and anions (chloride ions) also increased significantly with respect to the control group. The diuretic effect was comparable with that of the standard drug Furosemide. The increase of cations in the urine on treatment with Strychnos potatorum seed extract (SPSE) was dose-dependent. This effect supports the use of the Strychnos potatorum seeds as a diuretic in folk remedies.     

Ca2+, Mg2+, Li+, Na+, and K+ distributions in the headgroup region of binary membranes of phosphatidylcholine and phosphatidylserine as seen by deuterium NMR

The binding of calcium, magnesium, lithium, potassium, and sodium to membrane bilayers of 5 to 1 (M/M) 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl- 2-oleoylphosphatidylserine (POPS) was investigated by using deuterium nuclear magnetic resonance (2H NMR). Both lipids were deuteriated on their polar headgroups, and spectra were obtained at 25 degrees C in the liquid-crystalline phase as a function of salt concentration. The spectra obtained with calcium were correlated with 45CaCl2 binding studies to determine the effective membrane-bound calcium at low calcium binding, up to 0.78 calcium per POPS. Deuterium quadrupolar splittings of both POPC and POPS headgroups were shown to be very sensitive to calcium binding. The behavior of these two headgroups over a wide range of CaCl2 concentrations suggests that Ca2+ binding occurs in at least two steps, the first step being achieved with 0.5 M CaCl2, with a stoichiometry of 0.5 Ca2+ per POPS. Correlations of the deuterium Ca2+ binding data with related data obtained after incorporation of a cationic integral peptide showed that the effects of these two cationic molecules of the POPS headgroup are qualitatively similar, and provided further support for two-step Ca2+ binding to the POPC/POPS 5:1 membranes. The corresponding data obtained with magnesium, lithium, and potassium indicate that these cations interact with both the choline and serine headgroups. The amplitudes of headgroup perturbations could be partly correlated to the relative affinities of the metallic cations for the lipid membrane. The two-step binding described with Ca2+ appears to be relevant to the Mg2+ data, and in certain limits to the Li+ data. The data were interpreted in terms of conformational changes of the lipid headgroups induced by an electric field due to the charges of the membrane-bound metallic cations. A conformational change of the serine headgroup induced by the membrane-bound charges is proposed. We propose that the metallic cations can be differentiated on the basis of their respective spatial distribution functions relative to the choline and serine headgroups. According to this interpretation, the divalent cations Ca2+ and Mg2+ are more deeply buried in the membrane than monovalent Na+ and K+, the case of Li+ being intermediate of the latter two. This conclusion is discussed in relation to fundamental theories of the spatial distribution of ions near the interface between water and smooth charged solid surfaces.     

Effect of various ions, pH, and osmotic pressure on oxidation of elemental sulfur by Thiobacillus thiooxidans.

The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium). The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure.     

Relationship between ion distribution and membrane potential during a steady state

Equations were derived showing the relationship between the membrane potential and the quantities which influence it under steady state conditions. Essentially, the membrane potential is caused by the valence and concentration of the non-permeating ions. The permeating ions can modify the membrane potential by altering the relative concentration of the non-permeating ions with respect to the concentration of the permeating ions. For muscle, the sodium cations act as the non-permeating ions in the extracellular environment by the maintenance of some type of active metabolic process and large anions act as the non-permeating ions in the intracellular environment. Both of these non-permeating ions contribute about equally to the maintenance of the resting membrane potential. When the active metabolic process for sodium extrusion breaks down or when acids are added, the membrane potential should decrease. Water should enter the cell when the sodium metabolic process is diminished; water should leave the cell when acids are added. When acid is added, it is expected that the cations potassium and sodium will leave the cell with little or no shift of the chloride ions.     

Study on the diuretic activity of Strychnos potatorum Linn. seed extract in albino rats

Methanol extract of Strychnos potatorum Linn. seeds (SPSE) was evaluated for its diuretic activity in Wistar albino rats. The SPSE was administered at the graded doses of 200, 400, and 600 mg/kg body weight. The parameters which were taken into account during the experimental on each rat were: total urine volume (corrected for water intake during the test period), body weight before and after the experiment, and the concentration of sodium, potassium, and chloride ions in urine. The total urine volumes of the SPSE (600 mg/kg)-treated rats were evaluated nearly two and half fold then compared with the control (saline treated) group. Excretion of cations (sodium and potassium ions) and anions (chloride ions) also increased significantly with respect to the control group. The diuretic effect was comparable with that of the standard drug Furosemide. The increase of cations in the urine on treatment with Strychnos potatorum seed extract (SPSE) was dose-dependent. This effect supports the use of the Strychnos potatorum seeds as a diuretic in folk remedies.     

Effect of Various Ions, pH, and Osmotic Pressure on Oxidation of Elemental Sulfur by Thiobacillus thiooxidans

The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium). The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure.     

Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process

Sephadex G-10 gel sieving chromatography, Jones-Dole viscosity B coefficients, and solution neutron and X-ray diffraction are used to show that small ions of high charge density (e.g., sulfate, phosphate, the carboxylate, sodium, and fluoride) are strongly hydrated (kosmotropes) whereas large monovalent ions of low charge density (e.g., ammonium, chloride, potassium, and the positively charged amino acid side chains) are weakly hydrated (chaotropes). The heats of solution of the crystalline alkali halides are then used to show that only oppositely charged ions of equal water affinity spontaneously form inner sphere ion pairs, and that this controls ion binding to proteins. The net charge on a protein is a major determinant of its solubility. Finally, the surface potential difference and surface tension at an air-salt solution interface are used to generate a simple model for how ions affect protein stability and solubility through indirect interactions at the protein-solution interface. A few comments about small neutral osmolytes are also included.     

Molecular Model of a Cell Plasma Membrane With an Asymmetric Multicomponent Composition: Water Permeation and Ion Effects

We present molecular dynamics simulations of a multicomponent, asymmetric bilayer in mixed aqueous solutions of sodium and potassium chloride. Because of the geometry of the system, there are two aqueous solution regions in our simulations: one mimics the intracellular region, and one mimics the extracellular region. Ion-specific effects are evident at the membrane/aqueous solution interface. Namely, at equal concentrations of sodium and potassium, sodium ions are more strongly adsorbed to carbonyl groups of the lipid headgroups. A significant concentration excess of potassium is needed for this ion to overwhelm the sodium abundance at the membrane. Ion-membrane interactions also lead to concentration-dependent and cation-specific behavior of the electrostatic potential in the intracellular region because of the negative charge on the inner leaflet. In addition, water permeation across the membrane was observed on a timescale of ∼100 ns. This study represents a step toward the modeling of realistic biological membranes at physiological conditions in intracellular and extracellular environments.     

Effect of ions on antibacterial activity of human beta defensin 2

Human beta defensin 2 (HBD-2), the most recently discovered human defensin, has been considered to work as a host defense substance against microbial infection. Using Escherichia coli ATCC 25922, we investigated how some cations and anions influenced the antimicrobial activity of HBD-2. This activity, measured in 10 mM phosphate buffer at a concentration of 20 microg/ml, reduced significantly in the presence of 100 and 150 mM sodium or potassium chloride. The reduction was not significantly different when the total amounts of sodium and potassium ions were equal. The kind and the valence of anions (chlorine and sulfate ions) did not affect the bactericidal activity as long as the concentrations of sodium ions were equal. Divalent ions (calcium and magnesium ions) added to 10 mM of Tris buffer significantly inactivated HBD-2 at much lower concentrations (more than or equal to 0.01 mM and 0.05 mM, respectively) than the monovalent ions did. These findings suggest that HBD-2 kills the bacteria through at least two phases, which are affected independently by either monovalent or divalent ions and unaffected by anions.     

Effects of sodium ions and monensin on amylase secretion from rat parotid cells

The role of sodium ions in amylase secretion from rat parotid cells was studied using various Na+-free media and monensin. In a sucrose medium, amylase secretion was not stimulated by isoproterenol but was significantly stimulated by dibutyryl cAMP. In choline chloride and LiCl media, both isoproterenol and dibutyryl cAMP clearly evoked amylase release. Monensin itself elicited amylase secretion slightly, but significantly inhibited the secretion stimulated by isoproterenol or dibutyryl cAMP. The inhibitory effect of monensin was detectable even in choline chloride, LiCl and KCl media. These results indicate that sodium ions are not essential for amylase secretion from rat parotid cells and that the inhibitory effect of monensin is independent of influx of sodium ions or efflux of potassium ions.     

Air/water interface study of cyclopentane-containing archaeal bipolar lipid analogues

The synthesis of novel archaeal lipid analogues is described. The hydrophobic core of these tetraether bipolar lipids were based on a disubstituted 1,3-cyclopentane unit which was further equipped with mannosyl polar head groups. This hemimacrocylcic tetraether structure that can be compared to rare archaeal lipids permit to establish the behavior of such bipolar lipid at the air/water interface. The two oxygen atoms and the cyclopentane ring were found to be of importance on this behavior. Indeed, the air/water interface comparative study of tetraether- and diether-type lipids led to conclusions on a bent conformation of the tetraether at the air/water interface in the presence of a cyclopentane unit even if the presence of the two oxygen atoms favored an opened bent shape at the beginning of the compression.     

