Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments

Abstract

The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, betaIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and betaIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. betaIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.

Figure 1.

Na_v1.6, ankyrin-G, βIV spectrin, neurofascin, and NrCAM are targeted to axon initial segments of cerebellar Purkinje neurons. Sections of adult rat cerebellum were triple labeled with antibodies against calbindin (blue), ankyrin-G (green), and either Na_v1.6 (B), βIV spectrin (E), neurofascin (H), or NrCAM (K) (red). Composite images are shown in C, F, I, and L. Arrowheads indicate Purkinje cell initial segments. Bars, 10 μm.

Figure 4.

Targeting of βIV spectrin, Na_v1.6, NrCAM, and neurofascin to Purkinje neuron initial segments is disrupted in ankyrin-G cerebellum- specific knockout mice. Cerebellar sections from wild-type (A, D, G, J, and M) or cerebellar ankyrin-G knockout mice (B, C, E, F, H, I, K, L, N, and O) were double labeled with antibodies against calbindin (red in A–F; green in G–L) and either ankyrin-G (green in A–C), βIV spectrin (green in D–F), Na_v1.6 (red in G–I), NrCAM (red in J–L), or neurofascin (M–O). All images except M–O are composites. Bars: (A, B, and E) 5 μm; (C, D, F, G, I, and J–O) 10 μm; and (H) 25 μm.

Figure 2.

Targeting of ankyrin-G and βIV spectrin to Purkinje neuron initial segments precedes Na_v1.6 and neurofascin during development. Sections of P2 rat cerebellum were triple labeled with the same antibodies as described in the legend to Fig. 1. D-I show initial segments containing punctate Na_v1.6 (E) or neurofascin (H). D′–I′ show initial segments that do not contain Na_v1.6 (E′) or neurofascin (H′). Arrowheads mark Purkinje neuron axon initial segments. Bars, 10 μm.

Figure 3.

Na_v1.6 and neurofascin are clustered throughout the entire axon initial segment of Purkinje neurons by postnatal day 9. Sections of P9 rat cerebellum were triple labeled with the same antibodies as described in the legend to Fig. 1. Arrowheads indicate Purkinje cell initial segments. Bars, 10 μm.