Covariance of nonstationary sodium current fluctuations at the node of Ranvier.

A theory is presented which relates the nonstationary autocovariance (covariance) function to the kinetics of independently-gated ionic channels. The experimental covariance was calculated from ensembles of 256--504 current records elicited from single, voltage-clamped, frog myelinated nerve fibers. Analysis of the covariance shows that the decay of channels from conducting to nonconducting states proceeds more slowly late in a depolarization to near 0 mV, as compared with early in the same depolarization. This behavior is inconsistent with there being only one kinetic state corresponding to the open channel. The behavior can be explained by the existence of multiple kinetic states corresponding to the open channel, or, alternatively, by the existence of multiple, kinetically distinct populations of channels.     

Kinetic model of the gating system of the sodium channel in a Ranvier node membrane

A kinetic model of the sodium channel gating system consisting of four subunits with three states--closed (X), open (Y) and inactivated (Z)--is proposed. For the channel to conduct, all the four subunits must be in the open state. The transitions between states X and Y are independent, while those between states X and Z are coupled, so that for the particle considered transition of one of two neighbouring particles into state Z increases the activation energy of the step by kT. The model fits rather well to the experimental data.     

Initiation of sodium channel clustering at the node of Ranvier in the mouse optic nerve

Ion fluxes in mammalian myelinated axons are restricted to the nodes of Ranvier, where, in particular, voltage-gated Na⁺ channels are clustered at a high density. The node of Ranvier is separated from the internode by two distinct domains of the axolemma, the paranode and the juxtaparanode. Each axonal domain is characterized by the presence of a specific protein complex. Although oligodendrocytes and/or myelin membranes are believed to play some instructive roles in the organization of axonal domains, the mechanisms leading to their localized distribution are not well understood. In this paper we focused on the involvement of myelin sheaths in this domain organization and examined the distribution of axonal components in the optic nerves of wild type, hypomyelinating jimpy mice and demyelinating PLP transgenic mice. The results showed that the clustering of Na⁺ channels does not require junction-like structures to be formed between the glial processes and axons, but requires mature oligodendrocytes to be present in close vicinity.     

Single potassium channel conductance in the frog node of Ranvier.

The single K+-channel conductance was calculated from the variance of the spontaneous potassium noise currents in voltage clamped frog node. Essential for this calculation is the mean potassium conductance during the noise measurement. So far this quantity has been underestimated, apparently due to K+-ion accumulation. With the proper values, the single K+-channel conductance is an increasing function of membrane voltage.     

Nr-CAM and neurofascin interactions regulate ankyrin G and sodium channel clustering at the node of Ranvier.

Voltage-dependent sodium (Na(+)) channels are highly concentrated at nodes of Ranvier in myelinated axons and play a key role in promoting rapid and efficient conduction of action potentials by saltatory conduction. The molecular mechanisms that direct their localization to the node are not well understood but are believed to involve contact-dependent signals from myelinating Schwann cells and interactions of Na(+) channels with the cytoskeletal protein, ankyrin G. Two cell adhesion molecules (CAMs) expressed at the axon surface, Nr-CAM and neurofascin, are also linked to ankyrin G and accumulate at early stages of node formation, suggesting that they mediate contact-dependent Schwann cell signals to initiate node development. To examine the potential role of Nr-CAM in this process, we treated myelinating cocultures of DRG (dorsal root ganglion) neurons and Schwann cells with an Nr-CAM-Fc (Nr-Fc) fusion protein. Nr-Fc had no effect on initial axon-Schwann cell interactions, including Schwann cell proliferation, or on the extent of myelination, but it strikingly and specifically inhibited Na(+) channel and ankyrin G accumulation at the node. Nr-Fc bound directly to neurons and clustered and coprecipitated neurofascin expressed on axons. These results provide the first evidence that neurofascin plays a major role in the formation of nodes, possibly via interactions with Nr-CAM.     

