BetaIVSigma1 spectrin stabilizes the nodes of Ranvier and axon initial segments

Saltatory electric conduction requires clustered voltage-gated sodium channels (VGSCs) at axon initial segments (AIS) and nodes of Ranvier (NR). A dense membrane undercoat is present at these sites, which is thought to be key for the focal accumulation of channels. Here, we prove that betaIVSigma1 spectrin, the only betaIV spectrin with an actin-binding domain, is an essential component of this coat. Specifically, betaIVSigma1 coexists with betaIVSigma6 at both AIS and NR, being the predominant spectrin at AIS. Removal of betaIVSigma1 alone causes the disappearance of the nodal coat, an increased diameter of the NR, and the presence of dilations filled with organelles. Moreover, in myelinated cochlear afferent fibers, VGSC and ankyrin G clusters appear fragmented. These ultrastructural changes can explain the motor and auditory neuropathies present in betaIVSigma1 -/- mice and point to the betaIVSigma1 spectrin isoform as a master-stabilizing factor of AIS/NR membranes.     

CK2 accumulation at the axon initial segment depends on sodium channel Nav1

Accumulation of voltage-gated sodium channel Nav1 at the axon initial segment (AIS), results from a direct interaction with ankyrin G. This interaction is regulated in vitro by the protein kinase CK2, which is also highly enriched at the AIS. Here, using phosphospecific antibodies and inhibition/depletion approaches, we showed that Nav1 channels are phosphorylated in vivo in their ankyrin-binding motif. Moreover, we observed that CK2 accumulation at the AIS depends on expression of Nav1 channels, with which CK2 forms tight complexes. Thus, the CK2–Nav1 interaction is likely to initiate an important regulatory mechanism to finely control Nav1 phosphorylation and, consequently, neuronal excitability.     

betaIV spectrin is recruited to axon initial segments and nodes of Ranvier by ankyrinG

High densities of ion channels at axon initial segments (AISs) and nodes of Ranvier are required for initiation, propagation, and modulation of action potentials in axons. The organization of these membrane domains depends on a specialized cytoskeleton consisting of two submembranous cytoskeletal and scaffolding proteins, ankyrinG (ankG) and betaIV spectrin. However, it is not known which of these proteins is the principal organizer, or if the mechanisms governing formation of the cytoskeleton at the AIS also apply to nodes. We identify a distinct protein domain in betaIV spectrin required for its localization to the AIS, and show that this domain mediates betaIV spectrin's interaction with ankG. Dominant-negative ankG disrupts betaIV spectrin localization, but does not alter endogenous ankG or Na(+) channel clustering at the AIS. Finally, using adenovirus for transgene delivery into myelinated neurons, we demonstrate that betaIV spectrin recruitment to nodes of Ranvier also depends on binding to ankG.     

Disrupted axo-glial junctions result in accumulation of abnormal mitochondria at nodes of ranvier

Mitochondria and other membranous organelles are frequently enriched in the nodes and paranodes of peripheral myelinated axons, particularly those of large caliber. The physiologic role(s) of this organelle enrichment and the rheologic factors that regulate it are not well understood. Previous studies suggest that axonal transport of organelles across the nodal/paranodal region is locally regulated. In this study, we have examined the ultrastructure of myelinated axons in the sciatic nerves of mice deficient in the contactin-associated protein (Caspr), an integral junctional component. These mice, which lack the normal septate-like junctions that promote attachment of the glial (paranodal) loops to the axon, contain aberrant mitochondria in their nodal/paranodal regions. These mitochondria are typically large and swollen and occupy prominent varicosities of the nodal axolemma. In contrast, mitochondria located outside the nodal/paranodal regions of the myelinated axons appear normal. These findings suggest that paranodal junctions regulate mitochondrial transport and function in the axoplasm of the nodal/paranodal region of myelinated axons of peripheral nerves. They further implicate the paranodal junctions in playing a role, either directly or indirectly, in the local regulation of energy metabolism in the nodal region.     

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.     

Determinants of voltage-gated sodium channel clustering in neurons

In mammalian neurons, the generation and propagation of the action potential result from the presence of dense clusters of voltage-gated sodium channels (Nav) at the axonal initial segment (AIS) and nodes of Ranvier. In these two structures, the assembly of specific supra-molecular complexes composed of numerous partners, such as cytoskeletal scaffold proteins and signaling proteins ensures the high concentration of Nav channels. Understanding how neurons regulate the expression and discrete localization of Nav channels is critical to understanding the diversity of normal neuronal function as well as neuronal dysfunction caused by defects in these processes. Here, we review the mechanisms establishing the clustering of Nav channels at the AIS and in the node and discuss how the alterations of Nav channel clustering can lead to certain pathophysiologies.     

