Modification of sodium inactivation in myelinated nerve by Anemonia toxin II and iodate. Analysis of current fluctuations and current relaxations

(1) Na+ currents and Na+ current fluctuations were measured in single myelinated nerve fibres of Rana esculenta under voltage-clamp conditions. The process of Na+ inactivation was modified by external treatment with 7 microM Anemonia Toxin II or by internal application of 20 or 40 mM IO3(-). (2) At depolarization of 24 and 32 mV the spectral density of Na+ current fluctuations could be described as the sum of two contributions, Sh(f) and Sm(f), representing the spectrum from fluctuations of the inactivation (h) and activation (m) gates, respectively. At higher depolarizations of 40 and 48 mV the low frequency (h) fluctuations could be better fitted by the sum, Sh1(f)+Sh2(f), of two separate Lorentzian functions. (3) The Na+ current and the variance of Na+ current fluctuations between 150 and 450 ms after depolarization are increased by one order of magnitude after application of Anemonia Toxin II or IO3(-). (4) The kinetics of Na+ current inactivation were described as A1 x exp(-t/tau h1) + A2 x exp(-t/tau h2) + B. The constant, tau h1, of fast Na+ inactivation was the same in normal and modified nerve fibres. The slow inactivation time constant, tau h2, increased with increasing depolarizations in modified fibres but decreased under control conditions. In all cases tau h2 showed a similar voltage dependence as the time constant found by fitting the low frequency fluctuations of Na+ current with one Lorentzian function, Sh(f). (5) It is concluded that Anemonia Toxin II and IO3(-) modify a fraction of Na+ channels in an all-or-none manner. A lower limit of the number of modified Na+ channels is estimated from the Na+ current and the variance Na+ current fluctuations. 7 microM external Anemonia Toxin II modifies more than 17% and 20 or 40 mM internal IO3(-) more than 8% of all Na+ channels. The inactivation gates in modified channels experience an electric field different from that in normal fibres.     

Slow actions of hyperpolarization on sodium channels in the membrane of myelinated nerve

The mean sodium current, $\text{I}$, and the variance of sodium current fluctuations, var, were measured in myelinated nerve during a depolarization to $\text{V = 40}\text{mV}$ applied from the resting potential ($\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= 0}$) or from a hyperpolarizing holding potential $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$. From $\text{I}$ and var the relative variations in the number $\text{N}$ and the conductance γ of sodium channels following changes of the holding potential were calculated. Hyperpolarizing the membrane from $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= 0}$ to ?28 mV increased $\text{N}$ by a factor of 3.7, whereas γ decreased by a factor of 0.53. These actions of holding potential on sodium channels develop slowly since 500 ms prepulses to 0 or ?28 mV do not alter the values of $\text{N}$ and γ.     

Fluctuation analysis of Na+ channels modified by batrachotoxin in myelinated nerve

(1) Single myelinated nerve fibres of Rana esculenta were treated with the steroidal alkaloid batrachotoxin, and Na⁺ currents and Na⁺-current fluctuations were measured near the resting potential under voltage-clamp conditions. Between test pulses fibres were held at hyperpolarizing membrane potentials. (2) The spectral density of Na⁺-current fluctuations was fitted by the sum of a $\text{1}\text{f}$ component and a Lorentzian function. The time constant $\text{τ}{}_{\mathrm{<mtext>c</mtext>}}\text{= 1/(2π?}{}_{\mathrm{<mtext>c</mtext>}}\text{)}$ obtained from the corner frequency ?_c of the Lorentzian function approximately agreed with the activation time constant τ_m of the macroscopic currents. (3) The conductance γ of a single Na⁺ channel modified by batrachotoxin was calculated from the integral of the Lorentzian function and the steady-state Na⁺ current. At the resting potential V = O we obtained γ = 1.6 pS, higher γ-values of 3.2 and 3.45 pS were found at V = ?8 and ?16 mV, respectively. (4) The conductance of a modified Na⁺ channel is significantly lower than the values 6.4 to 8.85 pS reported in the literature for normal Na⁺ channels. Hence, our experiments are in agreement with the view that batrachotoxin acts in an ‘all-or-none’ manner on Na⁺ channels and creates a distinct population of modified channels.     

