Clustering of voltage-gated ion channels as an evolutionary trigger of myelin formation

Henrike Ohm, Simone Rey, Christian Klämbt

Neurons carry apical dendrites that perceive information and a basal axon that transmits the computed information towards its targets.The axon originates at the axon hillock which is followed by the axon initial segment.Here,action potentials are initiated that are based on millisecond long openings of specific voltagegated sodium and potassium channels that are conserved in all parahoxozoa (Placozoa, Cnidaria,Bilateria) (Li et al., 2015).This indicates that the basic principles in action potential generation and spreading are evolutionarily conserved.The conductance velocity of action potentials likely affects the evolutionary success of any animal species as it contributes, for example, to the success of escape responses.Physical laws state that axonal transduction velocity depends on the size of the axon.Alternatively, conductance speed is gained by arranging voltage-gated ion channels in spatially separated clusters.Such a distribution is thought to be a defining feature of the vertebrate nervous system and accumulations of voltage-gated ion channels are seen at the axon initial segment and the nodes of Ranvier.Together with intervening myelin, this enables saltatory transduction, which allows very fast conduction velocities.Surprisingly, recent work demonstrated a clustered distribution of voltagegated ion channels in the nervous system of the invertebrateDrosophila melanogaster(Rey et al.,2023).Channels are enriched at the axon initial segments of motor- and sensory neurons, cluster on a molecular scale with spacing of about 0.7 µm,supporting micro-saltatory conductance.Similar to in vertebrates, the positioning of ion channels is influenced by glia.Moreover, glia in adult flies form myelin-like structures next to the axon initial segments (Rey et al., 2023).Thus, the evolution of saltatory conductance is not specific to vertebrates but likely started before the separation of vertebrates and invertebrates.

Voltage-gated sodium and potassium ion channels are needed to transmit information along axons.Upon a uniform distribution of these channels,action potentials spread continuously along the axon, meaning that they have to be generated at every part of the membrane.This is not only a relatively slow form of conductance but it also requires a large amount of energy.This signaling mechanism is thought to be the primary conduction mode in the invertebrate nervous system and because these animals are generally small, slow conduction velocities can be tolerated.However, there are very large invertebrate species known, for example, female giant squid(Architeuthis dux) can reach a body size of 13 m– but it is currently unknown how these animals control the movement of their tentacles.In addition, text books state that invertebrate axons generally show only simple glial wraps.However,there are sporadic reports that invertebrates are able to form myelin-like structures (See Rey et al.,2023 for references), the most dramatic example being copepods that form a myelin-like coverage from axonal membranes (Wilson and Hartline,2011).But the question remains, why should invertebrates have myelin if they uniformly position voltage-gated ion channels along the axon?Vertebrates, in contrast, are known to locally position voltage-gated ion channels to reach high conductance velocities.The channels localize at the axon initial segment and the nodes of Ranvier which enables saltatory transduction.Such local clustering of ion channels comes with three major advantages compared to axons with uniformly distributed ion channels.First, the number of voltage-gated ion channels that are required for successful signal conductance is significantly lower.Second, channel clustering reduces metabolic costs linked to the maintenance of ion homeostasis.Finally, saltatory conduction is much faster compared to continuous conduction,which may have allowed the evolution of large animals (Zalc and Colman, 2000).In fact, the gain in conductance velocity is so strong, that Schwann cells can afford to actively keep axonal diameter small (Eichel et al., 2020).

In vertebrates, clustering of voltage-gated ion channels at the axon initial segment and the nodes of Ranvier coincides with the occurrence of myelin, a compact glial membrane stack that is formed around large diameter axons.It provides efficient electrical insulation to the axon and,equally important, keeps axons in some distance from each other.Gaps in the myelin sheath, the nodes of Ranvier, are characterized by a high density of voltage-gated ion channels.Although clusters of voltage-gated ion channels can form in the absence of glia, myelin-forming glial cells are able to influence the positioning of these channels by a variety of extracellular signals.The chicken or egg question is whether these two properties“clustered channels” and “myelin” have evolved independently from each other, or, differently phrased, who appeared first.Our recent finding that voltage-gated ion channels also form clusters in Drosophila corroborates related reports on clustered voltage-gated ion channels in Aplysia(Rey et al., 2023).Thus, we postulate that during evolution, the clustering of voltage-gated ion channels developed first.

