Kinetics of the slow variation of peak sodium current in the membrane of myelinated nerve following changes of holding potential or extracellular pH.

(1) Changes of the holding potential applied to the membrane of myelinated nerve fibres induced slow variations of the peak sodium current, which are super-imposed on the effect of sodium inactivation. (2) These slow variations are transitions between various steady levels of available sodium conductance. Their time course can be described by the function erfc (square root t/tau) where tau is the time and erfc the error function complement. The characteristic time tau lies in the range 2-4 min and depends on the membrane potential. (3) Changes of extracellular pH cause a rapid change of the peak sodium current followed by a slow variation as observed after changes of the holding potential. This slow variation can be prevented by applying simultaneously an appropriate change of the holding potential, e.g. the effect of changing pH from 7.3 to 5.3 is balanced by changing the potential from --70 to --55 mV. (4) The results are interpreted by postulating charged components diffusion slowly within the nodal membrane. Their transverse distribution controls the number of sodium channels available at a given membrane potential. The equivalence between change of pH and voltage is explained by assuming negative fixed charges at the outer surface of the membrane, which are protonated at low pH and thus affect the intrinsic membrane potential. (5) It is concluded that effects which are ascribed to the action of agents on individual sodium channels have to be corrected for variations in the number of available channels if these agents influence the intrinsic membrane potential, e.g. changes of extracellular pH.     

Minimizing the influence of the series resistance in potential clamped Ranvier nodes

Series resistance artifacts in ionic current measurements on single myelinated nerve fibres are commonly minimized by reducing sodium currents. Doing this some deviations from the predictions of the Hodgkin-Huxley-Frankenhaeuser formalism become evident. In the present investigation two methods to reduce sodium currents were used with and without compensated feedback to examine the influence of the nodal series resistance. Changing the availability of sodium permeability by appropriate prepulses peak sodium current-voltage relations obeyed the Hodgkin-Huxley-Frankenhaeuser formalism only provided the amount of compensated feedback was set to give minimum of the current-voltage relation near E = 0. For reduced sodium concentration in the bathing fluid the so-called independence principle predicts a shift of the minimum of the current-voltage relation on the potential axis in negative direction as compared to ordinary Ringer solution, if the effective series resistance is sufficiently small. This was confirmed by experiments only if the above mentioned amount of compensated feedback was used. The results suggest that the "E = 0"-criterion indicates optimum compensation of the influence of the series resistance.     

Steady state current rectification in potential clamped nodes of Ranvier (Xenopus laevis).

The membrane potential in single nodes of Ranvier was changed in rectangular pulse steps while the membrane currents, associated with the potential steps, were measured. Changes were made in the ionic composition of the external and the internal medium. The latter changes were obtained by free diffusion through a cut internode. The steady state currents, described on the basis of potassium and leak permeability, were affected by the solution composition in a characteristic way. Increased inside concentration of sodium and lithium caused a striking rectification of the outward steady state currents at large potential steps. Instantaneous potassium currents in high [K+]o at a second potential step to E approximately equal to minus 80 mV were not affected by [Na+]1. Neither [Na+]o nor [K+]i affected the potential at which this rectification appeared. Increased [K+]o shifted the region for rectification along the potential axis in positive direction. These findings form strict limitations for satisfactory models describing the mechanism for the steady state current in myelinated nerve.     

Inconsistent sodium current records derived on Ranvier nodes with a commercially available potential clamp device according to Nonner

We tried to reproduce some basic implications of the Hodgkin-Huxley-Frankenhaeuser formalism by measuring sodium currents in single myelinated nerve fibres with a commercially available version of the potential clamp device according to Nonner. The following contradictory observations were made: 1. The potential dependence of the time to peak sodium currents showed a discontinuity around the sodium equilibrium potential. 2. Defining the sodium permeability PNa by the constant field equation and fitting the peak PNa-voltage relation by a sigmoid function we obtained unbelievable high values of PNa at rest. 3. Testing PNa as calculated by the constant field equation by so-called "sodium tail current" experiments we obtained instantaneous changes of PNa. Summing up, neither the kinetics of sodium currents nor the constant field concept as tested with the equipment used seem to agree satisfactorily with the standard data of sodium currents in Ranvier nodes.     

