Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺² > Zn⁺² > Li⁺ > Ca⁺² > Mg⁺² > Cs⁺ > NH₄⁺ > Rb⁺ > K⁺ > Na⁺ and for anions (Na⁺ salts) the series is: I⁻ > ClO₄⁻ > ⁻SCN > Br⁻ ≈ NO₃⁻ > Cl⁻ ≈ ⁻OAc > F⁻ ≈ SO₄^− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK_as and not to an effect on the pH-independent second-order rate constant, (k_cat/K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK_a1 increases (from 3.7 to 4.9) and the apparent pK_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k_cat/K_m)^lim (= 9 × 10⁴ M^− 1 s^− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK_a of 3.7 and dissociated from the group with a pK_a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pK_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k_cat/K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced pK_a shifts, this provides a convenient test for the role of reverse protonation.     

Cation selectivity of the plasma membrane of tobacco protoplasts in the electroporated state

Cation selectivity of the cellular membrane of tobacco culture cells (cell line ‘bright yellow-2’) exposed to pulsed electric fields in the millisecond range was investigated. The whole cell configuration of the patch clamp technique was established on protoplasts prepared from these cells. Ion selectivity of the electroporated membrane was investigated by measuring the reversal potential of currents passing through field-induced pores. To this end the membrane was hyper- or depolarized for 10 ms (prepulse); subsequently the voltage was driven to opposite polarity at a constant rate (+ 40 or ? 40 mV/ms, respectively). The experiment was started by polarizing the membrane to moderately negative or positive voltages (prepulse potential ± 150 mV) that would not induce pore formation. Subsequently, an extended voltage range was scanned in the porated state of the membrane (prepulse potential ± 600 mV). IV curves in the porated and the non-porated state (obtained at the same prepulse polarity) were superimposed to determine the voltage at which both curves intersected (‘Intersection potential’). Using a modified version of the Goldmann–Hodgkin–Katz equation relative permeabilities to Ca^2 + and various monovalent alkali and organic cations were calculated. Pores were found to be fairly cation selective, with a selectivity sequence determined to be Ca^2 + > Li⁺ > Rb⁺ ≈ K⁺ ≈ Na⁺ > TEA⁺ ≈ TBA⁺ > Cl^?. Relative permeability to monovalent cations was inversely related to the ionic diameter. By fitting a formalism suggested by Dwyer at al. (J. Gen. Physiol. 75 (1980), 469–492) the effective average diameter of field induced pores was estimated to be about 1.8 nm. Implications of these results for biotechnology and electroporation theory are discussed.     

Arabidopsis KEA2, a homolog of bacterial KefC,encodes a K+/H+ antiporter with a chloroplast transit peptide

KEA genes encode putative K⁺ efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K⁺ efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast; however, a truncated AtKEA2 protein (AtsKEA2) lacking the N-terminal domain complemented disruption of the Na⁺(K⁺)/H⁺ antiporter Nhx1p to confer hygromycin resistance and tolerance to Na⁺ or K⁺ stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H⁺ exchange with preference for K⁺ = Cs⁺ > Li⁺ > Na⁺. When a conserved Asp⁷²¹ in transmembrane helix 6 that aligns to the cation binding Asp¹⁶⁴ of Escherichia coli NhaA was replaced with Ala, AtsKEA2 was completely inactivated. Mutation of a Glu⁸³⁵ between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K⁺/H⁺ antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids.     