Saxitoxin blocks batrachotoxin-modified sodium channels in the node of Ranvier in a voltage-dependent manner.

The inhibition by saxitoxin (STX) of single Na channels incorporated into planar lipid bilayers and modified by batrachotoxin (BTX) previously has been shown to be voltage dependent (Krueger, B.K.,J.F. Worley, and R. J. French, 1983, Nature [Lond.], 303:172-175; Moczydlowski, E., S. Hall, S. S. Garber, G. S. Strichartz, and C. Miller, 1984, J. Gen. Physiol., 84:687-704). We tested for such a voltage dependence of STX block of the Na current in voltage-clamped frog nodes of Ranvier. The block by STX of normal Na channels showed no modulation in response to maintained (20 s) changes of the membrane potential or to a train of brief pulses to potentials more positive than the holding potential. However, when the nodal channels were modified by BTX, the train of pulses produced a modulation of the block of the Na current by STX. The modulation of STX block depended on the voltage of the conditioning pulses and this voltage dependence agreed well with that predicted from the single channel studies over the membrane potential range used in those studies. In addition, we found that the voltage dependence of STX block was manifest only at potentials equal to or more positive than required to activate the channels. Most of the apparent differences among data from single channels in bilayers, equilibrium binding studies of STX, and the experiments described here are resolved by the hypotheses that (a) STX binding to open channels is voltage dependent, and (b) the affinities of STX for closed and inactivated channels are independent of voltage, equal, and less than the open channel affinity at potentials less than 0 mV. Whether these hypotheses apply to the STX block of all Na channels or just of BTX-modified channels remains to be determined.     

Decrease in maximal sodium permeability and slow sodium inactivation produced by trimecaine in the Ranvier node

The action of trimecaine on sodium permeability of the membrane was studied by the voltage clamp method in single nodes of Ranvier. Like procaine, trimecaine was shown to reduce maximal sodium permeability ( _Na) and to induce slow sodium inactivation. The slow inactivation arising in the presence of trimecaine was qualitatively very similar to that observed during the action of procaine or a high external potassium concentration. Dose-effect curves were obtained for _Na and slow inactivation and they showed that one molecule of trimecaine reacts with one sodium channel in the mechanism of both these effects. The local anesthetic has greater affinity for the receptor responsible for slow inactivation (equilibrium constants about 0.3·10^–3 M for _Na and 0.2·10^–14 M for slow inactivation.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 418–425, July–August, 1976.     

Effects of strychnine on the sodium conductance of the frog node of Ranvier

Strychnine blocks sodium conductance in the frog node of Ranvier. This block was studied by reducing and slowing sodium inactivation with scorpion venom. The block is voltage and time dependent. The more positive the axoplasm the greater the block and the faster the approach to equilibrium. Some evidence is presented suggesting that only open channels can be blocked. The block is reduced by raising external sodium or lithium but not impermeant cations. A quaternary derivative of strychnine was synthesized and found to have the same action only when applied intracellularly. We conclude that strychnine blocks sodium channels by a mechanism analogous to that by which it blocks potassium channels. The potassium channel block had previously been found to be identical to that by tetraethylammonium ion derivatives. In addition, strychnine resembles procaine and its derivatives in both its structure and the mechanism of sodium channel block.     

Kinetics of interaction of scorpion toxin with sodium channels in the Ranvier node membrane

The kinetics of binding the toxin ofButhus eupeus venom with sodium channels with a holding potential of –120 mV and subsequent dissociation of the toxin-channel complex during a shift of membrane potential (V_M) to between –60 and +120 mV were investigated by the voltage clamping method on the Ranvier node membrane. The rate of dissociation was shown to increase if V_M was shifted toward more positive values, exponentially with an e-fold increase every 32.3 mV. The results are in agreement with the hypothesis that dissociation of the toxin-channel complex during depolarization is determined by the difference between electrical energies of the inactivated states of normal and toxin-modified channels.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 12, No. 6, pp. 619–626, November–December, 1980.     

