CK2-regulated schwannomin-interacting protein IQCJ-SCHIP-1 association with AnkG contributes to the maintenance of the axon initial segment.

The axon initial segment (AIS) plays a central role in electrogenesis and in the maintenance of neuronal polarity. Its molecular organization is dependent on the scaffolding protein ankyrin (Ank) G and is regulated by kinases. For example, the phosphorylation of voltage-gated sodium channels by the protein kinase CK2 regulates their interaction with AnkG and, consequently, their accumulation at the AIS. We previously showed that IQ motif containing J-Schwannomin-Interacting Protein 1 (IQCJ-SCHIP-1), an isoform of the SCHIP-1, accumulated at the AIS in vivo. Here, we analyzed the molecular mechanisms involved in IQCJ-SCHIP-1-specific axonal location. We showed that IQCJ-SCHIP-1 accumulation in the AIS of cultured hippocampal neurons depended on AnkG expression. Pull-down assays and surface plasmon resonance analysis demonstrated that AnkG binds to CK2-phosphorylated IQCJ-SCHIP-1 but not to the non-phosphorylated protein. Surface plasmon resonance approaches using IQCJ-SCHIP-1, SCHIP-1a, another SCHIP-1 isoform, and their C-terminus tail mutants revealed that a segment including multiple CK2-phosphorylatable sites was directly involved in the interaction with AnkG. Pharmacological inhibition of CK2 diminished both IQCJ-SCHIP-1 and AnkG accumulation in the AIS. Silencing SCHIP-1 expression reduced AnkG cluster at the AIS. Finally, over-expression of IQCJ-SCHIP-1 decreased AnkG concentration at the AIS, whereas a mutant deleted of the CK2-regulated AnkG interaction site did not. Our study reveals that CK2-regulated IQJC-SCHIP-1 association with AnkG contributes to AIS maintenance. The axon initial segment (AIS) organization depends on ankyrin (Ank) G and kinases. Here we showed that AnkG binds to CK2-phosphorylated IQCJ-SCHIP-1, in a segment including 12 CK2-phosphorylatable sites. In cultured neurons, either pharmacological inhibition of CK2 or IQCJ-SCHIP-1 silencing reduced AnkG clustering. Overexpressed IQCJ-SCHIP-1 decreased AnkG concentration at the AIS whereas a mutant deleted of the CK2-regulated AnkG interaction site did not. Thus, CK2-regulated IQJC-SCHIP-1 association with AnkG contributes to AIS maintenance.

Tetrodotoxin-resistant voltage-gated sodium channel Nav 1.8 constitutively interacts with ankyrin G.

The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel Nav 1.8 is predominantly expressed in peripheral afferent neurons, but in case of neuronal injury an ectopic and detrimental expression of Nav 1.8 occurs in neurons of the CNS. In CNS neurons, Nav 1.2 and Nav 1.6 channels accumulate at the axon initial segment, the site of the generation of the action potential, through a direct interaction with the scaffolding protein ankyrin G (ankG). This interaction is regulated by protein kinase CK2 phosphorylation. In this study, we quantitatively analyzed the interaction between Nav 1.8 and ankG. GST pull-down assay and surface plasmon resonance technology revealed that Nav 1.8 strongly and constitutively interacts with ankG, in comparison to what observed for Nav 1.2. An ion channel bearing the ankyrin-binding motif of Nav 1.8 displaced the endogenous Nav 1 accumulation at the axon initial segment of hippocampal neurons. Finally, Nav 1.8 and ankG co-localized in skin nerves fibers. Altogether, these results indicate that Nav 1.8 carries all the information required for its localization at ankG micro-domains. The constitutive binding of Nav 1.8 with ankG could contribute to the pathological aspects of illnesses where Nav 1.8 is ectopically expressed in CNS neurons.

End-binding proteins EB3 and EB1 link microtubules to ankyrin G in the axon initial segment.