Selectivity of cation transport across lipid membranes by the antibiotic salinomycin

The ionophoric antibiotic salinomycin is in the phase of preclinical tests against several types of malignant tumors including breast cancer. Notwithstanding, the data on its ion selectivity, although being critical for its therapeutic activity, are rather scarce. In the present work, we studied the ability of salinomycin to exert cation/H⁺-exchange across artificial bilayer lipid membranes (BLM) by measuring electrical potential on planar BLM in the presence of a protonophore and fluorescence responses of the pH-sensitive dye pyranine entrapped in liposomes. The following order of ion selectivity was obtained by these two methods: K⁺ > Na⁺ > Rb⁺ > Cs⁺ > Li⁺. Measurements of the monovalent cation-induced quenching of fluorescence of thallium ions in methanol showed that salinomycin effectively binds potassium and calcium but poorly binds sodium and lithium ions. At high concentrations, salinomycin transports Ca²⁺ through membranes of liposomes and mitochondria, as measured by using the calcium-sensitive dye Fluo-5 N. The data obtained can be used in the mechanistic studies of the anti-tumor activity of salinomycin and its selective cytotoxicity towards cancer stem cells.     

BIOELECTRIC POTENTIALS IN VALONIA : II. EFFECTS OF ARTIFICIAL SEA WATERS CONTAINING LiCl,CsCl, RbCl,OR NH4Cl

In their influence on the P.D. across the protoplasm of Valonia macrophysa, Kütz., Li⁺ and Cs⁺ resemble Na⁺, while Rb⁺ and NH₄ ⁺ resemble K⁺. The apparent mobilities of the ions in the external surface layer of Valonia protoplasm increase in the order: Cs⁺, Na⁺, Li⁺ < Cl^- < Rb⁺ < K⁺ < NH₄ ⁺.     

Short-term mercury exposure on Na+/K+-ATPase activity and ionoregulation in gill and brain of an Indian major carp,Cirrhinus mrigala

Recently mercury pollution has been increased considerably in aquatic resources throughout the world and it is a growing global concern. In this study, the 96 h LC50 value of waterborne mercuric chloride for Cirrhinus mrigala was found to be 0.34 mg/L (with 95% confidence limits). Fingerlings of C. mrigala were exposed to 0.068 and 0.034 mg/L of mercuric chloride for 96 h to assess the Na⁺/K⁺-ATPase activity and ionoregulation (Na⁺, K⁺ and Cl^?) in gill and brain. Results showed that Na⁺/K⁺-ATPase activity and ionic levels (Na⁺, K⁺ and Cl^?) in gill and brain of fish exposed to different concentrations of mercuric chloride were found to be significantly (p < 0.05) decreased throughout the study period. Mercury inactivates many enzymes by attaching to sulfur atoms in which the enzyme Na⁺/K⁺-ATPase is highly sensitive to mercury. The inhibition of gill and brain Na⁺/K⁺-ATPase activity might have resulted from the physicochemical alteration of the membrane due to mercury toxicity. Moreover, inhibition of Na⁺/K⁺-ATPase may affect the ion transport and osmoregulatory function by blocking the transport of substances across the membrane by active transport. The present study indicates that the alterations in these parameters can be used in environmental biomonitoring of mercury contamination in aquatic ecosystem.     

Effects of alkali ions on the circadian leaf movements of Oxalis regnellii

The influence of alkali ions on the circadian leaf movements of Oxalis regnellii Mig. was investigated. Ions were given to the oscillating system via the transpiration stream of cut stalks in nutrient medium. Chloride solutions of Rb⁺, Cs⁺, Na⁺ and K⁺ were tested and the results compared to previously published LiCl-results. The period of the circadian leaf movements was unaffected by a continual addition of Na⁺ or K⁺ to the nutrient medium (at least up to 40 mM). Rb⁺, in the concentration of 2.5 or 5 mM, caused a shortening of the period when applied continuously. Rb⁺ concentrations up to 60 mM were tested. Cs⁺ ions caused only lengthenings of the circadian period. Cs⁺ concentrations up to 40 mM were tested. Cs⁺ resembled Li⁺ in producing period lengthenings, but was not as effective as Li⁺ when compared on a concentration basis. Toxicity of the effective ions was in the following order: Li⁺Cs⁺Rb⁺, Rb⁺ pulses (50 mM, 4 h) phase-shifted the rhythm and caused advances. A phase response curve was determined and the maximum steady state advances were of the order of 1 h. The dual effect of the Rb⁺ ions is discussed and is assumed to be due to two counteracting processes, exemplified by Rb⁺-sensitive ATPase-controlled pumping processes and protein synthesis. For comparison, the effects of Rb⁺ and Li⁺ in human depressive disorders is also discussed in relation to their influence on circadian systems. It is emphasized that Rb⁺ and K⁺ behave differently and are not interchangeable in their action on circadian systems.     

