Ba2+-Induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria)

Summary We studied the influence of mucosal Ba²⁺ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol. 47:77) transepithelial, mucosa towards serosa directed K⁺ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),I _sc (short-circuit current, K⁺ current), as well as the noise ofI _sc were recorded. Addition of millimolar concentrations of Ba²⁺ to the mucosal K⁺-containing solution resulted in a sudden but quickly reversible drop inI _sc.G andI _sc decreased continuously with increasing Ba²⁺ concentration, (Ba²⁺) _o . The apparent Michaelis constant of the inhibition by Ba²⁺ lies within the range 40–80 m. The apical membrane seems to remain permselective for K⁺ up to 500 m (Ba²⁺) _o . Higher (Ba²⁺) _o , however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K⁺-channel block by K⁺–Ba²⁺ competition. In the presence of Ba²⁺, the power spectrum of the K⁺ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K⁺-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K⁺ and can be depressed by addition of Cs⁺ ions, thus indicating that Ba²⁺ ions induce K⁺-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba²⁺) _o , shows a rise in the plateau values to a maximum around 60 m (Ba²⁺) _o , followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba²⁺-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba²⁺) _o with a tendency to saturate at higher (Ba²⁺) _o . Based on a three-state model for the K⁺ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba²⁺) computer calculations compared favorably with our results. The effect of Ba²⁺ could be explained by assuming reversible binding at the outer side of the apical K⁺ channel, thereby blocking the open channel in competition with K⁺. The association-dissociation of Ba²⁺ at its receptor site is thought to cause a chopping of the K⁺ current, resulting in modulated current fluctuations.     

Extracellular pH modulates the Ca2+ current activated by depletion of intracellular Ca2+ stores in human macrophages

Intracellular Ca²⁺ (Ca_i) signaling following the binding of surface receptors activates a Ca²⁺ permeable plasma membrane conductance which has been shown to be associated with store depletion in a number of cell types. We examined the activation of this conductance in human monocyte-derived macrophages (HMDMs) using whole-cell voltage-clamp techniques coupled with fura-2 microfluorimetry and characterized the importance of external pH (pH_o) as a modulator of current amplitude. Current activation was observed following experimental maneuvers designed to deplete intracellular Ca²⁺-stores including: (i) dialysis of the cell with 100 m inositol 1,4,5-triphosphate (IP₃), (ii) intracellular dialysis with high concentrations of the Ca²⁺ buffers EGTA and BAPTA, or (iii) exposure of the cell to the Ca²⁺-ATPase inhibitor thapsigargin (1 m). Currents associated with store depletion were inwardly rectifying with kinetics, inactivation, and selectivity that appeared similar irrespective of the mode of activation. Currents were Ca²⁺ selective with a selectivity sequence of Ca²⁺ > Sr²⁺ Mg²⁺ = Mn²⁺ = Ni²⁺. The Ca²⁺ influx current was modulated by changes in pH_o; modulation was not produced as a consequence of changes in internal pH (pH_i). External acidification led to a reversible reduction in current amplitude with a pKa at pH 8.2. Changes in pH_o alone failed to induce current activation. These observations are consistent with a scheme by which changes in pH_o, as would be encountered by macrophages at sites of inflammation, could change the time course and magnitude of the Ca_i transient associated with receptor activation by regulating the influx of Ca²⁺ ions.The authors wish to gratefully acknowledge the expert technical assistance of Weiwen Xie without whom the study could not have been completed. This work was supported by National Institutes of Health GM36823.     

The relationship between single-channel and whole-cell conductance in the T-type Ca2+ channel CaV3.1

