Ultraviolet-induced alterations of the sodium inactivation in myelinated nerve fibres.

Ultraviolet radiation irreversibly reduces the sodium permeability in nerve membranes and, in addition, induces a change of the potential dependence of the kinetic parameters of sodium inactivation in the node of Ranvier. This second ultraviolet effect shifts the kinetic parameters of sodium inactivation h infinity (V), alpha h (V), and beta h (V) to more negative potentials (no changes of the slopes of the curves). The amount of the displacement delta V along the potential axis is equal for the three parameters and depends on the ultraviolet dose. It is about delta V = --10 mV after an irradiation dose of 0.7 Ws/cm2 at 280 nm. Both ultraviolet-induced effects depend on membrane potential and on the wavelength of the applied radiation. But while the potential shift is enhanced at more negative holding potentials, the ultraviolet blocking is diminished and vice versa. Further, the ultraviolet-induced potential shift is greater at 260 nm than at 280 nm, whereas a maximum sensitivity of ultraviolet blocking is found at 280 nm. Therefore, the two radiation effects are the result of two separate photoreactions. For explanation of the radiation-induced potential shift it is assumed that ultraviolet radiation decreases the density of negative charges at the inner surface of the nodal membrane. From this hypothesis a value for the inner surface potential psii was derived. --19 mV less than or equal to psii less than or equal to --14 mV.     

Effects of some chemical reagents on sodium current inactivation in myelinated nerve fibers of the frog.

The effect of several chemical reagents on the sodium current was studied in voltage-clamped single nerve fibers of the frog. The oxidants halazone and hypochlorous acid drastically inhibited inactivation. Their effect was similar to that of chloramine T (Wang, 1984a). The curve relating the steady-state inactivation parameter h infinity to the conditioning potential E became nonmonotonic after treatment with the oxidants, i.e., dh infinity/dE greater than 0 for E greater than -20 mV. By contrast, the oxidants periodate, iodate, and hydrogen peroxide (applied for the same time, but at higher concentrations) merely produced a parallel shift of the h infinity(E) curve to more negative values of membrane potential. Diethylpyrocarbonate, a reagent that preferentially modifies histidine groups, had one marked effect: a strong shift of the h infinity(E) curve to more negative values of membrane potential. Almost no effect was observed after application of the tyrosine-reactive reagent N-acetylimidazole. Similarly, the arginine-reactive reagent glyoxal had only minor effects on the Na permeability. The results suggest that methionine is not critically involved in the kinetics of Na current inactivation. Similarly, an essential tyrosine or arginine residue seems to be unavailable to chemical reagents from outside on the frog node of Ranvier. Deduced from the reactivities of (some of) the reagents used, modification of membrane lipids is a tentative explanation for the effects observed on inactivation kinetics.     

The permeability of aconitine-modified sodium channels to univalent cations in myelinated nerve

1. Ionic currents through the sodium system of nodes of Ranvier treated with aconitine were measured under voltage clamp conditions in a Ringer solution containing Na+ or an equimolar amount of various test cations. 2. Average shifts in reversal potentials in nodes of Ranvier treated with aconitine with NH4+, Li+, K+, Rb+, Cs+ in place of Na+ in the Ringer solution are 7.6, --6.8, --25.0, --41.0 and --51.5 mV at 13--14degrees C. At 20--22degrees C the sequence of shifts is 7.5, --5.5, --13.5, --29.0 and --41.0 mV. For Tl+ the the average reversal potential shift is +3 mV at 20--22degrees C. 3. The slope of the instantaneous current-voltage relation at the reversal potential in nodes treated with aconitine changed with the various cations tested. The ratios are NH4+/Na+/K+/Rb+/Cs+/Li+ = 1.14 : 1.0 : 0.80 :0.67 :0.53 : 0.53. 4. Using a three energy barrier model some of the parameters for the aconitine-modified Na+ channels were estimated (Chizmadgev, Yu. A., Khodorov, B.I. and Aityan, S.Kh. (1974) Bioelectrochem. Bioenerg. 1, 301--312).     

