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Background: There is currently no effective treatment for promoting regeneration of injured nerves in patients who have sustained injury to the central nervous system such as spinal cord injury. Chondroitinase ABC is an enzyme, which promotes neurite outgrowth and regeneration. It has shown considerable promise as a therapy for these conditions. The aim of the study is to determine if targeting chondroitinase ABC expression to the neuronal axon can further enhance its ability to promote axon outgrowth. Long-distance axon regeneration has not yet been achieved, and would be a significant step in attaining functional recovery following spinal cord injury., Methodology/principal Findings: To investigate this, neuronal cultures were transfected with constructs encoding axon-targeted chondroitinase, non-targeted chondroitinase or GFP, and the effects on neuron outgrowth and sprouting determined on substrates either permissive or inhibitory to neuron regeneration. The mechanisms underlying the observed effects were also explored. Targeting chondroitinase to the neuronal axon markedly enhances its ability to promote neurite outgrowth. The increase in neurite length is associated with an upregulation of β-integrin staining at the axonal cell surface. Staining for phosphofocal adhesion kinase, is also increased, indicating that the β-integrins are in an activated state. Expression of chondroitinase within the neurons also resulted in a decrease in expression of PTEN and RhoA, molecules which present a block to neurite outgrowth, thus identifying two of the pathways by which ChABC promotes neurite outgrowth., Conclusions / Significance: The novel finding that targeting ChABC to the axon significantly enhances its ability to promote neurite extension, suggests that this may be an effective way of promoting long-distance axon regeneration following spinal cord injury. It could also potentially improve its efficacy in the treatment of other pathologies, where it has been shown to promote recovery, such as myocardial infarction, stroke and Parkinson's disease., Competing Interests: The authors have declared that no competing interests exist.

During amniote peripheral nervous system development, segmentation ensures the correct patterning of the spinal nerves relative to the vertebral column. Along the antero-posterior (rostro-caudal) axis, each somite-derived posterior half-sclerotome expresses repellent molecules to restrict axon growth and neural crest migration to the permissive anterior half-segment. To identify novel regulators of spinal nerve patterning, we investigated the differential gene expression of anterior and posterior half-sclerotomes in the chick embryo by RNA-sequencing. Several genes encoding extracellular matrix proteins were found to be enriched in either anterior (e.g. Tenascin-C, Laminin alpha 4) or posterior (e.g. Fibulin-2, Fibromodulin, Collagen VI alpha 2) half-sclerotomes. Among them, the extracellular matrix protein Fibulin-2 was found specifically restricted to the posterior half-sclerotome. By using in ovo ectopic expression in chick somites, we found that Fibulin-2 modulates spinal axon growth trajectories in vivo. While no intrinsic axon repellent activity of Fibulin-2 was found, we showed that it enhances the growth cone repulsive activity of Semaphorin 3A in vitro. Some molecules regulating axon growth during development are found to be upregulated in the adult central nervous system (CNS) following traumatic injury. Here, we found increased Fibulin-2 protein levels in reactive astrocytes at the lesion site of a mouse model of CNS injury. Together, these results suggest that the developing vertebral column and the adult CNS share molecular features that control axon growth and plasticity, which may open up the possibility for the identification of novel therapeutic targets for brain and spinal cord injury., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

A prominent anatomical feature of the peripheral nervous system is the segmentation of mixed (motor, sensory and autonomic) spinal nerves alongside the spinal cord. During early development their axon growth cones avoid the developing vertebral elements by traversing the anterior/cranial half of each somite-derived sclerotome, so ensuring the separation of spinal nerves from vertebral bones as axons extend towards their peripheral targets. A glycoprotein expressed on the surface of posterior half-sclerotome cells confines growth cones to the anterior half-sclerotomes by contact repulsion. A closely similar glycoprotein is expressed in avian and mammalian grey matter, where we hypothesize it may have evolved to regulate neural plasticity in birds and mammals., (© 2017 Anatomical Society.)

