Clustering Sodium Channels at the Node of Ranvier: Close Encounters of the Axon–Glia Kind
Authors: James L Salzer
Journal: Neuron (1997)

As nervous systems became increasingly complex and centralized, the need for efficient and rapid impulse conduction became pronounced. A solution to this problem, which independently appeared at least three times during evolution, was achieved by concentrating voltage-gated channels to unensheathed regions of the axon flanked by complex glial sheaths (11Hildebrand C Remahl S Persson H Bjartmar C Prog. Neurobiol. 1993; 40: 319-384Crossref PubMed Scopus (274) Google Scholar). This collaboration between glial cells and axons achieves its apotheosis in the node of Ranvier. This exquisitely specialized structure, which is regularly spaced along the length of myelinated nerve fibers, enables saltatory conduction in which action potentials "jump" from node to node. In addition to increasing the speed of conduction, nodal conduction results in enormous economies of energy and space. For example, the unmyelinated squid giant axon occupies 15,000 times the space of a comparably conducting myelinated mammalian nerve and uses several thousandfold more energy. With these important advantages, however, also came new vulnerabilities, reflected in the large number of human diseases that affect the myelin sheath and/or the node. Although the node was described more than 100 years ago (19Ranvier M.L Leçons sur l'histologie du système nerveux. F. Savy, Paris1878Google Scholar), details of its molecular organization have only recently begun to emerge. At the literal center of its structure and function is the remarkable enrichment of voltage-gated sodium channels in the nodal axolemma. This striking concentration of channels reflects interactions between axons and glial cells that are the subject of several recent investigations (7Davis J.Q Lambert S Bennett V J. Cell Biol. 1996; 135: 1355-1367Crossref PubMed Scopus (292) Google Scholar, 22Vabnick I Novakovic S.D Levinson S.R Schachner M Shrager P J. Neurosci. 1996; 16: 4914-4922Crossref PubMed Google Scholar, 8Deerinck T.J Levinson S.R Bennett G.V Ellisman M.H J. Neurosci. 1997; 17 (in press)PubMed Google Scholar, 14Kaplan M.R Meyer-Franke A Lambert S Bennett V Duncan I.D Levinson S.R Barres B.A Nature. 1997; 386: 724-728Crossref PubMed Scopus (267) Google Scholar). These studies provide new insights into the glial signals that regulate sodium channel clustering and the molecules that associate with and may promote such clusters. They also address whether these clusters determine the subsequent location of the nodes along the axon or, alternatively, are positioned by the glial cells. Nodes are the gaps between the much longer myelin segments, which are therefore referred to as internodes. The spacing of nodes, and the size of the internodes, like the thickness of the myelin sheath, vary with fiber diameter in a curvilinear relationship that is optimized for maximal conduction velocity (5Black, J.A., Sontheimer, H., Oh, Y., and Waxman, S.G. (1995). In The Axon, S. Waxman, J. Kocsis, and P. Stys, eds. (New York: Oxford University Press), pp. 116–143.Google Scholar). The size of the node is typically in the range of 1–2 μm, whereas that of internodes can be up to 1.5 mm or more, depending on the axon diameter and the specific fiber type. The structure of the node, and the flanking paranodal regions, are similar in the CNS and PNS (Figure 1) and entirely distinct from the internode under the compact myelin sheath (reviewed by11Hildebrand C Remahl S Persson H Bjartmar C Prog. Neurobiol. 1993; 40: 319-384Crossref PubMed Scopus (274) Google Scholar, 18Peters A Palay S.L Webster H.D The Fine Structure of the Nervous System, Third Edition. Oxford University Press, New York1991Google Scholar). The axon is exposed to the extracellular environment at the node. It is also typically constricted in its diameter, being reduced to as little as 20% of the diameter of the internode in large fibers. This decreased axon caliber reflects, in part, a higher packing density of neurofilaments in this region, which are less heavily phosphorylated and are transported more slowly (23Waegh S.M.D Lee V.M.-Y Brady S.T Cell. 1992; 68: 451-463Abstract Full Text PDF PubMed Scopus (645) Google Scholar). Vesicles, including synaptic vesicles, microtubules, and mitochondria, are also frequently increased at the node, suggesting there is a bottleneck of axonal transport in both directions (9Fabricius C Berthold C.H Rydmark M J. Neurocytol. 1993; 22: 941-954Crossref PubMed Scopus (46) Google Scholar) and local axonal–glial signaling. Freeze fracture studies (20Rosenbluth J J. Neurocytol. 