Kinetics of intramembrane charge movement and sodium current in frog node of Ranvier

Intramembrane charge movement (Q) and sodium current (INa) were monitored in isolated voltage-clamped frog nodes of Ranvier, ON charge movements (QON) for pulses from the holding potential (-100 mV) to potentials V less than or equal to 0 mV followed single exponential time courses, whereas two exponentials were found for pulses to V greater than or equal to 20 mV. The voltage dependence of both QON and its time constant tauON indicated that the two ON components resolved at V greater than or equal to 20 mV were also present, though not resolvable, for pulses to V less than or equal to 0 mV. OFF charge movements (QOFF) monitored at various potentials were well described by single exponentials. When QOFF was monitored at -30 or -40 mV after a 200-microsecond pulse to +20 mV and QON was monitored at the same potential using pulses directly from -100 mV, tauON/tauOFF = 2.5 +/- 0.3. At a set OFF potential (-90 to -70 mV), tauOFF first increased with increasing duration tON of the preceding pulse to a given potential (0 to +30 mV) and then decreased with further increases in tON. The declining phase of tauOFF followed a time course similar to that of the decline in QOFF with tON. For the same pulse protocol, the OFF time constant tauNa for INA also first increased with tON but then remained constant over the tON interval during which tauOFF and QOFF were declining. After 200- or 300-microsecond pulses to +20, +20, or +50 mV, tauOFF/tauNa at -70 to -90 mV was 1.2 +/- 0.1. Similar tauOFF/tauNa ratios were predicted by channel models having three identical charged gating particles that can rapidly and reversibly form an immobile dimer or trimer after independently crossing the membrane from their OFF to their ON locations.     

Ion-exchange at the node of Ranvier

Synopisis A study has been made of the cation-binding capacity of nodal regions of rat peripheral nerve fibres. Material within the nodal gap was found to bind ferric (Fe³⁺), barium (Ba²⁺) and potassium (K⁺) ions, the affinity for cations decreasing with ionic valency. The nature of the bond to presumed fixed anionic sites at the node was deduced to be electrostatic, the strength of the bond depending on the charge of the cation and also probably on its hydrated ionic diameter. Cation-exchange was demonstrated at the node histochemically. Material with ion-exchange properties is presumed to overlay the free surface of the electrically excitable axon membrane at the nodal gap. The effects of a variety of enzymes and organic solvents on cation uptake at the node were determined and suggestions are made as to the chemical nature of the anionic binding sites. The physiological implications of these findings are discussed within the framework of present concepts concerning the conduction of the nervous impulse.     

Temperature- and structure-dependent interaction of pyrethroids with the sodium channels in frog node of Ranvier

(1) The interaction of a series of pyrethroid insecticides with the Na+ channels in myelinated nerve fibres of the clawed frog, Xenopus laevis, was investigated using the voltage clamp technique. (2) Out of 11 pyrethroids 9 insecticidally active compounds induce a slowly decaying Na+ tail current on termination of a step depolarization, whereas the Na+ current during depolarization was hardly affected. These tail currents are most readily explained by a selective reduction of the rate of closing of the activation gate in a fraction of the Na+ channels that have opened during depolarization. (3) The rate of decay of the Na+ tail current varies considerably with pyrethroid structure. After alpha-cyano pyrethroids the decay is at least one order of magnitude slower than after non-cyano pyrethroids. The decay always follows a single-exponential time course and is reversibly slowed when the temperature is lowered from 25 to 0 degrees C. Arrhenius plots in this temperature range are linear. (4) These results indicate that the relaxation of the activation gate in pyrethroid-affected Na+ channels is governed by an apparent first order, unimolecular process and that the rate of relaxation is limited by a single energy barrier. Application of transition state theory shows that after alpha-cyano pyrethroids this energy barrier is 9.6 kJ/mol higher than after non-cyano pyrethroids. (5) Differences in rate of decay of the Na+ tail current account for the reported differences in repetitive nerve activity induced by various pyrethroids. In addition, the effect of temperature on the rate of decay explains the increase in repetitive activity with cooling.     

