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.     

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.     

Effects of chemical modification of carboxyl groups on the voltage-clamped nerve fiber of the frog

Summary Voltage-clamped single nerve fibers of the frogRana esculenta were treated with the carboxyl group activating reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of different primary amines and without added amine. Carboxyl groups form stable amide bonds with primary amines in the presence of EEDQ. EEDQ treatment reduced the sodium current considerably and irreversibly, regardless of the presence of a primary amine in the Ringer's solution. The potassium current was also reduced. After modification the reduced sodium currents inactivated slowly and incompletely. The descending branch of the sodium current-voltage relation,I _Na(E), was shifted along the voltage axis in the depolarizing direction. The size of the shift was strongly dependent on the amine present during modification with EEDQ. The voltage-dependence of sodium inactivation,h _x (E), was shifted to more positive values of membrane potential by EEDQ in the presence of ethylenediamine (11 mV) and glucosamine (3 mV). In contrast, a small shift to more negative potentials occurred in the presence of taurine (–3 mV) or without the addition of an amine (–2 mV). A tenfold increase of the calcium concentration still shifted theI _Na(E) andh _x (E) curves of the chemically modified fibers. However, these shifts were smaller than those observed on untreated fibers. The currents remaining after the modification were completely blocked by tetrodotoxin; no change of the reversal potential occurred.     

Conditioning prepulses and kinetics of potassium conductance in the frog node

Summary The kinetics of potassium conductance were analyzed in response to voltage-clamp steps with holding potential (–75 mV) as initial condition and after a positive prepulse to-wards +45 mV of 10-msec duration. As the potassium reversal potentialE _K altered during potassium current flow, a method to obtain the conductance independent ofE _K was used. Conductance kinetics at 15°C were analyzed according to the Hodgkin-Huxley (HH) model. The time constant of potassium activation, with holding potential as initial condition, is a monotonous decreasing function of membrane potential. Its value ofca. 9 msec at –50 mV decreases to 1 msec at +30 mV. Changes inE _K did not affect the voltage dependency of this time constant. The time constant of potassium deactivation, i.e. the off-response following a 10-msec prepulse towards +45 mV, shows a completely different voltage dependency. At a membrane potential of –90 mV it is approximately 2 msec and gradually increases for more positive voltages towards a maximum value of about 6 msec, that is reached between –5 and 0 mV. At still larger values of membrane voltage this time constant starts to fall again. It is concluded that a HH-model, as applied for a single population of potassium channels, has to be rejected. Computer simulations indicate that an extension to two populations of independent potassium channels, each with HH-kinetics, is also inconsistent with the observed results.     

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.     

Brevican distinctively assembles extracellular components at the large diameter nodes of Ranvier in the CNS

Brevican is known to be an abundant extracellular matrix component in the adult brain and a structural constituent of perineuronal nets. We herein show that brevican, tenascin-R (TN-R) and phosphacan are present at the nodes of Ranvier on myelinated axons with a particularly large diameter in the central nervous system. A brevican deficiency resulted in a reorganization of the nodal matrices, which was characterized by the shift of TN-R, and concomitantly phosphacan, from an axonal diameter-dependent association with nodes to an axonal diameter independent association. Supported by the co-immunoprecipitation results, these observations indicate that the presence of TN-R and phosphacan at nodes is normally brevican-dependent, while in the absence of brevican these molecules can also be recruited by versican V2. The versican V2 and Bral1 distribution was not affected, thus indicating a brevican-independent role of these two molecules for establishing hyaluronan-binding matrices at the nodes. Our results revealed that brevican plays a crucial role in determining the specialization of the hyaluronan-binding nodal matrix assemblies in large diameter nodes.     

Potassium currents in the giant axon of the crabCarcinus maenas

Summary Measurements were made of the kinetic and steadystate characteristics of the potassium conductance in the giant axon of the crabCarcinus maenas. These measurements were made in the presence of tetrodotoxin, using the feedback amplifier concept introduced by Dodge and Frankenhaeuser (J. Physiol. (London) 143:76–90). The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate potassium channels exist in these axons. The first potassium channel exhibited activation and fast inactivation gating which could be fitted using them ³ h, Hodgkin-Huxley formalism. The second potassium channel exhibited the standardn ⁴ Hodgkin-Huxley kinetics. These two postulated channels are blocked by internal application of caesium, tetraethylammonium and sodium ions. External application of 4 amino-pyridine also blocks these channels.     

