Accumulation Of Sodium Channels Research Articles

Structural and Functional Refinement of the Axon Initial Segment in Avian Cochlear Nucleus during Development.

The axon initial segment (AIS) is involved in action potential initiation. Structural and biophysical characteristics of the AIS differ among cell types and/or brain regions, but the underlying mechanisms remain elusive. Using immunofluorescence and electrophysiological methods, combined with super-resolution imaging, we show in the developing nucleus magnocellularis of the chicken in both sexes that the AIS is refined in a tonotopic region-dependent manner. This process of AIS refinement differs among cells tuned to different frequencies. At hearing onset, the AIS was ∼50 µm long with few voltage-gated sodium channels regardless of tonotopic region. However, after hatching, the AIS matured and displayed an ∼20-µm-long structure with a significant enrichment of sodium channels responsible for an increase in sodium current and a decrease in spike threshold. Moreover, the shortening was more pronounced, while the accumulation of channels was not, in neurons tuned to higher frequency, creating tonotopic differences in the AIS. We conclude that AIS shortening is mediated by disassembly of the cytoskeleton at the distal end of the AIS, despite intact periodicity of the submembranous cytoskeleton across the AIS. Importantly, deprivation of afferent input diminished the shortening in neurons tuned to a higher frequency to a larger extent in posthatch animals, with little effect on the accumulation of sodium channels. Thus, cytoskeletal reorganization and sodium channel enrichment at the AIS are differentially regulated depending on tonotopic region, but work synergistically to optimize neuronal output in the auditory nucleus.SIGNIFICANCE STATEMENT The axon initial segment (AIS) plays fundamental roles in determining neuronal output. The AIS varies structurally and molecularly across tonotopic regions in avian cochlear nucleus. However, the mechanism underlying these variations remains unclear. The AIS is immature around hearing onset, but becomes shorter and accumulates more sodium channels during maturation, with a pronounced shortening and a moderate channel accumulation at higher tonotopic regions. Afferent input adjusts sodium conductance at the AIS by augmenting AIS shortening (via disassembly of cytoskeletons at its distal end) specifically at higher-frequency regions. However, this had little effect on channel accumulation. Thus, cytoskeletal structure and sodium channel accumulation at the AIS are regulated differentially but work synergistically to optimize the neuronal output.

Loss of Neurofascin-186 Disrupts Alignment of AnkyrinG Relative to Its Binding Partners in the Axon Initial Segment

The axon initial segment (AIS) is a specialized region within the proximal portion of the axon that initiates action potentials thanks in large part to an enrichment of sodium channels. The scaffolding protein ankyrinG (AnkG) is essential for the recruitment of sodium channels as well as several other intracellular and extracellular proteins to the AIS. In the present study, we explore the role of the cell adhesion molecule (CAM) neurofascin-186 (NF-186) in arranging the individual molecular components of the AIS in cultured rat hippocampal neurons. Using a CRISPR depletion strategy to ablate NF expression, we found that the loss of NF selectively perturbed AnkG accumulation and its relative proximal distribution within the AIS. We found that the overexpression of sodium channels could restore AnkG accumulation, but not its altered distribution within the AIS without NF present. We go on to show that although the loss of NF altered AnkG distribution, sodium channel function within the AIS remained normal. Taken together, these results demonstrate that the regulation of AnkG and sodium channel accumulation within the AIS can occur independently of one another, potentially mediated by other binding partners such as NF.

Ankyrin G restricts ion channel diffusion at the axonal initial segment before the establishment of the diffusion barrier

In mammalian neurons, the precise accumulation of sodium channels at the axonal initial segment (AIS) ensures action potential initiation. This accumulation precedes the immobilization of membrane proteins and lipids by a diffusion barrier at the AIS. Using single-particle tracking, we measured the mobility of a chimeric ion channel bearing the ankyrin-binding motif of the Nav1.2 sodium channel. We found that ankyrin G (ankG) limits membrane diffusion of ion channels when coexpressed in neuroblastoma cells. Site-directed mutants with decreased affinity for ankG exhibit increased diffusion speeds. In immature hippocampal neurons, we demonstrated that ion channel immobilization by ankG is regulated by protein kinase CK2 and occurs as soon as ankG accumulates at the AIS of elongating axons. Once the diffusion barrier is formed, ankG is still required to stabilize ion channels. In conclusion, our findings indicate that specific binding to ankG constitutes the initial step for Nav channel immobilization at the AIS membrane and precedes the establishment of the diffusion barrier.