In Drosophila, the clustering of voltage-gated ion channels can be observed on two levels.First,channels are enriched at the axon initial segment of sensory as well as motor neurons (Rey et al.,2023).The length of the axon initial segments can vary depending on the neuronal subtype.For example, large caliber motor axons appear to have relatively short axon initial segments of about 50 µm length, whereas the axon initial segment of sensory neurons can reach 100 µm in length (Rey et al., 2023).The exact molecular determinants underlying this differential length of the axon initial segment are currently unknown.In addition to the local enrichment of voltage-gated ion channels along the length of the axon, we also noted a clustered distribution on the molecular scale(Figure 1A and B).Generally, individual channels or small groups of channels are separated by about 0.7 µm.Most importantly, the channels appear to be arranged in two distinct, opposing rows in motor axons which calls for specific cellular mechanisms that yet need to be identified in the future (Figure 1A and B).The channel clustering results in a gain of conduction speed.However, the local high density of voltage-gated ion channels results in a higher ion flow (= current) which generates a stronger electric field.A strong electric field might influence the gating probability of voltage-gated ion channels located on neighboring axons, causing the generation of an action potential here, too.This phenomenon is known as ephaptic coupling(Krnjevic, 1986).In some cases, ephaptic coupling is intended and beneficial, if a synchronized firing of multiple axons is required.However, it can also lead to unwanted signaling and thus impacts signaling precision (Kottmeier et al., 2020).Thus,the gain of conduction velocity comes along with the disadvantage of an elevated ephaptic coupling probability.To counteract this, axons have to be separated from each other, as the electric field decreases with the square of the distance.The generation of extracellular space not only physically separates axons to block ephaptic coupling, but it also helps to resolve the dilemma of ion homeostasis during action potential generation.

During the millisecond of voltage-gated ion channel opening setting off an action potential,about 104ions pass from the outside to the inside of the axon leading to a depolarization of the membrane potential (Milo and Phillips, 2016).Within the nervous system, individual axons and glial cells are generally only 20 nm apart and therefore the extracellular volume is dramatically small.Provided that a voltage-gated sodium channel would reside in an axonal area that is directly flanked by neighboring axons, the volume of a 100 nm radius would be:

The extracellular concentration of sodium ions is about 0.1 M, saying that 6.022 × 1022sodium ions are found in every liter or 1024nm3volume, or 6 sodium ions are found in a volume of 100 nm3.Thus, the number of sodium ions in this small volume covering a channel pore (4 × 104) is just enough to allow the generation of a few action potentials.Even if diffusion is able to replenish the ion concentration, sustained firing of action potentials appears difficult.

To solve this conundrum, invertebrates can form a lacunar system where glial cells establish a network of fine processes with a large extracellular space that embeds large caliber axons (Figure 1C).In adult flies we demonstrated that lacunae form next to axon segments enriched with clustered voltage-gated ion channels (Rey et al., 2023).The glial cells that mostly form these lacunar structures belong to the ensheathing or wrapping glial cell type similar to oligodendrocytes or Schwann cells.Their processes are characterized by a uniform width and often similar distances to each other.It will be interesting to see whether these processes express transporters and pumps needed to establish and maintain an ion reservoir.Within the lacunae, extracellular matrix depositions can be identified, which may further facilitate an ion storage or ion buffering function.Interestingly,ensheathing glial cells of the fly specifically express the enzymatic machinery needed to generate heparan or chondroitin sulfate proteoglycans(Pogodalla et al., 2021).These proteoglycans are able to bind ions and thus might contribute to ion storage.While in flies this has not yet been further analyzed, it is well known that perineuronal nets that are formed by chondroitin sulfate proteoglycans in the mammalian brain appear to act in ion buffering and participate in neuronal plasticity and function.Similarities between vertebrates and invertebrates may extend beyond the presence of related molecules used in ion homeostasis.Satellite glial cells form a sheath around frog dorsal root neurons which resemble the lacunar structures described in invertebrates.Moreover, Schwann cells cover the space above the node of Ranvier with numerous filopodia,again resembling the lacunar system (D’Este et al., 2017).In conclusion, both invertebrates and vertebrates need to regulate ion homeostasis during neuronal activity and thus the interstitial space and its buffering properties is highly relevant.In both animal clades, this relies on glial derived and possibly controlled extracellular volume filled with conserved ECM proteins.