In T-type Ca²⁺ channels, macroscopic I_Ba is usually smaller than I_Ca, but at high Ca²⁺ and Ba²⁺, single-channel conductance (γ) is equal. We investigated γ as a function of divalent concentration and compared it to macroscopic currents using Ca_V3.1 channels studied under similar experimental conditions (TEA_o and K_i). Single-channel current-voltage relationships were nonlinear in a way similar to macroscopic open-channel I/Vs, so divalent γ was underestimated at depolarized voltages. To estimate divalent γ, concentration dependence, i_Div, was measured at voltages <−50 mV. Data were well described by Langmuir isotherms with γ_max(Ca²⁺) of 9.5 ± 0.4 pS and γ_max(Ba²⁺) of 10.3 ± 0.5 pS. Apparent K_M was lower for Ca²⁺ (2.3 ± 0.7 mM) than for Ba²⁺ (7.9 ± 1.3 mM). A subconductance state with an amplitude 70% that of the main state was observed, the relative occupancy of which increased with increasing Ca²⁺. As predicted by γ, macroscopic G_maxCa was larger than G_maxBa at 5 mM (G_maxCa²⁺/Ba:²⁺1.43 ± 0.14) and similar at 60 mM (G_maxCa²⁺/Ba:²⁺1.10 ± 0.02). However, over the range of activation, I_Ca was larger than I_Ba under both conditions. This was a consequence of the fact that V_rev was more negative for I_Ba than for I_Ca, so that the driving force determining I_Ba was smaller than that determining I_Ca over the range of potentials in standard current-voltage relationships.     

Reaction of hydrogen peroxide with hexaaquacobalt(III) perchlorate

The reaction of with H₂O₂ in 1.0 M HClO₄/LiClO₄ was found to be first-order in both reactants and the [H⁺] dependence of the second-order rate constant is given by k_2obs = b/[H⁺], b at 25 °C is 26.4 ± 0.5 s⁻¹. The rate law shows a simple inverse dependence on [H⁺] that is consistent with a rapidly maintained equilibrium between and its hydrolyzed form Co(H₂O)₅(OH)²⁺, followed by the rate controlling step, i.e. oxidation of H₂O₂ by Co(H₂O)₅(OH)²⁺.     

Two cyano-bridged heterotrinuclear complexes built from [(Tp)Fe(CN)3] (Tp = hydrotris(pyrazolyl)borate): synthesis, crystal structures and magnetic properties

Using an anionic precursor [(Tp)Fe^III(CN)₃]⁻ (1) as a building block, two cyano-bridged centrosymmetric heterotrinuclear complexes, (2) and (3) (en = ethylenediamine), have been synthesized and structurally characterized. In each complex, [TpFe(CN)₃]⁻ acts as a monodentate ligand toward a central [Mn(C₂H₅OH)₄]²⁺ or [Ni(en)₂]²⁺ core through one of its three cyanide groups, the other two cyanides remaining terminal. The intramolecular Fe-Mn and Fe-Ni distances are 5.2354(4) and 5.0669(11) Å, respectively. The magnetic properties of complexes 2 and 3 have been investigated in the temperature range of 2.0-300 K. A weak antiferromagnetic interaction between the Mn(II) and Fe(III) ions has been found in complex 2. The magnetic data of 2 can be fitted with the isotropic Hamiltonian: where J and J′ are the intramolecular exchange coupling parameters between adjacent and peripheral spin carriers, respectively. This leads to values of J = −1.37 cm⁻¹ and g = 2.05. The same fitting method is applied to complex 3 to give values of J = 1.2 cm⁻¹ and g = 2.25, showing that there is a ferromagnetic interaction between the Fe(III) and Ni(II) ions.     

Microwave-assisted solubilization and solution properties of hyperbranched polysaccharide

A water-insoluble hyperbranched β-d-glucan (TM3a), extracted from sclerotia of Pleurotus tuber-regium, was treated by microwave exposure to improve its solubility in water. This method led to complete dissolution of the TM3a polysaccharide in 0.02 wt % aqueous NaN₃. Various treatment periods were tested, and optimal conditions corresponded to 35 s at 765 W. The solution properties of TM3a in water were studied systematically by using size-exclusion chromatography combined with laser light scattering, viscometry, and dynamic light scattering at 25 °C. The dependences of intrinsic viscosity ([η]), radius of gyration (), and hydrodynamic radius (R_h) on weight average molecular weight (M_w) for TM3a in 0.02 wt % aqueous NaN₃ at 25 °C were found to be , , and in the M_w range from 8.20 × 10⁵ to 4.88 × 10⁶. The fractal dimension, ratio of , and the <r²>_o/M_w value of TM3a were calculated and discussed. The results indicated that TM3a existed in a sphere-like conformation in 0.02 wt % aqueous NaN₃. Furthermore, by using transmission electron microscopy, we observed directly the spherical molecules of TM3a. This work gave valuable information on improvement of solubility and chain conformation characterization of the water-insoluble polysaccharide in water.     

