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

Abstract

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.

The potassium channel subunit K v β1 serves as a major control point for synaptic facilitation

“…Live-Cell Imaging. All live-imaging experiments were set up as previously described (26,60). Briefly, images were obtained using an Olympus microscope (IX-83) equipped with a 40× 1.35 numerical aperture (NA) oil immersion objective (UApoN40XO340-2) and captured with an IXON Ultra 897 EMCCD (Andor).…”

Neurofascin and Kv7.3 are delivered to somatic and axon terminal surface membranes en route to the axon initial segment

“…Neurofascin186 (Nfasc186) is a transmembrane protein with an essential role in maintaining the intactness of the AIS complex and in restricting AIS proteins to this specialized domain ( Alpizar et al, 2019 ; Boiko et al, 2007 ; Jenkins and Bennett, 2001 ; Zonta et al, 2011 ). Deletion of Nfasc186 in culture and in vivo causes the disintegration of the AIS with the loss of Nav, AnkG, βIV-Spectrin and Nr-CAM; the consequent disordered electrophysiology impairs motor learning ( Alpizar et al, 2019 ; Zonta et al, 2011 ).…”

The Ankyrin Repeat Domain Controls Presynaptic Localization of Drosophila Ankyrin2 and Is Essential for Synaptic Stability

“…The N-terminal ARD mediates membrane-association of Ankyrins and is essential for the subcellular localization and organization of transmembrane binding partners (Srinivasan et al, 1988; Davis and Bennett, 1994; Zhang et al, 1998; Devaux et al, 2003; Lemaillet et al, 2003; Kizhatil et al, 2007; Xu et al, 2007; Alpizar et al, 2019). Prior work largely focused on the organizational roles of giant Ankyrins at the AIS and nodes of Ranvier in vertebrate neurons.…”

“…Structurally, the ARD consists of 24 conserved ankyrin helix-turn-helix repeats that are each 33 amino acid (aa) long. Together, these repeats form a superhelical solenoid with an elongated conserved binding grove (Wang et al, 2014) that serves as a binding module for multiple, divergent transmembrane proteins including cell adhesion molecules (Davis and Bennett, 1994; Zhang et al, 1998; Kizhatil et al, 2007; Alpizar et al, 2019) and voltage gated sodium and Kv3.1 channels (Srinivasan et al, 1988; Devaux et al, 2003; Lemaillet et al, 2003; Xu et al, 2007). All giant Ankyrins bind CAMs of the L1 family (Chen et al, 2001; Hortsch et al, 2002; Godenschwege et al, 2006; Enneking et al, 2013; Wang et al, 2014; Siegenthaler et al, 2015) and this interaction is essential for the localization of giant Ankyrins to the AIS (Wang et al, 2014).…”