N1E-115-1

Live Labeling of the Axon Initial Segment via Unnatural Amino Acid Incorporation

The axon initial segment (AIS) is a specialized neuronal domain critical for action potential initiation and polarity maintenance. To enable live imaging of AIS components, Stajković et al. developed a method using unnatural amino acid (UAA) incorporation and click chemistry. This approach leverages the small size of UAAs and their flexible insertion sites to label large, structurally complex proteins without disrupting function.

They successfully labeled two major AIS proteins in primary neurons: the 186 kDa neurofascin-186 (NF186) and the 260 kDa voltage-gated sodium channel NaV1.6 (encoded by Scn8a), using conventional and super-resolution microscopy. To validate the technique, they assessed whether UAA tagging altered AIS structure or channel biophysics. Overexpression of clickable NaV1.6 variants did not significantly affect AIS length compared to untransfected neighboring neurons (Fig. 1A, B).

Electrophysiological analysis using whole-cell patch clamp in N1E-115-1 cells revealed that incorporation of the UAA (TCOA-Lys) had minimal impact on channel function. The NaV1.6^K1546TAG variant caused a small but significant depolarizing shift (+2.8 mV) in the fast inactivation curve, slightly slowed inactivation kinetics, and accelerated recovery (Fig. 1C, E). For the NaV1.6^K1425TAG variant, peak sodium current density was reduced compared to wild-type, while other gating parameters remained unchanged (Fig. 4D, F*). Transient transfection efficiency was improved by using a PiggyBac transposase system to stably integrate β-subunits and a P2A-linked eGFP reporter, enabling reliable recording of larger currents.

These results demonstrate that UAA-mediated click labeling provides a robust, minimally invasive strategy for live visualization and functional analysis of AIS proteins, facilitating studies of neuronal excitability and polarity.