Effects of laser-induced hyperthermia treatment on ionic permeability of myelinated nerve

Summary The effect of laser-induced hyperthermia on the ionic permeability of nerve membranes was studied using the nodes of Ranvier in amphibian myelinated nerve as a model. To effect a photothermal modification of nerve membrane functions, con trolled laser irradiation consisting of a 5-sec thermal pulse was applied to the nodal membrane, increasing the temperature to a maximum of 48–58°C at the node. Major electrophysiological changes observed in the nodal membrane following laser-induced hyperthermia were a differential reduction of the sodium and potassium permeability, an increase in the leakage current, and a negative shift on the potential axis of the steady-state Na inactivation. There was no significant change in the kinetics of ion channel activation and inactivation for treatments below 56°C. The results suggest that a primary photothermal damage mecha nism at temperatures below 56°C could be a reduction in the number of active Na channels in the node, rather than a change in individual channel kinetics, or in the properties of the lipid bilayer of intervening nerve membrane. A differential heat sensi tivity between the noninactivated and the inactivated Na channels is also suggested. For the treatments of 56°C and above, a signifi cant increase of membrane leakage current suggests an irrevers ible thermal damage to the lipid bilayer. This work was supported by the ONR/SDIO N00014-86-K-0188 Medical Free-Electron-Laser Program and the Columbus-Cabrini Foundation.     

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.     

Effects of glutaraldehyde on sodium channel activation and inactivation in frog nerve fiber

The effects of glutaraldehyde on sodium channel gating were investigated in the membrane of the node of Ranvier in frog nerve fiber. It was found that treating the membrane with glutaraldehyde slows the rate of inactivation, renders the inactivation curve considerably less steep, and leads to the appearance of a steady-state current component. It also decelerated the activation rate and reduced the slope of the central portion of the activation curve, which was shifted over to depolarization at the membrane. This produced no significant change in the effective charge in the effective charge of activation as determined from the limiting logarithmic slope of the activation curve. The mechanisms possibly underlying these changes in sodium channel gating are discussed.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 579–586, September–October, 1986.     

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.     

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.     

Quinidine blocking of sodium and potassium channels in myelinated nerve fibers

Experiments by the voltage clamp method showed that external application of quinidine (5 × 10^–5 M) to the Ranvier node membrane of the frog nerve fiber inhibitis both sodium and potassium currents. Blocking of the sodium current is considerably intensified by repetitive depolarization of the membrane (1–10 Hz); the rate of development of the block increases with an increase in stimulation frequency. After the end of stimulation the sodium current gradually returns to its initial level (with a time constant of the order of 30 sec at 12°C). Unlike repetitive depolarization with short (5 msec) stimuli, a prolonged shift (1 sec) of potential toward depolarization has no significant effect on quinidine blocking of the sodium current. Analysis of the current-voltage characteristic curves showed that quinidine blocks outward sodium current more strongly than inward. Batrachotoxin protects sodium channels against the blocking action of quinidine in a concentration of 10^–5 M. Inhibition of the outward potassium currents by quinidine is distinctly time-dependent in character: Initially the potassium current rises to a maximum, then falls steadily to a new stationary level. The results agree with the view that quinidine, applied externally, penetrates through the membrane in the basic form and blocks open sodium and potassium channels from within in the charged (protonated) form. The similarity in principle between the action of quinidine and local anesthetics on the sodium suggests that these compounds bind with the same receptor, located in the inner mouth of the sodium channel.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 324–330, May–June, 1982.     

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

The mean sodium current, I, and the variance of sodium current fluctuations, var, were measured in myelinated nerve during a depolarization to V = 40 mV applied from the resting potential (VH = 0) or from a hyperpolarizing holding potential VH = -28 mV. From I and var the relative variations in the number N and the conductance gamma of sodium channels following changes of the holding potential were calculated. Hyperpolarizing the membrane from VH = 0 to -28 mV increased N by a factor of 3.7, whereas gamma 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 N and gamma.     

Effects of diphenylhydantoin and phenobarbital on voltage-clamped myelinated nerve.

