Block of a Ca<Superscript>2+</Superscript>-activated Potassium Channel by Cocaine

The primary target for cocaine is believed to be monoamine transporters because of cocaine’s high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K⁺ channel in hippocampal neurons grown in culture (IC₅₀ = ∼30 μM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca²⁺-activated K⁺ channels are 261 ± 37 μM⁻¹s⁻¹ and 11451 ± 1467 s⁻¹. The equilibrium dissociation constant (K_B) for cocaine, determined from single-channel parameters, is 43 μM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca²⁺-dependent K⁺ channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.     

Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

Potassium conductances in isolated single cells from Xenopus laevis colonic epithelium

The patch-clamp technique was employed in whole cells to analyze K⁺ conductances of amphibian colonic cells. Xenopus laevis colonic epithelium was dissected, and single epithelial cells were isolated using Ca²⁺-free solution and mild enzyme treatment. Vital epithelial cells had a round shape, and a distinction between apical and basolateral poles was no longer possible. Their epithelial origin was, however, verified by antibodies against keratin. The average resting potential of the colonocytes was −37.6 ± 1 mV (n = 220) and the resulting membrane current was strongly potassium selective. Further characterization of this conductance was achieved by current-voltage relationship in the presence and absence of various K⁺ channel blockers. Barium and cesium showed pronounced voltage-dependent blockage, with interaction at about 35% inside the pores. Lidocain, as well as quinine and quinidine also blocked, but with different kinetics and binding characteristics. Both TEA and verapamil were ineffective. We also explored the effects of extra- (pH_o) and intracellular pH (pH_i) on the K⁺ conductance. An increase of pH_o, as well as pH_i, caused membrane hyperpolarization, and the shift of the current-voltage relationship indicates a stimulation of K⁺ channels by decreasing external and/or internal H⁺ concentration. The results provide the first whole-cell measurements on isolated amphibian colonic epithelial cells and demonstrate the presence of various K⁺ channel types in this preparation. Accepted: 15 January 1999     

Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (G_m), which in skeletal muscle fibers covers membrane conductances for K⁺ (G_K) and Cl⁻ (G_Cl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of G_K and G_Cl to inversely interfere with muscle excitability. However, whether regulation of G_K and G_Cl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of G_Cl and G_K with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of G_m in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in G_Cl of ∼70% caused G_m to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in G_K (∼14-fold) and G_Cl (∼3-fold) caused G_m to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, G_m recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (K_ATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl⁻ channel inhibitor) revealed that the decreased G_m during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated G_m during phase 2 reflected synchronized openings of ClC-1 and K_ATP channels. In conclusion, G_Cl and G_K are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.     

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na⁺/K⁺-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na⁺/K⁺-ATPase with the polyclonal antibody α (H-300) raised against the human α1-subunit of Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na⁺/K⁺-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na⁺/K⁺-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na⁺/K⁺-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 μmol g_FM⁻¹ h⁻¹) than in those of L. vulgaris (31.8 ± 3.3 μmol g_FM⁻¹ h⁻¹). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 μmol_ATP g_FM⁻¹ h⁻¹, respectively. High concentrations of Na⁺/K⁺-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO₂) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.     

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K⁺ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E_m) was not sensitive to the external K⁺ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K⁺-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E_rev) of the s-ORC conductance. The s-ORC was blocked by Ba²⁺ (K_1/2 = 0.3–1.3mM) and verapamil (K_1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba²⁺. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K⁺ concentration. The slow activation of the IRC (t_1/2 ≈ 0.5 s) and its negative activation potential (V_threshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes. Received: 28 March 1996 / Accepted: 11 November 1996     

Defensive Role of Quercetin Against Imbalances of Calcium,Sodium, and Potassium in Galactosemic Cataract

