A slow component in the gating current of the frog node of Ranvier

The slow component of the gating current on-response has been studied on voltage-clamped nodes of Ranvier of the frog Rana esculenta. At 0 mV and 20° C the charge and the time constant of the slow component were on average 37.1 fC and 0.39 ms, respectively. The slow component could be abolished by shifting the holding potential from its usual value (–100 mV) to –60 mV. Repolarization to –100 mV for 2 ms was sufficient for complete recovery of the slow component. The local anaesthetic benzocaine (1 mM) reduced the fast component (measured at 0 mV) on average to 54% and the slow component (also measured at 0 mV) to 70% of the control value. In the potential range from –60 to 60 mV the charge of the slow component increased slightly with voltage. No significant voltage dependence of its time constant was observed. The slow component most likely reflects charge movement between different open states; it does not seem to be related to inactivation of the sodium channels or activation of the potassium channels.     

Block of gating currents by ultraviolet radiation in the membrane of myelinated nerve

Summary The effect of ultraviolet radiation on the asymmetrical displacement currents in the membrane of the node of Ranvier was measured and compared with the ultraviolet blocking of the sodium current. Ultraviolet radiation irreversibly reduced the peak sodium current and the charge displaced during a depolarizing test pulse, the relative reduction being independent of potential. The ratio of the ultraviolet sensitivities of the sodium and the asymmetrical displacement currents is 2.3±0.2. This result suggests two independent identical gating particles per sodium channel in the membrane of myelinated nerve.Supported by Deutsche Forschungsgemeinschaft SFB 38 Membranforschung-Bonn-Bad Godesberg     

Reduced expression of Na(v)1.6 sodium channels and compensation by Na(v)1.2 channels in mice heterozygous for a null mutation in Scn8a

The voltage-gated sodium channel alpha subunit Na(v)1.6, encoded by the Scn8a gene, accumulates at high density at mature nodes of Ranvier of myelinated axons, replacing the Na(v)1.2 channels found at nodes earlier in development. To investigate this preferential expression of Na(v)1.6 at adult nodes, we examined isoform-specific expression of sodium channels in mice heterozygous for a null mutation in Scn8a. Immunoblots from these +/- mice had 50% of the wild-type level of Na(v)1.6 protein, and their optic-nerve nodes of Ranvier had correspondingly less anti-Na(v)1.6 immunofluorescence. Protein level and nodal immunofluorescence of the Na(v)1.2 alpha subunit increased in Scn8a(+/-) mice, keeping total sodium channel expression approximately constant despite partial loss of Na(v)1.6 channels. The results are consistent with a model in which Na(v)1.6 and Na(v)1.2 compete for binding partners at sites of high channel density, such as nodes of Ranvier. We suggest that Na(v)1.6 channels normally occupy most of the molecular machinery responsible for channel clustering because they have higher binding affinity, and not because they are exclusively recognized by mechanisms for transport and insertion of sodium channels in myelinated axons. The reduced amount of Na(v)1.6 protein in Scn8a(+/-) mice is apparently insufficient to saturate the nodal binding sites, allowing Na(v)1.2 channels to compete more successfully.     

Late sodium current in the node of Ranvier

Summary Voltage clamp experiments carried out on nodes of Ranvier of myelinated fibres ofRana esculenta showed that a small fraction of sodium channels fail to inactivate. Thus during long lasting depolarizing pulses there is a small Na-current superimposed on the leakage and potassium currents. This late Na-current appears more marked in sensory fibres than in motor ones.Supported by grants of the C.N.R.S. and D.G.R.S.T.     

Three functions of sodium channels in the toad node of Ranvier are altered by trimethyloxonium ions

Voltage-clamped nodes of Ranvier of the toadXenopus laevis were treated with trimethyloxonium ions (TMO) which are known to methylate carboxyl groups. TMO did not affect potassium channels but the sodium system was modified in three ways: a) the current was reduced, b) channels were rendered insensitive to tetrodotoxin (TTX) and c) the inactivation of all channels, TTX-resistant or not, was slowed in a potential range between 50 and 110 mV. Steady-state inactivation, however, was not changed. Presence of 100 nM TTX during TMO treatment prevented the production of TTX-resistant channels but did not hinder current reduction and slowing of inactivation. The TTX-resistant sodium channels were blocked by protons and had normal relative permeabilities to alkali metal ions. Repeated application of TMO further decreased the current and increased the relative amount of TTX-resistant channels; the slowing of inactivation, however, was quantitatively terminated after the first TMO treatment. It is concluded that the sodium channel contains at least three TMO-modifiable groups, which probably are carboxyl groups.     