Modification of sodium channels in myelinated nerve by Anemonia sulcata toxin II

1. Single myelinated nerve fibres of the frog, Rana esculenta, were investigated predominantly in voltage clamp experiments. 2. Sodium current (INa) inactivation was measured in the presence of 10 mM TEA to suppress IK. Inactivation was diphasic but complete in toxin-free solution; it was delayed and became incomplete in Anemonia sulcata toxin II (ATX II) leading to persistent INa flow even during long depolarizations. The effects were reversible. Activation was not affected. 3. The persistent INa component increased with increasing toxin concentration and saturated at ca. 15 microM. The lowest concentration yielding unequivocal effects in the voltage clamp was 0.5 microM. 4. The curve relating the steady-state inactivation parameter, h infinity to the conditioning potential V became non-monotonic in ATX II i.e. dh infinity/dV greater than 0 for V greater than 30 mV. 5. Inactivation could be formally described by a three-state model with two conducting (h2 and h2) and one closed state (x) in the sequence h1 in equilibrium x in equilibrium h2. 6. Ca2+ modifies h2(V) more than h1(V) whose reaction to Ca2+ is similar to h(V) in toxin-free solution. The Ca2+ effect is very rapid and reversible.     

Modification of ultraviolet radiation effects on the membrane of myelinated nerve fibers by sulfhydryl compounds

Summary The modification of the ultraviolet blocking of sodium channels and of the ultraviolet-induced potential shift of the gating parameters by means of the sulfhydryl compoundsl-cysteine and 2-mercaptoethanol was investigated in the node of Ranvier under voltage-clamp conditions. The UV wavelength was 280 nm. The radiation-induced potential shift of the voltage-dependent gating parameters was prevented or even reversed by the action of the sulfhydryl compounds (internal application), while the blocking effect was not affected. It is concluded that the two radiation effects are caused by two separate photoreactions. Internally applied N-ethylmaleimide, binding specifically to protein-SH groups, exhibits an effect similar to the ultraviolet-induced potential shift, without affecting the maximum sodium permeability. Therefore, the ultraviolet-induced potential shift might be caused by a photocatalyzed oxidation of —SH groups of membrane proteins changing the surface charge density at the inner side of the nodal membrane.     

Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin

(1) Na⁺ currents and Na⁺-current fluctuations were measured in myelinated frog nerve fibres at 15°C during 7.7 ms depolarizations to $\text{V = 40, 60}\text{and}\text{80}\text{mV}$. (2) The conductance γ of a single Na⁺ channel and the number $\text{N}{}_0$ of channels per node were calculated from ensemble average values of the mean Na⁺ current and the variance of Na⁺-current fluctuations. (3) For a hyperpolarizing holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ the mean values of the channel conductance and number were $\text{γ = 9.8}\text{pS}$ and $\text{N}{}_0\text{= 74 000}$. (4) After changing the holding potential to the resting potential ($\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= 0}$) the conductance γ increased by a factor of 1.37 whereas the number $\text{N}{}_0$ decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ increased γ by a factor of 1.55 and reduced $\text{N}{}_0$ by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na⁺ channels in the nodal membrane.     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. I. Formation of transmembrane channels in lipid bilayers

The addition of nanomolar amounts of a toxin preparation derived from the sea anemone Stoichactis helianthus to black lipid membranes increases their electrical conductance by one million-fold. In addition, the membranes become permeable predominantly to monovalent cations. The elevated bilayer conductance is voltage-dependent, and the current-voltage curves of these bilayers display rectification as well as a region of negative resistance. The membrane activity of the toxin is proportional to the third power of its concentration, and at very low concentrations the membrane conductance increases in discrete uniform steps. These observations indicate that the mechanism of toxin action involves the formation of transmembrane channels constructed by the aggregation of protein molecules which are inserted in the bilayer. The voltage-dependent membrane conductance arises from two distinct channel characteristics: (1) the unit conductance of individual channels is dependent on the polarity of applied voltage; (2) the number of ion-conducting channels is influenced by the polarity as well as the magnitude of applied potential. It is believed that these effects are due to the influence of an electric field on the insertion of toxin molecules into the bilayer or on their subsequent association with each other to produce channels. Partial chemical characterization of the toxin material has shown that the membrane active factor is a basic protein with a molecular weight of 17 500.     