Stimulus-dependent blockade of sodium channels of the Ranvier node membrane by the phenothiazine derivative ethmozine

The action of the antiarrhythmic drug ethmozine on sodium channels of the membrane was studied in experiments on single from Ranvier nodes by the voltage clamp method. Application of ethmozine to both the outer and the inner side of the membrane reduced the amplitude of the sodium current INa; the kinetics of this current and steady-state inactivation of the sodium channels were unchanged. Tonic and phasic (transient, stimulus-dependent) components can be distinguished in the ethmozine block of the sodium current. Tonic blockage of the sodium current develops slowly and can be potentiated by high-frequency stimulation of the membrane. The possible nature of the tonic block is discussed. The stimulus-dependent blockade of the sodium current deepens with an increase in the frequency and amplitude of depolarizing stimuli. Prolonged membrane depolarization does not evoke any additional blocking of the sodium current. It is concluded that the stimulus-dependent blockade is due to interaction between ethomizine and open sodium channels. Modification of the channels by batrachotoxin (preventing inactivation of the sodium channels) makes them insensitive to ethmozine. Increasing the potassium ion concentration on the outer side of the membrane was found to reduce the tonic effect of ethmozine and to potentiate the stimulus-dependent blockade. The action of ethmozine was compared with the effects of tertiary and quaternary local anesthetics.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 380–389, July–August, 1981.     

Effects of KC 3791 on sodium and potassium channels in frog node of Ranvier

Effects of a new antiarrhytmic compound KC 3791 on sodium (INa) and potassium (IK) currents were studied in frog myelinated nerve fibres under voltage clamp conditions. When applied externally to the node of Ranvier, KC 3791 (KC) at concentrations of 10(-5)-10(-4) mol.l-1 produced both tonic and cumulative (use-dependent) inhibition of INa. An analysis of the frequency-, voltage- and time dependence of cumulative block by KC suggested that this block resulted from a voltage-dependent interaction of the drug with open Na channels. The progressive decrease in INa during repetitive pulsing was due to accumulation of Na channels in the resting-blocked state: closing of the activation gate after the end of each depolarizing pulse stabilized the KC-"receptor" complex. To unblock these channels a prolonged washing of the node had to be combined with a subsequent repetitive stimulation of the membrane; this suggested that channel could not become cleared of the blocker unless the activation gate has opened. KC also proved to be capable of blocking open K channels at outwardly directed potassium currents (IK). This block increased during membrane depolarization. Unblocking of K channels after the end of a depolarizing pulse proceeded much faster than unblocking of Na channels under identical conditions. Cumulative inhibition of outward IK during high-frequency membrane stimulation was therefore readily reversible upon a decrease in pulsing frequency.     

Current-dependent inactivation induced by sodium depletion in normal and batrachotoxin-treated frog node of Ranvier