Effect of pH changes on after potentials of single frog Ranvier nodes

In experiments on single nodes of Ranvier of isolated frog nerve fibers a decrease in pH of the medium to 5 caused a small increase in after-depolarization but an increase in pH to 9 caused virtually no change in it. The after-hyperpolarization arising in potassium-free Ringer's solution was abolished by a decrease in pH but remained substantially unchanged as a result of an increase in pH; posttetanic hyperpolarization was reduced in amplitude and slightly increased in duration by a decrease in pH, but not appreciably changed by an increase in pH. It is concluded that the character of changes in after-potentials of single nodes of Ranvier of isolated nerve fibers in medium of different pH is entirely determined by the influence of this factor on the kinetics of the potassium permeability of the excitable membrane.I. N. Ul'yanov Pedagogic Institute, Ul'yanovsk. Translated from Neirofiziologiya, Vol. 8, No. 1, pp. 62–66, January–February, 1976.     

Comparative analysis of the effects of synthetic derivatives of batrachotoxin on sodium currents in frog node of Ranvier

1. In voltage-clamp experiments on frog myelinated nerve fibers, the effects of nine synthetic derivatives of batrachotoxin (BTX) obtained from 7,8-dihydrobatrachotoxinin A (DBTX-A) on Na+ currents (INa) have been investigated. 2. Both of 20 alpha-esters of DBTX-A with 2,4,5-trimethylpyrrol-3-carboxylic acid (DBTX-P) and benzoic acid (DBTX) at a 10(-5) M concentration caused modification of INa qualitatively similar to that induced by BTX. 3. The quaternary derivative of DBTX (QDBTX) produced such changes in INa only at a 5.10(-4) M concentration, apparently due to its much lower lipid solubility. 4. Replacement of a -CH2- by a -C = O. group in the homomorpholine ring near the tertiary nitrogen atom abolished the DBTX activity, strongly suggesting the necessity of tertiary nitrogen protonation for the toxin interaction with the channel receptor. 5. Transfer of an 11-hydroxygroup from the alpha- to the beta-position in the DBTX molecule did not decrease its activity in spite of the fact that in the beta-position this group is sterically very hindered. The activity of 11 beta-DBTX is at variance with the prediction of Codding's (1983) "oxygen triad" hypothesis. 6. DBTX-A and compounds obtained from DBTX by oxidation of the 11 alpha-hydroxygroup (K-DBTX), acetylation (Ac-DBTX), or reduction of the hemiketal moiety (H2DBTX) even at a concentration as high as 10(-3) M were able to modify only a very small fraction of the Na channels. However, a clear-cut reversible blocking action on both normal and modified Na channels was observed. 7. These results led us to conclude that BTX modifies the Na channels only in the charged form and hemiketal and 20 alpha-ester moieties provide adequate disposition of toxin on the receptor surface. The inability of H2DBTX, DBTX-A, and K-DBTX and Ac-DBTX to modify most of the Na channels can be explained by a low "probability of correct disposition" of these ligands on the receptor surface.     

Effects of silperisone on the excitation process in Ranvier nodes

The effect of silperisone on single intact Ranvier nodes of the toad Xenopus was investigated by adding it to the bathing medium. At 100 micromol/l the following fully reversible effects were observed: 1. The spike amplitude decreased in a frequency-dependent manner. 2. Both the sodium activation and the inactivation curves as well as the potential dependence of taum were slightly shifted in the negative direction, while tauh did not change. 3. The sodium permeability constant PNa decreased by 50%. 4. The potassium currents acquired a phasic time course previously described for certain psoralens. They reached a relative maximum and then approached a lower steady state value, kappa(infinity) with a time constant of about 5 ms. Concentration-related responses of PNa, PK and of k(infinity), yielded: 5. The apparent dissociation constant of block of PNa was 110 micromol/l. 6. PK proved not to be changed by silperisone in the concentration range tested, while the variable kappa(infinity) yielded a relation similar to that of PNa except that the apparent dissociation constant was 24 micromol/l. The phasic course of the potassium currents in the presence of silperisone may be due to an open channel blockade. In view of the similarities between the actions of silperisone and 5-methoxypsoralen, it is entirely conceivable that silperisone has potential for an antispastic drug, e.g., in demyelinating diseases like multiple sclerosis.     

The effect of tetramethylammonium on single sodium channel currents.