Galactosemic cataracts are characterized by electrolyte disturbances resulting in osmotic imbalance and loss of transparency. We have studied the defensive role of quercetin, a bioflavonoid, against the alterations of calcium (Ca²⁺), sodium (Na⁺), and potassium (K⁺) concentrations in galactose-induced cataract in a rodent model. The experimental study was conducted on weanling male Wistar rats with an average body weight of 34 ± 0.9 g. Different groups received normal stock AIN 93 diet (group A, n = 8), AIN 93 diet with 30% galactose (group B, n = 8), and AIN 93 diet with 30% galactose + quercetin at 400 mg/100 g diet (group C, n = 8). Aldose reductase activity and protein content and concentrations of Ca²⁺, Na+, and K⁺ were determined in normal and cataractous lenses. Treatment with quercetin resulted in a significant decrease in Na⁺ and Ca²⁺ and aldose reductase levels and an increase in K⁺ and protein levels in galactosemic cataractous lenses. These results imply that inclusion of quercetin contributes to lens transparency through the maintenance of characteristic osmotic ion equilibrium and protein levels of the lens.     

Outward K+ channels in Brassica chinensis pollen protoplasts are regulated by external and internal pH

Summary.  Patch-clamp whole-cell and single-channel recording techniques were used to investigate the regulation of outward K⁺ channels by external and internal protons in Brassica chinensis pollen protoplasts. Outward K⁺ currents and conductance were insensitive to external pH (pH_o) except at pH 4.5. Maximal conductance (G _max) for the outward K⁺ currents was inhibited at acidic external pH. Half-activation voltage (E _1/2) for the outward K⁺ currents shifted to more positive voltages along with the decrease in pH_o. E _1/2 can be described by a modified Henderson–Hasselbalch equation expected from a single titratable binding site. The activation kinetics of the outward K⁺ channels was largely insensitive to pH_o. An internal pH (pH_i) of 4.5 significantly increased outward K⁺ currents and conductance. G _max for the outward K⁺ currents decreased with elevations in pH_i. In contrast to the effect of pH_o, E _1/2 was shifted to more positive voltages with elevations in pH_i. The outward K⁺ currents, G _max and E _1/2 can be described by the modified Henderson–Hasselbalch equation. Furthermore, acidifying pH_i accelerated the activation of the outward K⁺ currents significantly. The differences in electro-physiological properties among previously reported and currently described plant outward K⁺ channels may reflect differences in the structure of these channels. Received May 7, 2002; accepted July 9, 2002; published online November 29, 2002     

Mode of depolymerisation of hemicellulose by various mannanases and xylanases in relation to their ability to bleach softwood pulp

Endo-mannanases and endo-xylanases cleave different heteromannans and xylans yielding mainly dimers and trimers of the corresponding sugars as end-products. However, in the early stages of hydrolysis, four purified mannanases and four xylanases from fungal and bacterial origin, examined in this study, showed a different pattern of released oligomers (determined up to the pentamers). Furthermore, some of these enzymes showed a preference for cleaving the polysaccharides in the middle of the chain while others acted more at the end. When the increase in the specific fluidity of mannan and xylan solutions per reducing sugar released (K _v) was measured against the bleaching effect of the enzymes on softwood kraft pulp, a correlation was found. A xylanase from Penicillium simplicissimum (K _v = 0.15 l mPa⁻¹s⁻¹g⁻¹) and a mannanase from Sclerotium rolfsii (K _v = 0.12 l mPa⁻¹s⁻¹g⁻¹) applied in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with hemicellulolytic enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution) gave a high brightness increase of 3.0% and 1.9% ISO respectively. A less significant brightness increase was obtained with enzymes showing lower K _v values, such as a xylanase from Schizophyllum commune (K_v = 0.051  l mPa⁻¹s⁻¹g⁻¹, 0.2% ISO) and a bacterial mannanase (K _v = 0.061 l mPa⁻¹s⁻¹g⁻¹,0.5% ISO). Received: 19 December 1996 / Received revision: 20 February 1997 / Accepted: 22 February 1997     

Effects of trans-pyridine ligands on the interactions of RuII and RuIII ammine complexes N7-coordinated to purine nucleosides and DNA

 DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, 4-bnpy) is highly selective for G⁷ with K _G=1.1×10⁴ to 2.8×10⁴, with the more hydrophobic Pyr ligands exhibiting slightly higher binding. A strong dependence on ionic strength indicates that ion-pairing with DNA occurs prior to binding. At μ=0.05, d[Ru^II-DNA]/dt=k[Ru^II][DNA], where k=0.17–0.21 M^–1s^–1 with the various Pyr ligands. The air oxidation of [(py)(NH₃)₄Ru^II] _n -DNA to [(py)(NH₃)₄Ru^III] _n -DNA at pH 6 occurs with a pseudo-first-order rate constant of k _obs=5.6×10^–4s^–1 at μ=0.1, T=25  °C. Strand cleavage of plasmid DNA appears to occur by both Fenton/Haber-Weiss chemistry and by base-catalyzed routes, some of which are independent of oxygen. Base-catalyzed cleavage is more efficient than O₂ activation at neutral pH and involves the disproportionation of covalently bound Ru^III and, in the presence of O₂, Ru-facilitated autoxidation to 8-oxoguanine. Disproportionation of [py(NH₃)₄Ru^III] _n -DNA occurs according to the rate law: d[Ru^II–G_DNA]/dt=k ₀[Ru^III–G_DNA]+k ₁[Ru^III–G_DNA][OH^–], where k ₀=5.4×10^–4s^–1 and k ₁=8.8 M^–1s^–1 at 25  °C, μ=0.1. The appearance of [(Gua)(py)(NH₃)₄Ru^III] under argon, which occurs according to the rate law: d[Ru^III–G]/dt=k ₀[Ru^III–G_DNA]+k ₁[OH^–][Ru^III–G_DNA] (k ₀=5.74×10^–5 s^–1, k ₁=1.93×10^–2M^–1s^–1 at T=25  °C, μ=0.1), is consistent with lysis of the N-glycosidic bond by Ru^IV-induced general acid hydrolysis. In air, the ratio of [Ru-8-OG]/[Ru-G] and their net rates of appearance are 1.7 at pH 11, 25  °C. Small amounts of phosphate glycolate indicate a minor oxidative pathway involving C4′ of the sugar. In air, a dynamic steady-state system arises in which reduction of Ru^IV produces additional Ru^II. Received: 11 November 1998 / Accepted: 3 March 1999     

Characterization of Regulatory Volume Behavior by Fluorescence Quenching in Human Corneal Epithelial Cells

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), τ_c = 19 min. In contrast, a hypotonic challenge induced a rapid (τ_c = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24°C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 μM), ouabain (1 mM), DIDS (1 mM), EIPA (100 μM), or Na⁺-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K⁺, Cl⁻ channel and K⁺-Cl⁻ cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 μM), high K⁺ (20 mM) or Cl⁻-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K⁺ and Cl⁻ channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na⁺-K⁺-2Cl⁻ cotransporter (Na⁺-K⁺-2Cl⁻) and the Na⁺-K⁺ pump. Inhibition of K⁺ and Cl⁻ channels and KCC but not Na⁺-K⁺-2Cl⁻ affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.     

Theoretical study of crown ethers with incorporated azobenzene moiety

A series of crown ethers containing the azobenzene moiety incorporated into crowns of various sizes [Cr(O₆)_, Cr(O₇) and Cr(O₈)] and their corresponding alkali metal cation (Li⁺, Na⁺, K⁺, Rb⁺) complexes have been studied theoretically. The density functional theory (DFT) method was employed to elucidate the stereochemical structural natures and thermodynamic properties of all of the target molecules at the B3LYP/6-31 G(d) and LANL2DZ level for the cation Rb⁺. The fully optimized geometries had real frequencies, thus indicating their minimum-energy status. In addition, the bond lengths between the metal cation and oxygen atoms, atomic torsion angles and thermodynamic energies for complexes were studied. Natural bond orbital (NBO) analysis was used to explore the origin of the internal forces and the intermolecular interactions for the metal complexes. The calculated results show that the most significant interaction is that between the lone pair electrons of electron-donating oxygens in the cis-forms of azobenzene crown ethers (cis-ACEs) and the LP* (1-center valence antibond lone pair) orbitals of the alkali-metal cations (Li⁺, Na⁺, K⁺ and Rb⁺). The electronic spectra for the cis-ACEs [cis-Cr(O₆), cis-Cr(O₇) and cis-Cr(O₈)] are obtained by the time-dependent density functional theory (TDDFT) at the B3LYP/6-31 G(d) level. The spectra of the cis-isomers show broad π → π* (S₀ → S₂) absorption bands at 310–340 nm but weaker n → π* (S₀ → S₁) bands at 480–490 nm. The calculated results are in good agreement with the experimental results.     