Inactivation of the potassium transport system of myelinated nerve in the presence of a cyclic ionophore

A potassium carrying ionophore dicyclohexano-18-crown-6 was found to affect the specific ionic currents in the node of Ranvier. Its presence caused an inactivation of the potassium permeability mechanism and a decrease of the sodium permeability. The rate of inactivation of the potassium permeability depended on crown ether concentration, on membrane potential and on temperature. At large concentrations, the rate of inactivation was of the same order of size as the normal rate of activation. The recovery from the inactivated state was slow, it took a few seconds, and recovery time depended on holding potential. The inactivation was caused by potential steps in positive direction. The un-complexed crown ether is uncharged and does not move due to a potential gradient. The mechanism by which the crown ether makes the inactivation potential dependent remains obscure. A few other ionophores were tested but they did not affect the membrane currents.     

Interaction of monovalent cations with tetrodotoxin and saxitoxin binding at sodium channels of frog myelinated nerve

Na currents and Na-current fluctuations were measured in myelinated frog nerve fibres to study interactions between monovalent externally applied cations and the binding of the Na-channel blockers tetrodotoxin (TTX) or saxitoxin (STX). Adding 110 mM NaCl to Ringer's solution increased the maximum peak Na conductance by a factor of 2.51 in the presence of 12 nM TTX and by a factor of 2.43 in the presence of 4 nM STX. According to the analysis of Na-current fluctuations this increase of the Na conductance is mainly caused by an increase of the number N of unblocked Na channels per node, while the conductance of a single channel saturates in the hyperosmolar solutions. The increase of N is interpreted by displacement of TTX or STX from Na channels by external Na⁺. Relief of TTX blockage was also observed by adding 100 mM chloride salts of Li⁺, hydrazine⁺, guanidine⁺ and K⁺ to Ringer, but not in Ringer + 110 mM tetramethylammonium chloride or 250 mM sucrose. The increase of N by the external cations is a saturating function of the permeability of the Na channel to these ions. The results are interpreted by a toxin receptor in a superficial prefilter to the Na channel, which contributes to cation discrimination at the outer channel region.     

Kinetic parameters of the ionic currents in myelinated axons: Characterization of temperature effects in a hibernator and a nonhibernator

Na⁺ and K⁺ currents were measured by the patch-clamp method in the paranodal region of single sciatic nerve fibres of rats and of warm-adapted and cold-adapted golden hamsters. Kinetic parameters and temperature dependence of the Na⁺ currents were determined. The time constant for activation (about 0.2 ms for rats and hamsters) as well as the time constant for inactivation (about 1.6 ms for rats and hamsters) at 15 °C and at −35 mV compared well with single fibre voltage-clamp data from the rat. Differences amongst the three groups of animals were not significant. The temperature coefficient, Q ₁₀, for the activation and the inactivation time constant as well as for the time-to-peak of the Na⁺ current ranged between 2.3 and 3.1. No data have previously been published on the temperature dependence of the delayed-rectifier K channels of mammalian nerve fibres. Most of the K⁺ current was carried by intermediate (K_I) and fast (K_F) K channels. Dendrotoxin block indicated that ≈55% of the K⁺ current was due to K_I channels, with no significant difference amongst the three groups of animals tested. The Arrhenius plot of the time constant of K⁺ current activation, τ_n, yielded a mean Q ₁₀ of 3.3 at −40 mV (4.0 at + 60 mV). No significant differences of the channel kinetics between rats, warm-adapted hamsters and cold-adapted hamsters were detected. We observed, however, a significant decrease of the Na channel density in the paranodal region of cold-adapted hamsters. Received: 10 June 1995/Received after revision: 3 November 1995/Accepted: 17 November 1995     

Temperature experiments on nerve and muscle membranes of frogs

The influence of temperature changes in the range of 25°C to –6°C on the time constants of Na activation (m) and inactivation (h) was studied in twitch muscle fibers and the node of Ranvier under voltage-clamp conditions. Arrhenius plots of m and h exhibit a change in activation enthalpy at temperatures below 10°C. Cooling and subsequent heating induce a hystersis in the temperature dependence of m and h Ni²⁺ and UO ₂ ²⁺ increase the hysteresis width. With fast temperature changes the gating kinetics relax to their new values more slowly than the temperature change. Hence, temperature must be changed more slowly than 5°C/min if an additional apparent hysteresis due simply to this relaxation is to be avoided. The data are explained by the hypothesis of a phase transition in the membrane lipids. This conception is favoured over a temperature-induced change in protein conformation, since the neutral local anaesthetic benzocaine shows use-dependent block as if low temperature restricted the access of the drug through the lipid phase to its receptor.Supported by grant NS 08174 from the U.S. Public Health Service and SFB 38 from Deutsche Forschungsgemeinschaft     