Acute effects of thyroid hormone on sodium currents in neonatal myocytes

Sodium currents and action potentials were recorded from myocytes of neonatal rats during acute exposure to thyroid hormone (5–20 nM). One to 5 minutes after addition of thyroid hormone to the bath, decay from peak Na current was slowed, with the fractional current flowing 20 ms after onset (relative to peak current) increasing from 6±5% to 17±13% (p<0.01, n=12). Action potential durations were increased from 55±14 to 86±36 msec (p<0.05, n=6). The effects of thyroid hormone were partially reversed by lidocaine (60 M, n=5), a specific blocker of a slow sub-population of Na channels. Thus thyroid hormone interacts directly with myocyte membrane, probably by slowing of inactivation of Na channels.Abbreviations and Symbols T3 3,5,3-triiodo-L-thyronine - I_Na current carried by sodium ions - I_T3 net current attributible to T3 treatment - I₂₀ % of peak current at 20 msec after current onset - ADP₉₀ Time required for action potential to return to within 10% of baseline level     

Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves

The node of Ranvier is a site for ionic conductances along myelinated nerves and governs the saltatory transmission of action potentials. Defects in the cross-bridging and spacing of the cytoskeleton are a prominent pathological feature in diseases of the peripheral nerve. Electron tomography was used to examine cytoskeletal–cytoskeletal, membrane–cytoskeletal, and heterologous cell connections in the paranodal region of the node of Ranvier in peripheral nerves. Focal attachment of cytoskeletal filaments to each other and to the axolemma and paranodal membranes of the Schwann cell via narrow cross-bridges was visualized in both neuronal and glial cytoplasm. A subset of intermediate filaments associates with the cytoplasmic surfaces of supramolecular complexes of transmembrane structures that are presumed to include known and unknown junctional proteins. Mitochondria were linked to both microtubules and neurofilaments in the axoplasm and to neighboring smooth endoplasmic reticulum by narrow cross-bridges. Tubular cisternae in the glial cytoplasm were also linked to the paranodal glial cytoplasmic loop juxtanodal membrane by short cross-bridges. In the extracellular matrix between axon and Schwann cell, junctional bridges formed long cylinders linking the two membranes. Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region are likely critical not only for scaffolding, but also for intracellular and extracellular communication.     

Pressure dependence of sodium gating currents in the squid giant axon

Asymmetric displacement currents, I_g, were measured in squid axons at different hydrostatic pressures, P, up to 60 MPa. Potassium and sodium currents were abolished by intracellular Cs⁺ and TEA⁺, by extracellular Tetrodotoxin (TTX), and by Na⁺ substitution with Tris⁺. The time course of Ig became progressively slower with increasing pressure, and the amplitude decreased. With appropriate scaling in time and amplitude, I_g records at any given P could be made to superimpose very well with those obtained at atmospheric pressure. The same scaling factors yielded a good superposition of all records obtained for voltage steps to membrane potentials in the range-30 to +42 mV. The ratio between the amplitude and time factors was larger than unity and increased with P, indicating a progressive decrease (up to 35% at 60 MPa) of the total charge displaced, Q, with no significant change in its voltage dependence. The time-scaling factor increased exponentially with P, as expected if all the steps involved in the opening of a sodium channel, and producing a major charge redistribution, have the same activation volume, V _g 17 cm³/mol. This value is roughly one-half of that characterizing the pressure dependence of sodium current activation, suggesting that some late, rate-limiting step in the opening of sodium channels has a large activation volume without being accompanied by an easily detected charge movement.Part of the decrease of Q with pressure could be attributed to an increase in sodium inactivation. However, we cannot exclude the possibility that there is a reversible reduction in the number of fast activating sodium channels, similar to the phenomenon that has been reported to occur at low temperatures (Matteson and Armstrong 1982).     