Minimal sodium channel pore consisting of S5-P-S6 segments preserves intracellular pharmacology

We studied the properties of a sodium channel comprised only of S5-P-S6 region of the rat sodium channel alpha-subunit Nav1.4 (micro1pore). Results obtained in HEK cell lines permanently transfected with the sodium channel alpha-subunit or with the micro1pore were compared with data of the native HEK cells. Sodium channel blockers, tetrodotoxin and tetracaine, protect cells transfected with the complete sodium channel against death produced by incubation with veratridine. Veratridine-induced cell death in cell lines expressing the micro1pore construct is antagonised by tetracaine, but not by tetrodotoxin. Whole-cell conductance also increases in the presence of veratridine in micro1pore transfected cells and tetracaine inhibits these currents. Our pharmacological and electrophysiological data suggest that micro1pore keeps binding sites for veratridine and tetracaine, but not for TTX, and reconstitutes the permeation pathway for Na+ ions.     

Removal of sodium channel inactivation in squid axon by the oxidant chloramine-T

We have investigated the effects of a mild oxidant, chloramine-T(CT), on the sodium and potassium currents of squid axons under voltage-clamp conditions. Sodium channel inactivation of squid giant axons can be completely removed by CT at neutral pH. Internal and external CT treatment are both effective. CT apparently removes inactivation in an irreversible, all-or-none manner. The activation process of sodium channels is little affected, as judged from the voltage dependence of peak sodium currents, the rising phase of sodium currents, and the time course of tail currents following the repolarization. The removal of inactivation by CT is pH-dependent; higher pH decreases the removal rate, whereas lower pH increases it. Internal metabisulfite, a strong reductant, does not protect inactivation from the action of external CT, nor does external metabisulfite protect from internal CT application. CT slightly depresses the peak potassium currents at comparable concentrations but has no apparent effects on their kinetics. Our results suggest that the neutral form of CT modifies an embedded methionine residue that is involved in sodium channel inactivation.     

Interaction of deoxycholate with the sodium channel of squid axon membranes

Deoxycholate can react with sodium channels with a high potency. The apparent dissociation constant for the saturable binding reaction is 2 microM at 8 degrees C, and the heat of reaction is approximately -7 kcal/mol. Four independent test with Na-free media, K-free media, tetrodotoxin, and pancuronium unequivocally indicate that it is the sodium channel that is affected by deoxycholate. Upon depolarization of the membrane, the drug modified channel exhibits a slowly activating and noninactivating sodium conductance. The kinetic pattern of the modified channel was studied by increasing deoxycholate concentration, lowering the temperature, chemical elimination of sodium inactivation, or conditioning depolarization. The slow activation of the modified channel can be represented by a single exponential function with the time constant of 1--5 ms. The modified channel is inactivated only partially with a time constant of 1 S. The reversal potential is unchanged by the drug. Observations in tail currents and the voltage dependence of activation suggest that the activation gate is actually unaffected. The apparently slow activation may reflect an interaction betweem deoxycholate and the sodium channel in resting state.     

Charged residues between the selectivity filter and S6 segments contribute to the permeation phenotype of the sodium channel

The deep regions of the Na(+) channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd(2+) block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd(2+) binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.     

Functional and evolutionary consequences of pyrethroid resistance mutations in S6 transmembrane segments of a voltage-gated sodium channel

Pyrethroids are a class of voltage-dependent sodium channel modifiers widely used as insecticides for control of disease vectors and agricultural pests. Many insect populations have developed resistance to pyrethroids linked to nervous system insensitivity and structural mutations in neuronal sodium channels. Pyrethroid resistant strains of the moth Heliothis virescens carry single point mutations leading to amino acid substitutions in either transmembrane segment I-S6 (V421M) or II-S6 (L1029H) of the para-homologous sodium channel. We analyzed the consequences of V421M and L1029H mutations constructed in the Drosophila para sodium channel heterologously expressed in Xenopus oocytes, and found that both mutations confer channel insensitivity to permethrin, with the L1029H mutation having a more pronounced effect. Both mutations also modify the intrinsic voltage-dependent gating properties of the channel, but L1029H less so than V421M. These results suggest that mutation V421M exacts a higher fitness cost than L1029H, providing a plausible explanation for genetic succession observed in field strains, where V421M was replaced by L1029H during the past decade.     

