Identification of a Conserved Ankyrin-binding Motif in the Family of Sodium Channel α Subunits

Abstract

Interactions with ankyrin G are crucial to the localization of voltage-gated sodium channels (VGSCs) at the axon initial segment and for neurons to initiate action potentials. However, the molecular nature of these interactions remains unclear. Here we report that VGSC-␣, but not -␤, subunits bind to ankyrin G using pull-down assays. Further dissection of this activity identifies a conserved 9-amino acid motif ((V/A)P(I/L)AXXE(S/D)D) required for ankyrin G binding. This motif is also required for the localization of chimeric neurofascin/sodium channel molecules to the initial segment of cultured hippocampal neurons. The conserved nature of this motif suggests that it functions to localize sodium channels to a variety of "excitable" membrane domains both inside and outside of the nervous system.The concentration of voltage-gated sodium channels (VGSCs) 1 into excitable membrane domains is crucial to information processing and transmission in the nervous system and to excitation/contraction coupling in muscle. Localized concentrations of VGSCs at the initial segment (IS) and the nodes of Ranvier are necessary for the initiation and propagation of action potentials through myelinated axons. However, mechanisms underlying the localization of VGSCs to these excitable membrane domains remain relatively unknown. In neurons, VGSCs exist as heterotrimers composed of a large pore-forming ␣ subunit associated with two smaller accessory ␤ subunits (reviewed in Ref. 1). At least 10 genes encoding putative ␣ subunits have been identified in mammals (reviewed in Ref.2) along with three ␤ subunit genes. ␤ subunits not only modulate the activity of ␣ subunits (3, 4) but also exhibit characteristics of cell adhesion molecules (CAMs) binding to extracellular matrix molecules such as tenascin-C and -R (5) and other CAMs such as neurofascin (6).Polarized localization of VGSCs in the axonal membrane requires interactions with members of the ankyrin family of peripheral membrane proteins (reviewed in Ref. 7). Ankyrins function as membrane-cytoskeleton adaptors that immobilize integral membrane proteins to the spectrin-based membrane skeleton. Ankyrins co-purify with and directly bind to purified VGSCs (8), and specific isoforms of the ankyrin G gene are concentrated with VGSCs at the IS (9), the nodes of Ranvier (9), and the neuromuscular junction (10). The functional importance of the interaction between ankyrin G and VGSCs is demonstrated in knockouts of ankyrin G in the mouse cerebellum. Purkinje cells from knockout animals are deficient in localized VGSC concentrations at the IS (11) and are unable to initiate action potentials (12).In addition to VGSCs, ankyrins also interact with a variety of other integral membrane proteins present at the node including the CAMs neurofascin and NrCAM (13,14). Interactions of ankyrins with VGSCs (15) and other integral membrane proteins are mediated through the N-terminal 90-kDa repeat or membrane-binding (MB) domain. The interaction between the ankyrin MB domain and neurofascin/NrCAM is depende...

Ankyrin-Dependent and -Independent Mechanisms Orchestrate Axonal Compartmentalization of L1 Family Members Neurofascin and L1/Neuron–Glia Cell Adhesion Molecule

“…The A/D PI of HA-NF FIGQA, in contrast, was 3.8 Ϯ 0.25 (mean Ϯ SEM), demonstrating significant axonal enrichment. Loss of IS localization of NF FIGQA was reported previously (Lemaillet et al, 2003), but axonal enrichment of the mutant was not noted. This might be attributable to the younger age of the neurons used in the other study.…”

Polarized localization of voltage-gated Na + channels is regulated by concerted FGF13 and FGF14 action

“…The cytoplasmic II-III linker within the pore-forming α-subunit of VGSCs contains a binding motif for the cytoskeletal scaffolding molecule ankyrin-G, and thereby serves as a critical determinant for localization within the AIS (10)(11)(12). Even though the ankyrin-G binding site in VGSCs is highly conserved between different VGSCs (13), their subcellular localization is not identical (6). This observation raises the question of whether there are additional regulators for the subcellular localization of VGSCs.…”

Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes

“…However, the most parsimonious interpretation of the data considered together is that Na v 1.5 associates with ankyrin-G and that ankyrin-G is required for cell surface expression of Na v 1.5 in cardiomyocytes. The ankyrin-G-binding motif (VPIAXX-ESD) is conserved among Na v 1.1, 1.2, 1.4, 1.5, and 1.6, and also is required for Na v channel targeting in both neurons (7,8). Therefore, an ankyrin-G-based mechanism for Na v channel targeting appears a conserved feature of both cardiomyocytes and neurons.…”