Synthesis and characterization of a high spin d Fe(II) complex with the tridentate ligand dipyrido(2,3-a:3,2-j)phenazine (dpop)

A new bis-(N-tridentate) Fe(II) complex [Fe(dpop^′)₂](PF₆)₂ (dpop^′=dipyrido(2,3-a:3^′,2^′-j)phenazine) was prepared and studied. The magnetic moment of the solid was determined as μ=5.2-4.9 BM and in CH₃CN solution as μ=4.9 BM and indicate the high spin Fe(II) state. The electronic absorption spectrum displays a broad weak absorption MLCT transition at 602 nm (ε=3.8×10³ M⁻¹ cm⁻¹), consistent with CT absorptions of other Fe(II) HS complexes. The cyclic voltammogram of the complex shows an irreversible Fe^2+/3+ oxidation at +1.55 V and two dpop^′0/−1 centered reductions at −0.20 and −0.59 V versus Ag/AgCl.     

Synthesis and characterization of Pt(II) and Pt(II)/Ru(II) complexes with the bidentate bridging ligand dipyrido(2,3-a:3′,2′-h)phenazine (dpop)

Three new complexes [Pt(dpop)(Cl)₂], [(Cl)₂Pt(dpop)Pt(Cl)₂] and [(bpy)₂Ru(dpop)Pt(Cl)₂](PF₆)₂ (dpop = dipyrido(2,3-a:3′,2′-h)phenazine) were prepared and studied. The electronic absorption spectra of the complexes display Pt dπ → dpop π* and Ru dπ → dpop π* MLCT transitions at longer wavelengths than for previously reported similar complexes. Results of cyclic voltammograms show reversible dpop centered reductions while for the mixed metal [(bpy)₂Ru(dpop)Pt(Cl)₂]²⁺ an irreversible Pt(II) oxidative wave precedes the Ru(II) oxidation/reduction couple. Spectroelectrochemical results show that all oxidative and reductive processes are completely reversible. The [(Cl)₂Pt(dpop)Pt(Cl)₂] complex cleaves in solution with pseudo-first order kinetics resulting in loss of the Pt dπ → dpop π* MLCT transition at 545 nm.     

Modification of membrane lipids. Phenethyl alcohol-induced alteration of lipid composition in Tetrahymena membranes

Direct measurement of the partition coefficient of $\text{n-}\text{hexane}$ into phosphatidylcholine and phosphatidylcholine-cholesterol bilayers showed that (a) isotropic liquids are not good models for lipid bilayers and (b), Regular Solution Theory cannot, in general, be applied to lipid bilayer membranes at temperatures above their phase transition. Theoretical and experimental evidence is given.     

Structural organization of the oxidizing side of photosystem II. Exogenous reductants reduce and destroy the Mn-complex in photosystems II membranes depleted of the 17 and 23 kDa polypeptides

Removal of 23 and 17 kDa water-soluble polypeptides from PS II membranes causes a marked decrease in oxygen-evolution activity, exposes the oxidizing side of PS II to exogenous reductants (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) Biochim. Biophys. Acta 765, 388–398) and alters a high-affinity binding site for Ca²⁺ in the oxygen-evolving complex (Ghanotakis, D.F., Topper, J.N., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 170, 169–173). We have examined further the state of the functional Mn complex in PS II membranes from which the 17 and 23 kDa species have been removed by high-salt treatment. These membranes contain a structurally altered Mn complex which is sensitive to destruction by low concentrations of NH₂OH which cannot, in native PS II membranes, cause extraction of functional Mn. In addition to NH₂OH, a wide range of other small (H₂O₂, NH₂NH₂, Fe²⁺) and bulky (benzidine, hydroquinone) electron donors extract Mn (up to 80%) from the polypeptide-depleted PS II preparations. This extraction is due to reduction of the functional Mn complex since light, which would generate higher oxidation states within the Mn complex, prevents Mn release by reductants. Release of Mn by reductants does not extract the 33 kDa water-soluble protein implicated in Mn binding to the oxidizing side of PS II, although the protein can be partially or totally extracted from Mn-depleted preparations by exposure to high ionic strength or to high (0.8 M) concentrations of Tris. We view our results as evidence for a shield around the Mn complex of the oxygen-evolving complex comprised of the 33 kDa polypeptide along with the 23 and 17 kDa proteins and tightly bound Ca²⁺.     