The early phase of sodium channel gating current in the squid giant axon

A fast component of displacement current which accompanies the sodium channel gating current has been recorded from the membrane of the giant axon of the squid Loligo forbesii. This component is characterized by relaxation time constants typically shorter than 25 µs. The charge displaced accounts for about 10% (or 2 nC/cm²) of the total displacement charge attributed to voltage-dependent sodium channels. Using a low noise, wide-band voltage clamp system and specially designed voltage step protocols we could demonstrate that this component: (i) is not a recording artifact; (ii) is kinetically independent from the sodium channel activation and inactivation processes; (iii) can account for a significant fraction of the initial amplitude of recorded displacement current and (iv) has a steady state charge transfer which saturates for membrane potentials above + 20 mV and below – 100 mV This component can be modelled as a single step transition using the Eyring-Boltzmann formalism with a quantal charge of 1 e^– and an asymmetrical energy barrier. Furthermore, if it were associated with the squid sodium channel, our data would suggest one fast transition per channel. A possible role as a sodium channel activation trigger, which would still be consistent with kinetic independence, is discussed. Despite uncertainties about its origin, the property of kinetic independence allows subtraction of this component from the total displacement current to reveal a rising phase in the early time course of the remaining current. This will have to be taken into account when modelling the voltage-dependent sodium channel.     

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.     

Displacement currents in sodium channels of the ranvier node membrane

The displacement current was recorded in the Ranvier node membrane ofRana ridibunda. This current was shown to be due to conversion of charges from the initial state in which they were when a high negative voltage was present on the membrane into the final state. The dependence of the displacement charge on the membrane potential and state of activation of the sodium channels suggests that the displacement current is connected with activation of the m gates of the sodium channels. Considering the density of the displaced charges, the density of the sodium channels can be estimated to be 5000 channels/µ².A. A. Ukhtomskii Institute of Physiology, A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 410–417, July–August, 1976.     

Slowing of sodium channel opening kinetics in squid axon by extracellular zinc

The interaction of Zn ion on Na channels was studied in squid giant axons. At a concentration of 30 mM Zn2+ slows opening kinetics of Na channels with almost no alteration of closing kinetics. The effects of Zn2+ can be expressed as a "shift" of the gating parameters along the voltage axis, i.e., the amount of additional depolarization required to overcome the Zn2+ effect. In these terms the mean shifts caused by 30 mM Zn2+ were +29.5 mV for Na channel opening (on) kinetics (t1/2 on), +2 mV for closing (off) kinetics (tau off), and +8.4 mV for the gNa-V curve. Zn2+ does not change the shape of the instantaneous I-V curve for inward current, but reduces it in amplitude by a factor of or approximately 0.67. Outward current is unaffected. Effects of Zn2+ on gating current (measured in the absence of TTX) closely parallel its actions on gNa. On gating current kinetics are shifted by +27.5 mV, off kinetics by +6 mV, and the Q-V distribution by +6.5 mV. Kinetic modeling shows that Zn2+ slows the forward rate constants in activation without affecting backward rate constants. More than one of the several steps in activation must be affected. The results are not compatible with the usual simple theory of uniform fixed surface charge. They suggest instead that Zn2+ is attracted by a negatively charged element of the gating apparatus that is present at the outer membrane surface at rest, and migrates inward on activation.     

Interaction of spin-labeled local anesthetics with the sodium channel of squid axon membranes

Summary The effects of spin-labeled local anesthetics on sodium currents of internally perfused squid axons were studied using the voltage-clamp technique. Internal application (10 m) of the most potent spin-labeled local anesthetic used in this study produced a small initial block of sodium currents. However, after sixty repetitive pulses (to +80 mV) given at 1 Hz, the sodium currents were drastically reduced. In addition to this frequency-dependent phenomenon, the anesthetic effect on the sodium currents was also sensitive to the voltage of the pulses. Both the frequency- and voltage-dependent properties remained intact after removal of sodium inactivation with pronase. The recovery of sodium currents from this frequency-dependent anesthetic effect followed a single exponential curve with a surprisingly long time constant of about 10 min. Such a long recovery time, which is longer than any known sodium inactivation process, led us to suggest that the recovery process represents the dissociation of drug molecules from their binding sites. We have also found that increasing hydrophobic character of the homologues series of spin-labeled local anesthetics enhances the frequency- and voltage-dependent block of sodium currents. This effect strongly suggests that hydrophobic interaction is an integral component of the binding site. These probes with their selective effects on the sodium currents, are expected to be highly useful in studying the molecular structure of the sodium channels.     

Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon

Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.