The mechanism of cation permeation in rabbit gallbladder

Summary The questions underlying ion permeation mechanisms, the types of experiments available to answer these questions, and the properties of some likely permeation models are examined, as background to experiments designed to characterize the mechanism of alkali cation permeation across rabbit gallbladder epithelium. Conductance is found to increase linearly with bathing-solution salt concentrations up to at least 400mm. In symmetrical solutions of single alkali chloride salts, the conductance sequence is K⁺>Rb⁺>Na⁺>Cs⁺∼Li⁺. The current-voltage relation is linear in symmetrical solutions and in the presence of a single-salt concentration gradient up to at least 800 mV. The anion/cation permeability ratio shows little change with concentration up to at least 300mm. Ca⁺⁺ reduces alkali chloride single-salt dilution potentials, the magnitude of the effect being interpreted as an inverse measure of cation equilibrium constants. The equilibrium-constant sequence deduced on this basis is K⁺>Rb⁺>Na⁺∼Cs⁺∼Li⁺. These results suggest (1) that the mechanism of cation permeation in the gallbladder is not the same as that in a macroscopic ion-exchange membrane; (2) that cation mobility ratios are closer to one than are equilibrium-constant ratios; (3) that the rate-limiting step for cation permeation is in the membrane interior rather than at the membrane-solution interface; and (4) that the rate-controlling membrane is one which is sufficiently thick that it obeys microscopic electroneutrality.     

Nigericin forms highly stable complexes with lithium and cesium

Nigericin is a monocarboxylic polyether molecule described as a mobile K⁺ ionophore unable to transport Li⁺ and Cs⁺ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li⁺, Cs⁺, Na⁺, Rb⁺, and K⁺. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li⁺ and Cs⁺ than for Na⁺, K⁺, and Rb⁺, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li⁺ and nigericin-Cs⁺ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na⁺, K⁺, and Rb⁺ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li⁺ and Cs⁺ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K⁺>Rb⁺>Na⁺.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia.     

Secretion of Na+, K+ and fluid by the Malpighian (renal) tubule of the larval cabbage looper Trichoplusia ni (Lepidoptera: Noctuidae)

The Malpighian (renal) tubules play important roles in ionic and osmotic homeostasis in insects. In Lepidoptera, the Malpighian tubules are structurally regionalized and the concentration of Na⁺ and K⁺ in the secreted fluid varies depending on the segment of tubule analyzed. In this work, we have characterized fluid and ion (Na⁺, K⁺, H⁺) transport by tubules of the larval stage of the cabbage looper Trichoplusia ni; we have also evaluated the effects of fluid secretion inhibitors and stimulants on fluid and ion transport. Ramsay assays showed that fluid was secreted by the iliac plexus but not by the yellow and white regions of the tubule. K⁺ and Na⁺ were secreted by the distal iliac plexus (DIP) and K⁺ was reabsorbed in downstream regions. The fluid secretion rate decreased > 50% after 25 μM bafilomycin A1, 500 μM amiloride or 50 μM bumetanide was added to the bath. The concentration of K⁺ in the secreted fluid did not change, whereas the concentration of Na⁺ in the secreted fluid decreased significantly when tubules were exposed to bafilomycin A1 or amiloride. Addition of 500 μM cAMP or 1 μM 5-HT to the bath stimulated fluid secretion and resulted in a decrease in K⁺ concentration in the secreted fluid. An increase in Na⁺ concentration in the secreted fluid was observed only in cAMP-stimulated tubules. Secreted fluid pH and the transepithelial electrical potential (TEP) did not change when tubules were stimulated. Taken together, our results show that the secretion of fluid is carried out by the upper regions (DIP) in T. ni Malpighian tubules. Upper regions of the tubules secrete K⁺, whereas lower regions reabsorb it. Stimulation of fluid secretion is correlated with a decrease in the K⁺/Na⁺ ratio.     