The chick embryo has provided a prominent model system for the study of segmental patterning in the nervous system. During early development, motor and sensory axon growth cones traverse the anterior/rostral half of each somite, so avoiding the developing vertebral components and ensuring separation of spinal nerves from vertebral bones. A glycoprotein expressed on the surface of posterior half-somite cells confines growth cones to the anterior half-somites by a contact repulsive mechanism. Hindbrain segmentation is also a conspicuous feature of chick brain development. We review how its contemporary analysis was initiated in the chick embryo, and the advantages the chick system continues to provide in its detailed elucidation at both molecular and neural circuit levels.

Background: There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location., Methodology/principal Findings: To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate., Conclusion/significance: Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

Background: The protein Nogo-A regulates axon growth in the developing and mature nervous system, and this is carried out by two distinct domains in the protein, Nogo-A-Δ20 and Nogo-66. The differences in the signalling pathways engaged in axon growth cones by these domains are not well characterized, and have been investigated in this study., Methodology/principal Findings: We analyzed growth cone collapse induced by the Nogo-A domains Nogo-A-Δ20 and Nogo-66 using explanted chick dorsal root ganglion neurons growing on laminin/poly-lysine substratum. Collapse induced by purified Nogo-A-Δ20 peptide is dependent on protein synthesis whereas that induced by Nogo-66 peptide is not. Nogo-A-Δ20-induced collapse is accompanied by a protein synthesis-dependent rise in RhoA expression in the growth cone, but is unaffected by proteasomal catalytic site inhibition. Conversely Nogo-66-induced collapse is inhibited ∼ 50% by proteasomal catalytic site inhibition., Conclusion/significance: Growth cone collapse induced by the Nogo-A domains Nogo-A-Δ20 and Nogo-66 is mediated by signalling pathways with distinguishable characteristics concerning their dependence on protein synthesis and proteasomal function.

We have carried out a series of experimental manipulations in the chick embryo to assess whether the notochord, neural tube and spinal nerves influence segmental patterning of the vertebral column. Using Pax1 expression in the somite-derived sclerotomes as a marker for segmentation of the developing intervertebral disc, our results exclude such an influence. In contrast to certain teleost species, where the notochord has been shown to generate segmentation of the vertebral bodies (chordacentra), these experiments indicate that segmental patterning of the avian vertebral column arises autonomously in the somite mesoderm. We suggest that in amniotes, the subdivision of each sclerotome into non-miscible anterior and posterior halves plays a critical role in establishing vertebral segmentation, and in maintaining left/right alignment of the developing vertebral elements at the body midline., (© 2012 The Authors. Journal of Anatomy © 2012 Anatomical Society.)

We have investigated the neurite growth-stimulating properties of euxanthone, a xanthone derivative isolated from the Chinese medicinal plant Polygala caudata. Euxanthone was shown to exert a marked stimulatory action on neurite outgrowth from chick embryo dorsal root ganglia explanted in collagen gels, in the absence of added neurotrophins. It was also shown to promote cell survival in explanted chick embryo ganglia, and to stimulate neurite outgrowth from isolated adult rat primary sensory neurons in vitro. The further finding that euxanthone stimulates neurite outgrowth from explants of chick embryo retina and ventral spinal cord suggests an action on signaling pathways downstream of neuronal receptors for specific neurotrophic factors. Consistent with this, euxanthone did not promote neurite outgrowth from non-transfected PC12 cells, or from PC12 cells transfected with TrkB or TrkC, under conditions in which these cells extended neurites in response to, respectively, the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3. Western blot analysis of euxanthone-stimulated dorsal root ganglion explants showed that expression of phospho-mitogen-activated protein (MAP) kinase was up-regulated after 1 h of euxanthone-treatment. Inhibition of the MAP kinase pathway using PD98059, a specific inhibitor of MAP kinase kinase, blocked all euxanthone-stimulated neurite outgrowth. However, analysis of phospho-Akt expression indicated that the phosphatidylinositol-3 kinase-Akt pathway, another major signaling pathway engaged by neurotrophins, is not significantly activated by euxanthone. These results suggest that euxanthone promotes neurite outgrowth by selectively activating the MAP kinase pathway.