1976; 5: 731-745Crossref PubMed Scopus (206) Google Scholar) have revealed that the nodal axolemma in both the CNS and PNS is enormously enriched in intramembranous particles (IMPs, >1200/μm2) compared to the internode (<100/μm2). These IMPs, which are associated with the outer leaflet (E face), are thought to correspond largely to sodium channels that are concentrated in the node (see below). A dense undercoating of 25–35 nm is subjacent to, but slightly separated from, the axolemma. A similar undercoating is also present in the initial segment of the axon, the site of action potential initiation. There are some important differences in the structure of the node in the CNS and PNS, reflecting their different cellular constituents. In the PNS, hundreds of specialized microvilli project from the outer collar of adjacent Schwann cells, interdigitate, and come into close proximity with the nodal axolemma of large fibers (18Peters A Palay S.L Webster H.D The Fine Structure of the Nervous System, Third Edition. Oxford University Press, New York1991Google Scholar). These finger-like projections of the Schwann cell are perpendicular to the node; in transverse EM sections through the node, they resemble petals of a sunflower radiating from a central axon. In contrast, in the CNS, one or more astrocytic processes frequently come into close proximity to the node. Recent evidence suggests that these processes arise from multifunctional astrocytes (6Butt A.M Duncan A Berry M J. Neurocytol. 1994; 23: 486-499Crossref PubMed Scopus (72) Google Scholar) rather than from a specialized population of astrocytes dedicated to contacting the node as previously believed. In addition, in the PNS, the basal lamina that surrounds Schwann cells is continuous across the node; central nodes are devoid of a well-formed basal lamina, although an ill-defined extracellular matrix has been observed in the nodal gap (11Hildebrand C Remahl S Persson H Bjartmar C Prog. Neurobiol. 1993; 40: 319-384Crossref PubMed Scopus (274) Google Scholar). Adjacent to the node, the compact myelin sheath opens up into a series of cytoplasmic loops that spiral around the axon. These paranodal loops are helically arranged with the outermost loops of the myelin sheath closest to the node. As many as 40 of these loops come into close apposition with, and physically invaginate, the axolemma (Figure 2). The resultant structure has been likened to a bolt (the axon) threaded into a nut (the paranodal loops; 18Peters A Palay S.L Webster H.D The Fine Structure of the Nervous System, Third Edition. Oxford University Press, New York1991Google Scholar). The axolemma comes into extremely close apposition with the paranodal loops of the glial cell to form a series of septate-like junctions that are detected as intermittent densities in longitudinal sections through the node (Figure 2). The molecular composition of these junctions has not yet been elucidated. Although the paranodal region is permeable to small molecular weight compounds and ions (12Hirano, A., and Llena, J.F. (1995). In The Axon, S. Waxman, J. Kocsis, and P. Stys, eds. (New York: Oxford University Press), pp. 49–67.Google Scholar), it may retard ionic movements sufficiently to facilitate saltatory conduction. The septate-like junctions have also been suggested (20Rosenbluth J J. Neurocytol. 1976; 5: 731-745Crossref PubMed Scopus (206) Google Scholar) to act as physical barriers that restrict the lateral diffusion of voltage-gated channels and maintain their unique distribution in the node and paranodes, as described further below. The molecular organization of the node is specialized for its function in impulse propagation. Estimates of the concentration of sodium channels in the node (∼1500/μm2) versus the internode (<25/μm2) based on electrophysiologic studies agree well with the number of IMPs in the node, suggesting that many of the IMPs correspond to sodium channels. Potassium channels have a complimentary distribution; they are essentially absent in the nodal axolemma, whereas in the paranodal axolemma and Schwann cell membranes at the node, there is a high concentration of delayed rectifier potassium channels (4Black J.A Kocsis J.D Waxman S.G Trends Neurosci. 1990; 13: 48-54Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 16Mi H Deerinck T.J Jones M Ellisman M.H Schwarz T.L J. Neurosci. 1996; 16: 2421-2429Crossref PubMed Google Scholar). The function of these potassium channels is unknown. They may contribute to the rapid repolarization of the action potential or play a role in buffering potassium ions at the node, although they are not ideally located for either function (16Mi H Deerinck T.J Jones M Ellisman M.H Schwarz T.L J. Neurosci. 