Crystal structure of Ankyrin-G in complex with a fragment of Neurofascin reveals binding mechanisms required for integrity of the axon initial segment.

The axon initial segment (AIS) has characteristically dense clustering of voltage-gated sodium channels (Nav), cell adhesion molecule Neurofascin 186 (Nfasc), and neuronal scaffold protein Ankyrin-G (AnkG) in neurons, which facilitates generation of an action potential and maintenance of axonal polarity. However, the mechanisms underlying AIS assembly, maintenance, and plasticity remain poorly understood. Here, we report the high-resolution crystal structure of the AnkG ankyrin repeat (ANK repeat) domain in complex with its binding site in the Nfasc cytoplasmic tail that shows, in conjunction with binding affinity assays with serial truncation variants, the molecular basis of AnkG-Nfasc binding. We confirm AnkG interacts with the FIGQY motif in Nfasc, and we identify another region required for their high affinity binding. Our structural analysis revealed that ANK repeats form 4 hydrophobic or hydrophilic layers in the AnkG inner groove that coordinate interactions with essential Nfasc residues, including F1202, E1204, and Y1212. Moreover, we show disruption of the AnkG-Nfasc complex abolishes Nfasc enrichment at the AIS in cultured mouse hippocampal neurons. Finally, our structural and biochemical analysis indicated that L1 syndrome-associated mutations in L1CAM, a member of the L1 immunoglobulin family proteins including Nfasc, L1CAM, NrCAM, and CHL1, compromise binding with ankyrins. Taken together, these results define the mechanisms underlying AnkG-Nfasc complex formation and show that AnkG-dependent clustering of Nfasc is required for AIS integrity.

Mind the Gap: Molecular Architecture of the Axon Initial Segment - From Fold Prediction to a Mechanistic Model of Function?

The axon initial segment (AIS) is a distinct neuronal domain, which is responsible for initiating action potentials, and therefore of key importance to neuronal signaling. To determine how it functions, it is necessary to establish which proteins reside there, how they are organized, and what the dynamic features are. Great strides have been made in recent years, and it is now clear that several AIS cytoskeletal and membrane proteins interact to form a higher-order periodic structure. Here we briefly describe AIS function, protein composition and molecular architecture, and discuss perspectives for future structural characterization, and if structure predictions will be able to model complex higher-order assemblies.

Trafficking of Kv2.1 Channels to the Axon Initial Segment by a Novel Nonconventional Secretory Pathway.

Kv2.1 is a major delayed-rectifier voltage-gated potassium channel widely expressed in neurons of the CNS. Kv2.1 localizes in high-density cell-surface clusters in the soma and proximal dendrites as well as in the axon initial segment (AIS). Given the crucial roles of both of these compartments in integrating signal input and then generating output, this localization of Kv2.1 is ideal for regulating the overall excitability of neurons. Here we used fluorescence recovery after photobleaching imaging, mutagenesis, and pharmacological interventions to investigate the molecular mechanisms that control the localization of Kv2.1 in these two different membrane compartments in cultured rat hippocampal neurons of mixed sex. Our data uncover a unique ability of Kv2.1 channels to use two molecularly distinct trafficking pathways to accomplish this. Somatodendritic Kv2.1 channels are targeted by the conventional secretory pathway, whereas axonal Kv2.1 channels are targeted by a nonconventional trafficking pathway independent of the Golgi apparatus. We further identified a new AIS trafficking motif in the C-terminus of Kv2.1, and show that putative phosphorylation sites in this region are critical for the restricted and clustered localization in the AIS. These results indicate that neurons can regulate the expression and clustering of Kv2.1 in different membrane domains independently by using two distinct localization mechanisms, which would allow neurons to precisely control local membrane excitability.SIGNIFICANCE STATEMENT Our study uncovered a novel mechanism that targets the Kv2.1 voltage-gated potassium channel to two distinct trafficking pathways and two distinct subcellular destinations: the somatodendritic plasma membrane and that of the axon initial segment. We also identified a distinct motif, including putative phosphorylation sites, that is important for the AIS localization. This raises the possibility that the destination of a channel protein can be dynamically regulated via changes in post-translational modification, which would impact the excitability of specific membrane compartments.

Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells.

Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, ß1, ß2, ß3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content.