Kinetic studies of the reduction of hexaaquacobalt(III) perchlorate by a cobaloxime, [Co(dmgBF2)2(H2O)2]

The reaction of with Co(dmgBF₂)₂(H₂O)₂ in 1.0 M HClO₄/LiClO₄ was found to be first-order in both reactants and the [H⁺] dependence of the second-order rate constant is given by k_2obs = b/[H⁺], b at 25 °C is 9.23 ± 0.14 × 10² s⁻¹. The [H⁺] dependence at lower temperatures shows some saturation effect that allowed an estimate of the hydrolysis constant for as K_a = 9.5 × 10⁻³ M at 10 and 15 °C. Marcus theory and the known self-exchange rate constant for Co(OH₂)₅OH^2+/+ were used to estimate an electron self-exchange rate constant of k₂₂ = 1.7 × 10⁻⁴ M⁻¹ s⁻¹ for .     

Methimazole complexes of platinum(II): Synthesis, characterization and redox behavior

A variety of platinum(II) complexes of methimazole (2-mercapto-1-methylimidazole; HImS = neutral form and ImS = thiolate form), coordinated in both thione and thiolate forms, have been isolated by reacting methimazole with [PtCl(terpy)]Cl (terpy = 2,2′:6′,2″ terpyridine), [PtCl₂(bipy)] (bipy = bipyridine), [PtCl₂(o-phen)] (o-phen = o-phenanthroline), [PtCl₂(CH₃CN)₂] and [PtCl₂(COD)] (COD = 1,5-cyclooctadiene). These complexes were characterized by electronic absorption, IR and NMR (¹H, ¹³C, ¹⁹⁵Pt) spectroscopies. Molecular structure of [Pt(bipy)(HImS)₂]Cl₂·3H₂O (3a·3H₂O) has been established by single crystal X-ray crystallography. Platinum thiolate complex, [Pt(ImS)₂(HImS)₂] (5), could be obtained by treatment of [Pt(HImS)₄]Cl₂ with sodium methoxide in methanol. The solution of 5 in organic solvents yielded bi- and tri-nuclear platinum complexes. The effect of diimine ligands on oxidation of methimazole moiety in the complexes has been studied by electrochemical oxidation and pulse radiolytic oxidation employing specific one-electron oxidant, radical.     

Rare earth complexation with 1,3,5-trideoxy-1,3,5-tris(2-hydroxybenzyl)amino-cis-inositol

The protonation constants of 1,3,5-trideoxy-1,3,5-tris(2-hydroxyl-benzyl)amino-cis-inositol (thci) in I = 1 M (NaClO₄) were determined to be: pK_a1 5.96 ± 0.03, pK_a2 7.21 ± 0.01, pK_a3 8.32 ± 0.07, pK_a4 8.95 ± 0.06. The solvent extraction studies were consistent with the formation of the Ln(thci)³⁺ and complexes. The log of the stability constants (log β₁ and log β₂) at 25 °C in 1 M (NaClO₄) at pH 4 for formation of these complexes are reported. Laser luminescence measurements of the ⁷F₀-⁵D₀ transition of Eu(III) complexed by thci indicated two species. The shifts in the peaks relative to that of Eu(aq)³⁺ were comparable to the values reported for other complexes of Eu(III) with organic ligands, but the intensities were greater. Luminescence lifetime measurements of the fluorescence spectra indicated that the complex has 5 inner sphere water molecules bound to the Eu(III) cation at pH 6.71-8.52. This was consistent with bidentate chelation of Eu(III) with each thci molecule. gaussian view energy calculations indicated bonding for M(III) to the amino and hydroxyl groups of the cyclohexanetriol and (2-hydroxybenzyl)amino moieties in the Ln(thci)³⁺ complex.     