Ionic mechanisms of the fast (nicotinic) phase of the acetylcholine response of identified Planorbarius corneus neurons

Responses to electrophoretic application of acetylcholine and suberyldicholine were investigated in identified neurons (LPed-2 and LPed-3) isolated from the left pedal ganglion ofPlanorbarius corneus. When microelectrodes filled with potassium chloride were used the reversal potentials of responses to acetylcholine and suberyldicholine were less negative than when microelectrodes filled with potassium sulfate were used; these reversal potentials were shifted toward depolarization if chloride ions in the medium were replaced by sulfate. These facts indicate that the responses in both LPed-2 and LPed-3 depend on chloride ions. Reversal potentials for acetylcholine and suberyldicholine in LPed-3 were virtually identical (–51 and –50 mV respectively), but in LPed-2 they differed significantly (–46 and –62 mV respectively). Replacement of sodium ions by Tris ions shifted the reversal potential for acetylcholine in LPed-2 toward hyperpolarization but did not change the reversal potential for suberyldicholine. Benzohexonium had the same action. The reversal potential for acetylcholine in medium with a reduced sodium concentration or in the presence of benzohexonium was the same as for suberyldicholine. It is concluded that on neuron LPed-2 acetylcholine activates both acetylcholine receptors which control conductance for chloride ions and acetylcholine receptors which change conductance for sodium ions, whereas suberyldicholine acts only on acetylcholine receptors responsible for the chloride conductance of the membrane.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 533–540, September–October, 1980.     

The effect of enternal sodium concentration upon sodium fluxes in Chironomus dorsalis (Meig.) and Camptochironomus tentans (Fabr.), and the effect of other ions on sodium influx in C. tentans.

In comparison with other freshwater animals, the sodium uptake mechanism in fourth instar larvae of both C. tentans and C. dorsalis has a moderate affinity for sodium. In both species half maximum influx (Km) occurs at about 0.57 mM-Na+ and is unaltered by salt depletion. Maximum influx is achieved in steady-state C. tentans at 1.9 mM-Na+, and in steady-state C. dorsalis at 3.0 mM-Na+. Both of these values increase on depletion. Efflux also appears to be saturable at higher external sodium concentrations. In C. tentans, sodium may be transported independently of chloride, although it seems likely that sodium movement is enhanced by chloride. Sulphate strongly inhibits sodium influx. Nitrate apparently inhibits sodium influx at low concentrations, but this inhibition is progressively overcome at external sodium concentrations approaching 4 mM. A number of cations interfere with sodium influx in depleted C. tentans, notably H+, Li+ and, to a lesser extent NH4+. It is suggested that these ions compete with sodium for carrier sites. Potassium is apparently transported independently of sodium.     

Permeation of large tetra-alkylammonium cations through mutant and wild-type voltage-gated sodium channels as revealed by relief of block at high voltage

Many large organic cations are potent blockers of K(+) channels and other cation-selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in the DEKA locus of voltage-gated Na(+) channels is an important determinant of Na(+)/K(+) discrimination, exclusion of Ca(2+), and molecular sieving of organic cations. In this study, we sought to determine whether the Lys(III) residue of the DEKA locus interacts with internal tetra-alkylammonium cations (TAA(+)) that block Na(+) channels in a voltage-dependent fashion. We investigated block by a series of TAA(+) cations of the wild-type rat muscle Na(+) channel (DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA(+) and larger TAA(+) cations block both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by large TAA(+) cations as revealed by a positive inflection in the macroscopic I-V curve at voltages greater than +140 mV. Paradoxically, relief of block at high positive voltage is observed for large (e.g., tetrapentylammonium) but not small (e.g., TEA(+)) symmetrical TAA(+) cations. The DEKA wild-type channel and the DERA mutant exhibit a similar relief-of-block phenomenon superimposed on background current rectification. The results indicate: (a) hydrophobic TAA(+) cations with a molecular diameter as large as 15 A can permeate Na(+) channels from inside to outside when driven by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important determinant of inward rectification and internal block in Na(+) channels. From these observations, we suggest that hydrophobic interfaces between subunits, pseudosubunits, or packed helices of P-region channel proteins may function in facilitating blocker access to the pore, and may thus play an important role in the blocking and permeation behavior of large TAA(+) cations and potentially other kinds of local anesthetic molecules.     

Osmotically induced removal of water from fungal cells as determined by a spin probe technique

Effects of physical environment on plasma membrane semipermeability and osmotic induction of changes in aqueous cytoplasmic volume were studied in vegetative and spore cells of a plant pathogenic fungus, Fusarium sulphureum. A direct method, employing a spin probe molecule that partitioned between intracellular aqueous and hydrophobic phases, allowed measurement of reversible water movement out of macroconidial cells and chlamydospores exposed to solutions of high osmolarity. Equilibrium distribution of the spin probe between intracellular aqueous and lipid phases was more rapid than movement of water in and out of the cells. The extent of water removal was exponentially dependent on osmotic strength. Some cells became irreversibly permeable to divalent cations on treatment with sodium chloride above 1.5 osmolar but addition of sucrose to the suspension medium at equivalent osmolar concentrations caused water removal without adversely affecting the viability. Sucrose also protected the plasma membrane against damage during freeze-drying. Induction of plasma membrane damage by osmotic shock or freeze-drying permitted rapid permeation of nickel ions. Neither slow equilibration of intracellular components with divalent paramagnetic cations nor partial permeability of damaged plasma membranes to these ions was observed.