In batrachotoxin (BTX)-treated frog node of Ranvier, in spite of a marked reduction in Na inactivation, the Na current still presents a time- and voltage-dependent inactivation that could induce a 50-60% decrease in the current. The inactivation was found to be modified by changing the amplitude of a conditioning pulse, adding tetrodotoxin in the external solution, or replacing NaCl with KCl in the external solution. Conditioning pulses were able to alter the reversal potential of the BTX-modified Na current (Vrev). Vrev was shifted toward negative values for inward conditioning currents and was shifted toward positive values for outward conditioning currents. The change in Vrev was proportional to the conditioning current amplitude. Large inward currents induced 15-25 mV shifts of Vrev. During a 10-20-ms depolarizing pulse, the inactivation and change in Vrev were proportional to the time integral of the current. For longer depolarizations, Vrev reached a steady state level proportional to the current amplitude. The conductance, as calculated from the current and the actual Vrev, showed an inactivation proportional to exp(Vrev F/RT). These observations suggest that the BTX-modified Na current induces a decrease in local Na concentrations, which results in an alteration of the driving force and the conductance. During a pulse that induced a large inward current, the Na space concentration [( Na]s) changed from 114 to 50-60 mM. In normal fibers, the reversal potential of Na current was also shifted toward negative values by a prepulse that induced a large inward current. The change in Vrev reached 5-15 mV, which corresponded to a decrease in [Na]s of 20-50 mM. This change in Vrev slightly altered the time course of Na current. On the basis of a three- compartment model (axoplasm-perinodal space-bulk solution), a Na permeability of the barrier between the space and the bulk solution (PNa,s) and a mean thickness of the space (theta) were calculated. The mean value of PNa,s was 0.0051 cm X s-1 in both normal and BTX-treated fibers, whereas the value of theta was 0.29 micron in BTX-treated fibers and 0.05 micron in normal fibers. When compared with the values calculated during K accumulation, PNa,s was 10 times smaller than PK,s and theta Na-BTX was equal to theta K.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blockade of sodium and potassium channels in the node of Ranvier by ajmaline and N-propyl ajmaline

The inhibition of sodium and potassium currents in frog myelinated fibres by ajmaline (AM) and its quaternary derivative, N-propyl ajmaline (NPA), depends on voltage-clamp pulses and the state of channel gating mechanisms. The permanently charged NPA and protonated AM interact only (or mainly) with open channels, while unprotonated AM affects preferently inactivated Na channels. Inhibition of Na currents by NPA and AM does not depend on the current direction and Na ion concentration in external or internal media. In contrast only the outward potassium currents can be blocked by NPA and AM; the inward potassium currents in high K+ ions external media are resistant to the blocking action of these drugs. The voltage dependence of ionic current inhibition by charged drugs suggests the location of their binding sites in the inner mouths of Na and K channels. Judging by the kinetics of current restoration after cessation of pulsing, the drug-binding site complex is much more stable in Na than in potassium channels. Batrachotoxin and aconitine, unlike veratridine and sea anemone toxin, decrease greatly the affinity of Na channel binding sites to NPA and AM. The effects of NPA and AM are compared with those of local anesthetics and other amine blocking drugs.     

Molecular constituents of the node of Ranvier

The interaction between neurons and glial cells that results in myelin formation represents one of the most remarkable intercellular events in development. This is especially evident at the primary functional site within this structure, the node of Ranvier. Recent experiments have revealed a surprising level of complexity within this zone, with several components, including ion channels, sequestered with a very high degree of precision and sharply demarcated borders. We discuss the current state of knowledge of the cellular and molecular mechanisms responsible for the formation and maintenance of the node. In normal axons, Na⁺ channels are present at high density within the nodal gap, and voltage-dependent K⁺ channels are sequestered on the internodal side of the paranode—a region known as the juxtaparanode. Modifying the expression of certain surface adhesion molecules that have been recently identified, markedly alters this pattern. There is a special emphasis on contactin, a protein with multiple roles in the nervous system. In central nervous system (CNS) myelinated fibers, contactin is localized within both the nodal gap and paranodes, and appears to have unique functions in each zone. New experiments on contactin-null mutant mice help to define these mechanisms.     

GPI-anchored proteins at the node of Ranvier

Contactin and TAG-1 are glycan phosphatidyl inositol (GPI)-anchored cell adhesion molecules that play a crucial role in the organization of axonal subdomains at the node of Ranvier of myelinating fibers. Contactin and TAG-1 mediate axo-glial selective interactions in association with Caspr-family molecules at paranodes and juxtaparanodes, respectively. How membrane proteins can be confined in these neighbouring domains along the axon has been the subject of intense investigations. This review will specifically examine the properties conferred by the lipid microenvironment to regulate trafficking and selective association of these axo-glial complexes. Increasing evidences from genetic and neuropathological models point to a role of lipid rafts in the formation or stabilization of the paranodal junctions.