Contributions of C3 and C4 plants to higher trophic levels in an Amazonian savanna

We studied the energy flow from C₃ and C₄ plants to higher trophic levels in a central Amazonian savanna by comparing the carbon stable-isotope ratios of potential food plants to the isotope ratios of species of different consumer groups. All C₄ plants encountered in our study area were grasses and all C₃ plants were bushes, shrubs or vines. Differences in δ¹³C ratios among bushes (xˉ = −30.8, SD = 1.2), vines (xˉ = −30.7, SD = 0.46) and trees (xˉ = −29.7, SD = 1.5) were small. However the mean δ¹³C ratio of dicotyledonous plants (xˉ = −30.4, SD = 1.3) was much more negative than that of the most common grasses (xˉ = −13.4, SD = 0.27). The insect primary consumers had δ¹³C ratios which ranged from a mean of −29.5 (SD = 0.47) for the grasshopper Tropidacris collaris to a mean of −14.7 (SD = 0.56) for a termite (Nasutitermes sp.), a range similar to that of the vegetation. However, the common insectivorous and omnivorous vertebrates had intermediate values for δ¹³C, indicating that carbon from different autotrophic sources mixes rapidly as it moves up the food chain. Despite this mixing, the frogs and lizards generally had higher values of δ¹³C (xˉ = −21.7, SD = 1.6; xˉ = −21.9, SD = 1.8, respectively) than the birds (xˉ = −24.8, SD = 1.8) and the only species of mammal resident in the savanna (xˉ = −25.4), indicating that they are generally more dependent on, or more able to utilise, food chains based on C₄ grasses. Received: 7 May 1998 / Accepted: 30 November 1998     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

Plasmalemmal voltage-activated K + currents in protoplasts from tobacco BY-2 cells: possible regulation by actin microfilaments?

Summary.  Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco ‘Bright Yellow-2’ cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K⁺ currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E_K when external [K⁺] was either 6 or 60 mM. Several channel blockers, including external Ba²⁺, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K⁺ current. Among the monovalent cations tested (NH₄ ⁺, Rb⁺, Li⁺, Na⁺), only Rb⁺ had appreciable permeation (P_Rb/P_K ⁼ 0.7). In addition, in 60 mM K⁺ solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K⁺ current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E_K, and other monovalent cations (NH₄ ⁺, Rb⁺, Li⁺, Na⁺) were not permeant. The inward current was blocked by external Ba²⁺ and niflumic acid. External Cs⁺ reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K⁺ current was generally small compared to the amplitude of the outward K⁺ current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K⁺ currents in tobacco ‘Bright Yellow-2’ cells. Received December 7, 2001; accepted April 15, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Pharmacologie et Physicochimie, UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur, 74 route du Rhin, BP 24, 67401 Illkirch, France. Abbreviations: TBY-2 Tobacco ‘Bright Yellow-2’; DHCB dihydrocytochalasin B; I_Kin inward rectifier K⁺ current; I_Kout outward K⁺ current; MFs microfilaments; MTs microtubules; NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid.     