1996; 16: 2421-2429Crossref PubMed Google Scholar). This highly asymmetric distribution of voltage-gated sodium and potassium channels is in striking contrast to their diffuse distribution in unmyelinated fibers (4Black J.A Kocsis J.D Waxman S.G Trends Neurosci. 1990; 13: 48-54Abstract Full Text PDF PubMed Scopus (111) Google Scholar). The filamentous network subjacent to the nodal membrane contains the cytoskeletal proteins spectrin and, notably, ankyrin. The ankyrin present at the node corresponds to 480 and 270 kDa alternatively spliced isoforms of the ankyrinG gene (15Kordeli E Lambert S Bennett V J. Biol. Chem. 1995; 270: 2352-2359Crossref PubMed Scopus (413) Google Scholar). The high concentration of ankyrin at the node may be functionally significant as many of the proteins that are concentrated at the node share an ability to bind to ankyrin with high affinity. Among these ankyrin binding proteins are the sodium channels. Additional ankyrin binding proteins concentrated at the node include the Na+/K+ ATPase, which presumably maintains ionic gradients within the axon, and specific isoforms of the neural cell adhesion molecules (CAMs), neurofascin and NrCAM (7Davis J.Q Lambert S Bennett V J. Cell Biol. 1996; 135: 1355-1367Crossref PubMed Scopus (292) Google Scholar). These cell adhesion molecules are part of a subfamily of immunoglobulin-like CAMs, which also includes the L1 adhesion molecule, each of which shares highly conserved cytoplasmic sequences that bind to ankyrin. All of these proteins, i.e., NrCAM and neurofascin, sodium channels, and ankyrinG are also enriched in the initial segment of axons, suggesting a functional relationship. The precise relationship of these individual molecular components to the clustering of sodium channels is not yet known. Potentially, a preformed cytoskeleton containing ankyrin and spectrin could target sodium channels and cell adhesion proteins to the node. Alternatively, extracellular interactions may regulate the clustering of the ankyrin binding CAMs, which sequentially recruit ankyrin and sodium channels to the node; conversely, ankyrin may function primarily to stabilize proteins that previously clustered by other mechanisms. A variety of other molecules are known to be specifically enriched in the glial membranes of the paranodal region where they contribute to its organization and structural integrity; other cell adhesion molecules have inconsistently been reported to be present at the node (see21Scherer S.S Microsc. Res. Tech. 1996; 34: 452-461Crossref PubMed Scopus (64) Google Scholar, for a brief review). These uncertainties about the molecular events that lead to channel clustering are reflected in broader developmental questions: what is the role of glial cells in this clustering, what is the nature of the signals that initiate sodium channel clustering, and is the location of these clusters specified by the axon (and intrinsic aspects of its cytoskeleton) or, alternatively, are such sites determined by glial contacts? These issues are central to a recent study of B. Barres and colleagues (14Kaplan M.R Meyer-Franke A Lambert S Bennett V Duncan I.D Levinson S.R Barres B.A Nature. 1997; 386: 724-728Crossref PubMed Scopus (267) Google Scholar). In this study, retinal neurons were grown in culture with or without optic nerve glia, and sodium channel distribution was analyzed by immunofluorescence. Channels were diffusely distributed in the neurons grown alone, or with astrocytes, whereas they were clustered in the oligodendrocyte cocultures. Conditioned media from oligodendrocytes, and extracts of optic nerve, induced clustering, suggesting that the oligodendrocyte signal may be soluble. The clusters colocalized with ankyrinG and were evenly distributed at ∼44 μm intervals along the length of the axon. Finally, in vivo, the development of sodium channel clusters was temporally correlated to the appearance of oligodendrocytes and significantly reduced in a hypomyelinating mutant rat characterized by a severe deficiency in the number and function of oligodendrocytes. These results suggest that oligodendrocytes have a principal role in clustering sodium channels and function in a paracrine manner. (These results do not preclude that this oligodendrocyte factor also functions by a juxtacrine mechanism, as many proteins exist in both soluble and membrane-associated forms). The identity of the oligodendrocyte clustering factor is not yet known. Initial characterization of this protein indicates that it is trypsin sensitive with a molecular weight >10 kDa (14Kaplan M.R Meyer-Franke A Lambert S Bennett V Duncan I.D Levinson S.R Barres B.A Nature. 