Protein kinase CK2 contributes to the organization of sodium channels in axonal membranes by regulating their interactions with ankyrin G

In neurons, generation and propagation of action potentials requires the precise accumulation of sodium channels at the axonal initial segment (AIS) and in the nodes of Ranvier through ankyrin G scaffolding. We found that the ankyrin-binding motif of Nav1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126). We showed that phosphorylation of these residues by protein kinase CK2 (CK2) regulates Nav channel interaction with ankyrins. Furthermore, we observed that CK2 is highly enriched at the AIS and the nodes of Ranvier in vivo. An ion channel chimera containing the Nav1.2 ankyrin-binding motif perturbed endogenous sodium channel accumulation at the AIS, whereas phosphorylation-deficient chimeras did not. Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons. In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

Ionic channels and neuropathic pain: Phisiopatology and applications

Neuropathic pain is defined by the International Association for Pain research as a pain associated to a primary lesion or a dysfunction of the central or peripheral system. Over the past few years the causes of the neuropathic pain were not known and there were not good treatments for it, now we have a better knowledge of the physiopathological aspects and there is a wider diffusion of the research for target aimed therapies. The physiologic genesis of nervous messages occurs exclusively in skin sensorial endings or in nerve tissues as a consequence of an adequate sensorial stimulus and depends on the quick variations of the electric potential difference at the endings of ionic membranes. These variations of even 500 V a second are possible because of the presence of ionic channels. In neuropathic pain impulses can be originated even from ectopic sites. Ectopic discharges originated in a peripheral neuropathic system have an important role in the early stage of neuropathic pain development in two different ways. First they give an excess of spontaneous and evoked electric impulses to the central nervous system, causing a primitive neuropathic pain signal; then the ectopic activity develops and maintains the central sensitisation process. All this amplifies the afferent signals deriving from residual efferents that go on innerving cutaneous areas damaged and partly disnerved, causing tactile allodynie. Sodium channels are the greatest responsible for electrogenesis, that is the basis of the action potential generation and its propagation. Action potential begins after a depolarization such that it could cause a membrane transitory modification, turning prevalently permeable to Na+ more than to K+ as during a release phase. Neuropathy generates a local accumulation of sodium channels, with a consequent increase of density. This remodel seems to be the basis of neuro hyperexecitably. Calcium channels have also an important role in cell working. Intracellular calcium increase contributes to depolarization processes, through kinase and determines the phosphorylation of membrane proteins that can make powerful the efficacy of the channels themselves. In the future new diagnostic opportunities of physiopathologist mechanism leading to neuropathic pain will allow treatments aimed at specific molecular changes of ionic channels.

Sodium Channel Blockers in Neuropathic Pain

Subtypes of tetrodotoxin resistant voltage-gated sodium channels are involved in the development of certain types of neuropathic pains. After nerve injury hyperexcitability and spontaneous firing develop at the site of injury and also in the dorsal root ganglion cell bodies. This hyperexcitability results at least partly from accumulation of sodium channels at the site of injury. The facts that these sodium channels seem to exist in peripheral nerves only and that they can be blocked at the resting state (use-dependent block) offer the possibility to develop drugs, which selectively block these damaged, overexcited nerves. At the moment no such drugs are available. However, some of the most potent drugs that are currently used to manage neuropathic pain e.g. amitriptyline and other tricyclic antidepressants, also block these channels in addition to having several other mechanisms of action. Also most anticonvulsants that are used to alleviate neuropathic pain are sodium channel blockers. Lidocaine, the prototype drug, has been shown to be effective in peripheral neuropathic pain. Its use is limited by the fact that it cannot be administered orally. An oral local anesthetic type sodium channel blocker, mexiletine is an antiarrhythmic agent that is effective in neuropathic pain. However, effective doses may be difficult to achieve because of adverse effects.

Novel clustering of sodium channel Na v1.1 with ankyrin-G and neurofascin at discrete sites in the inner plexiform layer of the retina

Voltage-gated sodium channels cluster at sites of action potential generation and propagation by interacting with partner proteins such as neurofascin, an adhesion molecule in the L1 family, and ankyrin-G, a spectrin-binding protein required for sodium channel accumulation at axon initial segments. Here, we describe in the inner plexiform layer of the retina a novel site of high-density sodium channel clustering, marked by ankyrin-G and neurofascin. The sodium channel isoform at this site is Na v1.1, instead of the Na v1.6 channels more commonly found in association with the clustering machinery. During development, Na v1.2 channels first associate with ankyrin-G in the inner plexiform layer but are later replaced by Na v1.1, similar to the switch from Na v1.2 to Na v1.6 at nodes of Ranvier and initial segments. This represents the first instance of high-density clustering of Na v1.1 channels, which may contribute to synaptic interactions among retinal neurons in the inner plexiform layer.

Accumulation of PN1 and PN3 sodium channels in painful human neuroma-evidence from immunocytochemistry.