Carbenoxolone-sensitive and cesium-permeable potassium channel in the rod cells of frog taste discs

The rod cells in frog taste discs display the outward current and maintain the negative resting potential in the condition where internal K⁺ is replaced with Cs⁺. We analyzed the properties of the Cs⁺-permeable conductance in the rod cells. The current–voltage (I/V) relationships obtained by a voltage ramp were bell-shaped under Cs⁺ internal solution. The steady state I/V relationships elicited by voltage steps also displayed the bell-shaped outward current. The activation of the current accelerated with the depolarization and the inactivation appeared at positive voltage. The gating for the current was maintained even at symmetric condition (Cs⁺ external and internal solutions). The wing cells did not show the properties. The permeability for K⁺ was a little larger than that for Cs⁺. Internal Na⁺ and NMDG⁺ could not induce the bell-shaped outward current. Carbenoxolone inhibited the bell-shaped outward Cs⁺ current dose dependently (IC₅₀: 27 μM). Internal arachidonic acid (20 μM) did not induce the linear current–voltage (I–V) relationship which is observed in two-pore domain K⁺ channel (K_2P). The results suggest that the resting membrane potentials in the rod cells are maintained by the voltage-gated K⁺ channels.     

Stabilities in nitromethane of alkali metal ion complexes with dibenzo-18-crown-6 and dibenzo-24-crown-8 and their transfer from nitromethane to other polar solvents

Stability constants for the 1:1 complexes of Na⁺, K⁺, Rb⁺, and Cs⁺ with dibenzo-18-crown-6 (DB18C6) and dibenzo-24-crown-8 (DB24C8) have been determined by conductometry at 25 °C in a poorly solvating solvent, nitromethane. For both the crown ethers, the stability constant decreases with increasing metal ion size, Na⁺ > K⁺ > Rb⁺ > Cs⁺, regardless of the size compatibility between the metal ions and the ligand cavities. A comparison of the results with those in several other solvents (S: acetonitrile, propylene carbonate, water, methanol, and N,N-dimethylformamide) leads to the conclusion that the selectivity sequence of these crown ethers in nitromethane agrees with the intrinsic one in the absence of a solvent. Transfer activity coefficients of the crown ethers and their complexes from nitromethane to S have been determined to evaluate the solute-solvent interactions. It is shown that DB24C8 shields the alkali metal ions more effectively from the solvents than DB18C6 because of the larger number of oxygen atoms and the more flexible structure of DB24C8. Regarding the complexation in nitromethane as a reference, the complex stability and selectivity in S are discussed. The selectivities of these crown ethers in water, methanol, and N,N-dimethylformamide, which apparently obey the size-fit concept, are largely due to the solvation of the free alkali metal ions.     

Negative conductance caused by entry of sodium and cesium ions into the potassium channels of squid axons

Internal Cs⁺, Na⁺, Li⁺, and, to a lesser degree, Rb⁺ interfere with outward current through the K pores in voltage clamped squid axons. Addition of 100 mM NaF to the perfusion medium cuts outward current for large depolarizations about in half, and causes negative conductance over a range of membrane voltages. For example, suddenly reducing membrane potential from +100 to +60 mv increases the magnitude of the outward current. Internal Cs⁺ and, to a small extent, Li⁺, also cause negative conductance. Na⁺ ions permeate at least 17 times less well through the K pores than K⁺, and Cs⁺ does not permeate measurably. The results strongly suggest that K pores have a wide and not very selective inner mouth, which accepts K⁺, Na⁺, Li⁺, Cs⁺, tetraethylammonium ion (TEA⁺), and other ions. The diameter of the mouth must be at least 8 A, which is the diameter of a TEA⁺ ion. K⁺ ions in the mouths probably have full hydration shells. The remainder of the pore is postulated to be 2.6–3.0 A in diameter, large enough for K⁺ and Rb⁺ but too small for Cs⁺ and TEA⁺. We postulate that Na⁺ ions do not enter the narrower part of the pore because they are too small to fit well in the coordination cages provided by the pore as replacements for the water molecules surrounding an ion.     