Polymorphism and weak antiferromagnetic interactions in dibromo[(−)-sparteine-N,N]copper(II)

Two polymorphic crystal structures of the title compound, dibromo[(−)-sparteine-N,N^′]copper(II), 1, were determined. The structures of two isomorphs of 1, 1a [orthorhombic, P2₁2₁2₁, a=11.0463(9) Å, b=11.9839(15) Å and c=12.7835(19) Å] and 1b [orthorhombic, P2₁2₁2₁, a=7.6779(9) Å, b=12.0927(14) Å and c=18.090(2) Å], are composed of the same basic structural unit, Cu(C₁₅H₂₆N₂)Br₂. The bond distances in the molecular structures of 1a and 1b are identical to each other within the esds. However, there are slight differences in the bond angles around the Cu(II) center and considerable differences in their packing structure. Crystal 1a exhibits weak anti-ferromagnetism (J=−1.89 cm⁻¹) as opposed to the magnetically isolated paramagnetism observed for the analogous dichloro[(−)-sparteine]copper(II), 2. The results of a magneto-structural investigation of 1a and 2, and other supporting evidence, suggest that the pathway for the weak antiferromagnetic super-exchange in 1a might be through a Cu-Br ? Br-Cu contact.     

A backflow of electrons around Photosystem II in Chlorella cells

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22°C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs · cm^?1 · s^?1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32°C between 1600 and 120 ergs · cm^?2 · s^?1. Under anerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs · cm^?2 · s^?1 at 22°C or about 1000 ergs · cm^?2 · s^?1 at 32°C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10^?8 M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.     

Modification of isolated α and β chains of human hemoglobin by the metal tetrasulfonated phthalocyanines. Studies on hybrid phthalocyanine-heme intermediates

α and β chains of hemoglobin have been modified with cobalt(II) tetrasulfonated phthalocyanine in place of heme. They display properties very similar to those of iron(II) phthalocyanine modified α and β chains. Mixed together they form tetrameric cobalt(II) phthalocyanine hemoglobin.Incorporation of Co(II)L into α and β globins results in stabilization of the protein structure, which is shown by a marked increase in its helicity content. Cobalt phthalocyanine substituted α and β chains are able to combine reversibly with oxygen giving more stable oxygenated species than their native analogues. The rate of both processes is lower in the case of the modified α chain. Recombination of the phthalocyanine α and β chains with the alternate heme containing chains give tetrameric hybrid hemoglobins. These comprise two phthalocyanine modified subunits and two heme containing subunits. The helicity content of the tetrameric hybrid hemoglobin calculated for one subunit is lower that the arithmetic mean of helicities for its isolated subunits. This suggests a destabilizing chain-chain interaction within the tetramer. Unlike in the separated subunits, oxygen binding by hybrid hemoglobins is irreversible. Deoxygenation by argon bubbling leads to the formation of inactive species which in oxygen atmosphere undergo irreversible oxidation with destruction of the complex.     

Coordination polymers of Cu(II) and Cd(II) with bifunctional chelates of the type dipicolylamino-alkylcarboxylate, (NC5H4CH2)2N(CH2)nCO2H (n = 1, 2, 3 and 4)

A series of bifunctional chelates of the type dipicolylamino-alkylcarboxylate (NC₅H₄CH₂)₂N(CH₂)_nCO₂H (n = 1-4; HL¹-HL⁴, respectively) has been prepared. Reactions of the ligands in aqueous methanol/N,N-dimethylformamide with the appropriate Cu(II) salts yielded the compounds [CuL¹](NO₃)·H₂O (1·H₂O), [CuL²(H₂O)]BF₄·H₂O (2·H₂O), [Cu(HL³)(SO₄)]₂ (3) and [CuL⁴(NO₃)]·MeOH (4·MeOH). While compounds 1, 2 and 4 are one-dimensional, the detailed connectivities within the chains are quite distinct, depending on factors such as alkyl chain length and ligation of aqua ligands or anionic components. In contrast to 1, 2 and 4, the structure of 3 is molecular, a binuclear assembly of edge-sharing Cu(II) ‘4+2’ distorted octahedra. The Cd(II) species, [{CdL²}₂(SO₄)]·4H₂O (5·4H₂O), prepared from HL² and CdSO₄·nH₂O in aqueous methanol/N,N-dimethylformamide, is two-dimensional, with a network constructed from binuclear units of seven coordinate Cd(II), , linked through bridging SO₄²⁻ groups to produce an assembly of linked hexagonal rings [{CdL²}₂(SO₄)]₆.     