Aberrant neurite growth is one of the neuropathological signatures of the Alzheimer's disease brain, both around amyloid plaques and in the cortical neuropil. Disruption of neuroinhibitory or repulsive growth and guidance signals, as well as of neurotrophic or permissive signals, may contribute to this dystrophic growth. Hence, therapeutic efforts directed exclusively at restoring neurotrophic activity are unlikely to meet with success. The molecular species responsible for neuroinhibitory effects in the Alzheimer's disease brain are beginning to be elucidated.

The notochord constitutes the main axial support during the embryonic and larval stages, and the arrangement of collagen fibrils within the notochord sheath is assumed to play a decisive role in determining its functional properties as a fibre-wound hydrostatic skeleton. We have found that during early ontogeny in Atlantic salmon stepwise changes occur in the configuration of the collagen fibre-winding of the notochord sheath. The sheath consists of a basal lamina, a layer of type II collagen, and an elastica externa that delimits the notochord; and these constituents are secreted in a specific order. Initially, the collagen fibrils are circumferentially arranged perpendicular to the longitudinal axis, and this specific spatial fibril configuration is maintained until hatching when the collagen becomes reorganized into distinct layers or lamellae. Within each lamella, fibrils are parallel to each other, forming helices around the longitudinal axis of the notochord, with a tangent angle of 75-80 degrees to the cranio-caudal axis. The helical geometry shifts between adjacent lamellae, forming enantiomorphous left- and right-handed coils, respectively, thus enforcing the sheath. The observed changes in the fibre-winding configuration may reflect adaptation of the notochord to functional demands related to stage in ontogeny. When the vertebral bodies initially form as chordacentra, the collagen lamellae of the sheath in the vertebral region are fixed by the deposition of minerals; in the intervertebral region, however, they represent a pre-adaptation providing torsional stability to the intervertebral joint. Hence, these modifications of the sheath transform the notochord per se into a functional vertebral column. The elastica externa, encasing the notochord, has serrated surfaces, connected inward to the type II collagen of the sheath, and outward to type I collagen of the mesenchymal connective tissue surrounding the notochord. In a similar manner, the collagen matrix of the neural and haemal arch cartilages is tightly anchored to the outward surface of the elastic membrane. Hence, the elastic membrane may serve as an interface between the notochord and the adjacent structures, with an essential function related to transmission of tensile forces from the musculature. The interconnection between the notochord and the myosepta is discussed in relation to function and to evolution of the arches and the vertebra. Contrary to current understanding, this study also shows that notochord vacuolization does not result in an increased elongation of the embryo, which agrees with the circular arrangement of type II collagen that probably only enables a restricted increase in girth upon vacuolization, not aiding elongation. As the vacuolization occurs during the egg stage, this type of collagen disposition, in combination with an elastica externa, also probably facilitates flexibility and curling of the embryo.

During the last century, experiments on the chick embryo established that the ballooning expansion of the early forebrain and midbrain vesicles is dependent on the underlying axial (notochordal) mesoderm. Transient separation of the early midbrain primordium from the notochord causes subsequent collapse of both midbrain and forebrain (telencephalic) vesicles, accompanied by pronounced folding of the neural epithelium. More recent experiments have shown that vesicle collapse is caused by defective Sonic Hedgehog (Shh) signaling from the notochord and floor plate. Separation of the notochord from the brain causes loss of ventral Shh expression, resulting in reduced cell proliferation and increased cell death in the expanding neural epithelium, and culminating in vesicle collapse. These experiments are reviewed here, and set in the context of other studies illustrating the wide range of molecular and cellular processes that cause abnormal brain morphogenesis when perturbed. We also speculate that variation in the regulation of signaling pathways such as Hedgehog may have played a significant part in generating rapid morphogenetic changes during the evolution of the vertebrate brain., ((c) 2005 Wiley Periodicals, Inc.)

During chick gastrulation, inhibition of BMP signaling is required for primitive streak formation and induction of Hensen's node. We have identified a unique secreted protein, Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family and is expressed in the primitive streak and Hensen's node. Grafts of cells expressing TSK in combination with the middle primitive streak induce an ectopic Hensen's node, while electroporation of TSK siRNA inhibits induction of the node. In Xenopus embryos, TSK can block BMP function and induce a secondary dorsal axis, while it can dorsalize ventral mesoderm and induce neural tissue in embryonic explants. Biochemical analysis shows that TSK binds directly to both BMP and chordin and forms a ternary complex with them. These observations indicate that TSK is an essential dorsalizing factor involved in the induction of Hensen's node.