Fenestration in the myelin sheath of nerve fibers of the shrimp: A novel node of excitation for saltatory conduction

Giant nerve fibers of the shrimp family Penaeidae conduct impulses at the velocity highest among all animal species (∼210 m/s; highest in mammals = 120 m/s). We examined these giant and other small nerve fibers morphologically using a differential interference contrast microscope as well as an electron microscope, and found a very specialized form of excitable membrane that functions as a node for saltatory conduction of the impulse. This node appeared under the light microscope as a characteristic pattern of concentrically aligned rings in a very small spot of the myelin sheath. The diameter of the innermost ring of the node was about 5 μm, and the distance between these nodes was as long as 12 mm. Via an electron microscope, these nodes were characterized by a complete lack of the myelin sheath, forming a fenestration that has a tight junction with an axonal membrane. Voltage clamp measurements by a sucrose gap technique demonstrated that the axonal membrane at these fenestration nodes is exclusively excitable and that the large submyelinic space is a unique conductive pathway for loop currents for saltatory conduction through such fenestration nodes. © 1996 John Wiley & Sons, Inc.     

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.     

二氧化硫代谢衍生物对大鼠海马CA1区神经元钠电流的影响

实验采用全细胞膜片钳技术 ,研究了SO2 代谢衍生物亚硫酸钠和亚硫酸氢钠 (两者分子比为 3∶1)对大鼠海马CA1区神经元钠电流的影响。结果表明 ,SO2 代谢衍生物可剂量依赖性地增大钠电流 ,剂量为 10和 10 0μmol/L时 ,钠电流分别增大 5 0 .5 9± 19.0 8%和 82 .0 6± 18.5 1%(n =15 ) ;此外还与电压呈依赖性关系 ,但不具有频率依赖性 ;10 μmol/LSO2 代谢衍生物不影响钠电流的激活过程 ,却非常显著地影响其失活过程 ,作用前后的半数失活电压分别为 - 6 9.71± 4.6 7和 - 5 3.2 7± 4.95mV (n =10 ,P <0 .0 1) ,但不改变失活曲线的斜率因子。实验结果提示 ,SO2 衍生物具有类似神经毒物的作用 ,大气SO2 污染可能与一些中枢神经系统疾病的发生有关。     

Effects of SO2 derivatives on sodium currents in acutely isolated rat hippocampal lead-exposed neurons

In this study, the effects of acute SO_2 derivatives and chronic lead exposure together on sodium cur-rents (INa) were investigated in acutely isolated rat hippocampal neurons by using the whole-cell patch clamp techniques. We found that chronic lead exposure hardly reduced the amplitudes of INa. In the normal condition, sodium current started to appear at around ?70 mV, and reached the peak current at around ?40 mV. After chronic lead exposure, the data changed to ?70 and ?30 mV. After adding SO2 derivatives, the data changed to ?80 and ?40 mV, respectively. SO_2 derivatives caused a significant in-crease of INa in hippocampal chronic-lead exposed neurons. Chronic lead exposure induced a right shift of the activation curve and a left shift of the inactivation curve of sodium channels. SO_2 derivatives caused negative shifts of the activation and inactivation curves of INa in hippocampal chronic-lead ex-posed neurons. Lead exposure put off the time reaching the peak of INa activation. SO_2 derivatives in-creased the time constants of inactivation after lead exposure. The interaction of lead and SO_2 deriva-tives with voltage-dependent sodium channels may lead to changes in electrical activity and contribute to worsening the neurotoxicological damage.     

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.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.     

The effects of sodium removal on the two types of calcium currents in single frog atrial cells.

The effects of sodium removal on the two types of calcium currents were studied in enzymatically dispersed frog (Rana esculenta) atrial myocytes with the single pipette patch-clamp technique. Reduction of calcium currents was recorded when NaCl was replaced either by TEACl, LiCl, TrisCl, cholineCl or by mannitol. An involvement of the Na-Ca exchange mechanism could be ruled out since the decrease was also observed after replacing external Ca with Ba. The slight shift of the apparent reversal potential recorded in our study suggests that the inward flow of Na ions through calcium channels does not contribute significantly to the L-type calcium current. Once again, the slight negative shift of the steady-state inactivation curve of the L-type calcium current cannot explain this decrease while no shift was recorded for the T-type calcium current. Even if a TTX-resistant Na current was recorded from a few cells this current cannot explain the decrease of calcium currents which was always observed upon sodium removal. To date we have no explanation for this effect.     