Cooperative effect of water molecules in the self-catalyzed neutral hydrolysis of isocyanic acid: a comprehensive theoretical study

The detailed reaction mechanism for the water-assisted hydrolysis of isocyanic acid, HNCO + (n + 1) H₂O → CO₂ + NH₃ + nH₂O (n = 0−6), taking place in the gas phase, has been investigated. All structures were optimized and characterized at the MP2/6-31 + G* level of theory, and then re-optimized at MP2/6-311++G**. The seven explicit water molecules participating in the hydrolysis can be divided into two groups, one directly involved in the proton relay, and the other located in the vicinity of the substrate playing the cooperative role by engaging in hydrogen-bonding to HN = C = O. Two possible reaction pathways, the addition of water molecule across the C = N bond or across the C = O bond, are discussed, and the former is proved to be more favorable energetically. Our calculations suggest that, in the most kinetically favorable pathway for the titled hydrolysis, three water molecules are directly participating in the hydrogen transfer via an eight-membered cyclic transition state, while the other four water molecules catalyze the hydrolysis of HN = C = O by forming three eight-membered cooperative loops near the substrate. This strain-free hydrogen-bond network leads to the best estimated rate-determining activation energy of 24.9 kJ mol⁻¹ at 600 K, in excellent agreement with the gas-phase kinetic experimental result, 25.8 kJ mol⁻¹.     

Evidence of Intergenomic Relationships in Triploid Hybrids of Coffee (<Emphasis Type="Italic">Coffea sp.)</Emphasis> as Revealed by Meiotic Behavior and Genomic in Situ Hybridization

Interspecific hybrids involving the cultivated C. arabica (2n = 4x = 44, E^aE^aC^aC^a) and two related diploid species (2n = 2x = 22), C. eugenioides (EE) and C. liberica (LL), were produced and analyzed for their relative genome affinity using different complementary approaches, including chromosome association analysis, genomic in situ hybridization (GISH) and pollen fertility. The mean arm pairing frequency (c) and the relative affinity index (x) of triploid hybrids with known genome combinations were used as a measure of chromosome homology. Triploid hybrids were highly sterile as a result of meiotic abnormalities (fertility ranged from 1 to 15 %). Nevertheless, all hybrids exhibited a significant occurrence of genome affinities (x = 0.96 for E^aC^aE and 0.81 for E^aC^aL). Further analysis using the GISH approach revealed that C. eugenioides was more closely related to C. arabica than to C. liberica, which was in agreement with the ancestral history of the allotetraploid C. arabica. The absence of incompatibility barriers at the stylar level in the flowers of the triploid hybrids indicates the possibility of desirable gene transfer through breeding strategies.     

Electrochemistry of the CuA domain of Thermus thermophilus cytochrome ba 3

 The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba ₃ has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)₂Cu]^+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°_rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)₂Cu]^+/0 electron transfer. An irreversible oxidation of [Cu(Cys)₂Cu]⁺ at 1 V vs NHE confirms that the Cu^II:Cu^II state of Cu_A is significantly destabilized relative to the Cu^II state of analogous blue-copper proteins. Received: 3 June 1996 / Accepted: 26 August 1996     

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K _D = [1.7 ± 0.2] × 10⁻¹⁰ M) and IgG3 (K _D = [1.1 ± 0.2] × 10⁻¹⁰ M) were similar to those of glycosylated rhFcγRI.     

Microsolvation effect and hydrogen-bonding pattern of taurine-water TA-(H2O)n (n = 1-3) complexes

The microsolvation of taurine (TA) with one, two or three water molecules was investigated by a density functional theory (DFT) approach. Quantum theory of atoms in molecules (QTAIM) analyses were employed to elucidate the hydrogen bond (H-bond) interaction characteristics in TA-(H₂O)_n (n = 1–3) complexes. The results showed that the intramolecular H-bond formed between the hydroxyl and the N atom of TA are retained in most TA-(H₂O)_n (n = 1–3) complexes, and are strengthened via cooperative effects among multiple H-bonds from n = 1–3. A trend of proton transformation exists from the hydroxyl to the N atom, which finally results in the cleavage of the origin intramolecular H-bond and the formation of a new intramolecular H-bond between the amino and the O atom of TA. Therefore, the most stable TA-(H₂O)₃ complex becomes a zwitterionic complex rather than a neutral type. A many-body interaction analysis showed that the major contributors to the binding energies for complexes are the two-body energies, while three-body energies and relaxation energies make significant contributions to the binding energies for some complexes, whereas the four-body energies are too small to be significant.