The vertebrates are defined by their segmented vertebral column, and vertebral periodicity is thought to originate from embryonic segments, the somites. According to the widely accepted 'resegmentation' model, a single vertebra forms from the recombination of the anterior and posterior halves of two adjacent sclerotomes on both sides of the embryo. Although there is supporting evidence for this model in amniotes, it remains uncertain whether it applies to all vertebrates. To explore this, we have investigated vertebral patterning in the zebrafish. Surprisingly, we find that vertebral bodies (centra) arise by secretion of bone matrix from the notochord rather than somites; centra do not form via a cartilage intermediate stage, nor do they contain osteoblasts. Moreover, isolated, cultured notochords secrete bone matrix in vitro, and ablation of notochord cells at segmentally reiterated positions in vivo prevents the formation of centra. Analysis of fss mutant embryos, in which sclerotome segmentation is disrupted, shows that whereas neural arch segmentation is also disrupted, centrum development proceeds normally. These findings suggest that the notochord plays a key, perhaps ancient, role in the segmental patterning of vertebrae.

During development of the amniote peripheral nervous system, the initial trajectory of primary sensory axons is determined largely by the action of axon repellents. We have shown previously that tissues flanking dorsal root ganglia, the notochord lying medially and the dermamyotomes lying laterally, are sources of secreted molecules that prevent axons from entering inappropriate territories. Although there is evidence suggesting that SEMA3A contributes to the repellent activity of the dermamyotome, the nature of the activity secreted by the notochord remains undetermined. We have employed an expression cloning strategy to search for axon repellents secreted by the notochord, and have identified SEMA3A as a candidate repellent. Moreover, using a spectrum of different axon populations to assay the notochord activity, together with neuropilin/Fc receptor reagents to block semaphorin activity in collagen gel assays, we show that SEMA3A probably contributes to notochord-mediated repulsion. Sympathetic axons that normally avoid the midline in vivo are also repelled, in part, by a semaphorin-based notochord activity. Although our results implicate semaphorin signalling in mediating repulsion by the notochord, repulsion of early dorsal root ganglion axons is only partially blocked when using neuropilin/Fc reagents. Moreover, retinal axons, which are insensitive to SEMA3A, are also repelled by the notochord. We conclude that multiple factors act in concert to guide axons in this system, and that further notochord repellents remain to be identified.

The mechanisms that coordinate the three-dimensional shape of the vertebrate brain during development are largely unknown. We have found that sonic hedgehog (Shh) is crucial in driving the rapid, extensive expansion of the early vesicles of the developing midbrain and forebrain. Transient displacement of the notochord from the midbrain floor plate resulted in abnormal folding and overall collapse of the vesicles, accompanied by reduced cell proliferation and increased cell death in the midbrain. Simultaneously, expression of Shh decreased locally in the notochord and floor plate, whereas overt patterning and differentiation proceeded normally. Normal midbrain expansion was restored by implantation of Shh-secreting cells in a dose-dependent manner; conversely, expansion was retarded following antagonism of the Shh signaling pathway by cyclopamine. Our results indicate that Shh signaling from the ventral midline is essential for regulating brain morphogenesis during early development.

The backbone or vertebral column is the defining feature of vertebrates and is clearly metameric. Given that vertebrae arise from segmented paraxial mesoderm in the embryo, this metamerism is not surprising. Fate mapping studies in a variety of species have shown that ventromedial sclerotome cells of the differentiated somite contribute to the developing vertebrae and ribs. Nevertheless, extensive studies in amniote embryos have produced conflicting data on exactly how embryonic segments relate to those of the adult. To date, much attention has focused on the derivatives of the somites, while relatively little is known about the contribution of other tissues to the formation of the vertebral column. In particular, while it is clear that signals from the notochord induce and maintain proliferation of the sclerotome, and later promote chondrogenesis, the role of the notochord in vertebral segmentation has been largely overlooked. Here, we review the established role of the notochord in vertebral development, and suggest an additional role for the notochord in the segmental patterning of the vertebral column.