Specific cation effect on quenching reactions of excited tris(α,α′-diimine) ruthenium(II) and chromium(III) complexes by cyanide complexes in aqueous solutions

Specific salt effects were studied on the quenching reaction of excited [Ru(NN)₃]²⁺ (NN=2,2′-bipyridine(bpy), 1,10-phenanthrorine(phen)) and [Cr(bpy)₃]³⁺ by [Cr(CN)₆]³⁻, [Fe(CN)₆]³⁻ and [Ni(CN)₄]²⁻ in aqueous solutions as a function of alkali metal ions which were added for adjustment of ionic strength. The quenching rate constants in [Ru(NN)₃]²⁺-[Cr(CN)₆]³⁻ and [Cr(bpy)₃]³⁺-[Cr(CN)₆]³⁻ systems are changed by the cations as Li⁺>Na⁺>K⁺≈Rb⁺≈Cs⁺. On the other hand, the rate constants in [Ru(NN)₃]²⁺-[Fe(CN)₆]³⁻ and [Ru(NN)₃]²⁺-[Ni(CN)₄]²⁻ systems, which are diffusion-controlled reactions, are not varied by the alkali metal cations. The obtained order (Li⁺>Na⁺>K⁺≈Rb⁺≈Cs⁺) of the quenching rate constant is quite different from salt effects, Li⁺<Na⁺<K⁺<Rb⁺<Cs⁺, which have been obtained in the electron transfer reactions between complex anions.     

Effects of potassium ion supplementation on survival and ion regulation in Gulf killifish Fundulus grandis larvae reared in ion deficient saline waters

Teleost fish often live in an environment in which osmoregulatory mechanisms are critical for survival and largely unknown in larval fish. The effects of a single important marine ion (K⁺) on survival and ion regulation of larval Gulf killifish, an estuarine, euryhaline teleost, were determined. A four-week study was completed in four separate recirculating systems with newly hatched larvae. Salinity in all four systems was maintained between 9.5 and 10‰. Two systems were maintained using crystal salt (99.6% NaCl) with K⁺ supplementation (1.31 ± 0.04 mmol/L and 2.06 ± 0.04 mmol/L K⁺; mean ± SEM), one was maintained with crystal salt and no K⁺ supplementation (0.33 ± 0.05 mmol/L K⁺), the fourth system was maintained using a standard marine mix salt (2.96 ± 0.04 mmol/L K⁺), the salt mix also included standard ranges of other ions such as calcium and magnesium. Larvae were sampled throughout the experiment for dry mass, Na⁺/K⁺-ATPase (NKA) activity, whole body ion composition, relative gene expression (NKA, Na⁺/K⁺/2Cl^? cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR)), and immunocytochemistry staining for NKA, NKCC, and CFTR. Larvae stocked into water with no K⁺ supplementation resulted in 100% mortality within 24 h. Mortality and dry mass were significantly influenced by K⁺ concentration (P ≤ 0.05). No differences were observed among treatment groups for NKA activity. At 1 dph NKA mRNA expression was higher in the 0.3 mmol [K⁺] group than in other treatment groups and at 7 dph differences in intestinal NKA and CFTR staining were observed. These data indicate that the rearing of larval Gulf killifish may be possible in ion deficient water utilizing specific ion supplementation.     

Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1

The wheat root high-affinity K⁺ transporter HKT1 functions as a sodium-coupled potassium co-uptake transporter. At toxic millimolar levels of sodium (Na⁺), HKT1 mediates low-affinity Na⁺ uptake while potassium (K⁺) uptake is blocked. In roots, low-affinity Na⁺ uptake and inhibition of K⁺ uptake contribute to Na⁺ toxicity. In the present study, the selectivity among alkali cations of HKT1 expressed in Xenopus oocytes and yeast was investigated under various ionic conditions at steady state. The data show that HKT1 is highly selective for uptake of the two physiologically significant alkali cations, K⁺ and Na⁺ over Rb⁺, Cs⁺ and Li⁺. In addition, Rb⁺ and Cs⁺, and an excess of extracellular K⁺ over Na⁺, are shown to partially reduce or block HKT1-mediated K⁺-Na⁺ uptake. Furthermore, K⁺, Rb⁺ and Cs⁺ also effectively reduce outward currents mediated by HKT1, thereby causing depolarizations. In yeast, HKT1 can produce high-affinity Rb⁺ uptake at approximately 15-fold lower rates than for K⁺. Rb⁺ influx in yeast can be mediated by the ability of the yeast plasma membrane proton pump to balance the 35-fold lower HKT1 conductance for Rb⁺. A model for HKT1 activity is presented involving a high-affinity K⁺ binding site and a high-affinity Na⁺ binding site, and competitive interactions of K⁺, Na⁺ and other alkali cations for binding to these two sites. Possible implications of the presented results for physiological K⁺ and Na⁺ uptake in plants are discussed.     

Ion and water balance in Gryllus crickets during the first twelve hours of cold exposure

Insects lose ion and water balance during chilling, but the mechanisms underlying this phenomenon are based on patterns of ion and water balance observed in the later stages of cold exposure (12 or more hours). Here we quantified the distribution of ions and water in the hemolymph, muscle, and gut in adult Gryllus field crickets during the first 12 h of cold exposure to test mechanistic hypotheses about why homeostasis is lost in the cold, and how chill-tolerant insects might maintain homeostasis to lower temperatures. Unlike in later chill coma, hemolymph [Na⁺] and Na⁺ content in the first few hours of chilling actually increased. Patterns of Na⁺ balance suggest that Na⁺ migrates from the tissues to the gut lumen via the hemolymph. Imbalance of [K⁺] progressed gradually over 12 h and could not explain chill coma onset (a finding consistent with recent studies), nor did it predict survival or injury following 48 h of chilling. Gryllus veletis avoided shifts in muscle and hemolymph ion content better than Gryllus pennsylvanicus (which is less chill-tolerant), however neither species defended water, [Na⁺], or [K⁺] balance during the first 12 h of chilling. Gryllus veletis better maintained balance of Na⁺ content and may therefore have greater tissue resistance to ion leak during cold exposure, which could partially explain faster chill coma recovery for that species.     

Differential tolerance to combined salinity and O2 deficiency in the halophytic grasses Puccinellia ciliata and Thinopyrum ponticum: The importance of K+ retention in roots

Saline environments of terrestrial halophytes are often prone to waterlogging, yet the effects on halophytes of combined salinity and waterlogging have rarely been studied. Either salinity or hypoxia (low O₂) alone can interfere with K⁺ homeostasis, therefore the combination of salinity or hypoxia is expected to impact significantly on K⁺ retention in roots. We studied mechanisms of tolerance to the interaction of salinity with hypoxia in Puccinellia ciliata and Thinopyrum ponticum, halophytic grasses that differ in waterlogging tolerance. Plants were exposed to aerated and stagnant saline (250 mM NaCl) treatments with low (0.25 mM) and high (4 mM) K⁺ levels; growth, net ion fluxes and tissue ion concentrations were determined. P. ciliata was more tolerant than T. ponticum to stagnant-saline treatment, producing twice the biomass of adventitious roots, which accumulated high levels of Na⁺, and had lower shoot Na⁺. After 24 h of saline hypoxic treatment, MIFE measurements revealed a net uptake of K⁺ (∼40 nmol m⁻² s⁻¹) for P. ciliata, but a net loss of K⁺ (∼20 nmol m⁻² s⁻¹) for the more waterlogging sensitive T. ponticum. NaCl alone induced K⁺ efflux from roots of both species, with channel blocker tests implicating GORK-like channels. P. ciliata had constitutively a more negative root cell membrane potential than T. ponticum (−150 versus −115 mV). Tolerance to salinity and hypoxia in P. ciliata is related to increased production of adventitious roots, regulation of shoot K⁺/Na⁺, and a superior ability to maintain negative membrane potential in root cells, resulting in greater retention of K⁺.     