Effects of chemical modification of amino groups by two different imidoesters on voltage-clamped nerve fibres of the frog

Voltage clamped single nerve fibres of the frogPanta esculenta were treated with the amino groups specific reagents ethyl acetimidate and isethionyl acetimidate. Ethyl acetimidate is lipid soluble, relatively non-polar and can penetrate a membrane. Isethionyl acetimidate is lipid-insoluble, polar and membrane-impermeant. Treatment with ethyl acetimidate caused an irreversible reduction of Na currents and a shift of the voltage dependence of the steady-state sodium inactivation,h (E), in the hyperpolarizing direction. The voltage dependence of sodium activation was much less affected and a small shift into the depolarizing direction was observed. By contrast, the non-permeant reagent did not reduce the sodium currents and the shifts of theh (E) curve were smaller than the shifts caused by ethyl acetimidate. Furthermore, a small shift of the voltagedependence of activation in the hyperpolarizing direction was observed. As the modification procedure with imidoesters does not cause a change of net charge, the results cannot be explained by an effect of surface charge modification, rather, they seem to be due to a direct effect of amino group modification on the voltage dependence of inactivation.Abbreviations TNBS Trinitrobenzenesulfonic acid - IAI isethionyl acetimidate - EAI ethyl acetimidate - MOPS morpholinopropanesulfonic acid - TEA tetraethylammonium - CHES (2-cyclohexylamino)-ethanesulfonic acid     

Na currents and action potentials in rat myelinated nerve fibres at 20 and 37° C

(1) Action potentials and membrane currents were recorded in single myelinated rat nerve fibres at 20 and 37° C. Three experiments were also performed in single cat nerve fibres. (2) K currents were blocked by internal CsCl and external TEA. The steady state and kinetic parameters of Na activation and inactivation were determined at both temperatures. (3) When the temperature was raised from 20 to 37° C, steady state Na activation,m (V), and inactivation,h (V), did not change significantly. (4) The time constant of Na activation, _m, was determined within the potential range of –40 to 125 mV at 20° C andV=40–60 mV at 37° C. The temperature coefficient, Q₁₀, of _m was 2.2. (5) The decay in the Na current was described by two exponentials at both temperatures. The amplitude of the slow phase was 1–10%. The time constant of the fast phase of Na inactivation, _h1, was determined at both temperatures within the potential range of –50 mV to 125 mV. The Q₁₀ of _h1 was 2.9 and did not depend on potential. (6) The Na equilibrium potential was 152 mV at 20° C and 144 mV at 37° C. The leakage conductance was 24 nS at 20° C and 43 nS at 37° C. These differences were interpreted as signs of fibre deterioration at higher temperature. (7) The results from the current and voltage clamp experiments performed in the cat nerve were essentially the same as those in the rat nerve fibres. (8) The action potentials computed on the basis of the voltage clamp results at 20° C were similar to the ones actually measured. This was also true for those action potentials predicted for 37° C on the basis of the 20° C data, theg _L andV _Na values measured at 37° C, and the Q₁₀ values of the time constants. (9) Steady state values and kinetic parameters of K permeability were adopted from the literature. As in the experiments the influence ofP _K on the shape of the predicted action potential was almost negligible at both temperatures.     

Effects of chemical modification of amino and sulfhydryl groups on the voltage-clamped frog node of Ranvier

Several reagents that react with sulfhydryl and amino groups were applied to voltage-clamped single nerve fibres of the frog. The fibres were exposed to comparable amounts of the chemical reagents for relatively short times. 3-(p-Hydroxyphenyl)propionic acid N-hydroxysuccinimide ester (HPPS), a substance which preferentially modifies amino groups, irreversibly reduced the size of the sodium and potassium current. The effect of HPPS on the Na current could be removed only partially by hyperpolarizing prepulses. N-ethylmaleimide (NEM), a reagent that preferentially reacts with sulfhydryl groups produced a small decrease of the sodium current which was removed almost completely by hyperpolarizing prepulses. NEM and HPPS shifted the voltage dependence of sodium inactivation,h (E), to more negative values of membrane potential, but had little effect on the time course of sodium activation and inactivation. Pretreatment of a fibre with NEM did not prevent the action of HPPS; however, pretreatment of a fibre with HPPS decreased considerably the shift of theh (E) curve caused by NEM. Our results suggest that modification of membrane bound amino groups affects the size of the ionic currents and the inactivation process. Although reagents that react with sulfhydryl groups were found to affect channel function, no definite evidence for the presence or absence of a functionally important sulfhydryl group on sodium channels has been obtained.Abbreviations NEM N-ethylmaleimide - MOPS morpholinopropanesulfonic acid - DTDP dithiodipyridine - SPDP N-succinimidyl-3-(2-pyridyldithio)propionate - HPPS 3-(p-hydroxyphenyl)propionic acid N-hydroxy-succinimide ester     