Here we present ideas connecting the behaviour of the cranial neural crest during development with the venerable, perhaps incorrect, view that gill-supporting cartilages of an ancient agnathan evolved into the skeleton of an early gnathostome's jaw. We discuss the pattern of migration of the cranial neural crest ectomesenchyme in zebrafish, along with the subsequent arrangement of postmigratory crest and head mesoderm in the nascent pharyngeal segments (branchiomeres), in diverse gnathostomes and in lampreys. These characteristics provide for a plausible von Baerian explanation for the problematic inside-outside change in topology of the gills and their supports between these 2 major groups of vertebrates. We consider it likely that the jaw supports did indeed arise from branchiomeric cartilages.

In higher vertebrates, the segmental organization of peripheral spinal nerves is established by a repulsive mechanism whereby sensory and motor axons are excluded from the posterior half-somite. A number of candidate axon repellents have been suggested to mediate this barrier to axon growth, including Sema3A, Ephrin-B, and peanut agglutinin (PNA)-binding proteins. We have tested the candidacy of these factors in vitro by examining their contribution to the growth cone collapse-inducing activity of somite-derived protein extracts on sensory, motor, and retinal axons. We find that Sema3A is unlikely to play a role in the segmentation of sensory or motor axons and that Ephrin-B may contribute to motor but not sensory axon segmentation. We also provide evidence that the only candidate molecule(s) that induces the growth cone collapse of both sensory and motor axons binds to PNA and is not Sema3A or Ephrin-B. By grafting primary sensory, motor, and quail retinal neurons into the chick trunk in vivo, we provide further evidence that the posterior half-somite represents a universal barrier to growing axons. Taken together, these results suggest that the mechanisms of peripheral nerve segmentation should be considered in terms of repellent molecules in addition to the identified molecules., (Copyright 2000 Academic Press.)

Neuropilin-1 (NP-1) is a component of the receptor for semaphorin3a (Sema3a), a member of a large family of molecules with widespread expression and demonstrable influence (via their ability to repel growing axons) on nervous system development. Recent studies have shown that some types of adult mammalian neurons retain the capacity to respond to Sema3a, particularly in relation to neuronal injury and regeneration. Although variations in expression of Sema3a mRNA have been revealed in neurons in both the central and peripheral nervous systems in this context, relatively little is known about NP-1 expression patterns. In this study we investigated the expression of NP-1 mRNA in adult dorsal root ganglion (DRG) neurons in intact and lesioned animals. We compared the effect of unilateral lesioning of the sciatic nerve or unilateral dorsal rhizotomy at lumbar levels L4/5, and bilateral dorsal funiculus lesioning at thoracic levels T10/11 on NP-1 mRNA expression in the cell bodies of lumbar DRGs. A significantly increased level of NP-1 mRNA expression was detected only following sciatic nerve lesioning (P < 0.001), but not after rhizotomy or dorsal funiculus lesioning. Furthermore, this upregulation was mainly confined to large diameter neurons of DRGs at lumbar levels L4/5, which provide the main sensory contribution to the sciatic nerve. These results suggest a role for NP-1 in the axonal response to peripheral nerve injury, which may be specific to a particular subset of primary sensory neurons., (Copyright 2000 Wiley-Liss, Inc.)

The secreted glycoprotein Sonic hedgehog (SHH), a vertebrate homologue of the Drosophila segment polarity gene Hedgehog, is essential for the development of diverse tissues during embryogenesis. Studies of SHH function during neural tube and somite development have focused on its role in specifying the dorsoventral polarity of these structures, but a recent report by Ahlgren and Bronner-Fraser(1) supports the possibility that SHH has additional functions in cell survival and cell proliferation. Perturbation of SHH signaling after the early dorsoventral specification of the cranial neural tube leads to increased cell death in both the neural tube and the neural crest. This implies that SHH is continually required as a trophic and/or mitogenic factor during brain development, and expands the variety of cellular responses to SHH signaling. BioEssays 22:499-502, 2000., (Copyright 2000 John Wiley & Sons, Inc.)