1997; 386: 724-728Crossref PubMed Scopus (267) Google Scholar). One potential candidate is tenascin-R, which is known to be present at the node and is secreted by oligodendrocytes (1Bartsch U Pesheva P Raff M Schachner M GLIA. 1993; 9: 57-69Crossref PubMed Scopus (101) Google Scholar). Of note in this regard is that an ancillary subunit of the sodium channel, β2, is homologous to the neural cell adhesion molecule contactin/F3/F11 (13Isom L.L Ragsdale D.S De Jongh K.S Westenbroek R.E Reber B.F Scheuer T Catterall W.A Cell. 1995; 83: 433-442Abstract Full Text PDF PubMed Scopus (390) Google Scholar), which is a known ligand for tenascin (17Norenberg U Hubert M Brummendorf T Tarnok A Rathjen F.G J. Cell Biol. 1995; 130: 473-484Crossref PubMed Scopus (56) Google Scholar). This originally raised the possibility (13Isom L.L Ragsdale D.S De Jongh K.S Westenbroek R.E Reber B.F Scheuer T Catterall W.A Cell. 1995; 83: 433-442Abstract Full Text PDF PubMed Scopus (390) Google Scholar), now confirmed (J. Srinivasan, 1997, Soc. Neurosci. abstract), that β2 binds to tenascin-C and tenascin-R. Tenascin-R could therefore have a role in targeting or stabilizing sodium channels at the node; its expression at relatively late stages of myelination (1Bartsch U Pesheva P Raff M Schachner M GLIA. 1993; 9: 57-69Crossref PubMed Scopus (101) Google Scholar) would seem to be more consistent with the latter possibility. Whatever this oligodendrocyte factor proves to be, its isolation is likely to provide important insights into the mechanisms of channel clustering. Potentially, this factor could be a multivalent ligand for one or more of the molecules present at the node and cross-link and thereby cluster nodal proteins. An alternative possibility is that the factor might activate intracellular signaling pathways. Of note, phosphorylation of the cytoplasmic domain of ankyrin binding CAMs, including neurofascin, regulates their binding to ankyrin and their lateral mobility (10Garver T.D Ren Q Tuvia S Bennett V J. Cell Biol. 1997; 137: 703-714Crossref PubMed Scopus (211) Google Scholar), suggesting that activation of kinases or phosphatases may regulate clustering of ankyrin complexes at the node. The absence of an effect of astrocytes on sodium channel distribution was unexpected in view of the close morphologic relationship of perinodal astrocyte processes, which had suggested a role in channel clustering (5Black, J.A., Sontheimer, H., Oh, Y., and Waxman, S.G. (1995). In The Axon, S. Waxman, J. Kocsis, and P. Stys, eds. (New York: Oxford University Press), pp. 116–143.Google Scholar). Indeed, in vivo studies have demonstrated that during development, astrocytic processes contact clusters of IMPs along the axon. However, recent studies indicate that astrocytes are not an invariant feature of all CNS nodes; many smaller CNS fibers appear to be devoid of such processes (3Bjartmar C Karlsson B Hildebrand C Brain. Res. 1994; 667: 111-114Crossref PubMed Scopus (14) Google Scholar). If the perinodal astrocytic processes have a dispensable function during node development, what then is their function? One possibility is that such processes help regulate the ionic environment of the node and contribute to its homeostasis consistent with the presence of voltage-gated channels in these cells (11Hildebrand C Remahl S Persson H Bjartmar C Prog. Neurobiol. 1993; 40: 319-384Crossref PubMed Scopus (274) Google Scholar). It is also possible that the processes are targetted via adhesive interactions to a preformed complex enriched in CAMs and stabilize its organization and demarcate the site of a future node. A surprising finding of Kaplan et al. is the uniform spacing of the clusters that formed on the retinal fibers in the absence of glial processes. These findings suggest that an intrinsic component of the axonal cytoskeleton may determine the spacing and future location of the channel–ankyrin complexes. The relationship between these channel clusters and the subsequent development of nodes is not addressed by this study, but they are inferred to represent future sites of nodes of Ranvier. On this point, the data are still inconclusive. While these clusters are uniform in vitro, the distribution of nodes along retinal ganglion axons in vivo is complex and not uniform (reviewed by11Hildebrand C Remahl S Persson H Bjartmar C Prog. Neurobiol. 