Inhibition of voltage-dependent Na+ and K+ currents by forskolin in nodes of Ranvier

The effect of forskolin on voltage-activated Na⁺ and K⁺ currents in nodes of Ranvier from the toad, Bufo marinus, has been examined using the vaseline-gap voltageclamp technique. Peak Na⁺ currents (I _Na) were reduced by 35% and the rate of decline of Na⁺ current during continuous depolarization was accelerated following treatment with 450 M forskolin. However, the voltage-dependence of steady-state inactivation as well as the rate of recovery from fast inactivation remained unchanged. Upon repetitive depolarization at 1–10 Hz, a further inhibition of I _Na (60%) was observed. This use-dependent or phasic inhibition recovers slowly at -80 mV ( 13 s) and had a voltage-dependence like that of activation of the Na conductance. Near maximal steady-state phasic inhibition occurred with depolarizing pulse durations of only 4 ms, consistent with a direct involvement of the open Na⁺ channel in the blocking process. Inhibition of the delayed K⁺ current (I _K) was characterized by a concentration-dependent reduction in steady-state current amplitude (IC₅₀ 80 M) and a concentration-independent acceleration of current inactivation. A similar inhibition of I _K was obtained with 1,9-dideoxyforskolin, a homolog which does not activate adenylate cyclase (AC). The results suggest that the inhibition of I _K and perhaps I _Na follows directly from drug binding and is not a consequence of AC activation.     

Changes in venous blood content from active and inactive hindlimb during isotonic exercise

Summary Venous blood samples were obtained from either exercising (n=9) or nonexercising (n=8) hindlimb during a progressive isotonic exercise in rabbits anesthetized with urethane and chloralose. Each experimental session consisted of 5-min non-exercise periods alternated with 6-min exercise periods, followed by a 10-min postexercise period. During each exercise period, stimulation of the distal stump of the right sciatic nerve at 1 Hz induced plantar flexions which lifted loads comparable to 2, 5, 8, 30, or 50% of an afterload at which only an isometric tension developed. Free-flowing venous blood samples were obtained before the first exercise period, during the last minute of each exercise period, and 10 min following the last exercise session. Increases in [Na⁺], [K⁺] and lactate concentration were obtained in blood from active limbs. Only lactate concentration increased in blood from non-exercising limbs, while [K⁺] decreased slightly. Inferences concerning the vascular volume response to this protocol would be quite different depending on the blood sampling site. Changes in blood from inactive tissue, further, may indicate only saturation of homeostatic mechanisms which normally compensate for vascular volume alterations initiated in active tissue.     

The effect of exercise on the venous blood ammonium concentration in man

Mild muscular exercise did not cause any significant rise in the ammonium concentration of venous blood draining the exercising forearm of control subjects or patients with cirrhosis. However, in both cirrhotic and non-cirrhotic subjects moderate exercise produced significant increases in venous blood ammonium values and these occurred earlier and were more prolonged in cirrhotic patients. Severe exercise caused larger increases in venous blood ammonium concentration in all subjects but there were no significant differences between the mean ammonium concentrations of the cirrhotic and control groups either before or after exercise. All ammonium values returned to their pre-exercise levels within half an hour of resting.The exact mechanism of these phenomena is not fully understood but they are of practical importance in the study of blood ammonium metabolism in normal subjects and in patients with cirrhosis of the liver. The importance of making subjects rest for at least 30 minutes before obtaining blood for ammonium determination is emphasized in order to obviate misleadingly high readings due to muscular activity.     

Gating kinetics of ATP-sensitive single potassium channels in myocardial cells depends on electromotive force

ATP-sensitive single-channel potassium currents were studied in the membrane of rat ventricular myocytes. With an internal K⁺ concentration of [K⁺]_i=140 mM, the outwardly directed currents saturated at 1.8 pA in the region of positive potentials independently of the external K⁺ concentration [K⁺]_o, whereas an increase in [K⁺]_i of up to 300 mM caused a positive shift in the region of current saturation (from +40 mV to +100 mV) and an increase in the level of the saturation up to 4 pA. The openings of the channels appeared in bursts. Gating kinetics within the bursts were investigated. It was shown that the channel mean open (_o) and closed (_c) times during a burst depended primarily on the electromotive force (V-V _k) for potassium ions. For different [K⁺]_o, _o was maximal and _c was minimal in the region of reversal potentials (V _rev); _o decreased and _c increased gradually with deviation ofV fromV _rev. Therefore we conclude that the gating properties of the ATP-sensitive K channels depend on the ion flux parameters.