Voltage-dependent Na conductance of rat myotubes was studied by patch recordings of single-channels. The patches were excised from the cell with the patch electrode, and the cytoplasmic surface was bathed in either CsF or tetramethylammonium (TMA)-F. Inward currents were examined from -20 to -50 mV. In this range Cs and TMA both appeared to be nearly impermeant, but TMA blocked the channel in a voltage-dependent manner. A first-order blocking site was located a maximum of 89% of the way through the membrane field from the cytoplasmic surface.     

Effects of external currents on duration and amplitude of normal and prolonged action potentials from single nodes of Ranvier

Duration and amplitude of normal and prolonged action potentials from single nodes of Ranvier vary as functions of potential changes induced by currents from an external source. The quantitative relations between externally applied potential and the resulting potential generated within the system are analyzed in order to obtain information about the kinetics of the electromotance,—potential,—and chemical changes taking place during excitation. The following preliminary conclusions are drawn: A depolarizing and a repolarizing process (positive and negative electromotance) increase and decrease with the potential. For a sudden potential displacement the negative electromotance reaches its new value at a faster rate than the positive electromotance. Since the individual values of the two electromotances depend on the potential and since they both generate a potential which is proportional to the difference of their absolute values, the values of either electromotance are determined by this difference as well as by any externally induced potential change.     

Immuno-ultrastructural localization of sodium channels at nodes of Ranvier and perinodal astrocytes in rat optic nerve

Immuno-electron microscopic localization of sodium channels at nodes of Ranvier within adult optic nerve was demonstrated with polyclonal antibody 7493. The 7493 antisera, which is directed against purified sodium channels from rat brain, recognizes a 260 kDa protein in immunoblots of the crude glycoprotein fraction from adult rat optic nerve. Intense immunoreactivity with 7493 antisera was observed at nodes of Ranvier. Axon membrane at the node was densely stained, whereas paranodal and internodal axon membrane did not exhibit immunoreactivity. The axoplasm beneath the nodal membrane displayed variable immunostaining. Neither terminal paranodal oligodendroglial loops nor oligodendrocyte plasmalemma were immunoreactive with 7493 antisera. However, perinodal astrocyte processes exhibited intense immunoreactivity with the anti-sodium channel antisera. Optic nerves incubated with pre-immune sera, or with 7493 antisera that had been pre-adsorbed with purified sodium channel protein, displayed no immunoreactivity. These results demonstrate localization of sodium channels at high density at mammalian nodes of Ranvier and in some perinodal astrocyte processes. The latter observation offers support for an active role for perinodal astrocyte processes in the aggregation of sodium channels within the axon membrane at the node of Ranvier.     

Gating current in the membrane of nodes of Ranvier under conditions of linear alteration of the membrane potential

Asymmetrical displacement currents (Id) in the frog Ranvier node (R. ridibunda) treated with tetrodotoxin and tetraethylammonium were studied by the use of ramp-voltage pulses. In some experiments both ramp- and step-voltage pulses were used. The net Id consists of two components, one of which Id (I) can be blocked by local anesthetic trimecaine, or inactivated with the 10 ms depolarizing prepulse sufficiently large to inactivate the sodium current in the same node (before Na+ removal and TTX application). Parameters of the steady state charge distribution are very close (though not identical) to that of the peak sodium permeability vs. potential relation. The charge carrying Id (I) is estimated as 0.3--0.5 of the net displaced charge. The results suggest that trimecaine- and inactivation-sensitive component of Id may be the true gating current of Na+ channels.     

Relation between the excitatory synaptic currents and clamped somatic potential in the model of neuron with nonlinear dendrites

The influence of the clamped somatic potential on the excitatory synaptic current (EPSC) was studied in the model of the dendrite with N-shaped instantaneous stationary current--voltage curve. Proximal EPSC diminish and become narrower with decreasing hyperpolarization or modest depolarization, distal EPSC increase and become wider, intermediately distant EPSC change insignificantly. Under increasing depolarization all the EPSC become significantly wider and larger. EPSC facilitate stable depolarization of the dendrites. When the dendrite is stable depolarized EPSC becomes very small and narrow, but it becomes larger and wider as the soma is hyperpolarized. EPSC becomes especially large and wide when the soma is hyperpolarized just to terminate the stable depolarization of the dendrite branch where the active synapses are located. The model explains certain phenomena which are difficult to understand by the theory of ohmic dendrites. New phenomena are predicted.     