The axon initial segment (AIS) plays a key role in maintaining the molecular and functional polarity of the neuron. The relationship between the AIS architecture and the microtubules (MTs) supporting axonal transport is unknown. Here we provide evidence that the MT plus-end-binding (EB) proteins EB1 and EB3 have a role in the AIS in addition to their MT plus-end tracking protein behavior in other neuronal compartments. In mature neurons, EB3 is concentrated and stabilized in the AIS. We identified a direct interaction between EB3/EB1 and the AIS scaffold protein ankyrin G (ankG). In addition, EB3 and EB1 participate in AIS maintenance, and AIS disassembly through ankG knockdown leads to cell-wide up-regulation of EB3 and EB1 comets. Thus, EB3 and EB1 coordinate a molecular and functional interplay between ankG and the AIS MTs that supports the central role of ankG in the maintenance of neuronal polarity.

Endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment.

The axonal initial segment is a unique subdomain of the neuron that maintains cellular polarization and contributes to electrogenesis. To obtain new insights into the mechanisms that determine protein segregation in this subdomain, we analyzed the trafficking of a reporter protein containing the cytoplasmic II-III linker sequence involved in sodium channel targeting and clustering. Here, we show that this reporter protein is preferentially inserted in the somatodendritic domain and is trapped at the axonal initial segment by tethering to the cytoskeleton, before its insertion in the axonal tips. The nontethered population in dendrites, soma, and the distal part of axons is subsequently eliminated by endocytosis. We provide evidence for the involvement of two independent determinants in the II-III linker of sodium channels. These findings indicate that endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment of hippocampal neurons.

Voltage-gated sodium channel organization in neurons: protein interactions and trafficking pathways.

In neurons, voltage-gated sodium (Nav) channels underlie the generation and propagation of the action potential. The proper targeting and concentration of Nav channels at the axon initial segment (AIS) and at the nodes of Ranvier are therefore vital for neuronal function. In AIS and nodes, Nav channels are part of specific supra-molecular complexes that include accessory proteins, adhesion proteins and cytoskeletal adaptors. Multiple approaches, from biochemical characterization of protein-protein interactions to functional studies using mutant mice, have addressed the mechanisms of Nav channel targeting to AIS and nodes. This review summarizes our current knowledge of both the intrinsic determinants and the role of partner proteins in Nav targeting. A few fundamental trafficking mechanisms, such as selective endocytosis and diffusion/retention, have been characterized. However, a lot of exciting questions are still open, such as the mechanism of differentiated Nav subtype localization and targeting, and the possible interplay between electrogenesis properties and Nav concentration at the AIS and the nodes.

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 Na(v)1.2 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 Na(v) 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 Na(v)1.2 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.

Dynamic compartmentalization of the voltage-gated sodium channels in axons.

One of the major physiological roles of the neuronal voltage-gated sodium channel is to generate action potentials at the axon hillock/initial segment and to ensure propagation along myelinated or unmyelinated fibers to nerve terminal. These processes require a precise distribution of sodium channels accumulated at high density in discrete subdomains of the nerve membrane. In neurons, information relevant to ion channel trafficking and compartmentalization into sub-domains of the plasma membrane is far from being elucidated. Besides, whereas information on dendritic targeting is beginning to emerge, less is known about the mechanisms leading to the polarized distribution of proteins in axon. To obtain a better understanding of how neurons selectively target sodium channels to discrete subdomains of the nerve, we addressed the question as to whether any of the large intracellular regions of Nav1.2 contain axonal sorting and/or clustering signals. We first obtained evidence showing that addition of the cytoplasmic carboxy-terminal region of Nav1.2 restricted the distribution of a dendritic-axonal reporter protein to axons of hippocampal neurons. The analysis of mutants revealed that a di-leucine-based motif mediates chimera compartmentalization in axons and its elimination in soma and dendrites by endocytosis. The analysis of the others generated chimeras showed that the determinant conferring sodium channel clustering at the axonal initial segment is contained within the cytoplasmic loop connecting domains II-III of Nav1.2. Expression of a soluble Nav1.2 II-III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, it is conceivable that concerted action of the two determinants is required for sodium channel compartmentalization in axons.

A targeting motif involved in sodium channel clustering at the axonal initial segment.

The sorting of sodium channels to axons and the formation of clusters are of primary importance for neuronal electrogenesis. Here, we showed that the cytoplasmic loop connecting domains II and III of the Nav1 subunit contains a determinant conferring compartmentalization in the axonal initial segment of rat hippocampal neurons. Expression of a soluble Nav1.2II-III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, this motif may play a fundamental role in controlling electrical excitability during development and plasticity.