Kinetic studies of tris(2,2′-bipyridine)iron(III) perchlorate with cobaloxime, [Co(dmgBF2)2(H2O)2]

The kinetics of the reduction of by Co(dmgBF₂)₂(H₂O)₂ in 0.041 M HNO₃/NaNO₃ was found to be first-order in both the oxidizing and reducing agents and the second-order rate constant is given by k_obs = k₁ + k₂K[Cl⁻], with k₁=1.59 × 10⁶ M⁻¹ s⁻¹and k₂K = 1.83 × 10⁸ M⁻² s⁻¹, at 25 °C. The term that is first-order in [Cl⁻] is attributed to the formation of an ion-pair between and Cl⁻. For k₁, the activation parameters ΔH* and ΔS* are 2.22 ± 0.02 kcal mol⁻¹ and −22.7 ± 0.8 cal mol⁻¹ K⁻¹, respectively. The self-exchange rate constant of k₂₂ ≈ 8.7 × 10⁻³ M⁻¹ s⁻¹ for was estimated using Marcus theory and the known self-exchange rate constant for .     

Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels

Ba²⁺ block of large conductance Ca²⁺-activated K⁺ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba²⁺ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine⁺ _o, 500 mM K⁺ _i, 10 μM Ba²⁺ _i, +30 mV, and 100 μM Ca²⁺ _i to fully activate the channel), Ba²⁺ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba²⁺ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba²⁺ blocks. Thus, the preopenings represent Ba²⁺-induced time-irreversible subconductance gating. The fraction of Ba²⁺ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba²⁺]_i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba²⁺ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba²⁺ _o and no added Ba²⁺ _i. Adding K⁺, Rb⁺, or Na⁺ to the external solution decreased the fraction of Ba²⁺ blocks with preopenings, with K⁺ and Rb⁺ being more effective than Na⁺. These results are consistent with models in which the blocking Ba²⁺ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba²⁺ blocking site and acts directly as the preopening gate.     

Thermodynamics and laser luminescence spectroscopy of binary and ternary complexation of Am, Cm and Eu with citric acid, and citric acid + EDTA at high ionic strength

The binary complexation of Am³⁺, Cm³⁺and Eu³⁺ with citrate has been studied at I = 6.60 m (NaClO₄), pcH 3.60 and in the temperatures range of 0-60 °C employing a solvent extraction technique with di-(2-ethylhexyl)phosphoric acid/heptane. Two complexes, MCit and , were formed at all temperatures. For the three metal ions, the log β₁₀₁ was between 5.9 and 6.2 and log β₁₀₂ between 10.2 and 10.6 at 25 °C. The thermodynamic parameters for the Am-Cit system have been calculated from the temperature dependence of the β₁₀₁ and β₁₀₂ values. Positive enthalpy and entropy values for the formation of both complexes are interpreted as due to the contributions from the dehydration of the metal ions exceeding the exothermic cation-anion pairing. The formation of the ternary complex M(EDTA)(Cit)⁴⁻ (M = Cm and Eu) was measured to have large stability constants (log β₁₁₁ between 20.9 and 24.4) at 25 and 60 °C. Time resolved laser luminescence spectroscopy and lifetime measurement data validated the nature of the complexes of Eu(III) formed in the presence of Cit and EDTA + Cit in 6.60 m (NaClO₄) solution.     

Luminescent metalloreceptors with pendant macrocyclic ionophore: Synthesis, characterization, electrochemistry and ion-binding study

Metalloreceptors containing ruthenium(II) bipyridine unit as fluorophore and pendant macrocyclic units as ionophore have been synthesized and their luminescence and electrochemical properties have been investigated. Ion-binding study of these fluoroionophore with the anions F⁻, Cl⁻, Br⁻, I⁻, , , , , CH₃COO⁻, and and cations Na⁺, K⁺, Mg²⁺, Ca²⁺, Zn²⁺, Ba²⁺, Sr²⁺ Cd²⁺, Hg²⁺, Pb²⁺ and Cu²⁺, monitored by luminescence and ¹H NMR spectral changes, reveal strong interactions of and F⁻ for 2 and 3 and of Cu²⁺ only for 3. Luminescence titrations for 2 and 3 have been carried out to determine binding constants (K_s), and the calculated values are in the range 2.85 × 10² to 4.48 × 10⁴ M⁻¹. The ¹H NMR spectral changes for 2 and 3 with the addition of increasing concentration of F⁻ and exhibit substantial low-field shift of the CONH proton indicating its involvement in complex formation with the anions. The adduct of 2 and 3 have been isolated and characterized by ¹H and ³¹P NMR, mass and IR spectroscopy. The results are discussed in light of selectivity, structures of the anion bound complexes and their luminescence property.     