1993; 40: 319-384Crossref PubMed Scopus (274) Google Scholar). As these fibers initially traverse the nerve fiber layer of the retina, a region that lacks oligodendrocytes, they are generally devoid of clustered IMPs, although some "hot spots" associated with astrocytic processes are present. In the optic nerve head, a transition zone that contains oligodendrocytes, myelin sheaths can be detected, but the spacing of nodes is heterogeneous and often quite small. Only in the optic nerve are nodes evenly distributed, but here they are spaced at 150–200 μm in the adult. This discrepancy in the spacing of nodes in vivo and the spacing of clusters in vitro may be partially explained by expansion of the optic nerve with development. It is also possible that the initial spacing of these clusters is regulated by axonal factors while final positioning of the nodes is specified by the growth of myelin sheaths. This assumes that a macromolecular complex containing ankyrin and transmembrane proteins can be repositioned. It is instructive in this context to compare these results to recent investigations of sodium channel localization in the PNS, which argue that glial processes may indeed be able to position such complexes. In a series of studies during development and remyelination, Shrager and his colleagues have analyzed the appearance of sodium channel clusters and typically find such aggregates to be in close proximity to the processes of Schwann cells (22Vabnick I Novakovic S.D Levinson S.R Schachner M Shrager P J. Neurosci. 1996; 16: 4914-4922Crossref PubMed Google Scholar). These clusters were only associated with Schwann cell processes that expressed MAG, a glial adhesion molecule that is expressed by Schwann cells that have formed at least one wrap around an axon prefatory to myelination. Pairs of clusters associated with neighboring Schwann cells were inferred to fuse as these glial processes subsequently approached each other at the nascent node. These results are consistent with earlier reports demonstrating that the appearance of IMP clusters in the axon were adjacent to Schwann cells that had begun to wrap axons (24Wiley-Livingston C Ellisman M.H Dev. Biol. 1980; 79: 334-355Crossref PubMed Scopus (75) Google Scholar) and suggest a critical role of the glial cell in determining the precise distribution of the nodes. Freeze fracture studies in the CNS have similarly demonstrated that clusters of IMPs appear prior to compact myelin formation near the edge of ensheathing glial processes (5Black, J.A., Sontheimer, H., Oh, Y., and Waxman, S.G. (1995). In The Axon, S. Waxman, J. Kocsis, and P. Stys, eds. (New York: Oxford University Press), pp. 116–143.Google Scholar). These results suggest a determinative role of glial cells. They are consistent with the significant repositioning of PNS nodes that occurs as myelin sheaths expand during development (2Berthold C.H Microsc. Res. Tech. 1996; 34: 399-421Crossref PubMed Scopus (15) Google Scholar) or contract following remyelination; in both cases, new nodes appear within previous internodal regions. A dissenting view is provided by studies of Ellisman and colleagues (8Deerinck T.J Levinson S.R Bennett G.V Ellisman M.H J. Neurosci. 1997; 17 (in press)PubMed Google Scholar). These authors have investigated sodium channel clusters in the merosin-deficient dystrophic mouse, which is characterized by proximal nerve roots devoid of Schwann cells. These investigators found, surprisingly, that in these proximal nerve roots, despite a severe deficiency of Schwann cells, sodium channels were still clustered. It may be that this clustering reflects the activity of Schwann cells previously associated with axons that were lost as development proceeded or, more likely, as the authors suggest, of soluble factors released by the few Schwann cells that persist on the outside of these nerve bundles in a manner analogous to the findings of 14Kaplan M.R Meyer-Franke A Lambert S Bennett V Duncan I.D Levinson S.R Barres B.A Nature. 1997; 386: 724-728Crossref PubMed Scopus (267) Google Scholar. These recent studies suggest that a glial signal promotes sodium channel clustering and that intrinsic axonal determinants specify the initial spacing of clusters. What remains to be resolved, in addition to the nature of the glial signal, is whether these clusters are positioned by glial processes. Potentially significant differences in the development and organization of CNS and PNS myelinated fibers may be reflected in distinct mechanisms of channel localization. Identification of the factor(s) that regulate clustering should provide insights not only into its mechanisms but also whether such mechanisms are conserved in the CNS and PNS. Detailed morphometric analysis of hypomyelinating mutations that specifically affect glial cells may further clarify whether the spacing of nodes is predetermined by the axon or is under glial control.