Inhibition of sodium currents in frog ranvier node treated with local anesthetics Role of slow sodium inactivation

Effects of different local anesthetics of sodium permeability were studied in single nerve fibres of frog by the method of voltage clamp. Inhibition of sodium current by externally applied tertiary anesthetics, procaine and trimecaine, was the sum of a potentially independent block (reduced $\text{P}{}_{\mathrm{rmNa}}$) and slow sodium inactivation with time constants ranging from tens to hundreds of ms depending on membrane potential (at room temperature). Externally applied uncharged benzocaine produced a potentially independent block only. According to dose-response curves both processes are one-to-one reactions. In the case of trimecaine equilibrium constant the reaction responsible for reduction of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}$ is about 0.3 mM, while that for slow inactivation is more than ten times less (0.02 mM). Increasing pH from 5.6 to 8.5 markedly accelerated the slow inactivation process at all potential values. Divalent cations Ca²⁺ and Ni²⁺ shifted the steady-state slow inactivation curve along the potential axis and simultaneously reduced slow inactivation at the saturation level. Permanently charged quaternary trimecaine was ineffective when applied externally. Internally applied tertiary anesthetics and quaternary trimecaine as well as externally applied quaternary derivative of lidocaine QX-572 produced a progressively irreversible block enhanced by depolarization and inhibition reversibly increased by repetitive short-term depolarization (frequency-dependent inhibition). Inhibition of sodium currents by repetitive stimulation observed also in the case of externally applied tertiary anesthetics is due mainly to slow inactivation. The data suggests the existence of several types of receptor sites through which local anesthetics exert their blocking action on sodium permeability.     

The effect of PKC activity on the TTX-R sodium currents from rat nodose ganglion neurons

To determine how protein kinase C (PKC) activity influences properties of the tetrodotoxin-resistant sodium current (TTX-R I(Na)) in neonatal rat nodose ganglion (NG) neurons, we assessed the effects of phorbol,-12-myristate,13-acetate (PMA), one of the PKC activators, and staurosporine, one of the PKC inhibitors, on the current. PMA (30 and 100 nM) induced an increase in the peak current amplitude of normalized current-voltage curves, a leftward shift in the potential for half activation (V(1/2)) of normalized conductance-voltage curves and a leftward shift of V(1/2) potential for steady-state inactivation curves. The effects of staurosporine (0.1 and 1 muM) on the peak current amplitude and the V(1/2) potential for activation were opposite compared with those seen after PMA application. Staurosporine (1 muM) antagonized PMA (100 nM)-induced modification of TTX-R I(Na). These results suggest that the basal TTX-R I(Na) obtained from neonatal NG neurons is controlled by the level of PKC activity.     

Effects of three alkoxypsoralens on voltage gated ion channels in Ranvier nodes.

The effects of the phototoxic K+- channel blockers 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP) on Ranvier nodes were compared to those of 5,8-diethoxypsoralen (5,8-EOP) by means of the Hodgkin-Huxley formalism. When these test substances were added individually to the bathing solution (8-MOP: 100 micromol/l; 5-MOP: 50 micromol/l; 5,8-EOP: 10 micromol/l) the following completely reversible effects were observed: 1. 8-MOP, caused a nearly potential-independent decrease of the sodium permeability, P'Na, by ca. 17%. 5-MOP and 5,8-EOP merely decreased the maximal value of P'Na, by ca. 12 and 8% respectively, whereas with weak depolarisations P'Na was unchanged. 2. In the tested potential range the potassium permeability, P'K, was caused to decrease by ca. 9% by 8-MOP, ca. 21% by 5-MOP and ca. 19% by 5,8-EOP. 3. The potassium currents acquired a phasic time course previously described for 8-MOP and 5-MOP. They reached a relative maximum and approached a lower steady-state value, kinfinity, with a time constant tauk at V = 120 mV of about 16 ms (8-MOP), 20 ms (5-MOP) and 94 ms (5,8-EOP). To obtain dose-response relations the drug-induced effects on peak P'K and on the steady state value, kinfinity, were measured. The corresponding apparent dissociation constants (in micromol/l) were 66.6 and 80.1 (for 8-MOP), 87.6 and 25.8 (for 5-MOP), and 13.5 and 6.5 (for 5,8-EOP). In view of the similarity of the actions of 5-MOP and 5,8-EOP as well as the fact that 5,8-EOP is not phototoxic, in future 5,8-EOP may well prove to be a particularly suitable K+-channel blocker for the symptomatic therapy of multiple sclerosis and other demyelinating diseases.