Diphenylhydantoin (DPH) and phenobarbital (PB) have a selective action in blocking spontaneous activity in nerves made hyperexcitable by lowering the calcium concentration of the bathing medium (Rosenberg, P. and Bartels, E. 1967 J. Pharmacol. Exp. Ther. 155, 532-544.). To investigate this further, we examined the action of DPH and PB on voltage-clamped single myelinated nerves at two different calcium concentrations. In 1.8 mM calcium Ringer, DPH reduced the sodium permeability (PNa) without affecting the potassium conductance (GK) or the voltage-dependent time constants of sodium activation (taum) and inactivation (tauh), and potassium activation (taun). PB was similar to DPH except that in addition to reducing PNa, it shifted taum in the direction of depolarization. When the calcium concentration was lowered to 0.36 mM, the curves relating taum and taun to membrane potential were shifted in the direction of hyperpolarization, as expected. However, the addition of DPH or PB reduced or abolished these shifts. It is suggested that both DPH and PB stabilize hyperexcitable membranes by an action on the parameter m, and that this may contribute to their antiepileptic action.     

Rapid and slow gating of veratridine-modified sodium channels in frog myelinated nerve

The properties of voltage-dependent Na channels modified by veratridine (VTD) were studied in voltage-clamped nodes of Ranvier of the frog Rana pipiens. Two modes of gating of VTD-modified channels are described. The first, occurring on a time scale of milliseconds, is shown to be the transition of channels between a modified resting state and a modified open state. There are important qualitative and quantitative differences of this gating process in nerve compared with that in muscle (Leibowitz et al., 1986). A second gating process occurring on a time scale of seconds, was originally described as a modified activation process (Ulbricht, 1969). This process is further analyzed here, and a model is presented in which the slow process represents the gating of VTD-modified channels between open and inactivated states. An expanded model is a step in the direction of unifying the known rapid and slow physiologic processes of Na channels modified by VTD and related alkaloid neurotoxins.     

Comparison of the effects of Anemonia toxin II on sodium and gating currents in frog myelinated nerve

Na+ and gating currents were measured in myelinated frog nerve fibres without and in the presence of 7 microM Anemonia toxin II in the extracellular solution. From the experiments, kinetic parameters of Na+ currents and of gating charge displacements during ('on' response) and after ('off' response) depolarizations were determined. The following parallel modifications of Na+ currents and charge displacements by Anemonia toxin II were observed: the toxin reduces the maximum Na+ permeability and the 'on' charge displacement; Na+ activation and 'on' charge displacement become faster; Na+ inactivation and the decline of the 'off' charge displacement with increasing pulse duration (charge immobilization) are prolonged; slow components of 'on' charge displacements are diminished. The observations support the notion that the fast 'on' charge displacement is connected with the process of Na+ activation, while Na+ inactivation is linked to charge immobilization. Our experiments suggest that slow 'on' charge displacements during longer depolarizations are correlated with the process of Na+ inactivation.     

Effects of some potassium channel blockers on the ionic currents in myelinated nerve

The effects of some potassium channel blockers on the ionic currents and on the so-called K(+)-depolarization in intact myelinated nerve fibres were studied. 4-AP, and in particular, Flaxedil, proved to be selective K(+)-current blockers. However, TEA, a crown ether (DCH18C6), a longchained triethylammonium compound (C10-TriEA), capsaicin, and the extract from the medicinal herb Ruta graveolens proved not to be selective K(+)-current blockers; they all block Na(+)-currents as well, although to a lesser extent. The sodium inactivation curve did not change under TEA and Flaxedil but was shifted on the potential axis in negative direction by DCH18C6, 4-AP, capsaicin and the Ruta extract whereas C10-TriEA caused a shift of both sodium inactivation and activation parameters in positive direction. Regarding to the kinetics of the persisting K(+)-current fraction, two different kinds of blockade were found: 1. Unchanged K(+)-kinetic which is typical for the effects of TEA, 4-AP, Flaxedil, and C10-TriEA. 2. Clearly changed K(+)-kinetic, characterized by K(+)-transients; which is typical for the effects of capsaicin and in particular, for those of DCH18C6 and of the Ruta extract. The possibly different modes of action of both groups of blockers are discussed in terms of current models for the action of potassium channel blockers.