The axolemmal distribution and density of voltage-gated sodium channels largely determines the electrical excitability of sprouting neurites. Recent evidence suggests that accumulation of sodium channels at injured axonal tips may be responsible for ectopic axonal hyperexcitability and the resulting abnormal sensory phenomena of pain and paresthesias. For future improvement in pain management it is necessary to identify structurally significant generators of autorhythmicity. A first step in this regard will be to determine the predominant types of sodium channels in injured axons. The opportunity to test human specimens from painful and non-painful neuroma is of great value. We employed immunocytochemical methods to investigate if two types of highly specific voltage-gated sodium channel subtypes could be detected in sections of human neuroma. Both subtypes of sodium channels PN1 and PN3 accumulated abnormally in human neuromas. The immunoreactive pattern was more pronounced in painful neuromas. This is in contrast to previous reports that focused either on PN1 or PN3 as main generators of hyperexcitability induced pain. Both, PN1 and PN3 seem to be involved in hyperexcitability induced pain. It can be expected that a variety of other highly specific voltage gated sodium channel subtypes will be detected in regenerating peripheral nerve in the near future, which contribute to the development of neuropathic pain states. Thus, in order to therapeutically control hyperexcitability induced neuropathic pain, it might be worthwhile to develop pharmaceuticals that can selectively block different sodium channel subtypes and subunits.A review of the role of sodium channels in neuropathic pain is implemented in the discussion.

Mechanism-based treatment principles of neuropathic pain

Traditionally, neuropathic pain has been classified due to aetiology of nerve damage-traumatic, inflammatory or metabolic, for instance. Based on this classification, pain therapy often is insufficient. Recent research revealed different mechanisms, which are responsible for the generation of pain after nerve lesion. These mechanisms seem to be independent of aetiology of the nerve damage. The most important mechanisms are accumulation of sodium channels on injured nerves, pathological sympatho-afferent coupling, disinhibition of nociception and central or peripheral nociceptive sensitisation. Each individual mechanism could be treated specifically by current available drugs, or by non-drug therapy. However, future research has to focus on exploring tools to recognise individual pain mechanisms in single patients. Thereby treatment will become more effective.

Sodium channels and pain.

Although it is well established that hyperexcitability and/or increased baseline sensitivity of primary sensory neurons can lead to abnormal burst activity associated with pain, the underlying molecular mechanisms are not fully understood. Early studies demonstrated that, after injury to their axons, neurons can display changes in excitability, suggesting increased sodium channel expression, and, in fact, abnormal sodium channel accumulation has been observed at the tips of injured axons. We have used an ensemble of molecular, electrophysiological, and pharmacological techniques to ask: what types of sodium channels underlie hyperexcitability of primary sensory neurons after injury? Our studies demonstrate that multiple sodium channels, with distinct electrophysiological properties, are encoded by distinct mRNAs within small dorsal root ganglion (DRG) neurons, which include nociceptive cells. Moreover, several DRG neuron-specific sodium channels now have been cloned and sequenced. After injury to the axons of DRG neurons, there is a dramatic change in sodium channel expression in these cells, with down-regulation of some sodium channel genes and up-regulation of another, previously silent sodium channel gene. This plasticity in sodium channel gene expression is accompanied by electrophysiological changes that poise these cells to fire spontaneously or at inappropriate high frequencies. Changes in sodium channel gene expression also are observed in experimental models of inflammatory pain. Thus, sodium channel expression in DRG neurons is dynamic, changing significantly after injury. Sodium channels within primary sensory neurons may play an important role in the pathophysiology of pain.

Sodium channel accumulation in humans with painful neuromas.

Painful neuromas from 16 patients were examined using site-specific antisodium channel antibodies employed in immunocytochemical and radioimmunoassay methods. Normal sural nerves from six of these patients served as controls. Immunocytochemistry showed abnormal segmental accumulation of sodium channels within many axons in the neuromas. Dens immunolocalization was especially apparent within the axonal tips. Radioimmunoassay confirmed a significantly greater density of sodium channels in the neuromas as compared with the sural nerves. Thus, sodium channel accumulate abnormally within the axons of neuromas in humans. This alteration of the sodium channels may underlie the generation of axonal hyperexcitability and the resulting abnormal sensory phenomena (pain and paresthesias), which frequently occur after peripheral nerve injury.

Sodium channels accumulate at the tips of injured axons.

The axolemmal distribution of voltage-gated sodium channels largely determines the regions of axonal electrical excitability. Using a well-characterized anti-sodium channel antibody, we examined peripheral nerve fibers focally injured by exposure to the neurotoxic agent, potassium tellurite (K2TeO3). Immunocytochemical and radioimmunoassay data showed a focal accumulation of sodium channels within the tips of injured axons. The major increase in sodium channel concentration occurred between 7 and 11 days after toxin exposure; however, immunocytochemically, excess sodium channels persisted in several axonal endings for a much longer time. The accumulation of sodium channels at injured axonal tips may be responsible, in part, for ectopic axonal excitability and the resulting abnormal sensory phenomena (especially pain and paresthesias) which frequently complicate peripheral nerve injury in humans.