In Drosophila, the formation of lacunae not only provides an ion reservoir but also spatially separates axon initial segments and thus, reduces the likelihood of ephaptic coupling among axonal segments rich in voltage-gated ion channels.When channel density decreases, a lacunar system is not needed anymore.In consequence, glial processes forming the lacunae collapse to form myelin-like membrane sheets that cover large caliber axons (Figure 1D).In contrast to the “real”myelin found in vertebrates, Drosophila myelin-like structures are not concentric around an axon but rather stacks of cell processes flap around axons.Moreover, myelin-like structures are found for only short stretches and are not found along the entire length of the axon.Here they may help on the one hand to stabilize the flanks of the lacunae by providing adhesive functions and on the other hand may participate in ensuring the correct localization of voltage-gated ion channels.In the absence of axon contacting glia, neurons localize these channels all over the axonal membrane and in fact upregulate the expression of voltagegated sodium channels (Rey et al., 2023).How glial cells are able to influence the expression of the respective mRNA or its stability remains unknown.In Drosophila, myelin-like structures appear as a consequence of having a lacunar system, and the lacunar system forms in regions with very high voltage-gated ion channel density.Thus, Drosophila development may depict the different stages of myelin evolution.Early on, during larval stages, the voltage-gated ion channel density is low.They still organize in clusters but no lacunae form.In the adult, voltage-gated ion channel density is very high and lacunae form which is accompanied by myelin-like structures at their distal end (Figure 1).The notion that the formation of voltage-gated ion channel clusters is evolutionarily older than the formation of myelin is also supported by the finding that unmyelinated axons of vertebrates show a node-like clustering (Pristerà et al., 2012).Moreover, channel clustering occurs prior to myelination.In mammals, clustering of voltagegated sodium channels is mediated by the ankyrin-G scaffolding protein.Although the anchor motif for sodium channel clustering evolved early in the chordate lineage and is absent in many invertebrates, including Drosophila, axon initial segments are still found in flies (Hill et al., 2008;Rey et al., 2023).

Invertebrates provided fruitful models in neuroscience in the past.Our understanding of neurogenesis, axon pathfinding, action potential electrophysiology of the neuron and even learning and memory greatly benefitted from work on flies,worms, squids, and sea snails.The elucidation of the distribution of voltage-gated ion channels and the formation of myelin-like structures now adds another facet to the picture of an amazingly conserved nervous system.

In conclusions, the recent work not only provides a new entry point to the dissection of action potential propagation but may also allow seeking for molecules that trigger myelin formation.Flies can in principle form myelin-like membrane stacks and modern gain as well as loss of function genetics in the future will help to identify genes relevant for myelin formation.

We are grateful to all our colleagues from Institut für Neuro-und Verhaltensbiologie for many discussions.

This work was supported by the Deutsche Forschungsgemeinschaftthrough funds to CK(SFB 1348,B5,Kl 588/29).

Henrike Ohm, Simone Rey,Christian Klämbt*

Institut für Neuro- und Verhaltensbiologie,Münster, Germany

Figure 1 ｜Clusters of voltage-gated ion channels in Drosophila and myelin formation.

*Correspondence to:Dr.Christian Klämbt,klaembt@uni-muenster.de.

https://orcid.org/0000-0002-6349-5800(Christian Klämbt)

Date of submission:August 25, 2023

Date of decision:October 12, 2023

Date of acceptance:October 26, 2023

Date of web publication:December 11, 2023

https://doi.org/10.4103/1673-5374.389636 How to cite this article:Ohm H,Rey S,Klämbt C(2024)Clustering of voltage-gated ion channels as an evolutionary trigger of myelin formation.Neural Regen Res 19(8):1631-1632.

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