The study of spinal nerve trajectories in higher vertebrate embryos has revealed an inherent polarity within somites along the antero-posterior axis, and provides a simple system in which to study the factors that influence axon pathfinding. We argue that the orientation of spinal axons is determined by the simultaneous operation of two distinct guidance mechanisms, contact repulsion and chemorepulsion. Motor and sensory axons traverse the anterior half of each somite because they are excluded by contact repulsion from the posterior half-somite, and the molecular nature of several candidate contact repellents is reviewed. In contrast, we find that the dorsoventral trajectory of primary sensory axons is oriented by diffusible repellents originating from the notochord medially and dermamyotome laterally. In this system, therefore, repulsion by surrounding tissues ('surround-repulsion') is the main force directing axon growth in three dimensions.

Recent evidence suggests that the oligodendrocyte cell loss observed in multiple sclerosis sufferers is in part mediated by apoptosis. Here we use a human cell line, MO3.13, as a model system to investigate the biochemical processes involved in oligodendroglial cell death. Treatment with staurosporine kills both naive and differentiated cells in a dose-dependent manner; however, much higher concentrations of staurosporine are required to kill differentiated cells compared to their naive progenitors. Dying cells displayed the typical morphological characteristics of apoptosis, including cell shrinkage and chromatin condensation. Biochemical analysis showed that caspases, a group of enzymes intimately involved in the execution of apoptosis, are activated in both naive and differentiated cells. Western blotting analysis revealed that similar subsets of caspase enzymes were operating and that the substrate cleavage patterns were identical in both naive and differentiated cells. Treatment of MO3.13 cells with the general caspase inhibitor zVAD-FMK protected them from toxin-induced cell death. These results indicate that when an oligodendroglial human cell line is exposed to toxin it dies in an apoptotic manner. In addition, we show that cells can be protected from toxin-induced death using an appropriate inhibitor., (Copyright 1999 Wiley-Liss, Inc.)

During development of the vertebrate visual system, retinal ganglion cell (RGC) axons follow a precise path toward their midbrain targets. Although much is known about the cues that direct RGC axons once they have left the optic disc, less is known about the guidance of axons at earlier stages, when RGCs first send out their axons to navigate within the developing retina. Using collagen gel coculture experiments, we find that the embryonic lens produces a powerful diffusible repulsive activity for RGC axons. We also find that this activity is localized to the lens epithelium and not the lens fiber layer, while the pigmented epithelium and vitreous humour are devoid of activity. The further observation that the lens also chemorepels primary sensory axons, but does not repel olfactory bulb axons, shows that this activity is specific for subsets of axons. Our experiments have excluded two candidate repellents for RGC axons (collapsin-1/sema III and chondroitin sulfate proteoglycans). These results implicate the lens in the earliest stages of RGC axon guidance. One function of the lens repellent may be to prevent aberrant targeting toward the lens, and it may also be involved in the directional guidance of RGC axons toward the optic disc., (Copyright 1999 Academic Press.)

In the voltage-gated ion channels of every animal, whether they are selective for K+, Na+ or Ca2+, the voltage sensors are the S4 transmembrane segments carrying four to eight positive charges always separated by two uncharged residues. It is proposed that they move across the membrane in a screw-helical fashion in a series of three or more steps that each transfer a single electronic charge. The unit steps are stabilized by ion pairing between the mobile positive charges and fixed negative charges, of which there are invariably two located near the inner ends of segments S2 and S3 and a third near the outer end of either S2 or S3. Opening of the channel involves three such steps in each domain.

A model of the voltage-gated sodium channel is put forward suggesting that the four S4 voltage-sensors behave as screw-helices making a series of discrete transitions that carry one elementary charge for each notch of the screw helix. After the channel has been activated by the first two steps R in equilibrium with P in equilibrium with A in all four domains, followed by a voltage-independent rearrangement, it is opened by a third cooperative step A in equilibrium with B in domains I, II and III in conjunction with hydration. Inactivation is a voltage-dependent process controlled by the third step A in equilibrium with I in sensor IVS4, and the closing of the channel is brought about its dehydration. From the inactivated steady state the channel may be reopened by a fourth step, I in equilibrium with C in sensor IVS4 and rehydration. The computed kinetics of the model are shown to conform closely with those observed experimentally.