Synthesis, double stranded DNA-binding and photocleavage studies of a functionalized ruthenium(II) complex with 7,7′-methylenedioxyphenyldipyrido[3,2-a:2′,3′-c]-phenazine

A new Ru(II) complex [Ru(phen)₂(mdpz)]²⁺ (phen = 1,10-phenanthroline, mdpz = 7,7′-methylenedioxyphenyl-dipyrido-[3,2-a:2′,3′-c]phenazine) has been synthesized and characterized in detail by elemental analysis, mass spectrometry and ¹H NMR spectroscopy. The interaction of the complex with calf thymus DNA was investigated by spectroscopic and viscosity measurements. The results suggest that the complex binds to DNA via an intercalative mode and serves as a molecular “light switch” for DNA. Moreover, the complex has been found to promote the photocleavage of plasmid DNA pBR322 under irradiation at 365 nm. The mechanism studies reveal that singlet oxygen (¹O₂) plays a significant role in the photocleavage.     

Block and inactivation of sodium channels in nerve by amino acid derivatives. II. Dependence on temperature and drug concentration.

The interaction of n-propylguanidinium (nPG) with sodium channels has been further characterized. From experiments at varying temperatures, the Q10 for the sodium current decay time constant in the two [Na+] gradients is 2.6-2.9 independent of drug. Testing several nPG concentrations we find that peak sodium current declines sharply with [nPG] at all levels, but the decay time constant approaches an asymptote above 4 mM. No "hooks" in sodium tail currents are seen. If the sodium current is allowed to decay completely before repolarization no tail current is observed. We have developed a kinetic model in which nPG acts at a single site within the sodium channel. Reaction of nPG with its receptor requires two steps. Fitting the temperature data shows that the first step involves diffusion of the drug to the site and close association with it. The second step may include molecular reorganization of the complex. The rate constants for the reaction are all simple exponential functions of voltage. Using them, the model successfully predicts decay time constants and peak currents, and their dependence on potential, [Na+] gradient, temperature, and nPG concentration. The results are consistent with the idea that an arginine residue may be closely associated with inactivation.     

Identification of cis/trans isomers of bis(acetylacetonato)nickel(II) complexes in solution based on electronic spectra

Electronic spectra of Ni(acac)₂ were studied in acetone, DMF, and some other solvents for the purpose of identifying the cis/trans isomers from the spectra (acac⁻ = acetylacetonate anion). The spectral components were investigated in the spin-allowed transition bands, and a relationship was found between the spectral pattern and the cis/trans isomers. According to this relationship, it was concluded that the cis isomer was formed in DMF and in N-methylformadide, whereas the trans isomer was formed in acetone and in pyridine. Based on the DFT computation, the cis-[Ni(acac)₂(DMF)₂] was found to be stabilized by intramolecular hydrogen bonds between acetylacetonate and DMF.     

Studies of copper(II) and nickel(II) complexation by bis(1,4,7-triazacyclononane) ligands containing alkene and alkyne bridging groups

The copper(II) and nickel(II) complexes of three new 1,2-bis(1,4,7-triazacyclononane) ligands containing unsaturated four carbon bridging groups is studied by continuous variation UV-Vis spectroscopic and pH potentiometric equilibrium experiments. The cis-butene-2 (L_C) linked ligand may form monomeric MN₆-type complexes while the trans-butene-2 (L_T) and butyne-2 (L_Y) ligands are prevented by their stereochemistry from forming monomeric complexes and form oligomeric complexes. It is determined that the stability of the CuL_C²⁺ complex is not appreciably different from the oligomeric complexes of L_T and L_Y. Single-crystal X-ray structure determinations are made on three square pyramidal Cu₂L⁴⁺ complexes: [Cu₂L_CCl₄] (1), [Cu₂L_YCl₄] (2), and [Cu₂L_T(NO₃)₂(H₂O)₂](NO₃)₂ (3). The structure of [Ni₂(L_C)₂](ClO₄)₄ · 2H₂O (4) is a binuclear dimer that contains two nickel(II) ions sandwiched between two ligands, indicating that bis([9]aneN₃) ligands with four linker atom chains may form either monomeric or oligomeric structures.