Lanthanide complexation with 1,3,5-triamino 2,4,6-trihydroxycyclohexane

The protonation constants of 1,3,5-triamino-2,4,6-trihydroxycyclohexane (taci), at 25 °C in I = 1.00 M (NaClO₄) were determined to be: pKa₁, 5.57 (0.08); pKa₂, 7.45 (0.02); pKa₃, 9.05 (0.04). The log of the stability constants, log β₃₀₂, at 25°C in I = 1.00 M (NaClO₄) for formation of were measured by potentiometry to be: Nd(III), 25.33 (0.09); Eu(III), 26.42 (0.06); Tm(III), 30.07 (0.10); Lu(III), 33.68 (0.07) ; Y(III), 28.59 (0.07). ¹H NMR spectra were consistent with formation of a single complex from pcH 6 to 10. Laser fluorescence measurements of the ⁷F_o-⁵D_o transition of Eu(III) complexed by taci indicated a single complexed species. The shift in this peak relative to that of Eu³⁺(aq) was significantly greater than the values reported for the complexes of other organic ligands with Eu(III). Luminescence lifetime measurements indicated two water molecules bound to each of the Eu(III) cations in the taci complex.     

Single-channel K+ currents inDrosophila muscle and their pharmacological block

Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba²⁺, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The K_ST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K _d 35mm), and was the most sensitive to Ba²⁺ (complete block at 10^–4 m). A Ca²⁺-activated potassium channel. K_CF 72pS (130/130mm KCl), was gated open at>10^–8 m Ca²⁺, was the least sensitive to Ba²⁺ (K _d of 3mm) and TEA (K _d of 100mm), and was not affected by quinidine. K₂ was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba²⁺ and 25mm TEA. A fourth channel type, K₃, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba²⁺ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba²⁺ and quinidine were reversible in all channels studied. The actions of TEA and Ba²⁺ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba²⁺ decreased channel open probability.     

Synthesis of bimetallic fulvalene and bis(cyclopentadienyl)methane derivatives of niobium and tantalum tetracarbonyl

Ring coupled bimetallic derivatives (μ-η^5:5-C₅H₄C₅H₄)[Nb(CO)₄]₂ and [μ-CH₂(η⁵-C₅H₄)₂][M(CO)₄]₂, where M = Nb and Ta have been prepared. The molecular structures of the latter two compounds have been determined: , triclinic, , a = 8.028(2) Å, b = 11.414(1) Å, c = 12.711(2) Å, α = 75.020(8)°, β = 80.34(2)°, γ = 79.46(2)°, V = 1097.3(4) ų, Z = 2, R(F) = 2.79%; [μ-CH₂(η⁵-C₅H₄)₂][Ta(CO)₄]₂, triclinic, , a = 7.815(3) Å, b = 10.275(4) Å, c = 13.135(4) Å, α = 104.25(3)°, β = 100.26(4)°, γ = 96.86(3)°, V = 991.2(6) ų, Z = 2, R(F) = 3.00%.     

Two-dimensional oxides constructed from octamolybdate clusters and M/tetrapyridylpyrazine subunits (M = Co, Ni: n = 2; M = Cu: n = 1)

The hydrothermal reactions of MoO₃, tetra-2-pyridylpyrazine (tpyprz) and M(CH₃CO₂)₂ · 2H₂O (M = Co, Ni) yielded the two-dimensional oxides [M₂(tpyprz)(H₂O)₂Mo₈O₂₆] · xH₂O [M = Co, x = 1.8 (1); M = Ni, x = 0.6 (2)]. However, the reaction of (NH₄)₆Mo₇O₂₄ · 4H₂O, tpyprz and Cu(CH₃CO₂)₂ · H₂O produced [{Cu₂(tpyprz)}₂Mo₈O₂₆] · 2H₂O (3 · 2H₂O). The isomorphous structures of 1 and 2 are constructed from clusters linked through {M₂(tpyprz)(H₂O)₂}⁴⁺ subunits into two-dimensional networks. While the structure of 3 is also two-dimensional, the molybdate building block is present as the δ-isomer and the secondary-metal/ligand component consists of a one-dimensional chain. The structure of 3 is compared to that of the previously reported three-dimensional material [{Cu₂(tpyprz)}₂Mo₈O₂₆] · 7H₂O which contains clusters and structurally distinct chains.