FGF13 modulates the gating properties of the cardiac sodium channel Nav1.5 in an isoform-specific manner

FGF13 (FHF2), the major fibroblast growth factor homologous factor (FHF) in rodent heart, directly binds to the C-terminus of the main cardiac sodium channel, Na_V1.5. Knockdown of FGF13 in cardiomyocytes induces slowed ventricular conduction by altering Na_V1.5 function. FGF13 has five splice variants, each of which possess the same core region and C terminus but differing in their respective N termini. Whether and how these alternatively spliced N termini impart isoform-specific regulation of Na_V1.5, however, has not been reported. Here, we exploited a heterologous expression to explore the specific modulatory effects of FGF13 splice variants FGF13S, FGF13U and FGF13YV on Na_V1.5 function. We found these three splice variants differentially modulated Na_V1.5 current density. Although steady-state activation was unaltered by any of the FGF13 isoforms (compared to control cells expressing Nav1.5 but not expressing FGF13), open-state fast inactivation and closed-state fast inactivation were markedly slowed, steady-state availability was significantly shifted toward the depolarizing direction, and the window current was increased by each of FGF13 isoforms. Most strikingly, FGF13S hastened the rate of Na_V1.5 entry into the slow inactivation state and induced a dramatic slowing of recovery from inactivation, which caused a large decrease in current after either low or high frequency stimulation. Overall, these data showed the diversity of the roles of the FGF13 N-termini in Na_V1.5 channel modulation and suggested the importance of isoform-specific regulation.     

Effects of arachidonic acid and the other long-chain fatty acids on the membrane currents in the squid giant axon

Summary The effects of arachidonic acid and some other long-chain fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular application of the test substances. The effects of these acids, which are usually insoluble in solution, were examined by using -cyclodextrin as a solvent. -cyclodextrin itself had no effect on the excitable membrane. Arachidonic acid mainly suppresses the Na current but has little effect on the K current. These effects are completely reversed after washing with control solution. The concentration required to suppress the peak inward current by 50% (ED₅₀) was 0.18mm, which was 10 times larger than that of medium-chain fatty acids like 2-decenoic acid. The Hill number was 1.5 for arachidonic acid, which is almost the same value as for medium-chain fatty acids. This means that the mechanisms of the inhibition are similar in both long- and medium-chain fatty acids. When the long-chain fatty acids were compared, the efficacy of suppression of Na current was about the same value for arachidonic acid, docosatetraenoic acid and docosahexaenoic acid. The suppression effects of linoleic acid and linolenic acid on Na currents were one-third of that of arachidonic acid. Oleic acid had a small suppression effect and stearic acid had almost no effect on the Na current. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (Hodgkin, A.L., Huxley, A.F. (1952)J. Physiol (London) 117:500–544) and the change in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m ) for the Na current against membrane potential and the time constant of activation ( _m ) were shifted 10 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation ( _h ) has almost unaffected by application of these fatty acids.     

Effects of external K concentration on the electrogenicity of the insulin-stimulated Na,K-pump in frog skeletal muscle

Summary Insulin hyperpolarized the membrane of frog skeletal muscle by stimulating the electrogenic Na,K-pump. At external K concentrations of 1, 2, 5 and 10mm, both the insulin-induced hyperpolarization and the insulin-stimulated ouabain-sensitive Na efflux (an index of Na, K-pump activity) were observed. By increasing the external K concentration, the insulin-stimulated Na efflux increased, but the magnitude of the insulin-induced hyperpolarization decreased; i. e., although the activity of the insulin-stimulated Na,K-pump increased, on the contrary, the magnitude of the hyperpolarization decreased. To clarify the causes of this phenomenon, the specific membrane resistance was measured and found to decrease upon increasing the external K concentration.One of the reasons for the decrease in magnitude of the hyperpolarization is the decrease in the specific membrane resistance. However, the decrease in magnitude of the hyperpolarization with a rise of the external K concentration, which increased the insulin-stimulated Na,K-pump activity, cannot be explained only by the decrease in the specific membrane resistance. It is suggested that the decrease in magnitude of the hyperpolarization is mainly caused by a decrease in the electrogenicity of the insulin-stimulated Na,K-pump upon an increase in the external K concentration. The conclusion of the present study is that the electrogenicity of the insulin-stimulated Na,K-pump in muscles is variable and decreases with increasing the external K concentration.