Contribution of residues in second transmembrane domain of ASIC1a protein to ion selectivity

Acid-sensing ion channels (ASICs) are proton-gated cation-selective channels expressed in the peripheral and central nervous systems. The ion permeation pathway of ASIC1a is defined by residues 426–450 in the second transmembrane (TM2) segment. The gate, formed by the intersection of the TM2 segments, localizes near the extracellular boundary of the plasma membrane. We explored the contribution to ion permeation and selectivity of residues in the TM2 segment of ASIC1a. Studies of accessibility with positively charged methanethiosulfonate reagents suggest that the permeation pathway in the open state constricts below the gate, restricting the passage to large ions. Substitution of residues in the intracellular vestibule at positions 437, 438, 443, or 446 significantly increased the permeability to K⁺ versus Na⁺. ASIC1a shows a selectivity sequence for alkali metals of Na⁺>Li⁺>K⁺≫Rb⁺>Cs⁺. Alanine and cysteine substitutions at position 438 increased, to different extents, the relative permeability to Li⁺, K⁺, Rb⁺, and Cs⁺. For these mutants, ion permeation was not a function of the diameter of the nonhydrated ion, suggesting that Gly-438 encompasses an ion coordination site that is essential for ion selectivity. M437C and A443C mutants showed slightly increased permeability to K⁺, Rb⁺, and Cs⁺, suggesting that substitutions at these positions influence ion discrimination by altering molecular sieving. Our results indicate that ion selectivity is accomplished by the contribution of multiple sites in the pore of ASIC1a.     

Application of seaweeds for the removal of lead from aqueous solution

Ten different seaweed species were compared on the basis of lead uptake at different pH conditions. The brown seaweed, Turbinaria conoides, exhibited maximum lead uptake (at pH 4.5) and hence was selected for further studies. Sorption isotherms, obtained at different pH (4–5) and temperature (25–35 °C) conditions were fitted using Langmuir and Sips models. According to the Langmuir model, the maximum lead uptake of 439.4 mg/g was obtained at optimum pH (4.5) and temperature (30 °C). The Sips model better described the sorption isotherms with high correlation coefficients at all conditions examined. Various thermodynamic parameters such as ΔG°, ΔH° and ΔS° were calculated indicating that the present system was a spontaneous and endothermic process. Through potentiometric titrations, number of binding sites (carboxyl groups) and pK₁ were determined as 4.1 mmol/g and 4.4, respectively. The influence of co-ions (Na⁺, K⁺, Mg²⁺ and Ca²⁺) on lead uptake was well pronounced in the case of divalent ions compared to monovalent ions. The solution of 0.1 M HCl successfully eluted all lead ions from lead-loaded T. conoides biomass. The regeneration experiments revealed that the alga could be successfully reused for five cycles without any loss in lead biosorption capacity. A glass column (2 cm i.d. and 35 cm height) was used to study the continuous lead biosorption performance of T. conoides. At 25 cm (bed height), 5 ml/min (flow rate) and 100 mg/l (initial lead concentration), T. conoides exhibited lead uptake of 220.1 mg/g. The column was successfully eluted using 0.1 M HCl, with elution efficiency of 99.7%.